Pesticides Chlorpyrifos Research Articles

Effect of chlorpyrifos on the earthworm Eudrilus euginae and their gut microbiomeby toxicological and metagenomic analysis.

The earthworms are important soil invertebrates and play a crucial role in pedogenesis. The application of pesticides and prolonged exposure to pesticides causes mortality of earthworms apart from profoundly affecting the resident gut microbiome. The microbiome plays a significant effect on the metabolic processes associated with earthworms. The pesticide Chlorpyrifos (CPF) was studied for its toxicity on Eudrilus euginae by toxicity studies. The LC50 value of filter paper contact test and acute toxicity test was 3.8mg/mL and 180mg/kg. The prolonged exposure of earthworms to pesticide on reproductive toxicity resulted in the mortality of earthworms and absence of cocoon formation. Further, the effects of CPF on the whole gut microbiome of E. euginae was analyzed using a long amplicon Nanopore sequencing. Results indicated no fluctuations with Firmicutes and Bacteroidetes, that were found to be dominant at bacterial phyla level while at the genus level, remarkable differences were noticed. Clostridium dominated the earthworm gut prior to CPF exposure while Bacillus dominated after exposure. Similarly, the fungal members such as Ascomycota and Basidiomycota were observed to dominate the gut of earthworm at the phyla level before and after exposure to CPF. In contrast, Clavispora (65%) was the dominant genus before CPF exposure and Taloromyces (42%) dominated after the CPF exposure. Our study demonstrates the effect of CPF on the mortality of E. euginae while the amplicon sequencing established the unique microbiome of the gut in response to the CPF exposure.

The pace-of life explains whether gills improve or exacerbate pesticide sensitivity in a damselfly larva

Trait-based approaches are promising to make generalizations about the sensitivity of species and populations to pesticides. Two traits that may shape the sensitivity to pesticides are the surface area (related to pesticide uptake) and the metabolic rate (related to pesticide elimination). We compared the sensitivity of damselfly larvae to the pesticide chlorpyrifos and how this was modified by loss of external gills (autotomy, reducing the surface area) in both fast pace-of-life (high metabolic rate) and slow pace-of-life (low metabolic rate) populations of Ischnura elegans. The slow-paced populations were more sensitive to the pesticide than the fast-paced populations in terms of survival, growth and energy metabolism. This suggests the higher metabolic rate of fast-paced populations enabled a faster pesticide elimination. Pesticide exposure also reduced heat tolerance, especially in slow-paced larvae under hypoxia. Gill loss had opposite effects on pesticide sensitivity in slow- and fast-paced populations. In slow-paced larvae, gill loss lowered the sensitivity to the pesticide, while in fast-paced larvae, gill loss increased the sensitivity. This difference likely reflects the balance between the roles of the gills in pesticide uptake (more detrimental in slow-paced populations) and oxygen uptake (more important in fast-paced populations). Our results highlight the need to consider trait interactions when applying trait-based approaches to predict the sensitivity to pesticides.

Chlorpyrifos pesticide promotes oxidative stress and increases inflammatory states in BV-2 microglial cells: A role in neuroinflammation

The exposure to environmental stressors, such as organophosphate (OP) pesticides, has been associated with the development of neurodegenerative diseases. Chlorpyrifos (CPF) is the worldwide most used OP pesticide and one of the most hazardous pesticides as it can cross the blood-brain barrier. Since studies evaluating the effects of CPF on brain immune cells are scarce, this research investigated the oxidative and inflammatory responses of CPF exposure in murine microglial cells. BV-2 cells were exposed to different concentrations of CPF pesticide (0.3–300 μM). CPF induced activation of microglial cells, confirmed by Iba-1 and CD11b marking, and promoted microglial proliferation and cell cycle arrest at S phase. Moreover, CPF exposure increased oxidative stress production (NO, MDA, and O2∙), and upregulated pro-inflammatory cytokines (IL-1β and NLRP3) genes expression in BV-2 cells. Overall, data showed that CPF exposure, at the lowest concentrations, acted by promoting pro-oxidative and pro-inflammatory states in microglial cells. These results provide important information on the potential role of microglial activation in CPF-induced neuroinflammation and add to the expanding knowledge on the neurotoxicity of OP.

