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Article

Behavioural and Biochemical Responses of Freshwater Bivalve Anodonta marginata Exposed to Dichlorvos

by
Abdullahi Bala Alhassan
1,* and
Mohammed Othman Aljahdali
2,*
1
Department of Biology, Faculty of Life Sciences, Ahmadu Bello University, Zaria 810001, Nigeria
2
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Water 2024, 16(24), 3572; https://doi.org/10.3390/w16243572
Submission received: 12 November 2024 / Revised: 6 December 2024 / Accepted: 9 December 2024 / Published: 12 December 2024
(This article belongs to the Section Biodiversity and Functionality of Aquatic Ecosystems)
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Abstract

:
The use of pesticides for the prevention and eradication of a variety of pests has been on the increase, hence the need for investigations on their impact on the environment and non-target organisms. Fractions of the 24 h LC50 of dichlorvos in the form of 1 2 (LC50), 1 4 (LC50), 1 8 (LC50) and 1 16 (LC50) were determined to achieve varying concentrations for this study, and ten Anodonta marginata were placed in each aquarium for the definitive test, with each treatment concentration set up in triplicates. The setup was monitored daily for four days (96 h) for changes in behavioural and biochemical responses. Behavioural responses such as opening of the shell, extension of the foot, complete shell closure, and activity of enzymes such as AChE and GSH were determined as endpoint biomarkers in A. marginata tissue. An analysis of variance was used to determine significant variations in behavioural responses, survival, GSH and AChE content in bivalves across varying concentrations of dichlorvos. The exposure of A. marginata to varying concentrations (0, 10, 20, 40 and 70 mg/L) of dichlorvos for 96 h led to an LC50 value of 4.79 mg/L when compared to exposure concentrations. There was a significant (p < 0.05) variation in biochemical responses and opening of the shell as a behavioural response in A. marginata across varying concentrations of dichlorvos with time, with the highest percentage shell opening and GSH activity recorded at the highest concentration (70 mg/L) of dichlorvos and time (96 h). In contrast, AChE activity and percentage survival of A. marginata were lowest at the highest concentration of dichlorvos, confirming dichlorvos as an AChE inhibitory organophosphate pesticide. There is a need for proper monitoring and management of pesticide contamination in order to protect freshwater ecosystems.

