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Article

Behavioral and Biochemical Effects of Glyphosate-Based Herbicide Roundup on Unionid Mussels: Are Mussels Good Indicators of Water Pollution with Glyphosate-Based Pesticides?

by
Agnieszka Drewek
1,
Jan Lubawy
2,
Piotr Domek
1,
Jan Polak
3,
Małgorzata Słocińska
2,
Aleksandra Dzięgelewska
2 and
Piotr Klimaszyk
1,*
1
Department of Water Protection, Adam Mickiewicz University, Uniwersytetu Poznańskiego 6, 61-614 Poznan, Poland
2
Department of Animal Physiology and Developmental Biology, Adam Mickiewicz University, 61-614 Poznan, Poland
3
Faculty of Mechanical Engineering, Poznan University of Technology, 60-965 Poznan, Poland
*
Author to whom correspondence should be addressed.
Water 2024, 16(13), 1882; https://doi.org/10.3390/w16131882
Submission received: 30 May 2024 / Revised: 26 June 2024 / Accepted: 28 June 2024 / Published: 1 July 2024
Browse Figures
Versions Notes

Abstract

:
The behavioral (filtration activity) and biochemical (oxidative stress) effects of Roundup 360 Plus (active substance glyphosate) herbicide on two species of unionid mussels, Unio tumidus (Philipsson, 1788) and Anodonta anatina (L.), were evaluated at concentrations ranging from 15 to 1500 μg L−1 of glyphosate for five days. During all experiments, we did not record the mortality of the studied mussel species. Exposure to Roundup herbicide induced dose-dependent filtration disruptions in both U. tumidus and A. anatina. Exposure of the mussels to a low and environmentally relevant concentration 15 µg glyphosate L−1 resulted in a slight (<20%) and temporary decrease in mean valve dilation. Exposure of the mussels to Roundup at relatively high concentrations caused drastic and prolonged shell closure and a reduction in the mussel shell opening rate. Exposure of both mussel species to herbicide resulted in oxidative stress; an increase in superoxide dismutase enzymatic activity was detected. The most significant increase in SOD activity was observed after the exposure to the highest Roundup concentration. However, no correlation between the Roundup concentration and enzymatic activity was found. The use of unionid mussels to detect environmentally relevant concentrations of Roundup, as a part of biological early warning system for pollution, is limited, but they can serve to detect the incidental pollution of aquatic ecosystems with high concentrations of this herbicide.

