In-Vivo Ecotoxicological Impact of the Insecticide Indoxacarb on Aquatic Crustaceans

doi:10.21203/rs.3.rs-3243894/v1

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In-Vivo Ecotoxicological Impact of the Insecticide Indoxacarb on Aquatic Crustaceans

https://doi.org/10.21203/rs.3.rs-3243894/v1

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Indoxacarb, an oxadiazine-class of sodium channel blocker insecticide (SCBI), demonstrates remarkable efficacy in combating a diverse array of agricultural pests. Its distinctive neurotoxic mechanism of action disrupts synaptic transmission, presenting a valuable tool for integrated pest management strategies that simultaneously mitigate environmental risks. Brine shrimp (Artemia sp), a zooplanktonic crustacean is one of the most widely occurring euryhaline organisms. It is being extensively used in various bioassay research and applied toxicology experiments, most importantly in the assessment of ecotoxicological impact of xenobiotics. Brine shrimp lethality assay is an important tool for the preliminary cytotoxicity assay of chemicals based on the ability to kill their laboratory cultured larvae (nauplii). The study was conducted to evaluate the in-vivo ecotoxicological effect of indoxacarb, a sodium-channel blocking insecticide (SCBI) on aquatic non-target Artemia species. Insects are the most important target species for indoxacarb. The present toxicological study has estimated an LC 50 value of 5.13 µg/ml with a confidence interval in the range of 0.502 to 0.917 in Artemia sp. The results indicate that indoxacarb possesses a high risk of ecotoxicity to crustaceans when used as an agricultural pesticide.

Indoxacarb

pesticide

ecotoxicity

sodium channel

brine shrimp lethality

Artemia, a zooplanktonic crustacean is one of the most widely occurring euryhaline organisms (Nunes et al., 2005). It is being extensively used in various bioassay research and applied toxicology experiments. Propagation of Artemia is very simple and cost effective which makes it a very convenient and simple organism for laboratory purposes. Despite the extensive pieces of literature on artemia, a standardized protocol for acute toxicity testing was developed only in the 1980’s (Sarah et al., 2017). Recent researches focus more on the use of this species for monitoring ecotoxicology. Additionally, the laboratories focusing on their research in environmental issues utilize Artemia sp. tests just as frequently as the medical, pharmaceutical, and food industries. Artemia bioassays are mainly employed for the preliminary toxicity screening of various plant extracts (Ghosh et al., 2015), fungal toxins (Harwig & Scott, 1971), cyanobacterial toxins (Lee et al., 1999), heavy metals (Reddy & Osborne, 2020), pesticides (Michael et al., 1956) and recently for nanoparticles also (Ates et al., 2020).

Indoxacarb, a novel member of the oxadiazine class of insecticides, functions via its active metabolite, which targets voltage-gated sodium channels in insects. This interaction obstructs the initiation of action potential generation within the nerve fibres, effectively impeding neural communication, leading to insecticidal activity (Mudaraddi et al., 2012), and is used in agriculture and horticulture against lepidopteran and coleopteran pests (Mccann et al., 2001). Despite their effectiveness, the broad category of pyrazoline insecticides has encountered obstacles to successful commercialization due to concerns surrounding their environmental persistence, bioaccumulation, and non-target toxicity. These effects can potentially impact birds, fish, and beneficial insects, thereby raising significant ecological and environmental concerns. The mode of action of indoxacarb is largely derived from the studies on pyrazoline (also known as dihydropyrazole), which acts by blocking the initiation of action potential generation in nerve fibres thereby blocking the voltage-gated sodium channels. The selective toxicity of indoxacarb towards pests is due to the formation of this primary active metabolite DCJW ((S)-methyl 7- chloro − 2,5 -dihydro − 2- [[(methoxycarbonyl) [4-(trifluoromethoxy) phenyl] amino] carbonyl] indeno [1,2-e] [1,3,4]oxadiazine- 4a(3H)-carboxylate whereas in higher animals’ its inactive metabolites are formed which will be excreted via alternate routes (Tsurubuchi & Kono, 2003). Consequently, this compound demonstrates a higher degree of selective toxicity towards insects as compared to mammals, offering an advantageous safety profile from an environmental and ecological perspective. However, despite its selective toxicity, this pesticide may still pose detrimental effects on aquatic ecosystems when utilized in agricultural settings. Leaching into water bodies could lead to the inadvertent extermination of beneficial organisms, thereby disrupting the ecological balance and overall health of these aquatic environments. Therefore, a research investigation was undertaken to elucidate the impact of this compound on Artemia, an exemplar aquatic organism. The study sought to quantify the degree of toxicity inflicted upon this species, thereby contributing to a more comprehensive understanding of the environmental implications associated with the use of this pesticide.