1. Introduction

Freshwater ecosystems are important for biodiversity, as they provide habitats for several aquatic organisms, maintaining balances in trophic interactions for the sustenance of global ecological balance [1]. However, aquatic ecosystems are faced with increasing anthropogenic pollutants such as dichlorvos, which are a threat to the pristine nature of this ecosystem and their biodiversity [2]. Particularly, pesticides such as dichlorvos, are widely used organophosphate, making the list of the anthropogenic pollutants of major concern worldwide. Dichlorvos is known for its acute toxicity and environmental persistence, causing a significant risk to non-target aquatic organisms such as bivalves [3]. Freshwater bivalves, including Anodonta marginata, serve as a very good biomonitor due to their sensitivity to pollutants and their role as filter feeders and provision of ecosystem services such as nutrient cycling in aquatic ecosystems, thus reflecting ecosystem health [4]. Behavioural and biochemical responses in bivalves provide valuable insights into the acute and chronic effects of xenobiotics, bridging the gap between environmental exposure and disruptions in biosystem functions [5]. Globally, the adverse impacts of pollution from pesticides have raised concerns over freshwater ecosystem resilience, hence the need for studies that assess the biochemical and behavioural effects of dichlorvos exposure [6].
Pesticides are chemical compounds used to prevent and eradicate a variety of pests that are persistent in agriculture, forestry and households [7]. Extensive agricultural activities and indiscriminate use of pesticides have led to contamination of the aquatic environment by direct application of pesticides, as well as surface runoff and wind-borne drifts that carry these toxic substances to various water sources [8]. As a result, there has been a gradual increase in the concentration of pesticides and their metabolites in water columns and benthic sediments, thus causing severe contamination of the aquatic environment [9,10,11]. Chronic or acute exposure to pesticides may cause deleterious problems for non-target organisms, leading to altered or disrupted biochemical processes. There is also often a high expenditure of energy for the removal of the toxicants and repair of damaged processes [12].
Dichlorvos (2,2-dichloroethyl dimethyl phosphate) is an organophosphate insecticide widely used in agriculture practices and has been detected in surface waters receiving runoff from agricultural and non-agricultural areas [13]. Dichlorvos has been used, mainly in agriculture, for the proper storage of grain and control of pests in livestock and households [14]. Previous studies have revealed a higher concentration of dichlorvos than the sum of all other organophosphate pesticides monitored [15] and was above the permissible concentration for the protection of water wildlife in countries like the United States (US), Argentina, Europe, and China [16]. This gives an insight into its occurrence as, no doubt, a serious problem, and hence the need for the establishment of environmental benchmarks for organophosphate pesticides such as dichlorvos through the utilisation of toxicological data. The control of dichlorvos through monitoring of the environment involves regulatory restrictions to reduce its usage and put in place strategies to prevent agricultural runoff into water bodies [17]. Introducing bioremediation using microorganisms with the potential to degrade organophosphate pesticides such as dichlorvos has been effective in reducing its environmental impact [18]. Adsorption techniques, like activated carbon or biochar usage, can help remove dichlorvos from aquatic systems [6]. It is noteworthy that integrated pest management reduces reliance on chemical pesticides by combining biological and cultural control methods; this is a preventive strategy against excessive utilisation of dichlorvos [19].
Neurotoxic pesticides such as dichlorvos can damage the health of non-target aquatic organisms; affect the nervous system, behaviour, growth and proliferation of nerve cells, degenerate cell mitochondria; and cause apoptotic pathway interference [20]. The behavioural changes include valve closure, filtration rate, adhesion to the surface and burrowing into sediment, which in turn affect bivalves’ survival. Herbert et al. [21] reported that aquatic macroinvertebrates exhibit immobility and detachment from surfaces following acute, brief exposure to sublethal concentrations of neurotoxic pesticides, hence promoting downstream drift.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are important enzymes involved in cholinergic signalling regulation in organisms [22,23]. AChE is found primarily in neural tissues, synapses and red blood cells, and it rapidly hydrolyses acetylcholine, a neurotransmitter that terminates nerve impulses in order to ensure proper neuromuscular function [22]. In contrast, BuChE is primarily present in the liver tissue, plasma and other non-neural tissues and has a broader substrate specificity, with a secondary role in hydrolysing acetylcholine [24]. Organophosphate pesticides inhibit both AChE and BuChE by binding irreversibly, leading to the accumulation of acetylcholine and then nerve overstimulation [25]. While inhibition of AChE is known to be directly associated with neurotoxic effects, BuChE is often considered a biomarker for pesticide exposure because it serves as a scavenger for toxic compounds, reducing their inhibitory effect on AChE [25]. The measurement of AChE and BuChE activity is used widely in toxicological assessments to evaluate the neurotoxic effect of contaminants [26]. AChE builds up excessively in synapses, resulting in clinical signs of OP pesticide poisoning [27]. AChE and BuChE are irreversibly inhibited by organophosphate compounds [28]. This leads to an excessive build-up of AChE and the paralysis of cholinergic transmission in the central nervous system, autonomic ganglia, parasympathetic nerve endings, some sympathetic nerve endings and the neuromuscular junction [29].
This study aims to evaluate the behavioural and biochemical responses of Freshwater bivalve A. marginata exposed to dichlorvos. Understanding these responses in A. marginata will provide important data for the assessment of ecotoxicological risk and management of water quality.