1. Introduction

Studies on the potential consequences of pesticide contamination on nontarget organisms are numerous and have shown various effects on model organisms living in aquatic environments [1]. Even at very low concentrations, pesticides have lethal effects on aquatic organisms, and at sublethal concentrations, they may can cause behavioral changes [2], reduced growth and developmental rates [3], and an increased frequency of developmental abnormalities [4]. On the other hand, some studies have demonstrated no visible effect of different pesticides on aquatic biota [1]. Moreover, pesticides can increase the effects of other biotic and abiotic stress factors occurring in aquatic ecosystems [5,6].
Glyphosate [(N-phosphonomethyl-glycine; CAS number: 1071-83-6)] is the primary nonselective active ingredient of various pesticides used for the control of weeds in agricultural, urban, and household settings. The biological activity of glyphosate (GLY) involves the inhibition of enolpyruvylshikimate-3-phosphate (EPSP) synthetase, which is not present in animals. Therefore, it is regarded as potentially nonharmful for animals. When GLY reaches soils, due to its high affinity for soil organic matter, it becomes less mobile and less biologically unavailable [7,8,9]. GLY is rapidly degraded by microbial soil communities to sarcosine or aminomethylphosphonic acid (AMPA) [10]. However, due to its high solubility, terrestrial-targeted GLY may be transported by surface runoff to surface waters and leached through soil particles to groundwater. In water, GLY easily undergoes ionization and, as an anion, is strongly absorbed by sediments. This makes it particularly toxic to bottom dwellers and species periodically inhabiting the bottom zone of water bodies [2]. Moreover, to control weeds, GLY herbicides are sometimes applied directly to freshwater ecosystems [9,11,12]. There is a high variability in glyphosate levels across the world’s freshwater ecosystems. The maximum concentration of GLY (about 100 mg L−1) was found in the agricultural region of Argentina, which is a large glyphosate user [13]. Although GLY is frequently recorded in surface waters, its concentrations are usually much lower and even in agricultural areas do not exceed 10 mg L−1 [14]. In trophic diversified lakes of China, the maximum concentration of glyphosate did not exceed 3 µg L−1 [15]. In urban areas of Australia, the highest concentration of glyphosate in surface waters was below 15 µg L−1, while in rural areas GLY was not detected [16]. On the global market, GLY is available as a number of formulations, of which the most popular is Roundup® (RDP). All these products contain different concentrations of GLY and auxiliary components–surfactants that increase the effectiveness of the herbicide [17]. Many studies have shown that in the case of GLY-based herbicides, surfactants are largely responsible for their toxic effects on humans [18,19]. This also applies to other animals, including aquatic organisms. Toxic effects on aquatic organisms are caused by both glyphosate and surfactants, and the magnitude of these impacts, in addition to the pesticide concentration, also depend on a number of environmental factors, such as temperature and pH [11,20,21].
Bivalves, as benthic filtrators, are a group of aquatic organisms that are most strongly exposed to the presence of glyphosate-based pesticides and their metabolites in aquatic ecosystems. They are among the most sensitive groups of freshwater animals and strongly respond to environmental changes [22,23,24]. Despite their wide distribution, estimates indicate that 40% of species are either near-threatened, threatened, or on the brink of extinction [23]. The decline in freshwater mussels, especially those belonging to the order Unionoida, is a global phenomenon, occurring with varying intensities across almost all areas of their distribution [25]. The primary cause of this phenomenon is broadly defined as habitat destruction [26], accounting for more than 75% of cases of drastic population reduction or complete extinction. Within this category, the most influential factors include habitat pollution and the resulting deterioration in water quality, habitat fragmentation, changes in hydrological regimes, and alterations in watershed structure. The global decline in freshwater mussel populations is crucial because they are regarded as key species and environmental engineers that enable the stable functioning of aquatic ecosystems [27]. This function primarily arises from their feeding behavior, as they filter suspensions from the water column, reducing the abundance of phytoplankton, bacteria, and fine particulate organic matter [22]. In particular, species with large body sizes (e.g., Unionidae) exhibit high filtration efficiency [28]. Mussels also serve as essential hosts for the development of eggs and offspring of several bitterling fish species (Rhodeus sp.). The stable functioning of populations of these fish species depends on the presence of freshwater mussels [29].
Mussels are considered as ideal biological indicators in the monitoring of anthropogenic pollution trends [30,31] because they meet almost all required criteria for a useful bioindicator species. Mussels have wide distribution range, well-recognized biology, low mobility, an ability to provide an early alert, a homogeneous response to pollutants, and identifiable toxic effects associated with the degree of pollution [32,33,34,35,36,37,38,39].
A method employed for detecting water pollution (especially incidental events) is the analysis of mussel respiration/filtration behavior. This method is based on the observation and analysis of valve movement, because mussels typically keep their shells open most of the time to breathe and filter suspended matter from the water, closing them for an extended period in response to stressors. The magnitude of the reduction in shell opening and the duration of this reaction is dependent on stress intensity [34,40,41,42].
The aim of this study was to analyze the effect of the glyphosate-based herbicide Roundup 365 Plus on the filtration activity of two widely distributed species of bivalves of the genus Unionidea: Anodonta anatina and Unio tumidus. It was also determined whether exposure of mussels to different doses of RDP would result in increased symptoms of oxidative stress. The results reveal whether the bivalves are sensitive to RDP at environmentally relevant concentrations and may be useful for detecting the incidental pollution of aquatic environments caused by this herbicide.

