Behavioral Responses of Unio tumidus Freshwater Mussels to Neonicotinoid Pesticide Contamination

Abstract

This investigation examined the behavioral responses of freshwater mussels to neonicotinoid pesticide exposure, a widely implemented agricultural crop protection agent. The study systematically evaluated the behavioral modifications of U. tumidus under controlled laboratory conditions, with particular emphasis on shell dynamics and activity patterns during both acute (2.5 h) and prolonged (20 h) exposure to imidacloprid at 50 µg/L concentration. The experimental findings revealed consistent and statistically significant behavioral alterations. Upon initial pesticide exposure, specimens exhibited an immediate reduction in shell aperture, followed by a sustained period of enhanced opening amplitude. Activity metrics demonstrated acute elevation immediately post-exposure, indicative of stress-induced responses, subsequently achieving homeostatic equilibrium before declining in later temporal phases. These behavioral modifications demonstrated statistical significance across all four experimental iterations, indicating a robust and reproducible stress response pattern. This study demonstrated that exposure to high concentrations of the neonicotinoid pesticide imidacloprid significantly affects the freshwater mussel causing significant, repeatable changes in mussel behavior: initial shell closure followed by prolonged opening and increased activity, indicating stress and subsequent toxic effects.

1. Introduction

The widespread use of pesticides in modern agriculture has undoubtedly increased crop yields and food production efficiency. These substances are widely used to control pests and increase yields, but their excessive and irrational application has led to significant contamination of both terrestrial and aquatic ecosystems [1]. The use of pesticides in agriculture has been increasing from year to year, particularly in more economically developed countries. Pesticides have brought many benefits to agriculture and industry, but their harmful effects on humans and animals have always been a source of concern. The overuse of pesticides and their long-term impact on the environment continues to pose significant challenges [2]. Pesticides can be found everywhere in honey, fruits, vegetables, cereals, and drinking water, but also in human hair, which is a particular threat to human health. They are also a threat to aquatic ecosystems and biodiversity. When pesticides enter water bodies through runoff, leaching, or atmospheric deposition, they pose a serious threat to non-target organisms. Excessive concentrations of these chemicals can disrupt fundamental ecological processes, leading to changes in community structures and, in severe cases, the collapse of aquatic populations [3,4]. The impact of pesticides extends to various trophic levels, affecting not only primary consumers such as invertebrates but also the higher trophic levels that depend on them. Therefore, understanding the effects of pesticide exposure on sensitive aquatic species is essential for assessing ecosystem health and developing sustainable management strategies [5].

Neonicotinoid pesticides are extensively used directly on crops to protect plants, as well as for seed treatments, leaf spraying, and soil drenching. Due to the high water solubility of neonicotinoids, concerns have arisen about the risk of their entry into water bodies and the environment, posing threats to aquatic organisms [6,7]. Under simulated environmental conditions, neonicotinoids are readily leached from soil, plants, and seeds into water [8,9]. It has been established that neonicotinoids infiltrate watercourses through direct leaching into groundwater, which then enters surface waters through the decomposition of treated plants in waterways, direct contact with dust stirred up during the sowing of treated seeds, and drift of sprays into water bodies [10,11]. The main effects resulting from exposure to imidacloprid include negative impacts on the central nervous system by altering levels of metabolites such as nucleotides and amino acids, reducing filtration and the exchange of choline and acetylcholine [12], as well as decreasing heart rate and gill ventilation, which lead to death. This impact varies and depends on the species of the contaminated animal but also on the concentration of this pesticide [13].

Molluscs, representing one of the most species-rich phyla in the animal kingdom (over 130,000 known extant species), play an important role in environmental biomonitoring. Particularly bivalves, constituting about 15% of all mollusc species (20,500 species), and gastropods (110,000 species) demonstrate exceptional qualities as indicator organisms. The sedentary lifestyle of adult specimens enables local monitoring and also limits their ability to escape from contaminated areas. It should also be noted that bivalves not only accumulate substances from food but also from water, which is their living environment, resulting in accumulation through absorption by their entire body. The biology and ecology of these organisms are well understood, and their monitoring has a long history, notably including the “Mussel Watch” program, which was initiated in 1986 [14,15]. Benthic macroinvertebrates, particularly bivalve mollusks, have proven to be valuable bioindicators due to their sensitivity to a range of pollutants, including pesticides, heavy metals, microplastic, wastewater, and nutrient enrichment [16,17,18,19,20,21,22,23]. Mussels respond to water quality changes through observable behaviors such as shell opening and closing, which can be monitored in real time using valvometry [24]. As filter feeders, mussels are particularly susceptible to various organic and inorganic pollutants dissolved in the water [25].