2. Materials and Methods

2.1. Procurement of Dichlorvos, Collection and Acclimatisation of A. marginata

Dichlorvos are commonly used on farms and hence available in outdoor pesticide companies. The one used in this study was procured at Bim 33 International Limited, Kaduna, Nigeria.
A total of 200 bivalves were collected from a freshwater reservoir using an Ekman grab (model number 923), measuring 19 cm by 14 cm with an area of 0.0266 m2, and were kept in a glass aquarium measuring 80 cm × 40 cm × 40 cm in dimension for 30 days to acclimatise [30]. Before the toxicity tests, a pre-screening procedure was conducted prior to exposure to confirm the absence or presence of dichlorvos in the bivalves initially collected from the freshwater reservoir. The bivalves were randomly selected, and whole-body tissue was analysed for dichlorvos [31] using gas chromatography coupled with mass spectrometry (GC-MS), model no. GC 7890B, MSD 5977A, thereby validating the baseline conditions for the acute toxicity studies.

2.2. Toxicity Tests

A range finding test was conducted for 24 h to determine the most appropriate dose levels for the main study [32]. A total of 5 dichlorvos concentrations were selected from [33,34] to determine the 24 h LC50. However, for the definitive test, fractions of the 24 h LC50 in the form of 1 2 (LC50), 1 4 (LC50), 1 8 (LC50) and 1 16 (LC50) was determined to achieve varying concentrations for this study [30]. In addition, ten A. marginata were placed in each aquarium, with each treatment concentration set up in triplicates. The setup was monitored daily for four days (96 h) for changes in behavioural responses, mortality, pH, EC, TDS and temperature.

2.3. Determination of Behavioural and Biochemical Marker

2.3.1. Opening of Shell and Extension of the Foot

Opening of the shell and complete shell closure were all monitored by physical observation of the bivalves in clear and transparent aquaria daily for 96 h [30,35]. The behavioural response was observed and recorded every 12 h over the 96 h exposure period [36], and percentages were calculated [34].

2.3.2. Reduced Glutathione (GSH)

GSH is widely used as a biomarker of oxidative stress in organisms exposed to contaminants such as dichlorvos. It plays a key role in oxidative stress defence, where changes in GSH levels are an indication of its impact on cellular redox balance and detoxification processes [37], hence the need for its utilisation in this study.
Bivalve tissues were homogenised in ice-cold buffer [1 M sodium phosphate monobasic (NaH2PO4) and sodium phosphate dibasic (Na2HPO4) pH 7.4] to ensure efficient extraction of GSH. The homogenates were centrifuged at 10,000 rpm for 5 to 10 min, and the supernatants were collected for analysis. The assay was conducted according to the manufacturer’s protocol. The determination of GSH was based on the principle that the ratio of reduced glutathione (GSH) to oxidised glutathione (GSSG) serves as a primary dynamic indicator of cellular redox status. Measuring both the levels of GSH and GSSG and their ratio within cells reflects the cell’s redox state. To ensure the samples were free from contamination, deionised water was used to clean and rinse all glassware and equipment thoroughly, and the process of homogenisation and centrifugation was conducted under sterile conditions with the use of fresh, high-purity reagents to reduce the risk of contamination that could interfere with the measurement of GSH. DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)) reacts with GSH to form a complex that exhibits a characteristic absorption peak at 412 nm. The absorbance at this wavelength is directly proportional to the GSH content in the sample.

2.3.3. Acetylcholinesterase (AChE)

The AChE content in A. marginata exposed to dichlorvos was determined using an Enzyme-Linked Immunosorbent Assay (ELISA) kit (Cat No.: ER0461-CM) and following the manufacturer’s instructions. The tissue of A. marginata was homogenised in ice-cold buffer [1 M (NaH2PO4) and (Na2HPO4) pH 7.4], the homogenates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were collected for further analysis. The concentration of salt in [1 M (NaH2PO4) and (Na2HPO4)] solution is 1 mole per litre of [(NaH2PO4) and (Na2HPO4)]. The manufacturer’s instructions were followed when performing the analysis using the ELISA microplate reader. Solutions of standards were added to the wells of the microtiter plate pre-coated with an anti-AChE antibody, followed by incubation for 1 hr under room temperature to allow the AChE in the samples to bind to the antibodies. After the plate was washed twice to remove the unbound substances, an enzyme-linked secondary antibody was added and incubated for about 30 min. Then, another washing was performed, and after that, a substrate solution was added to the ELISA kit wells and incubated for 15 min to develop the presence of colour; then, the reaction was stopped by adding a stop solution, and the absorbance was measured at 450 nm using a Multiskan SkyHigh Microplate Reader (Model number: A51119700C). The readings of the absorbance were compared to a standard calibration curve prepared with known concentrations of AChE in order to determine the AChE content in the samples, and they were expressed in pg/mL.

