Nanoformulation of Cartap Hydrochloride and its Effect on Growth and Physiology of Anabaena Variabilis ARM 441

Abstract

Insecticides are extensively applied in crop production to combat crop loss due to pest infestation and sometimes at higher concentration than their recommended doses, which has become an environmental hazard. Sustainable agricultural practices highlight minimal application of insecticides at low concentration. Use of controlled release formulations (CRFs) of pesticides in which active compound is associated with inert materials has emerged as an appealing alternative. Cartap hydrochloride is a moderately hazardous nereistoxin analogue insecticide that is predominantly applied in paddy fields of India, at a recommended dose of 10 µg ml-1 to kill chewing and sucking insect pests of rice crop. Nanoformulation of cartap hydrochloride using two natural polymers chitosan and alginate was done and study its release kinetics under in vitro conditions. Prior to that overall impact of cartap hydrochloride induced stress response and physiology of Anabaena variabilis ARM 441 were studied under pure conditions, a filamentous heterocystous cyanobacterium commonly used as algal biofertilizer in paddy cultivation.

Anabaena sp. could tolerate commercial grade insecticide up to 30 µg ml-1. However, at the recommended dose of 10 µg ml-1, it caused reduction in algal growth, total nitrogen and heterocyst frequency by 47.28, 24.29 and 17.72% respectively, as well as photosynthetic pigments under pure culture conditions. Increased concentrations of cartap resulted in a decrease in total protein content while increasing carbohydrate content. Scanning electron micrographs revealed cell rupture and breakage in filaments due to cartap exposure with the formation of akinetes. Cartap hydrochloride induced stress, since levels of superoxide dismutase, peroxidase and catalase were increased by 108.57, 187.5 and 117% respectively. Generation of superoxide radicals and hydrogen peroxide were also increased by 152.48 and 34% respectively. Lipid peroxidation was increased by 31.03%, whereas there was decline in ascorbate content by 48.45%, however the glutathione content was increased by 128.57%. The effect of cartap was strongest on NiR activity, followed by GS activity, and NR activity, among these three enzymes. To check whether the cyanobacterium was resistant to cartap or utilized it intracellularly, cyanobacterium was grown in medium supplemented with cartap ranging from 5, 10, 30 µg ml-1. The cyanobacterium removed cartap by 67, 55.8, and 50.43% in 5, 10, 30 µg ml-1 after 24 hours. Increase in osmolytes such as proline from 8.6 to 32.8% and sucrose from 61.22 to 90.13% indicates their possible role in overcoming cartap induced oxidative stress and can be helpful in assessing its detrimental effect on Anabaena variabilis ARM 441, since cyanobacterial biofertilizers are purposely used in paddy fields as nitrogen contributors.

In order to reduce toxicity of cartap and for its controlled delivery, two types of insecticidal nanoformulations were developed using chitosan tripolyphosphate and chitosan alginate with help of ionic gelation and polyelectrolyte complexation method. These nanoformulations were characterized by dynamic light scattering (DLS), field emission scanning electron microscope (FESEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectra. The FESEM images revealed that the size of chitosan tripolyphosphate (CS-TPP) nanospheres (nps) was in range of 117.01-185.27 nm, whereas cartap hydrochloride entrapped chitosan tripolyphosphate (C-CS-TPP) nanospheres had a size of 163.50-276.74 nm. However, the size of chitosan alginate (CS-ALG) nanospheres (nps) was in range of 101.66 -158.33 nm, whereas cartap hydrochloride entrapped chitosan alginate (C-CS-ALG) nanospheres had a size of 107.58 -173.07 nm as revealed by FESEM. FTIR results confirmed loading of cartap hydrochloride into both type of nanospheres. The nanospheres showed encapsulation efficiency of 86.1% and 76.19% in C-CS-TPP and C-CS-ALG and were stable for 30 days at ambient temperature. In-vitro release kinetics of insecticide from nanospheres followed a non Fickian diffusion mechanism in C-CS-TPP but super case II transport mechanism was followed by C-CS-ALG nanospheres.

With the application of such control release nanoformulations, it is possible to reduce the frequency of field application of insecticide due to its slow release to the target organism, which is economical as well as environmentally safe. Encapsulating cartap hydrochloride in a single carrier system can have prolong activity against targeted organisms at low doses of insecticide, and will also allow to proliferate cyanobacteria in paddy fields because of slow release of insecticide without negatively affecting the cyanobacterial flora. These nanoformulations because of their biodegradability, biocompatibility, greater stability, low toxicity and simple synthesis process, offers valuable tool for pesticide delivery systems.