Unio tumidus, a widespread species in Europe’s freshwater bodies, is particularly well suited for biomonitoring [26,27,28,29]. Its sedentary lifestyle and filter-feeding behavior make it highly responsive to environmental disturbances, while its abundance allows for its use in ecotoxicological studies without impacting local populations. Although U. tumidus has been shown to be sensitive to various pollutants [17,18,19,28,30] and it is regarded as a valuable bioindicator of environmental pollution [31], its reaction to pesticides remains relatively understudied [18,28,30]. The majority of research on neonicotinoid toxicity has focused on pollinators, particularly bees [32]. However, in recent years, there has been growing attention to the effects of neonicotinoids on aquatic organisms [32,33,34,35]. Despite this increased interest, data on both acute and chronic toxicity of neonicotinoids for aquatic organisms remain limited, with imidacloprid being the most extensively studied compound in this context [34,36].

Given the increasing detection of neonicotinoids in aquatic environments and potential adverse effects on non-target organisms, it is necessary to investigate how U. tumidus responds behaviorally to imidacloprid exposure. Such studies may provide insights into the sublethal effects of neonicotinoids on freshwater mussels and enhance the utility of U. tumidus as a bioindicator species.

This study investigates the impact of pesticides on aquatic organisms. Specifically, we aimed to evaluate the effects of imidacloprid, a neonicotinoid pesticide, on the behavior of freshwater mussels (Unio tumidus). We examined both short- and long-term behavioral responses to pesticide exposure, hypothesizing that (1) imidacloprid would significantly alter bivalve behavior and (2) behavioral responses would evolve over time following exposure. We hypothesize that: imidacloprid exposure will significantly alter the behavior of U. tumidus by affecting shell movement dynamics and activity levels due to its neurotoxic effects. By investigating these hypotheses, the study seeks to fill the existing research gap and contribute valuable data on the sublethal effects of neonicotinoids on freshwater bivalves. The findings will have implications for environmental monitoring and risk assessment protocols, emphasizing the role of U. tumidus as a reliable bioindicator species for detecting pesticide contamination in aquatic ecosystems.

2. Materials and Methods

2.1. Materials and Model Organism

Mussels (Unio tumidus) were collected from Lake Wolsztyńskie in the Wolsztyn community, approximately 80 km from the biomonitoring laboratory at Poznań University of Life Sciences, Faculty of Environmental and Mechanical Engineering, Department of Ecology and Environmental Protection. Samples were gathered using rakes at depths of 0.5 to 1.0 m. Mature mussels (3–6 cm in length) were selected using vernier calipers. After collection, the mussels were manually cleaned and stored in a 10 L mobile fridge filled with 8 L of lake water to maintain optimal conditions during transport.

Upon arrival at the laboratory, mussels were transferred to an acclimatization aquarium for two weeks to minimize stress and allow adaptation to laboratory conditions before use in experimental studies. This step ensured the organisms’ well-being, as mussels are highly sensitive to environmental changes.

2.2. Construction of the Monitoring System

A standard Biological Early Warning System (SYMBIO) was employed in this study, with its design illustrated in Figure 1. Shell movements were monitored using Hall sensors, which measure magnetic field changes caused by the movement of a ferrite magnet (8 mm diameter) attached to one shell half. The sensors recorded continuous data on shell opening levels, which were processed and visualized by dedicated software. Calibration established the minimum and maximum shell-opening ranges for each individual mussel.

For experiments, eight mussels were placed in a 150 × 25 × 25 cm aquarium filled with 60 L of aerated commercially distributed spring water. Water parameters were maintained at a temperature of 14–16 °C, pH of 8.0–8.4, and dissolved oxygen (DO) levels of 7.8–9.1 mg/L. An air pump ensured adequate aeration, and an aquarium chiller regulated the water temperature. Control conditions were defined as five days in clean water at 10–12 ± 1 °C, during which mussel behavior remained stable.

2.3. Pesticides Tested

This study utilized imidacloprid, a neonicotinoid pesticide, to assess behavioral responses in mussels. Four experimental series were conducted:

Experiment 1: 26 January 2023

Experiment 2: 7 July 2023

Experiment 3: 29 August 2023

Experiment 4: 3 January 2024

During experiments, imidacloprid was introduced at a concentration of 50 µg/L. The system continuously monitored shell movements, with data recorded 50 times per minute, amounting to approximately 72,000 records daily per individual. Short-term reactions were analyzed over 2.5 h, with a 0.5 h control period and 2 h following pesticide exposure. Long-term reactions were evaluated over 20 h, with a 5 h control period and 15 h of pesticide exposure.