In Vitro Antioxidant Activity of Litsea martabanica Root Extract and Its Hepatoprotective Effect on Chlorpyrifos-Induced Toxicity in Rats.

In Thailand, people in the highland communities whose occupational exposure to pesticides used the root of Litsea martabanica as a detoxifying agent. However, the scientific data to support the traditional use of this plant are insufficient. This study aimed to evaluate the antioxidant activity and anti-pesticide potential of L. martabanica root extract. Antioxidant properties were investigated by 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, superoxide radicals scavenging assay, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric reducing antioxidant power (FRAP), and total phenolic content determination. In all assays, L. martabanica extracts and their fractions exhibited high antioxidant activities differently. The water extract is traditionally used as a detoxifying agent. Therefore, it was chosen for in vivo experiments. The rats received the extract in a way that mimics the traditional methods of tribal communities followed by chlorpyrifos for 16 days. The results showed that acetylcholinesterase activity decreases in pesticide-exposed rats. Treatment with the extract caused increasing acetylcholinesterase activity in the rats. Therefore, L. martabanica extract may potentially be used as a detoxifying agent, especially for the chlorpyrifos pesticide. The antioxidant properties of L. martabanica may provide a beneficial effect by protecting liver cells from damage caused by free radicals. Histopathology results revealed no liver cell necrosis and showed the regeneration of liver cells in the treatment group. L. martabanica extract did not cause changes in behavior, liver weight, hematological and biochemical profiles of the rats.

Microbiological quality and determination of pesticide residues in the lettuce produced in Bahía Blanca's horticultural belt, Argentina

The aims of this research were 1) to find out the possible microbiological contamination and residues of pesticides (chlorpyrifos and cypermethrin) in lettuce grown in Bahía Blanca’s horticultural belt, and bacteriological quality of waters used for irrigation and post-harvest washing, 2) to investigate and compare the internalization capacity of a strain of Escherichia coli isolated from irrigation water with another hospital uropathogenic strain. Some strains of bacteria can internalize into the plant tissue. This capability presents food safety threats as they cannot be removed with standard sanitation practices. To determine bacteriological quality of lettuce, E. coli, Salmonella spp and E. coli O157: H7/NM (n = 10) were investigated. Bacteriological quality of water was determined by quantification of fecal coliforms/100 mL (n = 20). Pesticides were determined by using an improved and validated QuEChERS method combined with GC-MSD. Irrigation water in 19/20 samples did not present health risk. Bacteriological quality of lettuce met demands in 9/10 samples. Pesticides were detected in half of the samples, values exceeding the FAO recommendations. An effort is strongly recommended to implement both, best phytosanitary practices and monitoring programmes. Uropathogenic strain was able to internalize into plant tissue, while the nonpathogenic one was not.

NMR-Based Metabolomics Approach to Explore Brain Metabolic Changes Induced by Prenatal Exposure to Autism-Inducing Chemicals

NMR offers the unique potential to holistically screen hundreds of metabolites and has already proved to be a powerful technique able to provide a global picture of a wide range of metabolic processes underlying complex and multifactorial diseases, such as neurodegenerative and neurodevelopmental diseases. The aim of this study was to apply an NMR-based metabolomics approach to explore brain metabolic changes in both male and female rats induced by prenatal exposure to two chemicals associated with autism disorders-the organophosphorus pesticide chlorpyrifos (CPF) and the antiepileptic drug valproic acid (VPA)-at different postnatal ages. Depending on the age and on the brain region (hippocampus and cerebellum), several metabolites were shown to be significantly affected by exposure to both compounds. The evaluation of the spectral profiles revealed that the nervous-system-specific metabolite N-acetylaspartate (NAA), amino acid neurotransmitters (e.g., glutamate, glutamine, GABA, glycine), pyroglutamic acid, unsaturated fatty acids, and choline-based compounds are discriminant biomarkers. Additionally, metabolic changes varied as a function of age, but importantly not of sex.

