Acute Toxicity of Malathion, Permethrin, and Roundup on the Tropical Freshwater Shrimp Xiphocaris elongata (Guérin-Méneville, 1855)

Abstract

Urban and agricultural runoffs can transport contaminants and pesticides into freshwater ecosystems, particularly in the developing tropics. For instance, organophosphate and pyrethroids pesticides, such as Roundup, Malathion, and Permethrin, have been found in tropical streams. The uncontrolled application of these pesticides has become a growing concern due to their adverse effects on various non-targeted organisms. Unfortunately, most studies have focused on a few selected model species, ignoring the effects on other non-target organisms, which may play an important role in tropical lotic ecosystems. In addition, the biological characteristics of aquatic crustaceans, including their morphology, physiology, and behavior, make them susceptible to toxic chemicals. For this reason, this study used the widely distributed freshwater shrimp Xiphocaris elongata as a model organism to determine the acute toxicity of Permethrin, Malathion, and Roundup. Our results show that the proportion of mortality of X. elongata in each concentration group became progressively higher as the concentration of exposure increased. We also found that the synthetic pyrethroid Permethrin was the most toxic pesticide tested, with a median lethal concentration (LC₅₀) value for 96 h of 3.96 × 10⁻⁶ µg·L⁻¹, followed by organophosphate Malathion (8.87 µg·L⁻¹) and Roundup (748.92 µg·L⁻¹). Experiments with this freshwater shrimp showed a good control performance and reproducibility for the tested pesticides. This study demonstrated that X. elongata is a suitable test organism that can be a representative bioindicator of pesticide toxicity in tropical streams.

1. Introduction

Pesticide contamination has become a growing concern in the tropics due to its adverse effects on a variety of non-targeted organisms. Faunal assemblages of lotic ecosystems are threatened by anthropogenic activities, including pesticide pollution [1,2]. Urban and agricultural runoff can transport contaminants and pesticides into streams [3,4]. Pesticides, however, are expected to have a much higher effect on aquatic environments because these are the ultimate recipients of contaminants [5,6]. The adverse effects of pesticides can vary depending on the organism and the chemical’s mode of action. Most ecotoxicological studies have focused on a few selected model species, ignoring the effects on other non-target organisms that may play important roles in tropical lotic ecosystems [7,8].

Organophosphate and pyrethroids pesticides, such as Roundup, Malathion, and Permethrin, have been present in many tropical freshwater environments [9,10]. For years, Roundup has been a common organophosphate pesticide that controls weeds in cultivated croplands. However, due to its toxicity to humans, it has recently been banned in several countries [11]. The primary mode of action is in the shikimate pathway that links primary and secondary metabolisms [12]. Specifically, glyphosate inhibits the activity of the enzyme 5-enolpyuvylshikimate-3-phosphate synthase (EPSPS) found in plants and acetylcholinesterase (AChE) enzyme activity in freshwater shrimp [13]. The exposure of glyphosate to non-target organisms is of great concern to aquatic toxicologists because of their extensive use, high water solubility, and slow degradation rate in freshwater environments [14].

Permethrin is a synthetic pyrethroid used in agricultural and residential zones to control a wide range of insects. The application of Permethrin is increasing because of its use in mosquito control programs [15]. Toxicological data for Permethrin are available for aquatic organisms, but the information is needed to apply to tropical regions [7,15]. Synthetic pyrethroids induce a continuous series of nerve impulses that disrupts the normal functioning of the nervous system. The mode of action is related to the inhibition of sodium channels in nerve membranes and acetylcholinesterase (AChE) enzyme activity [16,17].

Malathion is a common organophosphate insecticide used to control mosquitoes and fruit flies. As an organophosphate insecticide, Malathion inhibits acetylcholinesterase (AChE). Acetylcholinesterase is a critical enzyme for the normal function of the nervous system [18,19]. The inhibition of this enzyme can result in the accumulation of the neurotransmitter acetylcholine in the synaptic gap, leading to disruption of the nervous system [16]. Previous studies found that Malathion is highly toxic to many fish and aquatic invertebrates [18,19,20,21,22,23,24]. In addition, the morphology, physiology, and behavior of aquatic invertebrates, such as crustaceans, make them particularly susceptible to toxic chemicals [23]. Because of those biological characteristics, this study used the widely distributed tropical freshwater shrimp Xiphocaris elongata (Guérin-Méneville, 1855) [24] as a model organism to determine the acute toxicity of Permethrin, Malathion, and Roundup.