2.4. Experimental Setup and Behavioral Assessment

Each mussel was mounted on a methacrylate holder and equipped with a magnet and Hall sensor, which detected shell movements by measuring changes in magnetic field strength. The collected data were processed to calculate the degree of shell opening (average percentage) and activity levels (average standard deviation in 1 min intervals). Behavioral responses were compared between control conditions and pesticide exposure periods.

The setup ensured precise monitoring and analysis of mussel behavior under controlled conditions, enabling detailed assessments of the effects of imidacloprid. No mortality was observed during the acclimatization or experimental periods, and all mussels were of uniform size (6 ± 1 cm length, 3.5 ± 0.5 cm width) and estimated age (6–9 years).

The significance of differences between the calculated means was tested based on ANOVA, and differences between individual intervals were assessed using Tukey’s HSD post hoc test. STATISTICA 13.0 software was used for statistical analyses.

3. Results

3.1. Analysis for 5 min Intervals

In the experiment, the change in mussel behavior under the influence of a high concentration of the pesticide (imidacloprid at a concentration of 50 µg/L) was analyzed in 5 min intervals. Changes in the average level of mussel shell opening and the intensity of these changes were observed in control conditions and after the pesticide was administered (

Table 1. Average shell opening (%) and mussel activity (average standard deviation in 1 min intervals) for 5 min intervals; control in time intervals 1–6, pesticide application in intervals 7–30.

Interval Number	Experiment 1		Experiment 2		Experiment 3		Experiment 4
	Opening (%)	Activity	Opening (%)	Activity	Opening (%)	Activity	Opening (%)	Activity
1	43.16	0.09	43.75	0.09	43.16	0.00	43.09	0.14
2	43.00	0.15	43.72	0.14	42.82	0.30	43.16	0.12
3	43.43	0.08	43.43	0.16	43.13	0.07	42.79	0.27
4	43.32	0.06	42.97	0.13	43.38	0.03	42.46	0.16
5	42.54	0.12	43.03	0.16	43.45	0.10	42.62	0.13
6	41.72	0.23	42.78	0.53	43.58	0.08	43.07	0.08
7	39.17	0.35	38.76	0.38	35.64	1.22	42.69	0.26
8	37.55	0.35	38.35	0.60	34.43	0.54	41.33	0.27
9	44.09	0.77	40.48	2.44	41.89	1.81	46.51	2.37
10	51.22	0.93	55.10	1.97	58.68	2.63	53.62	1.53
11	69.68	1.10	67.76	1.10	75.88	1.36	61.74	1.05
12	73.32	0.96	71.83	0.90	77.10	1.06	66.17	0.75
13	73.72	0.95	72.06	0.81	76.29	0.85	69.03	1.05
14	73.81	0.79	78.48	0.49	77.11	0.39	71.65	0.80
15	74.75	0.69	79.22	0.36	77.83	0.62	71.45	0.41
16	76.37	0.78	79.08	0.27	77.04	1.11	70.92	0.61
17	77.67	0.80	80.14	0.23	79.16	0.45	70.50	0.56
18	78.61	0.50	81.77	0.33	78.81	0.48	69.32	0.60
19	79.20	0.53	81.85	0.19	79.66	0.33	65.97	1.14
20	80.76	0.18	82.97	0.30	77.78	1.35	65.94	0.52
21	81.17	0.40	83.29	0.17	79.49	0.37	66.08	0.35
22	81.84	0.18	84.03	0.23	79.09	0.18	67.14	0.19
23	82.26	0.17	84.24	0.26	78.36	0.26	67.05	0.27
24	82.30	0.25	84.50	0.28	77.91	0.62	67.26	0.55
25	82.44	0.13	85.05	0.29	77.66	0.56	67.10	0.69
26	82.55	0.21	85.14	0.33	77.89	0.18	67.94	0.18
27	81.94	0.29	85.59	0.22	77.04	0.36	68.11	0.25
28	81.82	0.29	86.32	0.36	76.70	0.34	68.18	0.07
29	81.89	0.33	86.10	0.24	76.99	0.32	68.36	0.04
30	81.34	0.23	85.53	0.22	76.45	0.22	67.98	0.31

).

The significance of differences between the calculated means was tested based on ANOVA (

Table 2. Analysis of variance for average shell opening and average clam activity in 5 min intervals; significant effects at p < 0.01 are marked in red, MS—the mean sum of squares, SS—the sum of squares.