2. Materials and Methods

2.1. Pesticides Used

Commercially acquired Roundup 360 Plus (Bayer, Warsaw, Poland) was used in the surveys. The solution contained glyphosate potassium salt (CAS-70901-12-1) and 5EO isotridecyletherpropylamin, a POEA surfactant (CAS-68478-96-6), imitating realistic environmental pollution. All concentrations used in the experiments were recalculated to show the amount of glyphosate in the solutions. We used a range of concentrations, starting from 15 µg GLY L−1, which is recorded in aquatic ecosystems [13,43], and concentrations ten and a hundred times greater (150 and 1500 µg GLY L−1).

2.2. Mussel Collection and Preparation for Experiments

This study was carried out on two species of bivalve mollusks from the Unionidae family: Anodonta anatina and Unio tumidus. Animals and water were collected in 2021 from a mesotrophic mid-forest seepage lake (N 52°42′34.46″, E 16°08′34.76″) where the catchment areas have never been treated with glyphosate-based herbicides. The experiments were conducted between May 2021 and September 2021. Before the experiments, the mussels were acclimatized for at least 14 days in laboratory conditions, kept in dechlorinated and oxygenated water (16 °C), and fed on dry Chlorella sp. and Spirulina sp. commercial mix (1:1, w/w) weekly. The individuals selected for the surveys were of similar size and weight (A. anatina 7.4 ± 0.6 cm long; and 34.2 ± 5.1 g; U. tumidus 5.4 ± 0.3 cm long and 20.6 ± 2.3 g).

2.3. Monitoring of Mussel Filtration Behavior

The filtration behavior surveys were based on the opening of individual mussel valves (according to Kramer and Foekema [44]). In each experiment, 8 individuals of one species were placed in a 20 L throughflow, aerated water tank, filled with conditioned and filtered (net mesh size of 20 μm) lake water. The water temperature during the experiments was kept constant at 16 ± 0.5 °C.
The monitoring system consisted of 4 components: sensors, data-reading processing module, database, and web interface (Figure 1). The system used linear Hall effect sensors coupled with pill neodymium magnets to measure the valve position. Each mussel was attached by one valve with nontoxic glue to a dedicated stand, and a neodymium pill magnet was glued to the second valve. Watertight sensors were mounted to stands, allowing manual adjustment of relative positions between the sensor and magnet (Figure 2). The data-reading processing module was hardware that collected and transformed analog signals coming from the sensors and sent persisting data into the database. The data from each sensor were recorded every 30 s. The module was designed to be weatherproof, allowing it to be located in proximity to the water tank. The web interface was used for reading and transferring data from the database.
Three experiments were conducted for each mussel species. In every experiment, individuals were exposed to one of three RDP concentration (15 µg GLY L−1, 150 µg GLY L−1, or 1500 µg GLY L−1) and the behavioral response was observed. Each experiment consisted of three subsequent stages: calibration, control period, and the study period.
At the beginning of each experiment, the system was calibrated for 24 h. During calibration for each mussel specimen, extreme values of valve position were determined, and it was assumed that maximum value (magnet close to the sensor) indicated a closed shell (0%), and the minimum value (magnet far from the sensor) indicated 100% open valves (Figure 2). In the subsequent 24 h, the filtration behavior of each mussel in conditions without RDP was noted (control period). After that time, the pesticide was applied. Observations of mussel filtering behavior were conducted for the next 120 h–5 days (the study period).