2.4. Determination of Dichlorvos Concentrations in A. marginata After 96 h Exposure

A total of 1–2 g of whole-body tissue of A. marginata after 96 h exposure, from each concentration, was utilised for the analysis of dichlorvos using gas chromatography coupled with mass spectrometry (GC-MS), model no. GC 7890B, MSD 5977A [31], to determine the actual concentrations. The implementation of quality control for the GC-MS analysis includes readings of control standards and a procedural blank analysed for every set of 10 samples, the utilisation of independent control standards (other than the ones for calibration) and a coefficient of correlation R ≥ 0.99, together with calibration curve represented in the graphical form [38].

2.5. Data Analyses

The initial data generated for survival, behavioural response, GSH and AChE were subjected to Leven’s homogeneity of variance and a Shapiro–Wilk test to test for homogeneity and normality of the data. One-way analysis of variance (ANOVA) was used to determine significant variations in GSH and AChE content in the bivalves across varying concentrations of bivalves, while two-way ANOVA was used to determine significant variations in behavioural response and survival of the A. marginata across varying concentrations of dichlorvos and time. Where significant differences existed, Duncan’s Multiple Range Test (DMRT) was used to separate the mean values at p ˂ 0.05. A principal component analysis was used to determine the influence of dichlorvos on behavioural response, survival, GSH and AChE content in A. marginata. A cluster heat map was used to determine the relationship between behavioural response, survival, GSH and AChE in A. marginata exposed to varying concentrations of dichlorvos.
R. Statistical software (v. 4.3.1) for Windows was used for analysis.

3. Results

3.1. Screening of Bivalves for Dichlorvos and Lethal Toxicity

The result of the pre-screening of A. marginata revealed the concentration of dichlorvos in the bivalves to be below the detectable limit; however, the post-screening results revealed dichlorvos concentrations ranging from BDL in the control group to the highest actual concentration (6.35 mg/L) at the highest nominal concentration (70 mg/L) of exposure (Table 1). The exposure of A. marginata to varying concentrations (0, 10, 20, 40 and 70 mg/L) of dichlorvos for 96 h led to an LC50 value of 4.79 mg/L when compared to the exposure concentrations (Table 1). The LC50 (4.79 mg/L) was about 48% of the lowest concentration (10 mg/L) of exposure and about 7% of the highest concentration (70 mg/L) of exposure. As revealed in Table 1 with the log-transformed concentrations of dichlorvos, the LC50 values guide and give insight into the mortality and survival of A. marginata at the different concentrations of exposure.

3.2. Behavioural Response/Shell Opening

The exposure to varying concentrations of dichlorvos across time caused a clear change in the opening, closing and foot extension of A. marginata (Figure 1). Our findings revealed that the toxic effects of dichlorvos on the freshwater mussel, A. marginata, vary in terms of their impact on behaviour, using percentage shell opening as an index, biochemical changes and the rapid onset of death.
There was a significant (p < 0.05) variation in the opening of the shell as a behavioural response in A. marginata across varying concentrations of dichlorvos with time (Figure 1A–D).
However, it is important to note that the highest percentage shell opening was recorded at the highest concentration (70 mg/L) of dichlorvos and time (96 h) (Figure 1D). Although, even for the other concentrations of dichlorvos, the highest percentage shell opening was recorded at 96 h exposure (Figure 1D). Additionally, the lowest percentage shell opening was recorded for the control treatment, even though no shell opening was observed at 24 h (Figure 1A). It is important to note that at 20 mg/L of dichlorvos, no significant variation in percentage shell opening was observed between 24 and 48 h of exposure (Figure 1A,B).