Elimination of chlorpyrifos from plastic bags used in the protection of banana and plantain clusters

ABSTRACT In Colombia, plastic bags containing the pesticide chlorpyrifos protect the growing bunches of bananas and plantains from insects. When the bananas are harvested, the plastic bags are removed. This plastic, which may contain chlorpyrifos residues, is sometimes reused or recycled for other uses such as toy manufacturing. This is prohibited by environmental authorities in Colombia. The paper reports a study into the elimination of residual chlorpyrifos from such bags by means of hydrolysis and photolysis. About 100% of the chlorpyrifos was removed.

Biodegradation pathway of the organophosphate pesticides chlorpyrifos, methyl parathion and profenofos by the marine-derived fungus Aspergillus sydowii CBMAI 935 and its potential for methylation reactions of phenolic compounds

The indiscriminate use of organophosphate pesticides causes serious environmental and human health problems. This study aims the biodegradation of chlorpyrifos, methyl parathion and profenofos with the proposal of new biodegradation pathways employing marine-derived fungi as biocatalysts. Firstly, a growth screening was carried out with seven fungi strains and Aspergillus sydowii CBMAI 935 was selected. For chlorpyrifos, 32% biodegradation was observed and the metabolites tetraethyl dithiodiphosphate, 3,5,6-trichloropyridin-2-ol, 2,3,5-trichloro-6-methoxypyridine, and 3,5,6-trichloro-1-methylpyridin-2(1H)-one were identified. Whereas 80% methyl parathion was biodegraded with the identification of isoparathion, methyl paraoxon, trimethyl phosphate, O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate, 1-methoxy-4-nitrobenzene, and 4-nitrophenol. For profenofos, 52% biodegradation was determined and the identified metabolites were 4-bromo-2-chlorophenol, 4-bromo-2-chloro-1-methoxybenzene and O,O-diethyl S-propylphosphorothioate. Moreover, A. sydowii CBMAI 935 methylated different phenolic substrates (phenol, 2-chlorophenol, 6-chloropyridin-3-ol, and pentachlorophenol). Therefore, the knowledge about the fate of these compounds in the sea was expanded, and the marine-derived fungus A. sydowii CBMAI 935 showed potential for biotransformation reactions.

Microplastics, chlorpyrifos and their mixtures modulate immune processes in the terrestrial crustacean Porcellio scaber

Microplastics and agrochemicals are common pollutants in terrestrial ecosystems. Their interaction during coexistence in soils may influence their fate and adverse effects on terrestrial organisms. The aim of this study was to investigate how the exposure to two types of microplastics; polyester fibres, and crumb rubber; induce changes in immune parameters of Porcellio scaber and if the co-exposure of microplastics affects the response induced by the organophosphate pesticide chlorpyrifos. A number of immune parameters, such as total haemocyte count, differential haemocyte count, and phenoloxidase-like activity were assessed. In addition, the acetylcholinesterase (AChE) activity in the haemolymph was evaluated as a measure of the bioavailability of chlorpyrifos. After three weeks of exposure, the most noticeable changes in the measured immune parameters and also a significantly reduced AChE activity were seen in chlorpyrifos-exposed animals. Both types of microplastic at environmentally relevant concentrations caused only slight changes in immune parameters which were not dependent on the type of microplastic, although the two types differed significantly in terms of the chemical complexity of the additives. Mixtures of chlorpyrifos and microplastics induced changes that differed from individual exposures. For example, alterations in some measured parameters suggested a reduced bioavailability of chlorpyrifos (AChE activity, haemocyte viability) caused by both types of microplastics exposure, but the increase of haemocyte count was promoted by the presence of fibres implying their joint action. In conclusion, this study suggests that immune processes in P. scaber are slightly changed upon exposure to both types of microplastics and microplastics can significantly modulate the effects of other co-exposed chemicals. Further research is needed on the short-term and long-term joint effects of microplastics and agrochemicals on the immunity of soil invertebrates.

Acute Changes in Thyroid Hormone Levels among Thai Pesticide Sprayers.