Experimental organisms

Artemia sp. cysts were purchased locally and were stored in a cool and dry place. In this experiment, approximately 500 milligrams of the cysts were placed into a glass receptacle containing saltwater, formulated by dissolving 9 grams of uniodized table salt in one litre of uncontaminated tap water. Adequate aeration and agitation were ensured by connecting an aerator to the jar, with the end of the airline from an air pump inserted into the base of the jar. A 60-Watt bulb, serving as a light source, was positioned over the glass container. Following an incubation period of 24–36 hours at room temperature (between 25–30°C), under conditions of robust aeration and continuous illumination, the hatched larvae (nauplii) were successfully hatched and observed to be swimming at the surface of the jar.

Post hatching, nauplii were collected by discontinuing the aerator and the lamp. The unfilled eggs floated, enabling the brine shrimp to concentrate within the water column, thus facilitating easy collection. Throughout the duration of the study, the nauplii were not provided with any external nourishment, as they could survive for up to 48 hours without food, drawing sustenance from the yolk sac. Potential contributing factors to the nauplii mortality could have been the impact of the test substance or malnutrition. To ensure that the mortality observed was an effect of the test compound, a control sample containing only nauplii, without the presence of any compound, was simultaneously employed.

Assessment of acute toxicity of indoxacarb on brine shrimp larvae

In order to estimate the acute toxicity of indoxacarb, stock solution of the same was prepared at a concentration of 100 µg/ml by dissolving 0.1 mg indoxacarb in 1 ml distilled water and serial dilutions were made at 50, 37.5, 25, 12.5, 10, 6.25, 5, 3.125, and 1.5625 µg/ml from the stock solution. Six each of nauplii were transferred into a six-well plate using a Pasteur pipette and were exposed to different concentrations of indoxacarb. Three replicates were taken for each concentration. Following exposure to the test compound for 1 hour, the nauplii were visually inspected under a microscope. Individuals displaying no observable movement, even after gentle prodding with pipette, were classified as dead (Sarah et al., 2017). The percentage of mortality was calculated after 1 hour by counting the number of dead nauplii and those who survived using the formula mentioned below:

Percentage mortality = \(\frac{Numer of dead nauplii}{Total number of nauplii in a well}\) x 100

From the percentage mortality, the acute LC₅₀ was calculated by non-linear dose response analysis with percent mortality plotted against log dose using GraphPad Prism V 5.0.

The mean mortality percent (± SEM) in artemia induced by indoxacarb is depicted in Table 1. It can be observed that the compound did not produce any mortality till 3.125 µg/mL while it produced 100 per cent mortality at 37.5 µg/mL concentration in brine shrimp. These data imply that the compound exhibits substantial lethality, thus potentially posing a significant risk to aquatic crustaceans.

Table 1

Mean per cent mortality of brine shrimp larvae (nauplii) with ascending concentrations of indoxacarb with three replicates (with n = 6)
Concentration of indoxacarb (µg/mL)	Mean(± SEM) percent mortality of nauplii
1.5625	0 ± 0
3.125	0 ± 0
5	28.57 ± 0
6.25	42.59 ± 3.46
10	65 ± 8.9
12.5	78.33 ± 8.25
25	95.83 ± 2.95
37.5	100 ± 0
50	100 ± 0
100	100 ± 0

Dose response curve and log dose versus response curve were created using the individual data of each replicate and their graphs are plotted in Fig. 1. The LC50 of indoxacarb was calculated using in-built function of GraphPad Prism V 5.0.

The present toxicological study on Artemia sp. using indoxacarb, a sodium channel blocking insecticide (SCBI) showed a mean log LC₅₀ value of 0.87 ± 0.06 and LC₅₀ value of 7.391µg/mL with a confidence interval in the range of 5.485 to 9.958 µg/ml. The determination of the LC₅₀ value and plotting of the graph was done using GraphPad Prism V 5.0.

Artemia sp. can be used as an appropriate testing organism for a preliminary toxicity screening for most of the compounds since it represents a unique ecosystem (Johari et al., 2019). In this study, the graph clearly indicates a concentration dependent effect on mortality of Artemia sp. using serial dilutions of indoxacarb. The acute effect of pesticides on species other than the targets is indicated by a steady increase in mortality rate in the early stages as observed in the study. The study is significant in the sense that it produces acute toxicity even in an untargeted organism. The aforementioned discovery emphasizes the necessity of comprehensive assessments of toxicity in additional targeted and nontargeted species, with concurrent use of several creatures, representative of various trophic levels (Nunes et al., 2005). It is debatable whether various test species are appropriate for aquatic toxicology screening applications.