3.3. Survival Response

The survival of A. marginata determined after exposure dichlorvos in this study puts into consideration mortality recorded as an effect. The variation in survival of A. marginata across varying concentrations of dichlorvos with time was significant (p < 0.05) (Figure 2A–D). During exposure of the bivalves to dichlorvos, the highest percentage survival (100%) was recorded at 10 mg/L and 20 mg/L of dichlorvos at 24 h (Figure 2A), while the lowest percentage survival, 20%, was recorded in the treatment with 70 mg/L of dichlorvos at 96 h (Figure 2D). Additionally, at 70 mg/L, percentage survival significantly decreased at 72 and 96 h (Figure 2C,D), having 30% and 20% survival, respectively, while 90% and 80% survival were recorded for 24 and 48 h, respectively (Figure 2A,B). Percentage survival at 40 mg/L was 80% for 24 and 48 h, while for 72 and 96 h, it recorded 50% and 40% survival, respectively. However, at 20 mg/L, the percentage survival was the same for 72 and 96 h (90%) (Figure 2C,D).

3.4. Acetylcholinesterase (AChE) and Reduced Glutathione (GSH) Activity

A. marginata exposed to varying concentrations of dichlorvos recorded AChE activity of 323.74 Pg/mL, 313.20 Pg/mL, 302.78 Pg/mL, 275.123 Pg/mL and 228.47 Pg/mL at the control and with concentrations of 10, 20, 40 and 70 mg/L of dichlorvos, respectively. The significantly highest activity recorded was in the control treatment, and the lowest activity was recorded in the treatment with the highest concentration (70 mg/L) of dichlorvos (Figure 3A). The AChE activity decreased in a concentration-dependent pattern, with the activity significantly decreasing as the concentration of dichlorvos increased (Figure 3).
GSH activity in the tissue of A. marginata due to the effect of dichlorvos recorded in this study is presented in Figure 3B. GSH activity was significantly (p < 0.05) different across varying concentrations of dichlorvos. GSH activity was at its maximum (2.35 U/mg protein) at 70 mg/L of dichlorvos and was at its minimum (0.65 U/mg protein) at 40 mg/L, while the activities at the control, 10 and 20 mg/L of dichlorvos were not significantly different even though there was an initial increase from the control to the 10 mg/L concentration and a sudden fall at the 20 mg/L concentration (Figure 3B).

3.5. Correlation Between Behavioural Responses and Biochemical Markers

The correlation results between behavioural responses and biochemical markers are presented in Figure 4 and Figure 5. In Figure 4, the cluster heat map revealed a strong (heat map value ≥ 250) influence of varying concentrations of dichlorvos on AChE, more than any other parameter (Figure 4). This has shown the effect of a decreasing pattern of AChE with an increasing pattern of dichlorvos concentration, especially at the highest concentration of 70 mg/L. Increasing concentration also influences the opening of the shell, as shown at concentrations of 10, 20, 40 and 70 mg/L. Noteworthy is the horizontal clustering of 40 and 70 mg/L of dichlorvos together at the highest similarity level, forming a single group, while 0, 10 and 20 mg/L cluster together to form a single group too at the highest similarity level, even though they separated at the next similarity level after the highest, with 10 and 20 mg/L forming a single group. This group formation and preferences are relative to the potency or effect of the dichlorvos concentration on behavioural responses and biochemical markers. For vertical clustering, AChE alone forms a separate group from the other parameters, while GSH, survival and shell opening form another group together. However, GSH detached and formed its group at the next cluster similarity level after the former higher similarity level.
The principal component analysis biplot accounting for a total variation of 96.6% (PC1 = 74.1%, PC2 = 22.5%) revealed a strong positive correlation between survival and AChE. It was influenced by the control (0 mg/L) and 10 mg/L concentration treatments but negatively correlated with GSH and shell opening, as influenced positively by the dichlorvos concentrations of 40 and 70 mg/L (Figure 5). Additionally, GSH activity was revealed to be positively correlated with shell opening. That relationship was influenced by 40 and 70 mg/L of dichlorvos.