This study aimed to determine the LD₅₀ of Malathion, Permethrin, and Roundup pesticides on the tropical shrimp X. elongata. Xiphocaris is an endemic shredder shrimp in the Caribbean and plays a fundamental role in organic matter processing in Caribbean tropical streams [25,26,27]. Xiphocaris is a slender shrimp adapted to walk delicately on the substrate and swim on the water column. Due to its cosmopolitan distribution in the Caribbean area, it is an ideal animal to use as a model organism. The findings will provide a scientifically significant contribution to environmental toxicology and the protection of aquatic ecosystems.

2. Materials and Methods

2.1. Collection and Acclimation of Freshwater Shrimp

Adults of X. elongata (post-orbital length larger than 13.0 mm) were collected at Río Sabana near Sabana Field Research Station (18°19′29″ N, 65°43′47″ W) in Luquillo (elevation 113 above sea level; Figure 1), Puerto Rico. All shrimp were collected during the high-rainfall season (May–November) of the year 2019. Freshwater shrimp were collected using an electrofishing backpack (Model 12-B, Smith-Root, Vancouver, Washington, DC, USA) [28]. Collections consisted of five upstream electrofishing passes in each sampling reach (10 m). Hand nets were used to collect the organisms. The habitats sampled included riffles, runs, pools, and aquatic vegetation. The collected shrimp were identified according to [24,25,26]. Each shrimp’s post-orbital length (POL) was measured from the post-orbital region to the end of the carapace with a dial caliper (0.01 mm precision). The measurement from the tip of the rostrum was not used because the length of X. elongata varies depending on the presence of fish predators. The gravid condition (presence of eggs on the abdomen) and the sexual maturity of each shrimp were determined (post-orbital length larger than 13.0 mm). Acclimation was performed, where the organisms were maintained for at least one week in aerated water, at 20 °C, and with a photoperiod of 12 h:12 h (light:dark). Shrimp were fed daily with fish flakes.

2.2. Acute Toxicity Tests

Acute toxicity tests were performed to determine the effect of pesticides on the survival of X. elongata. Toxicities were determined by applying a static procedure without renewal of test solutions. All experiments were conducted in glass aquaria with a 5 L capacity, to which the pesticides were added at different concentrations. Toxicities were expressed based on the concentration of the active ingredient rather than the formulation.

Ten test concentrations of pesticides and controls were used for each bioassay (Malathion, μg/L—1.18, 1.77, 2.66, 3.99, 5.98, 8.97, 13.5, 20.2, 30.3, and 45.4; Permethrin, μg/L—1.90 × 10⁻⁷, 3.90 × 10⁻⁷, 5.90 × 10⁻⁷, 9.90 × 10⁻⁷, 1.00 × 10⁻⁶, 2.00 × 10⁻⁶, 5.00 × 10⁻⁶, 9.00 × 10⁻⁶, 1.90 × 10⁻⁵, and 3.90 × 10⁻⁵; Roundup, μg/L—68.1, 153.2, 229.8, 344.8, 517.2, 775.9, 1163.8, 1745.8, 2618.6, and 3927.9). The pesticide concentrations were obtained by dilutions of the Roundup Super Concentrate^®® (50.2% glyphosate isopropilamine salt), Southern Ag Malathion^®® (50% Malathion), and Martins Permethrin SFR^®® (36.8% Permethrin). These chemicals represent the most common pesticides in use on the island.