2.4. Oxidative Stress Analyses

Mussel gill excision for an oxidative stress marker analyses was performed after the behavioral experiments. The specimens were cleaned of surface contaminants, first in tap water and then in deionized water, and then they were dried using paper tissues. Excised gills intended for biochemical analyses were shock-frozen in liquid nitrogen. Gills samples (~0.5 g) were homogenized in chilled 0.1% (w/v) trichloroacetic acid (TCA) using a hand mortar (Kimble Chase, NJ, USA). After centrifugation (12,000× g, 20 min, 4 °C), the obtained supernatant was collected, and the protein concentration was measured using infrared spectrometer (Direct Detect, Merck, Darmstadt, Germany) according to methods described previously by Szymczak-Cendlak et al. [45]. Next, the samples were subjected to colorimetric assays for thiobarbituric acid reactive substances (TBARS) and superoxide dismutase (SOD).
The level of lipid peroxidation was determined by measuring the concentration of compounds reacting with thiobarbituric acid (TBARS) using a TBARS Assay Kit (700870, Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. One hundred microliters of the sample obtained as described above was combined with 100 µL of 10% trichloroacetic acid (TCA) and 800 µL of the coloring reagent (thiobarbituric acid (TBA), acetic acid, sodium hydroxide) in a 2 mL capacity vial. The vials were then placed in boiling water. After 1 h, they were placed on ice and incubated for 10 min to stop the reaction. After centrifugation for 10 min at 1600× g at 4 °C, the absorbance of the samples was measured at a wavelength of 530 nm using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Santa Clara, CA, USA). The results were calculated from the standard curve of malondialdehyde (MDA) and are presented as µM MDA per µg of protein.
A Superoxide Dismutase Assay Kit (706002, Cayman Chemical) was used to determine the activity of superoxide dismutase (SOD). To measure SOD activity, 10 µL of the sample obtained as described above was combined with 200 µL of the radical detector (tetrazolium salt diluted in 50 mM Tris-HCl, pH = 8.0 containing 0.1 mM diethylenetriaminepentaacetic acid (DPTA) and 0.1 mM hypoxanthine) in a 96-well microplate. To initiate the reaction, 20 µL of xanthine oxidase was added to each well, and the plate was incubated for 30 min at room temperature on a shaker. After incubation, the absorbance of the samples was read at 540 nm using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Santa Clara, CA, USA). The results were calculated from the standard curve of SOD isolated from bovine erythrocytes and are presented as units of SOD activity per milliliter per µg of protein (U/mL/µg).

2.5. Statistical Analysis

Differences in mussel activity between the control and study periods were compared by one-way ANOVA. Dunn’s multiple comparisons test was employed as a post-hoc analysis for testing the significance of differences between the control period and subsequent days of the experiment The significance of differences of results of oxidative stress surveys was determined with the non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons test. Analyses were performed using the Statistica 12 (TIBCO Software Inc., Santa Clara, CA, USA).

