# Chemical residues in tomato plant tissue and in vitro toxicology profiles of Nemarioc-AL and Nemafric-BL phytonematicides on Raw 264.7 macrophage cells

## TL;DR

This study examines the residue levels and toxicity of two phytonematicides in tomato plants and macrophage cells.

## Contribution

The study provides new data on cucurbitacin residue accumulation and cytotoxicity of phytonematicides on macrophage cells.

## Key findings

- Nemarioc-AL and Nemafric-BL phytonematicides leave cucurbitacin residues in tomato fruits, with sandy loam soil showing the highest accumulation.
- Low concentrations of the phytonematicides are non-toxic to Raw 264.7 macrophage cells, but higher concentrations are cytotoxic.
- Apoptosis was observed at high concentrations of the phytonematicides, suggesting potential non-target effects.

## Abstract

A shift from synthetic chemical nematicides to bionematicides in plant protection has led to the development of Nemarioc-AL and Nemafric-BL phytonematicides to manage plant parasitic nematodes. However, there is a lack of information on the accumulation of cucurbitacin residues and the cytotoxicity of phytonematicides on non-target entities. The aims of the study were to determine the cucurbitacin residue accumulation after the application of Nemarioc-AL and Nemafric-BL phytonematicides in tomato fruits and to determine their cytotoxic effects on a eukaryotic (Raw 264.7 cell line) model system. Two separate trials for Nemarioc-AL phytonematicide and Nemafric-BL phytonematicide, each applied at 3%, were conducted concurrently on sandy loam, dark soil, red soil, silt soil, sandy soil, and sandy loam (+). Each trial was arranged in a randomized complete block design (RCBD) and replicated six times. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assay was used to assess the cytotoxicity of Nemarioc-AL and Nemafric-BL phytonematicides, and the Annexin-V and DAPI apoptosis assay was performed on Raw 264.7 macrophage cells. In Nemarioc-AL phytonematicide-treated soil type, the highest accumulation of cucurbitacin B residues in fruits was observed on sandy loam (+) (37.1 ng/g), followed by red soil with 27.0 ng/g and then sandy soil with 21.7 ng/g, and dark soil showed the least at 20.3 ng/g. The phytonematicides were non-toxic at lower concentrations, ≤1.25 mg/mL. However, the higher concentrations (>1.25 mg/mL) of phytonematicides exhibited cytotoxic effects on the Raw 264.7 cell line, with 50% cell viability in comparison with curcumin (100 μM). The IC50 values for Nemarioc-AL and Nemafric-BL phytonematicides on Raw 264.7 cell lines were 0.55 and 1.6 mg/mL, respectively. Similar to the MTT viability assay, the Annexin-V and DAPI apoptosis assay did show that the low concentrations of phytonematicides (0.313 mg/mL) had no signs of apoptosis or necrosis; however, high concentrations (10 mg/mL) had signs of apoptosis as opposed to necrosis. Therefore, the products can be used at lower concentrations to manage nematodes and avoid the toxicity of the products.