Experiment	Variable	SS	MS	SS	MS	F	p
1	Opening	45,125	1556.1	235.9	1.97	791.6	<0.01
	Activity	14	0.5	14.3	0.12	4.2	<0.01
2	Opening	49,838	1718.6	358.2	2.98	575.7	<0.01
	Activity	40	1.4	11.5	0.10	14.7	<0.01
3	Opening	42,122	1452.57	554.5	4.62	314.3	<0.01
	Activity	51	1.77	53.3	0.44	4.0	<0.01
4	Opening	19,899	686.2	116.2	0.97	708.6	<0.01
	Activity	37	1.3	15.8	0.13	9.7	<0.01

). Significant effects were demonstrated for each experiment with respect to both average opening and activity (p < 0.05)

In Experiment 1, before the application of the pesticide (imidacloprid 50 µg/L), the average shell opening level ranged from 41.72% to 43.43% (intervals 1–6 in Figure 2 and Table 1). After the application of imidacloprid, the average shell opening level decreased to 37.55% within the first 10 min, and then strongly increased over the next 20 min, reaching 73.32%. Later, the opening level continued to slightly increase, reaching 82.55% in the 19th interval after pesticide application, which is after 1 h and 35 min. In subsequent intervals, the shell opening level remained high, only slightly decreasing to 81.34%.

In Experiment 2, before the application of the pesticide, the average shell opening level ranged from 42.78% to 43.75% (intervals 1–6 in Figure 2 and Table 1). After the application of imidacloprid, the average shell opening level decreased to 38.35% within the first 5 min and then began to strongly increase over the next 20 min, reaching 71.83%. The opening level continued to increase but to a much lesser extent, reaching 85.14% in the 19th interval after pesticide application, which is 1 h and 35 min later. In subsequent intervals, the shell opening level remained high at 86.32%, and then decreased to 85.53% in interval 30.

Experiment 3, before the application of the pesticide, the average shell opening level was very stable and ranged from 42.82% to 43.58% (intervals 1–6 in Figure 2 and Table 1). After the application of imidacloprid, the average shell opening level decreased to 34.43% within the first 10 min and then began to strongly increase over the next 20 min, reaching 77.10%. The opening level continued to increase but to a much lesser extent, reaching 79.66% in the 19th interval after pesticide application, which is 1 h and 35 min later. In subsequent intervals, the shell opening level remained high, ranging from 76.70% to 79.49%, and then decreased to 76.45% over an interval of 30 min.

In Experiment 4, before the application of the pesticide, the average shell opening level ranged from 42.62% to 43.16% (intervals 1–6 in Figure 2 and Table 1). After the application of imidacloprid, the average shell opening level decreased to 41.33% within the first 5 min and then began to strongly increase over the next 30 min, reaching 71.65%. In subsequent intervals, the shell opening level remained high, gently oscillating above 65.94%.

The post hoc analysis using Tukey’s test allowed us to verify the significance of the average clam opening level for 5 min time intervals. The change in shell opening level after pesticide application was significant in each experiment for both shell opening and consequent activity (p < 0.01).

In Experiment 1, during the control period, clams exhibited consistent behavioral activity, with an average minute standard deviation ranging from 0.06 to 0.23 (Figure 3 and Table 1). Following the introduction of the pesticide, there was a noticeable increase in activity, with the standard deviation rising to 1.10 after 25 min. This elevated activity persisted above 0.5 for the next 40 min, eventually settling into a gentle oscillation slightly higher than the control period levels.

In Experiment 2, clams showed steady behavioral activity during the control period, with measurements ranging from 0.09 to 0.53 (Figure 3 and Table 1). Post-pesticide introduction, activity significantly increased, and the average minute standard deviation climbed to 2.44 within 15 min. It remained high, between 0.23 and 1.97, for the following 45 min before stabilizing at a lower range of 0.17 to 0.36, still above pre-pesticide levels.

In Experiment 3, during the control period, a consistent behavioral activity of clams was observed (Figure 3 and Table 1), determined based on the average 5 min standard deviation, which ranged from 0.00 to 0.30. After the introduction of the pesticide, a marked increase in activity was observed, and the average minute standard deviation rose, reaching 2.63 after 20 min. For the next 50 min, it remained within a broad range of 0.39 to 1.36, and then gently oscillated at a lower level but higher than during the control period.

In Experiment 4, during the control period, a consistent behavioral activity of clams was observed, ranging from 0.08 to 0.27 (Figure 3 and Table 1). After the introduction of the pesticide, a marked increase in activity was observed, and the average minute standard deviation rose, reaching 2.37 after 15 min. For the next 50 min, it remained at a very high level with fluctuations exceeding 1.0, and then decreased but oscillated within a broad range from 0.04 to 0.69, most often exceeding the level before the pesticide was administered.