3. Results

3.1. Mussel Filtration Behavior

During the control period, when the mussels were not exposed to the herbicide, the average shell opening was high for both mussel species, ranging from 62% to almost 80% (Figure 3). During the control, individuals snapped their valves intermittently, but the shells were never completely closed.
Exposure to RDP herbicide induced dose-dependent filtration abnormalities in both species. During the survey with the lowest concentration of herbicide (15 µg GLY L−1), the application of the RDP induced a response—a reduction in the valves opening (Figure 3). The average shell dilation of the A. anatina population decreased from almost 70% to 43%. These values remained for approximately 1.5 h, after which the average opening of the shells began to slowly increase. In particular, for specimens of A. anatina, the response to the appearance of the herbicide varied. Three individuals did not react significantly, while one completely closed its shell. On the second day after the herbicide exposure, the A. anatina filtration activity decreased, and the average shell opening was slightly lower than 40%. On subsequent days, no differences were noted compared to the control (Figure 4). The response of U. tumidus to the lower concentrations of RDP was less distinct. After herbicide application, the average shell opening decreased from 70% to 60% and then 55%, and remained at this level for a few hours. However, the lowest average opening was lower (47%) during the control period. The lowest average filtering activity was observed on the second day after herbicide administration, with a valve opening of lower than 40% (Figure 3). Despite the fact that the differences in the average opening of the shells on individual days of the experiments were low, statistical analysis revealed a significant difference between the control and the study period (Figure 4).
Exposure to RDP at concentrations of 150 and 1500 µg GLY L−1 induced much more rapid and prolonged disruption of filtration activity in both A. anatina and U. tumidus (Figure 3). Exposure to RDP, at a concentration of 150 µg GLY L−1, induced a sharp reduction in A. anatina filtration activity. The average shell opening decreased from 82 to 14% in the first few minutes. Most individuals stopped filtration completely. This state continued for a few hours, after which the average shell opening gradually increased. The average shell opening on the second day after RDP administration was approximately 40%, and on the following days, it was approximately 60%. A similar trend was found for U. tumidus, although here, the reduction in filtration activity was somewhat less intense than that for A. anatina. The minimum average valve opening (24%) was recorded approximately one hour after the RDP exposure. Statistical analysis revealed a significant difference in shell opening between the control and the study periods (H = 368.7 p < 0.001 and H = 584.9 p < 0.001, respectively) for A. anatina and U. tumidus. A post hoc Dunn test revealed significantly lower mussel activity in each day after exposure to RDP (Figure 4). The addition of RDP at a ten-times greater concentration (1500 µg GLY L−1) resulted in a rapid and long-lasting reduction in filtration activity in both mussel species tested. A few minutes after herbicide administration, the shells of all individuals of A. anatina closed completely. In the first 24 h after exposure, the maximum average opening did not exceed 20%. For the next 2 days, the filtration activity of A. anatina was low (most individuals remained closed), and the average shell opening of the test population did not exceed 30%. In the following days, the mussel activity increased to approximately 40%. U. tumidus responded similarly to exposure to the highest concentration of RDP. Just a moment after adding RDP to the water, most individuals closed their shells, and the minimum average shell opening was 10%. During the first two days after exposure, most of the individuals reduced their filtration completely, and then, the bivalves gradually began to open their shells, reaching an opening of approximately 60% on the last day of the experiment. Similar to the ten-times lower concentration, statistical analysis (ANOVA and post hoc Dunn test) revealed a significant difference in shell opening between the control and each day after exposure to RDP for both species (Figure 4).

3.2. Oxidative Stress

Exposure of both studied mussel species to RDP resulted in an increase in SOD enzymatic activity (Figure 5). In the case of A. anatina, the greatest increase in SOD activity compared to that in the control group was noted after exposure to the lowest RDP concentration of (15 µg GLY L−1), and the average level of superoxide dismutase activity in gills tissue was more than 40% greater (Figure 5). At higher concentrations of the RDP, the differences compared to those in the control group were less significant. Additionally for U. tumidus, exposure to the pesticide resulted in increased levels of SOD activity. In this species, the most significant increase in SOD activity was observed after the exposure to the highest RDP concentration (Figure 5). Statistical analysis did not reveal significant differences in SOD activity between the control and test groups. However, for the lowest dose for A. anatina and the highest for U. tumidus, the post hoc test values were p = 0.0542 and p = 0.0580, respectively.
A. anatina showed elevated mean values of the oxidative injury marker (TBARS) when exposed to RDP at a concentration of 150 mg GLY L−1. When the mussels were exposed to a concentration of 1500 mg GLY L−1, the TBARS was 150% greater than that in the control group. The opposite trend was observed for U. tumidus. For this species, TBARS reached its highest value when exposed to the lowest concentration of the RDP (approximately 70% higher than that in the control). Exposure to higher RDP concentrations resulted in a decrease in the TBARS marker value, and at the highest RDP concentration, the level of the oxidative stress marker was comparable to that observed in the control group (Figure 5). Similar to those for SOD, the mean TBARS values in groups did not significantly differ among the groups.