4. Discussion

4.1. Dichlorvos Toxicity and Effect on Behavioral Responses in A. marginata Shell Opening

In this study, the LC50 value of 4.79 mg/L for dichlorvos represents the lethal concentration for 50% of the population and is significant as it is 48% of the lowest exposure concentration (10 mg/L) and merely 7% of the highest (70 mg/L). In addition, the log-transformed concentrations underscore the acute toxicity of dichlorvos, emphasising its potential risk to the freshwater bivalve population. In contrast with our findings, previous research by Shiry et al. [39] on freshwater bivalve exposed to pesticide reported 85.20 mg/L 96 h LC50 value, possibly due to the use of a different pesticide (diazone) that may have had a different mode of action and potency to mortality and behavioural responses on the bivalves.
The significant variation in behavioural changes across varying concentrations of dichlorvos over time in this study gives insight into A. marginata’s sensitivity to dichlorvos and suggests possible potential implications for the overall health and ecological dynamics of bivalve abundance and diversity [40,41], especially at the highest concentrations of dichlorvos. This could also provide an understanding of the occurrence of the lowest percentage of shell opening and no shell opening observed at the 24 h mark, highlighting the absence of immediate adverse effects under normal conditions [20]. This result agrees with the findings of Hartmann et al. [42] on the behavioural responses of freshwater mussels Unio tumidus to pesticide contamination. Jakubowska et al. [36] also report a similar finding in their work on a freshwater bivalve Anodonta cygnea, exposed to pesticide.
Pesticides have been reported to be among the major stressors found in aquatic ecosystems, especially those located in and around areas that are prone to agricultural activities [43]. The issue can be serious as negative impacts of pesticides due to contamination can change the entire setting or pristine nature of the trophic chains of aquatic ecosystems at large [42].
Of significance, no significant variation in percentage shell opening noted between 24 and 48 h of exposure to dichlorvos at 20 mg/L can be due to a temporal aspect of behavioural responses, providing valuable insight into the dynamics of bivalve reactions to specific concentrations of dichlorvos. This could suggest potential acclimatisation or delayed effects at lower concentrations [44]. Notably, our results support and build upon the existing body of literature, emphasising the importance of considering both concentration and exposure duration in assessing the behavioural impacts of pesticides on bivalve populations [45].

4.2. Survival of A. marginata in Response to Dichlorvos

The rate of survival of the bivalves as a response to pollutants in this study can be attributed to the varying concentrations of dichlorvos. The maximum percentage survival in the control treatment could be as a result of the absence of dichlorvos in the control treatment. However, in the 10 and 20 mg/L treatments, the lower potency of the treatments at 24 h could also be the reason for the maximum survival observed. This could also be attributed to the higher percentages of survival at the lower concentrations. This is in agreement with the study of Maxio et al. [46] in a work that studied the survival, growth and physiology of bivalves and reported higher survival rates in control and lower treatment concentrations. The lower survival percentage for 70 mg/L at 96 h could, however, be associated with the effect of the highest concentration of the dichlorvos and time. Additionally, the gradual reduction in survival with time observed can be attributed to the long-term effect of the dichlorvos on A. marginata. Boldina-Cosqueric et al. [47] reported a similar trend of survival in Sinonovacula constricta in long-term stress conditions.