Post hoc analysis using Tukey’s test allowed us to verify the significance of the mean activity of mussels in 5 min time intervals. The change in mussel activity after pesticide application was significant in each experiment (p < 0.01) with respect to the first time interval, but further oscillations did not show significance anymore.

The behavioral responses of Unio tumidus to imidacloprid exposure reveal a sophisticated and reproducible stress response pattern, characterized by an initial protective phase followed by prolonged compensatory behavior, thereby validating this species’ potential as a sensitive bioindicator of aquatic ecosystem perturbation.

3.2. Analysis for Hourly Intervals

In the experiment, the change in clam behavior under the influence of a high concentration of pesticide (imidacloprid at a concentration of 50 µg/L) was analyzed in 60 min intervals. Changes in the average level of clam opening and the intensity of these changes were observed under control conditions and after pesticide administration (

Table 3. Average shell opening (%) and mussel activity (average standard deviation in 1 min intervals); 1 h intervals; control in time intervals 1–5, pesticide application in intervals 6–21.

Interval Number	Experiment 1		Experiment 2		Experiment 3		Experiment 4
	Opening (%)	Activity	Opening (%)	Activity	Opening (%)	Activity	Opening (%)	Activity
1	30.79	0.23	33.90	0.40	33.33	0.24	31.46	0.18
2	35.59	0.16	42.48	0.22	29.79	0.26	33.41	0.15
3	42.18	0.12	42.05	0.18	26.58	0.04	36.20	0.26
4	41.76	0.13	41.51	0.15	26.84	0.05	35.41	0.12
5	42.53	0.11	40.62	0.17	30.49	0.15	35.66	0.14
6	64.16	0.75	41.89	0.57	41.94	0.77	55.97	0.93
7	81.63	0.27	76.20	0.45	71.55	0.58	60.16	0.29
8	79.42	0.32	81.22	0.30	70.31	0.28	59.82	0.22
9	79.26	0.34	79.13	0.74	69.08	0.25	60.35	0.27
10	74.61	0.29	82.59	0.32	65.16	0.31	61.75	0.17
11	66.01	0.22	82.25	0.15	59.32	0.28	61.28	0.18
12	58.98	0.23	80.99	0.14	57.27	0.34	60.74	0.18
13	52.17	0.23	79.86	0.13	64.10	0.48	60.23	0.11
14	44.48	0.16	78.65	0.11	63.14	0.54	56.14	0.18
15	37.70	0.31	74.77	0.23	52.50	0.47	44.86	0.20
16	27.53	0.14	70.69	0.08	45.01	0.40	44.38	0.13
17	25.67	0.19	72.40	0.16	35.72	0.33	37.32	0.11
18	18.69	0.09	72.32	0.37	27.89	0.24	37.49	0.11
19	28.03	0.19	62.85	0.11	24.05	0.00	37.10	0.19
20	27.79	0.18	28.10	0.16	24.12	0.00	36.93	0.20
21	27.44	0.18	34.28	0.14	24.15	0.00	35.05	0.27

).

The significance of differences between the calculated means was tested based on ANOVA (

Table 4. Analysis of variance for average shell opening and average clam activity in 1 h intervals; significant effects at p < 0.01 are marked in red, MS—the mean sum of squares, SS—the sum of squares.

Experiment	Variable	SS	MS	SS	MS	F	p
1	Opening	490,951	24,547.6	21,404.9	17.28	1420.9	<0.01
	Activity	23	1.1	64.1	0.05	22.0	<0.01
2	Opening	472,585	23,629.3	15,618.5	12.61	1874.5	<0.01
	Activity	35	1.8	164.1	0.132	13.3	<0.01
3	Opening	382,888	19,144.4	17,771.9	14.34	1334.7	<0.01
	Activity	52	2.6	185.7	0.15	17.2	<0.01
4	Opening	168,440	8422.0	8140.9	6.57	1281.8	<0.01
	Activity	35	1.8	104.3	0.08	21.0	<0.01

). Significant effects were demonstrated for each experiment concerning both average opening and activity (p < 0.05).

In Experiment 1, before the pesticide application, the average shell opening level ranged from 30.79% to 42.53% (intervals 1–5 in Table 3 and Figure 4). After the application of imidacloprid, the average shell opening level began to increase significantly, reaching a maximum value of 81.63% in the second hour. In the following hours, a decrease in the shell opening level was observed initially, the decrease was slight for three hours, then it became more pronounced, reaching a minimum shell opening level of 18.69% 13 h after the pesticide application (interval 18, Figure 4 and Table 4). In the subsequent hours, the shell opening level remained low, fluctuating between 27.44% and 28.03%

In Experiment 2, before the pesticide application, the average shell opening level ranged from 33.90% to 42.48% (intervals 1–5 in Table 3 and Figure 4). After the application of imidacloprid, the behavioral response was immediate, but the average shell opening level only significantly increased in the second hour. The maximum shell opening value, 82.59%, was reached in the fifth hour after the pesticide application. In the following hours, a gradual decrease in the shell opening level was observed initially, the decrease was slight for eight hours, then it dropped sharply over the next two hours to 28.10% (interval 20, Table 3 and Figure 4). In the next hour, it slightly increased again.