4. Discussion

Over the past few decades, the impact of GLY and GLY-based herbicides on diverse aquatic vertebrates and invertebrates has been studied. Exposure to GLY has been associated with various alterations in fish, including disruptions in hematologic and biochemical processes, genotoxic effects, histopathological lesions, immunotoxic reactions, and cardiotoxic responses. In invertebrate species, GLY exposure has been linked to changes in biochemical processes within tissues, developmental shifts, alterations in behavior, modifications in hemolymph composition, impacts on reproductive systems, and a decrease in cholinesterase activity [2,12,17,20,46,47]. It is worth noting that most of the observations come from acute toxicity tests, where specimens were exposed to doses of GLY that exceeded environmentally relevant concentrations. There are limited data on the chronic effects of glyphosate on nontarget aquatic organisms and the effects of this herbicide on entire aquatic biocoenoses. In this study, the herbicide Roundup 360 Plus, which is a mixture of GLY (sodium salt) and surfactant POEA, was used. Exposure to the mixture of POEA and GLY increases both the bioaccumulation of GLY in aquatic organisms and the magnitude of its toxic effects. POEA increases the permeability of cells, enhancing the transport and accumulation of GLY [17,21,48]. Compared to those of GLY, the lethal concentrations (LC50) of the POEA+GLY mixture are many times lower for fish and amphibians [20,46,49,50,51].
For both studied mussel species, changes in filtration behavior were observed after subchronic exposure to the glyphosate-based herbicide Roundup 360 Plus. The degree of reduction in filtration activity and the time over which this response occurred depended on the dose of herbicide used. When exposed to the lowest and environmentally relevant concentration of 15 µg GLY L−1, the reduction in the average opening of the shells of both mussel species was slight and short-lasting. Chmist et al. [42] also reported no significant reduction in the filtration activity of unionid mussels exposed to similar concentrations of the pesticides DDT, thiacloprid, and tebuconazole. However, subchronic exposure to the herbicide lenacil and the fungicide tebuconazole (both at a concentration of 10 µg L−1) reduced the average opening of U. tumidus populations from approximately 60% to 12% and 20%, respectively. At the same time, the maximum reduction in shell opening was observed for lenacil on day 3 after pesticide administration and for tebuconazole on the first day but several hours after exposure. Exposure to comparable concentrations of the pyrethroid pesticide cyphermetrin did not result in a reduction in the filtration activity of the marine bivalve Mytillus galloprovincialis. The first symptoms of avoidance behavior were found when individuals were exposed to a concentration of 100 µg L−1 [52]. At comparable concentrations of Roundup herbicide (150 µg L−1), the response of both A. anatina and U. tumidus was rapid and prolonged. After approximately one day for the first species and two days for the second species, the filtration activity gradually increased to pre-herbicide levels. The reason for this trend is likely that mussels have the ability to reduce glyphosate loads in the water [53,54]. With decreasing water pollution, the energetically costly cessation of respiration and feeding [44] results in more losses than the toxic effects of the herbicide. Exposure of the mussels to RDP at a concentration of 1500 µg GLY L−1 caused drastic and prolonged shell closure. The reduction in the mussel shell opening rate was also notable. A similar mussel reaction to pesticide exposure was also reported by Chmist et al. [42]. Reduced locomotor activity in mussel shells after a long period of valve shutting is associated with adductor muscle fatigue [42,55,56]. During all tests, we did not observe mussel mortality or complete relaxation of the adductor muscles, symptoms that are observed with prolonged exposure of mussels to toxicants [42].