4.3. Effect of Dichlorvos on Acetylcholinesterase (AChE) and Reduced Glutathione (GSH) Activity

AChE has been reported to be the target enzyme for organophosphate pesticides in biosystems; its inhibition can lead to the accumulation of a neurotransmitter called acetylcholine, leading to mortality in organisms. The highest activity recorded in all the controls when compared to the treatment groups is a result of the absence of dichlorvos in the control group. Within the group, however, the inhibition of AChE was highest in the treatment with 70 mg/L dichlorvos, which had the highest concentration of the exposure pesticide, revealing the negative effect of the pesticide, especially at high concentrations, which is also linked to the inhibition of AChE, which can lead to an accumulation of acetylcholine and mortality. Our findings are congruent with those of Choi et al. [48], who used AChE as a potential biomarker of pesticide exposure in the manila clam, Ruditapes philippinarum, in Korea. Elsewhere, previous studies [3,39] also reported similar findings involving the inhibition of AChE as a result of organisms such as bivalve exposure to pesticides.
The activities of reduced glutathione in A. marginata were influenced by the varying concentrations of dichlorvos. The highest GSH activity recorded at 70 mg/L can be the result of oxidative stress due to exposure to high concentrations of dichlorvos. The lowest concentrations of GSH recorded at 40 mg/L of dichlorvos, which was a sudden drop after the initially higher concentration of GSH at 10 and 20 mg/L of dichlorvos, can be attributed to the organism’s response to the effect of dichlorvos and biosystem adjustment in cellular activity in order to combat the negative effect of the pesticide. The slight rise in the GSH from the control treatment to treatment with 10 mg/L dichlorvos can be linked to the adaptive nature of the organism to harsh environments, as these organisms have been described to be able to withstand harsh conditions in the aquatic environment [49]. Dichlorvos has been reported to cause irregularity in GSH activities in organisms; hence, the sudden rise and fall in GSH activity can be linked to exposure to this pesticide [50].

4.4. Influence of Dichlorvos on Behavioural Responses and Biochemical Markers

The influence of dichlorvos on AChE, GSH and other related behavioural responses in A. marginata was revealed and demonstrated in this study. The inhibition pattern of AChE by dichlorvos can be due to its effect and possible accumulation of acetylcholine, which can result in neurotoxicity, as shown by the marked decrease in AChE activity at higher concentrations [51]. This possible neurotoxicity in A. marginata caused primarily by dichlorvos may lead to disruptions of the normal neural transmission and muscle function, explaining the observed index used as behavioural changes in this study, which is the shell opening [52]. The correlation between increased dichlorvos concentration with reduced survival and elevated GSH levels can be a reflection of the stress response of the organism. However, increased GSH, an antioxidant, is an indication of oxidative stress due to dichlorvos exposure [53]. Also, the clustering of the 40 and 70 mg/L concentrations and their association with higher effects of dichlorvos underscores the potency of dichlorvos.
The positive correlation between survival and AChE activity at lower dichlorvos concentrations revealed by PCA aligns with the report of Montory et al. [54], who reported similar findings on the effects of pesticides on AChE, physiological performance and survival rates. Conversely, the positive correlation between GSH and shell opening at higher concentrations is an indication of stress response impacting the bivalve’s behaviour, as seen in other studies [53]. These findings are significant for monitoring the environment and regulating the use of pesticides together with the protection of non-target organisms in aquatic ecosystems. The strong positive influence of dichlorvos on A. marginata agrees with previous research on the neurotoxic effects of organophosphates through species-specific differences, suggesting the need for advanced ecological assessments [3,55].

5. Conclusions

This study reveals the significant impact of dichlorvos on the freshwater bivalve A. marginata through behavioural and biochemical assessments. The LC50 value of 4.79 mg/L over 96 h highlights the pesticide’s lethality, even at low concentrations. Behavioural changes, particularly in shell opening, were evident, with the highest activity observed at 70 mg/L and 96 h. Survival rates were lowest at the highest concentrations and longest exposure times. Biochemical markers such as acetylcholinesterase (AChE) and reduced glutathione (GSH) showed concentration-dependent responses, with AChE activity decreasing significantly and GSH activity varying. Correlation analyses indicated a strong relationship between behavioural changes and biochemical responses, particularly an inverse relationship between AChE activity and dichlorvos concentration. The principal component analysis further showed a positive correlation between survival and AChE and a negative correlation with GSH and shell opening, influenced by higher dichlorvos concentrations. These findings underscore the detrimental or chronic effects of dichlorvos on A. marginata, providing essential insights for environmental monitoring and regulatory policies. Future research should focus on long-term exposure and recovery studies to better understand the chronic effects and potential resilience in A. marginata.