The objective of this study was to investigate the relationship of acute pesticide exposures and acute changes in thyroid hormones among Thai farmers. We recruited 78 farmers, who were scheduled to spray insecticides (chlorpyrifos and/or cypermethrin) or herbicides (paraquat and/or glyphosate). On the day before spraying, farmers collected their first morning void urine and went for blood collection. On the spray day, urine samples were collected at end of the spraying event and they were interviewed with questionnaires. The next morning, the first morning void urine and blood samples were collected. Blood samples were analyzed for thyroid hormones. Urine samples were analyzed for the metabolites of the pesticide sprayed. The results showed that the thyroid hormones, free triiodothyronine (FT3) and total triiodothyronine (T3) were significantly reduced as urinary chlorpyrifos metabolite increased the day after spraying. Total thyroxine (T4) significantly increased as cypermethrin metabolites increased the day after spraying. T4 significantly increased as urinary glyphosate levels increased; however, FT3 and T3 decreased significantly as urinary paraquat levels increased the day after spraying. These findings suggest that acute exposures to the pesticides chlorpyrifos, cypermethrin, paraquat and glyphosate can produce acute effects on the hypothalamic–pituitary–thyroid (HPT) axis, acutely altering thyroid hormone levels.

Rapid detection of chlorpyrifos pesticide residue in tea using surface-enhanced Raman spectroscopy combined with chemometrics

Surface enhanced Raman spectroscopy based on rapid pretreatment combined with Chemometrics was used to determine chlorpyrifos residue in tea. Au nanoparticles were used to as enhance substrate. Different dosages of PSA and NBC were investigated to eliminate the tea substrate influence. Competitive adaptive reweighted sampling (CARS) was used to optimize the characteristic peaks, and compared to full spectra variables and the experiment selected variables. The results showed that PSA of 80 mg and NBC of 20 mg was an excellent approach for rapid detecting. CARS - PLS had better accuracy and stability using only 1.7% of full spectra variables. SVM model achieved better performance with R2p = 0.981, RMSEP = 1.42 and RPD = 6.78. Recoveries for five unknown concentration samples were 98.47 ~ 105.18% with RSD − 1.53% ~ 5.18%. T-test results showed that t value was 0.720, less than t0.05,4 = 2.776, demonstrating that no clear difference between the real value and predicted value. The detection time of a single sample is completed within 15 min. This study demonstrated that SERS coupled with Chemometrics and QuEChERS may be employed to rapidly examine the chlorpyrifos residue in tea towards its quality and safety monitoring.

MINIATURIZED LIQUID-LIQUID EXTRACTION WITH LOW-TEMPERATURE PARTITIONING TECHNIQUE (LLE/LTP) FOR PESTICIDE ANALYSIS IN A BIOLOGICAL MATRIX BY GAS CHROMATOGRAPHY

The liquid-liquid extraction with low-temperature partitioning technique (LLE/LTP) was miniaturized to determine the pesticides chlorpyrifos, bifenthrin, λ-cyhalothrin, and deltamethrin in a biological matrix, by gas chromatography. The method was proposed for fetal-bovine serum samples (500 μL), by using a full factorial design (23) with one center point. The evaluated factors included the volume of water added to the sample (1000 μL), volume of extraction solvent (500 μL) and stirring time (60 s) – a cleanup step was not necessary. The method was validated for the main figures of merit. The results indicated that the LLE/LTP/GC/ECD miniaturized method was efficient in extracting pesticides from blood-serum samples (75 to 115%). It showed both selectivity and sensitiveness, with limits of quantification (LOQ) ranging from 8 at 17 μg L-1. Once validated, the method was applied to blood-serum samples of rural workers from a city in the Brazilian state of Minas Gerais. In that case, the presence of chlorpyrifos was detected in two of the samples.

Chlorpyrifos in environment and food: a critical review of detection methods and degradation pathways.