Adult freshwater shrimp (POL > 13.0 mm) were selected from the acclimation tanks and distributed randomly into exposure tanks. The exposure tanks (N = 25; cubic dimensions: length 15.4 cm, height 15.4 cm, and depth 15.4 cm) were filled with 2.0 L of dechlorinated water, pesticide, and shrimp (1 shrimp per tank). Dead shrimp were counted and removed from the tanks every 24 h (24, 48, 72, and 96 h), and the alive organisms continued in the experiment until we reached 96 h of observations. This procedure was repeated every 24 h. The control and the exposure tanks were kept under similar conditions of light, temperature, and constant aeration during the experiment. If the control tanks’ mortality rate reached 10% during the first 24 h, the experiments had to be reset. One hundred shrimp (1 replica = 25 shrimp; total of 4 replicas = 100 shrimp) were used for each concentration group and the control. The organisms were not fed during the test period. After pesticide exposure, mortality was recorded between 24 h and 96 h.

2.3. Statistical Analyses

Lethal concentration (LC₅₀) estimates were determined using probit analyses. Dose–response results were modeled in JMP Pro version 17.1-2023 Software (SAS Institute Inc., Cary, NC, USA) [29]. Individual mortality per dose was used in the probit analyses to predict the median lethal concentrations. The goodness-of-fit test for normal distribution showed that the data were normally distributed. One-way analysis of variance (ANOVA) was used to compare the mortality rates among different test groups. A post hoc Tukey’s test was used to identify organophosphate and pyrethroids pesticide tests with significant differences (p < 0.05).

3. Results

The proportion of mortality of X. elongata in each concentration group became progressively higher as the concentration of exposure increased. The mortality rates observed in acute toxicity tests for Permethrin (ANOVA, F(_9,39) = 66.13; p < 0.001), Malathion (ANOVA, F(_9,39) = 84.35; p < 0.001), and Roundup (ANOVA, F(_9,39) = 107.64; p < 0.001) were statistically different (

Table 1. Median lethal concentration (LC₅₀) estimates for Permethrin, Malathion, and Roundup after 96 h of exposure. CI: confidence interval; df: degrees of freedom. Asterisks indicate significant difference, *** p < 0.001.

Pesticide	LC50 µg·L−1 (95% CI)	df	F	p
Permethrin	3.96 × 10−6 (4.49 × 10−6–4.52 × 10−6)	9	66.13	<0.001 ***
Malathion	8.87 (8.31–9.49)	9	84.35	<0.001 ***
Roundup	748.92 (701.01–802.45)	9	107.64	<0.001 ***

). Median lethal concentration (LC₅₀) values for 24 h and 96 h for Permethrin were 6.91 × 10⁻⁶ µg·L⁻¹ and 3.96 × 10⁻⁶ µg·L⁻¹, respectively (Table 1; Figure 2 and Figure 3). However, malathion median lethal concentration values for 24 h and 96 h were 13.44 µg·L⁻¹ and 8.87 µg·L⁻¹, respectively (Table 1; Figure 4 and Figure 5). The least toxic pesticide was glyphosate, with LC₅₀ values of 1156.4 µg·L⁻¹ and 748.92 µg·L⁻¹ for 24 h and 96 h, respectively (Table 1; Figure 6 and Figure 7).