The growth phase of Artemia sp. after hatching undergoes rapid morphological changes and they reach the adult stage in just 12–15 days. The genus of Artemia consists of both reproductive and parthenogenic species (Browne, 1980). Artemia sp. presents a highly advantageous model for toxicity studies due to several factors, including the obviation of vertebrate animal welfare issues, the relative simplicity of laboratory culture, a comprehensive understanding of its biology, and the convenience of its wide geographic distribution. Further enhancing its suitability are its easy propagation and small size, as well as its adaptation to a broad spectrum of water salinity levels ranging from 5 g/L to 300 g/L and temperatures spanning from 6°C to 40°C. This is in addition to its short life cycle, the commercial availability of its cysts, and the viability of these cysts when stored under appropriate conditions. (Madhav et al., 2017). Artemia, serving as a substantial nutritional resource for juvenile fish, is endorsed by the Organization for Economic Cooperation and Development (OECD) for incorporation in juvenile fish diets, as per OECD guideline 210, 1992 (OECD 210, 1992). Therefore, Artemia, being a prey species for fish, constitutes a potential vector for bioaccumulation, thereby facilitating the transference of toxins through the gastrointestinal systems of fish and crustacean species. This phenomenon could precipitate potential risks related to cumulative consumption over extended periods.

The study findings elucidate that indoxacarb exhibits substantial toxicity to aquatic crustaceans even at minimal concentration of 7.4 µg/mL. Insecticides can lead to significant ecotoxicity, with impacts that can extend across multiple levels of biological organization. Key consequences may include loss of biodiversity due to decline or extinction of non-target species, disruption of food chains resulting from an imbalance in prey and predator populations, and pollinator decline which has far-reaching effects on both wild and crop plants. Additionally, the phenomenon of bioaccumulation and biomagnification can lead to toxin concentration in apex predators, affecting their health and survival. Insecticides can also degrade soil health by affecting beneficial soil organisms and nutrient cycles, reduce water quality through leaching into water bodies, and promote the development of resistance in target populations, exacerbating environmental impacts (Goulson, 2013). Hence, responsible use of insecticides, considering possible alternatives, and taking measures to minimize non-target exposure and environmental contamination is of utmost importance. The fraction of this compound that seeps into the environment post-application in agricultural or animal husbandry practices poses a significant hazard to aquatic organisms, particularly invertebrates. Despite its relative low toxicity in mammals, indoxacarb presents considerable risk to aquatic invertebrates, thereby highlighting the necessity of evaluating its ecotoxic potential prior to its utilization as an agricultural pesticide.

The present study concludes that the estimated LC 50 value of indoxacarb was 5.13 µg/ml with a confidence interval in the range of 0.502 to 0.917 in Artemia sp. The results indicate that indoxacarb possesses a high risk of ecotoxicity to aquatic crustaceans when used as an agricultural pesticide, when it leaches into aquatic ecosystems.

Acknowledgements

Funding provided by Kerala State Council for Science Technology and Environment vide “KSCSTE/919/2022-SRSLS Development of an eco-friendly smart organogel based drug delivery system for administration of antibiotics in aquaculture and evaluation of its efficacy as targeted drug delivery system in fishes” is thankfully acknowledged. Authors are thankful to the facilities provided by Kerala Veterinary and Animal Sciences University.

Authors’ contributions

Suresh N Nair conceptualised and designed the study, Aswathy
P.U, and Archana Raj conducted the experiments, Suresh N Nair analysed the data, Aswathy
P.U, and Archana Raj wrote the initial draft, Suresh N Nair edited the manuscript. Finally, all the authors have read and finalised the manuscript.