Glyphosate or commercial formulations such as Roundup have been shown to cause oxidative stress responses in a variety of aquatic animals. This effect is most likely caused by the ability of glyphosate to chelate minerals, particularly manganese. This may lead to a deficiency of Mn in mitochondria, which is a key component of the SOD [57]. Superoxide dismutase is one of the main antioxidant enzymes that alternately catalyzes the dismutation of the superoxide (O2•−) anion radicals into molecular oxygen and hydrogen peroxide (H2O2). This makes it an important enzyme in the oxidative stress response. During normoxia, there is a steady-state balance between reactive oxygen species (ROS) and cellular antioxidant systems. However, when this finely tuned balance is disrupted, oxidative stress occurs. The increased production of ROS occurs when animals are exposed to xenobiotics and/or environmental factors, which can result in the onset of cellular dysfunction and apoptosis [58]. SODs are thought to provide a first line of defense against O2•−, which is the major ROS produced by mitochondrial respiration and various metabolic reactions. Previous studies have shown that exposure to GLY-based herbicides can induce oxidative stress in aquatic organisms. Exposure of the fish Prochilodus lineatus to increasing concentrations of RDP enhanced ROS generation and simultaneously inhibited antioxidant activity [59], and similar effects were observed in Danio rerio after exposure to 10 mg/L of glyphosate and Roundup [60]. In mollusks such as the Pacific oyster Crassostrea gigas, exposure to GLY can cause changes in xenobiotic detoxification activity [61] or increased glutathione-s-transferase (GST) and alkaline phosphatase (ALP) activities, as observed in the mussel Limnoperna fortune [62]. Similarly, GLY decreases SOD activity in the smooth scallop Flexopecten glaber [63]. However, for the lowest and highest doses of GLY for A. anatina and U. tumidus, respectively, the statistics were slightly below the significance limit. Overall, we did not observe significant changes in SOD activity or oxidative damage, as measured by lipid peroxidation. Our results could be explained by the fact that SOD enzymes are not the only enzymes in the whole antioxidant machinery. Similar results to ours were obtained for L. fortune, in which catalase, SOD, and acetylocholine esterase activities did not differ between the exposed and nonexposed individuals. Additionally, no oxidative damage to lipids, which was measured in the same way as in this study using TBARS, was observed in response to glyphosate [64]. However, the activities of other detoxifying and antioxidant enzymes of this mussel were significantly increased, namely, GST, carboxylesterases (CES), and the general metabolism enzyme ALP [64]. Therefore, in A. anatina and U. tumidus, these enzymes are mainly involved in the response to glyphosate.
Most mussel biological early warning systems (BEWS) are based on changes in shell opening [65]. In our study, both mussel species exhibited reduced valve opening after exposure to the Roundup at a dose of 15 µg GLY L−1 (and the difference from the control period was statistically significant). However, at the lowest tested RDP concentration, the reaction was too weak to be useful as an alarm indicator in BEWS. The most defined reaction to the exposure to toxicants, regarded as an alarm level, is a decrease in the average valve opening of mussels below 20% or a reduction in opening of more than 25% within 15 min after administration of the toxicant compared to the average opening one hour before exposure [44,66]. The second parameter, however, is not applicable in BEWS due to the unknown time of the onset of the toxic substance. Exposure of A. anatina and U. tumidus to higher concentrations of Roundup caused a rapid and prolonged response in filtration rate (Table 1). Although the mussels did not show a significant response to environmentally relevant concentrations of Roundup, the study demonstrated that BEWS systems based on these mussel species can be useful for detecting incidental water pollution caused by high doses of glyphosate-based herbicides.