In Experiment 3, before the pesticide application, the average shell opening level ranged from 26.58% to 33.33% (intervals 1–5 in Table 3 and Figure 4). After the application of imidacloprid, the behavioral response was immediate, and the average shell opening level increased, reaching a maximum value of 71.55% in the second hour. In the following hours, a gradual decrease in the shell opening level was observed, with one fluctuation, ultimately reaching a level of 24.05% (interval 19, Table 3 and Figure 4). In the subsequent hours, this level remained very similar.

In Experiment 4, before the pesticide application, the average shell opening level ranged from 31.46% to 36.20% (intervals 1–5 in Table 3 and Figure 4). During the first two hours after the application of imidacloprid, the average shell opening level of the mussels increased significantly, reaching 60.16%. Over the next six hours, the shell opening level remained similarly high, reaching its maximum of 61.75% in the fifth hour after the pesticide application. Then, over the next eight hours, the shell opening level decreased to 37.32% (interval 17, Table 3 and Figure 4). In the subsequent hours, the shell opening level remained at a similarly low level.

The post hoc analysis using Tukey’s test allowed for the verification of the significance of the average activity shell opening level for 1 h time intervals. The change in shell opening level after pesticide application was significant in each experiment (p < 0.01). The later oscillations also showed significance in most of the following intervals.

In Experiment 1, during the control period, the behavioral activity of mussels ranged between 0.11 and 0.23 (Table 3 and Figure 5). Following the introduction of the pesticide, a significant increase in activity was observed, reaching 0.75 within the first hour and remaining above 0.2 for the next 7 h. In subsequent intervals, mussel activity decreased and stayed below 0.20, with the exception of a sporadic increase to 0.31 in the tenth hour after pesticide application.

In Experiment 2, during the control period, mussel behavioral activity varied between 0.15 and 0.40 (Table 3 and Figure 5). After the pesticide was introduced, a marked increase in activity was observed, peaking at 0.74 in the fourth hour, and subsequently remaining at a low level below 0.2, apart from three peaks (0.32, 0.23, and 0.37).

In Experiment 3, during the control period, the visible behavioral activity of mussels varied within relatively wide ranges of 0.04–0.26 (Table 3 and Figure 5). After the introduction of the pesticide, a significant increase in activity was observed, with the average minute standard deviation rising to 0.77 within the first hour and remaining high for the next hour (0.58). In the subsequent intervals, mussel activity fluctuated widely, but 14 h after the pesticide application, it decreased sharply, and the activity of all individuals almost ceased.

In Experiment 4, during the control period, with slight changes in the average shell opening level of mussels, the average standard deviation ranged from 0.12 to 0.26 (Table 3 and Figure 5). After the introduction of the pesticide, a one-hour increase in activity to a level of 0.93 was observed, which then clearly decreased to 0.29 in the next hour. In the subsequent intervals, this activity gently oscillated at the level observed before the pesticide application.

A post hoc analysis using Tukey’s test was conducted to assess the significance of the average activity of mussels for 1 h time intervals. Pesticide application caused a significant change in activity (p < 0.01) with respect to the following interval. In the subsequent interval, a significant decrease in activity was also observed (Experiments 1, 2, and 4). Further oscillations were no longer significant.

The data revealed that exposure to imidacloprid induced significant alterations in both the magnitude and temporal dynamics of shell movements. These modifications manifested as complex, time-dependent responses that reflected both immediate defensive mechanisms and longer-term physiological adaptations to chemical stress.

4. Discussion

The conducted experimental research allowed us to achieve the aim of the study; to demonstrate changes in the behavioral reaction of the molluscan under the influence of pesticides. Studies have shown changes in mussel behavior at different time intervals. Statistical analysis confirmed the relevance of the observations made. Both hypotheses were verified positively by proving that the inflow of pesticides has a significant effect on bivalves’ behaviors.