Author Contributions

Conceptualisation, A.B.A. and M.O.A.; methodology, A.B.A. and M.O.A.; software, A.B.A.; validation, A.B.A. and M.O.A.; formal analysis, A.B.A.; investigation, A.B.A. and M.O.A.; resources, A.B.A. and M.O.A.; data curation, A.B.A.; writing—original draft preparation, A.B.A. and M.O.A.; writing—review and editing, A.B.A. and M.O.A.; visualisation, A.B.A. and M.O.A.; supervision, A.B.A. and M.O.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors acknowledge with thanks R.I. Sha’aba of the Department of Biology, Ahmadu Bello University Zaria, for providing a space at the freshwater bivalve laboratory, A.B.U., Zaria, for the experimental setup and also harvesting and analysis of the samples.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 16 03572 g001
Figure 1. Changes in shell opening as an index of behavioural response in A. marginata exposed to dichlorvos after (A) 24 h, (B) 48 h, (C) 72 h and (D) 96 h.
Figure 1. Changes in shell opening as an index of behavioural response in A. marginata exposed to dichlorvos after (A) 24 h, (B) 48 h, (C) 72 h and (D) 96 h.
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Figure 2. Changes in the survival of A. marginata exposed to dichlorvos after (A) 24 h, (B) 48 h, (C) 72 h and (D) 96 h.
Figure 2. Changes in the survival of A. marginata exposed to dichlorvos after (A) 24 h, (B) 48 h, (C) 72 h and (D) 96 h.
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Figure 3. Effect of varying concentrations of dichlorvos on (A) AChE content and (B) GSH activity in A. marginata. * Significantly different from control (p ˂ 0.05); a, b and c are the rankings for mean separation, indicating significant variation between the mean values (p ˂ 0.05).
Figure 3. Effect of varying concentrations of dichlorvos on (A) AChE content and (B) GSH activity in A. marginata. * Significantly different from control (p ˂ 0.05); a, b and c are the rankings for mean separation, indicating significant variation between the mean values (p ˂ 0.05).
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Figure 4. Cluster heat map for the relationship between behavioural responses and biochemical markers in A. marginata exposed to varying concentrations of dichlorvos.
Figure 4. Cluster heat map for the relationship between behavioural responses and biochemical markers in A. marginata exposed to varying concentrations of dichlorvos.
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Figure 5. Influence of dichlorvos on biochemical markers and behavioural response indices in A. marginata.
Figure 5. Influence of dichlorvos on biochemical markers and behavioural response indices in A. marginata.
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Table 1. The 96 h LC50 values of dichlorvos, percentage mortality and survival of A. marginata.
Table 1. The 96 h LC50 values of dichlorvos, percentage mortality and survival of A. marginata.
Conc. (mg/L)Log of Conc.Actual Conc. (mg/L)Total No. of Bivalve ExposedMortality% MortalitySurvival% SurvivalPSLC50
00BDL100010100BDL *4.79
1010.2410440660
201.3011.9410110990
401.6022.5710660440
701.8456.3510880220
Notes: BDL = below detectable limit; BDL * = concentration of dichlorvos determined before exposure; PS = pre-screening of A. marginata for dichlorvos.
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Alhassan, A.B.; Aljahdali, M.O. Behavioural and Biochemical Responses of Freshwater Bivalve Anodonta marginata Exposed to Dichlorvos. Water 2024, 16, 3572. https://doi.org/10.3390/w16243572

AMA Style

Alhassan AB, Aljahdali MO. Behavioural and Biochemical Responses of Freshwater Bivalve Anodonta marginata Exposed to Dichlorvos. Water. 2024; 16(24):3572. https://doi.org/10.3390/w16243572

Chicago/Turabian Style

Alhassan, Abdullahi Bala, and Mohammed Othman Aljahdali. 2024. "Behavioural and Biochemical Responses of Freshwater Bivalve Anodonta marginata Exposed to Dichlorvos" Water 16, no. 24: 3572. https://doi.org/10.3390/w16243572

APA Style

Alhassan, A. B., & Aljahdali, M. O. (2024). Behavioural and Biochemical Responses of Freshwater Bivalve Anodonta marginata Exposed to Dichlorvos. Water, 16(24), 3572. https://doi.org/10.3390/w16243572

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