5. Conclusions

Subchronic exposure of unionid mussels results in a dose-dependent behavioral response. Environmentally relevant doses of Roundup herbicide cause minor and short-lasting disturbances in the filtration behavior of the surveyed mussel species. Higher RDP doses, which are sometimes recorded in freshwater ecosystems, cause a drastic reduction in the filtration activity of A. anatina and U. tumitus. This demonstrates that the incidental pollution of aquatic ecosystems caused by GLY-based herbicides can threaten bivalve populations. Exposure of bivalves to RDP results in changes in oxidative stress markers, although the results obtained were not conclusive. A comparison of the two tested species reveals that behavioral changes are more severe in A. anatina, which may suggest that this species is more sensitive to Roundup contamination.
Since GLY is globally used, and mussels are key elements for the stable functioning of aquatic ecosystems, the issue of the effects of this herbicide on mussels should be comprehensively recognized. The toxic effects of chronic exposure of mussels to glyphosate and the rate and level of accumulation of glyphosate and its metabolites (e.g., AMPA) in tissues should be identified. Also, the data on the potential effects of GLY-based herbicides on juvenile mussel stages: glochidia, juvenile, or sub-adult individuals, which are more sensitive to environmental alteration, are still lacking.

Author Contributions

A.D. (Agnieszka Drewek): conceptualization, methodology, resources, validation, formal analysis, investigation, data curation, and writing—original draft preparation; J.L.: methodology, validation, formal analysis, investigation, data curation, and writing—original draft preparation; P.D.: resources and investigation; J.P.: methodology, software, and writing—original draft preparation; M.S.: validation, formal analysis, and investigation; A.D. (Aleksandra Dzięgelewska): resources and investigation; P.K.: conceptualization, methodology, resources, validation, formal analysis, investigation, data curation, writing—original draft preparation, supervision, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors would like to thank Kaira Laurell Kamke for the proof reading of the final version of the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 16 01882 g001
Figure 1. Scheme of mussel activity testing system: (A) mussel with magnet and Hall sensor (B) data processing module with software (C) computer.
Figure 1. Scheme of mussel activity testing system: (A) mussel with magnet and Hall sensor (B) data processing module with software (C) computer.
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Water 16 01882 g002
Figure 2. The set of U. tumidus mussels with magnets and Hall sensors.
Figure 2. The set of U. tumidus mussels with magnets and Hall sensors.
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Water 16 01882 g003
Figure 3. The average valves opening in control period and after mussel exposure to different doses of Roundup herbicide (the red dashed line marks the beginning of the exposure).
Figure 3. The average valves opening in control period and after mussel exposure to different doses of Roundup herbicide (the red dashed line marks the beginning of the exposure).
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Figure 4. The average valves opening (bars) and SE (whiskers) in control and subsequent experiment days along with Dunn test results. (* p < 0.05, ** p < 0.005, *** p < 0.001).
Figure 4. The average valves opening (bars) and SE (whiskers) in control and subsequent experiment days along with Dunn test results. (* p < 0.05, ** p < 0.005, *** p < 0.001).
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Water 16 01882 g005
Figure 5. The average (box) and SE (whisker) values of superoxide dismutase (SOD) activity and lipid peroxidation level (TBARS) in A. anatina (green) and U. tumidus (blue) gill tissues after the exposure to different concentration of Roundup pesticide.
Figure 5. The average (box) and SE (whisker) values of superoxide dismutase (SOD) activity and lipid peroxidation level (TBARS) in A. anatina (green) and U. tumidus (blue) gill tissues after the exposure to different concentration of Roundup pesticide.
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Table 1. Lowest average valve opening after exposure to Roundup (LOE) and reduction in average valve opening in 15 min after herbicide exposure to 1 h before exposure (BAR).
Table 1. Lowest average valve opening after exposure to Roundup (LOE) and reduction in average valve opening in 15 min after herbicide exposure to 1 h before exposure (BAR).
A. anatinaU. tumidus
15
µg GLY L−1		150
µg GLY L−1		1500
µg GLY L−1		15
µg GLY L−1		150
µg GLY L−1		1500
µg GLY L−1
LOE (%)44103532714
BAR (%)197588154975
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Drewek, A.; Lubawy, J.; Domek, P.; Polak, J.; Słocińska, M.; Dzięgelewska, A.; Klimaszyk, P. Behavioral and Biochemical Effects of Glyphosate-Based Herbicide Roundup on Unionid Mussels: Are Mussels Good Indicators of Water Pollution with Glyphosate-Based Pesticides? Water 2024, 16, 1882. https://doi.org/10.3390/w16131882

AMA Style

Drewek A, Lubawy J, Domek P, Polak J, Słocińska M, Dzięgelewska A, Klimaszyk P. Behavioral and Biochemical Effects of Glyphosate-Based Herbicide Roundup on Unionid Mussels: Are Mussels Good Indicators of Water Pollution with Glyphosate-Based Pesticides? Water. 2024; 16(13):1882. https://doi.org/10.3390/w16131882

Chicago/Turabian Style

Drewek, Agnieszka, Jan Lubawy, Piotr Domek, Jan Polak, Małgorzata Słocińska, Aleksandra Dzięgelewska, and Piotr Klimaszyk. 2024. "Behavioral and Biochemical Effects of Glyphosate-Based Herbicide Roundup on Unionid Mussels: Are Mussels Good Indicators of Water Pollution with Glyphosate-Based Pesticides?" Water 16, no. 13: 1882. https://doi.org/10.3390/w16131882

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