4. Discussion

This study assessed the acute toxicity of Permethrin, Roundup, and Malathion for the tropical freshwater shrimp Xiphocaris elongata. To evaluate the biological outcome of such exposure, it is necessary to determine lethal and sublethal concentrations for organisms. Due to the high costs and long duration of chronic tests, there is a need to perform more acute toxicity research. One advantage of using acute toxicity data is that they have been proven to be highly accurate in making long-term predictions [20]. Permethrin was the most toxic pesticide tested for our model organism, followed by Malathion and Roundup. Similarly, Sánchez-Fortun and Barahona [30] found that the aquatic invertebrates most sensitive to pyrethroid pesticides are surface-dwelling insects, mayfly nymphs, and crustaceans. Among crustacean species, Daphnia magna Straus, 1820, Daphnia pulex Leydig, 1860, Palaemonetes pugio Holthuis, 1949, and Macrobrachium rosenbergii De Man, 1879, have been used as model organisms in aquatic toxicology [31,32,33]. However, the disadvantage of using those species is that they are laboratory specimens and are not present in tropical freshwater ecosystems. In tropical regions, aquatic organisms are often exposed to pesticides and other contaminants in different stages of their lifecycle; for this reason, there is a need to develop model organisms for this geographic region, such as we have carried out here with the tropical freshwater shrimp Xiphocaris elongata. Freshwater shrimp in tropical regions have a complex lifecycle—they are amphidromous.The diadromous lifecycle is observed in many tropical and subtropical freshwater caridean shrimp: it comprises adults residing, reproducing, and releasing small embryos (larvae) in freshwater habitats, while their offspring undergo an extended larval development phase in marine environments. After 90 days in the marine or estuarine environment (depending on the species), the larvae metamorphose into juveniles after several molts. During the movement of the larvae or the juvenile from the upper to the lower parts of the stream or contrariwise, these stages are exposed to localities with different land uses (e.g., industrial, agriculture, residential, and recreational). Studies in urban and forested streams in Puerto Rico demonstrated that the different land uses have a direct influence on the freshwater shrimp, and watersheds with low urban land uses had more diversity and density [34,35]. Cruz-Rosa and Perez-Reyes [36] reported malformations in the larvae of a freshwater shrimp as a result of exposure to nanoparticles of TiO₂. This demonstrated the high sensitivity of the larvae to water pollution in the freshwater ecosystem.

Shrimp are highly susceptible to pyrethroids pesticides [37,38]. Even at sublethal concentrations, significant behavioral changes in shrimp feeding behavior and locomotion activities could affect their survival. In this study, Permethrin’s median lethal concentration (LC₅₀) value for 96 h exposure was 6.91 × 10⁻⁶ µg·L⁻¹. A study about the effect of Permethrin on the grass shrimp P. pugio showed it was acutely toxic at 0.21 µg·L⁻¹ [39]. Another study found that the freshwater prawns Palaemonetes argentinus Nobili, 1901, are susceptible to pyrethroids, with a median lethal concentration of 0.0031 µg·L⁻¹ [40]. This study also determined that Malathion was highly to moderately toxic, with a 96 h LC₅₀ value of 13.44 µg·L⁻¹. Similar results were found in studies with Malathion, where the shrimp exhibited severe toxicities: P. pugio (LC₅₀ of 38.19 µg·L⁻¹) [41], Macrobrachium nipponense (De Haan, 1849; LC₅₀ of 61.27 µg·L⁻¹) [42], Macrobrachium dayanum (Henderson, 1893; LC₅₀ of 8.0 µg·L⁻¹) [43], and Macrobrachium ferreirai Kensley & Walker, 1982 [44].

Few studies have evaluated the toxicity of glyphosate on freshwater organisms [45,46,47]. In this study, the Roundup-glyphosate median lethal concentration value for 96 h was 748.92 µg·L⁻¹. Mensah et al. [48] also investigated the acute toxicity of glyphosate on freshwater shrimp Caridina nilotica Roux, 1926. Their results showed a median lethal concentration of 25.3 mg·L⁻¹. The low toxicity of glyphosate exposure may be because the shikimic acid pathway is absent in animals. However, some studies demonstrated that high concentrations could alter animal mitochondrial activity by uncoupling oxidative phosphorylation during cellular respiration [47].

In summary, the tropical shrimp X. elongata is an ecologically important organism in freshwater environments that can be exposed to synthetic pyrethroid and organophosphate pesticides. This study demonstrated that X. elongata is a suitable test or model organism that can be a representative bioindicator of pesticide toxicity in tropical freshwater environments. Similar results were observed in a study where AChE activity was used as a biomarker to detect and assess organophosphate and pyrethroid pesticide exposure using X. elongata as a model to assess the risk of pesticide contamination in tropical freshwater environments [49]. Experiments with this freshwater shrimp species showed a good control performance and reproducibility for the different pesticides tested. Future studies should evaluate sublethal effects using environmentally relevant concentrations of specific pesticides and mixtures.