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest

Ates, M., Danabas, D., Ertit Tastan, B., Unal, I., Cicek Cimen, I. C., Aksu, O., Kutlu, B., & Arslan, Z. (2020). Assessment of Oxidative Stress on Artemia salina and Daphnia magna After Exposure to Zn and ZnO Nanoparticles. Bulletin of Environmental Contamination and Toxicology, 104(2), 206–214. https://doi.org/10.1007/s00128-019-02751-6
Browne, R. A. (1980). Competition Experiments Between Parthenogenetic and Sexual Strains of the Brine Shrimp , Artemia Salina Author ( s ): Robert A . Browne Published by : Ecological Society of America Stable URL : http://www.jstor.org/stable/1937409 COMPETITION EXPERIMENTS B. America, 61(3), 471–474.
Ghosh, A., Banik, S., & Islam, M. A. (2015). In vitro thrombolytic, anthelmintic, anti-oxidant and cytotoxic activity with phytochemical screening of methanolic extract of Xanthium indicum leaves. Bangladesh Journal of Pharmacology, 10(4), 854–859. https://doi.org/10.3329/bjp.v10i4.23829
Goulson, D. (2013). An overview of the environmental risks posed by neonicotinoid insecticides. Journal of Applied Ecology, 50(4), 977–987. https://doi.org/10.1111/1365-2664.12111
Harwig, J., & Scott, P. M. (1971). Brine shrimp (Artemia salina L.) larvae as a screening system for fungal toxins. Applied Microbiology, 21(6), 1011–1016. https://doi.org/10.1128/aem.21.6.1011-1016.1971
Johari, S. A., Rasmussen, K., Gulumian, M., Ghazi-Khansari, M., Tetarazako, N., Kashiwada, S., Asghari, S., Park, J. W., & Yu, I. J. (2019). Introducing a new standardized nanomaterial environmental toxicity screening testing procedure, ISO/TS 20787: aquatic toxicity assessment of manufactured nanomaterials in saltwater Lakes using Artemia sp. nauplii. Toxicology Mechanisms and Methods, 29(2), 95–109. https://doi.org/10.1080/15376516.2018.1512695
Lee, T.-H., Chen, Y.-M., & Chou, H.-N. (1999). Toxicity assay of cyanobacterial strains using Artemia salina in comparison with the mouse bioassay. Acta Zoologica Taiwanica, 10(1), 1–8.
Madhav, M. R., David, S. E. M., Kumar, R. S. S., Swathy, J. S., Bhuvaneshwari, M., Mukherjee, A., & Chandrasekaran, N. (2017). Toxicity and accumulation of Copper oxide (CuO) nanoparticles in different life stages of Artemia salina. Environmental Toxicology and Pharmacology, 52, 227–238.
Mccann, S. F., Annis, G. D., Shapiro, R., Piotrowski, D. W., Lahm, G. P., Long, J. K., Lee, K. C., Hughes, M. M., Myers, B. J., Griswold, S. M., Reeves, B. M., March, R. W., Sharpe, P. L., Lowder, P., Barnette, W. E., & Wing, K. D. (2001). The discovery of indoxacarb : oxadiazines as a new class of pyrazoline-type insecticides. 164(October 2000), 153–164.
Michael, A. S., Thompson, C. G., & Abramovitz, M. (1956). Artemia salina as a test organism for bioassay. Science, 123(3194), 464.
Mudaraddi, T. Y., Potadar, R. R., & Kaliwal, B. B. (2012). Indoxacarb induces liver oxidative stress in Swiss Albino Mice. European Journal of Experimental Biology, 2(1), 180–186.
Nunes, B., Carvalho, F., & Guilhermino, L. (2005). Acute toxicity of widely used pharmaceuticals in aquatic species: Gambusia holbrooki, Artemia parthenogenetica and Tetraselmis chuii. Ecotoxicology and Environmental Safety, 61(3), 413–419. https://doi.org/10.1016/j.ecoenv.2004.08.010
OECD 210, (1992). Test No. 210: Fish, Early-Life Stage Toxicity Test. Test No. 210: Fish, Early-Life Stage Toxicity Test, July, 1–24. https://doi.org/10.1787/9789264070103-en
Reddy, S., & Osborne, W. J. (2020). Heavy metal determination and aquatic toxicity evaluation of textile dyes and effluents using Artemia salina. Biocatalysis and Agricultural Biotechnology, 25, 101574.
Sarah, Q. S., Anny, F. C., & Misbahuddin, M. (2017). Brine shrimp lethality assay. Bangladesh Journal of Pharmacology, 12(2), 186–189. https://doi.org/10.3329/bjp.v12i2.32796.
Tsurubuchi, Y., & Kono, Y. (2003). Modulation of sodium channels by the oxadiazine insecticide indoxacarb and its N-decarbomethoxylated metabolite in rat dorsal root ganglion neurons. Pest Management Science, 59(9), 999–1006. https://doi.org/10.1002/ps.652.

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In-Vivo Ecotoxicological Impact of the Insecticide Indoxacarb on Aquatic Crustaceans

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Abstract

Figures

Introduction

Materials and methods

Experimental organisms

Assessment of acute toxicity of indoxacarb on brine shrimp larvae

Results

Discussion

Conclusion

Declarations

References

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