In four experiments examining the short-term response of mussels to imidacloprid (50 µg/L), distinct, repeatable patterns of behavioral changes were observed, including both shell opening degree and activity level. In all experiments, immediately after pesticide application, a sharp decrease in shell opening degree was observed. This demonstrates that pesticides trigger an immediate biological response, and the reaction changed over time, confirming mussel stress responses to various types of pollution [16,18,27]. These organisms react this way to stress or attempt to protect themselves from a new environmental factor. This decrease was short-lived, typically lasting several minutes before the next stage of response occurred. Healthy and strong mussels open their shells for feeding and gas exchange. When exposed to pollutants, their typical response is closure [18,23], which protects their soft tissues from harmful substances. However, exposure to highly toxic substances can cause abnormal functioning of the adductor muscles responsible for shell closure. Substances such as dichlorvos (concentrations 0.1 and 1 μg/L) [22] and DDVP (concentrations 10 and 100 μg/L) caused paralysis of these muscles, which may be due to their prolonged contraction, but this process is not fully understood and requires further research.

After the initial closing phase, the degree of shell opening in the conducted experiments increased rapidly. Within 20–30 min after the application of imidacloprid, the shell opening level reached significantly higher values than those observed under control conditions. This increase can be considered a specific stress response caused by the presence of the pesticide. Subsequently, the degree of opening stabilized at a high level, higher than before the pesticide application. The increase in shell opening reached maximum values within the first 2–5 h. The opening level increased to about 60–80%, which was significantly higher than the control period values (about 30–40%). After the growth period, a stabilization of the elevated shell opening was observed, with the highest values maintained in various experiments within 3–8 h. After the stabilization period, a gradual decrease in shell opening was noted in all experiments. After 10–13 h from the pesticide application, the opening level decreased to values close to or even lower than the control period, oscillating at a low level (about 18–37%) for the last hours of the study. This phenomenon indicates the cessation of stress after exposure to the pesticide.

For some mussel species, such as mussels (Mytilidae), which produce byssal threads, shell opening and the associated formation of byssal threads are considered good indicators for monitoring poor mussel health, which can be linked to reduced vitality and negative impacts on their survival [31]. According to the literature, among the studied mussels, endosulfan was found to be the most toxic among various pesticides tested by Roberts [37], causing a reduction in the number of byssal threads by up to 50% after 24 h of exposure at a concentration of 0.45 mg/L. The reduction in the number of byssal threads is also confirmed by studies on imidacloprid. This example can be linked to the reaction of mussels in our experiment, which initially closed, and then opened more than under control conditions.

In terms of activity after pesticide application, a rapid increase in mussel activity was observed. Activity, measured as the standard deviation of the average opening measured for 1 min intervals, showed that mussels quickly opened and closed. In each experiment, behavioral activity increased rapidly, reaching its maximum within the first few to several minutes after pesticide application. After reaching the peak level, activity began to decrease just as quickly but remained slightly higher than during the control period for the next several minutes (about 0.2–0.3) within 2–4 h after pesticide application. Subsequently, activity oscillated at a level higher than during the control period for several hours but gradually decreased. In the later hours, especially after 10–14 h from application, mussel activity decreased significantly. This decrease indicates the cessation of stress after exposure to the pesticide.

The literature indicates that mussel responses to pesticides can vary depending on many factors, such as substance concentration, exposure time, or the specific characteristics of the studied species [25]. Nevertheless, there are studies that can help understand these reactions, especially in the context of increased mussel activity in the initial phase of pesticide exposure. Studies indicate that the initial phase of exposure to harmful chemical substances can cause increased mobility or escape attempts [38], before a decrease in activity due to the toxic effects of the harmful substance. Similar reactions regarding changes in mussel behavior, such as filtration, activity, and avoidance of harmful factors, were noted during studies on Anodonta anatina mussels exposed to salt. The mussels showed an initial increase in activity, trying to escape from the stressor, which can be an indicator of toxicity in ecological studies, especially in freshwater mussels [25].

During the study of the impact of neonicotinoids on the behavior of land mollusks Helix aspersa and squids, it was confirmed that this species exhibited a decrease in overall activity, which may indicate a characteristic response of mollusks to the administered poison and can be associated with nervous system damage and an increased risk of death [39].

Studies on the activity and increased movements of Dreissena polymorpha mussels have shown that chemical substances can cause an initial increase in activity before toxins lead to a decrease in overall motility and survival. This suggests that mussels may respond with a short-term increase in activity as a reaction to chemical stressors [40], before the effects of pollution become apparent [41,42].

Statistical analysis—both ANOVA and Tukey’s post hoc analyses showed that short-term and long-term changes in both shell opening levels and activity were statistically significant (p < 0.01) in all experiments. All four studies demonstrated that the mussel response is highly repeatable. The significance of these results suggests that the observed reactions are repeatable and consistent, confirming the impact of imidacloprid as a factor causing changes in mussel behavior. Some behavioral traits indeed differed; for instance, the percentage of shell opening in Experiment 3 was noticeably distinct. However, the response to the pesticide remained repeatable across all experiments.

Behavioral responses of Unio tumidus in the presence of the pesticide confirmed observations by other researchers regarding the impact of neonicotinoids on the activity of the enzyme acetylcholinesterase present in mussel gills [43]. Acetylcholinesterase is the most important cholinesterase, breaking down acetylcholine, a substance that causes muscle contraction and transmits impulses between neurons in the nervous system. Exposure of the mussel Mytilus galloprovincialis to high concentrations of imidacloprid significantly reduced the activity of acetylcholinesterase [44]. The study showed that imidacloprid lowers AChE activity, which can disrupt nervous functions by causing improper regulation of the neurotransmitter acetylcholine. This effect would also confirm the behavioral response of the swollen river mussel Unio tumidus to high concentrations of imidacloprid.

Exposure of the mussel Mytilus galloprovincialis to a mixture of neonicotinoids impacts mussel health, with an emphasis on molecular and biochemical changes in these organisms. The study uses transcriptomic (gene expression analysis) and proteomic (protein analysis) approaches to identify changes in response to chemical stress induced by pesticides [44].

In the laboratory experiments conducted, we studied the response of mussels to imidacloprid at a concentration of 50 µg/L, which is high but can be found in the environment. The concentration of neonicotinoid pesticides in natural aquatic ecosystems varies and ranges from 0.1 to 100 µg/L, and their presence has been documented across five continents [7]. It is important to note that in some agricultural catchments, this concentration may even exceed levels considered lethal [38]. The highest values reported in the literature are 320 µg/L [7]. Thus, the results of our studies demonstrate the real-world implications of pesticide pollution.

Our laboratory findings reveal significant implications for real-world mussel populations in pesticide-contaminated water bodies. Mussels exposed to imidacloprid exhibit stress and behavioral changes that disrupt essential functions such as feeding and respiration, potentially reducing their survival rates. Prolonged stress responses suggest energy depletion and physiological damage, which can impair growth and immune function, particularly in areas with recurring contamination. Given the ecological importance of mussels, their decline could destabilize aquatic ecosystems by disrupting nutrient cycling, water filtration, and food webs, leading to broader impacts on biodiversity.

The research we conducted was based on a single species of mussel. This introduces a certain limitation in the interpretation of the obtained conclusions when referring to the threats of the tested insecticide to existing ecosystems. The justification for using Unio tumidus is that it is common, is not protected, occurs in clean water reservoirs, is sensitive to changes in the quality of environmental water [17,18,19,28,30], and its collection does not threaten biodiversity. The species Unio tumidus is widely used in biomonitoring, and its use is scientifically supported [26,27,28,29]. In the future, we plan to conduct similar comparative experiments with other species of mussels, but the scale of these experiments may be smaller because we will only be looking for confirmation of the observed phenomena.

The risk of imidacloprid contamination [45] indicates the importance of incorporating mussels into monitoring programs as bioindicators for assessing water quality and the ecological impact of agricultural practices and beyond, as the application of neonicotinoids is much broader. Other studies also show that these compounds affect many aquatic organisms, including amphibians [46], aquatic arthropods [47], aquatic vertebrates [48], and fish [49], and also impact entire ecosystems [50]. There is also a lack of research on the effects of these pesticides on humans and higher vertebrates over a longer period of time. Regulators should consider setting restrictions on the use of neonicotinoids, as their ability to bioaccumulate can reveal long-term ecological consequences for entire trophic chains, including humans [51].

5. Conclusions

Neonicotinoids like imidacloprid significantly impact aquatic ecosystems, with Unio tumidus serving as a reliable bioindicator due to its measurable behavioral responses.

This study demonstrated that exposure to high concentrations of the neonicotinoid pesticide imidacloprid significantly affects the freshwater mussel. The introduction of imidacloprid caused significant, repeatable changes in mussel behavior: initial shell closure followed by prolonged opening and increased activity, indicating stress and subsequent toxic effects.

Experiment findings were consistent across multiple experiments, showing minimal seasonal variation and reinforcing the reliability of U. tumidus as a test organism.

This study underscores the importance of behavioral monitoring for assessing pesticide risks and highlights the need for further research on long-term and multi-pesticide impacts. Future research should focus on studies of long-term exposure and recovery to gain a deeper understanding of the chronic effects and potential resilience of Unio tumidus.

The observed changes in shell opening and activity levels indicate a clear stress response, which highlights the potential ecological risks posed by such contaminants in aquatic environments. The consistent patterns across multiple experiments underscore the reliability of these behavioral indicators in assessing water quality and the health of aquatic ecosystems.