Chlorpyrifos (CP) is a class of organophosphorus (OP) pesticides, which find extensive applications as acaricide, insecticide and termiticide. The use of CP has been indicated in environmental contamination and disturbance in the biogeochemical cycles. CP has been reported to be neurotoxic and has a detrimental effect on immunological and psychological health. Therefore, it is necessary to design and develop effective degradation methods for the removal of CP from the environment. In the past few years, physicochemical (advanced oxidation process) and biological treatment approaches have been widely employed for the pesticide removal. However, the byproducts of this process are more toxic than the parent compound and along with an incomplete degradation of CP. This review focuses on the toxicity of CP, the sources of contamination, degradation pathways, physicochemical, biological, and nano-technology based methods employed for the degradation of CP. In addition, consolidated information on various detection methods and materials used for the detection have been provided in this review.

Matrix-dispersed magnetic molecularly-imprinted polyaniline for the effective removal of chlorpyrifos pesticide from contaminated water.

We report a new adsorbent nanocomposite material based on matrix-dispersed superparamagnetic iron oxide nanoparticles (SPIONs) in molecularly-imprinted polyaniline for the removal of chlorpyrifos (CPF), a hazardous organophosphate pesticide, from water. The synthesized magnetic molecularly-imprinted polymer (MMIP) was characterized by FTIR spectroscopy, XRD, magnetic susceptibility, DLS, zeta potential measurement, SEM and high-resolution TEM imaging. The average size of the naked SPIONs ranges from 15 to 30 nm according to the high-resolution TEM analysis. Moreover, the adsorption kinetics, thermodynamic parameters (ΔG, ΔH and ΔS), adsorption isotherms and rebinding conditions were investigated in detail. The proposed MMIP has an imprinting factor of 1.64. In addition, it showed a high experimental adsorption capacity of 1.77 mg g−1 and a removal efficiency of nearly 80%. The fabricated MMIP material demonstrated excellent magnetic susceptibility allowing for easy separation using an external magnetic field. The adsorption mechanism of CPF onto the MMIP adsorbent followed the second-order kinetics model and fitted to the Temkin adsorption isotherm. By studying the adsorption thermodynamics, negative ΔG values (−1.955 kJ mol−1 at room temperature) were obtained revealing that the adsorption process is spontaneous. Furthermore, the maximum adsorption capacity was obtained at room temperature (ca. 303 K), neutral pH and using a high CPF concentration.

Analytical and Bio-Analytical Method Development and Validation of Chlorpyrifos Pesticide by RP-HPLC Method

Analytical and Bio-Analytical Method Development and Validation of Chlorpyrifos Pesticide by RP-HPLC Method

Sex and Exposure to Postnatal Chlorpyrifos Influence the Epigenetics of Feeding-Related Genes in a Transgenic APOE Mouse Model: Long-Term Implications on Body Weight after a High-Fat Diet.

Developmental exposure to toxicants and diet can interact with an individual’s genetics and produce long-lasting metabolic adaptations. The different isoforms of the apolipoprotein E (APOE) are an important source of variability in metabolic disorders and influence the response to the pesticide chlorpyrifos (CPF). We aimed to study the epigenetic regulation on feeding control genes and the influence of postnatal CPF exposure, APOE genotype, and sex, and how these modifications impact on the metabolic response to a high-fat diet (HFD). Both male and female apoE3- and apoE4-TR mice were exposed to CPF on postnatal days 10–15. The DNA methylation pattern of proopiomelanocortin, neuropeptide Y, leptin receptor, and insulin-like growth factor 2 was studied in the hypothalamus. At adulthood, the mice were given a HFD for eight weeks. The results highlight the importance of sex in the epigenetic regulation and the implication of CPF treatment and APOE genotype. The body weight progression exhibited sex-dimorphic differences, apoE4-TR males being the most susceptible to the effects induced by CPF and HFD. Overall, these results underscore the pivotal role of sex, APOE genotype, and developmental exposure to CPF on subsequent metabolic disturbances later in life and show that sex is a key variable in epigenetic regulation.

Erratum — Remediation of Wastewater from Chlorpyrifos Pesticide by Nano-Gold Photocatalyst

Erratum — Remediation of Wastewater from Chlorpyrifos Pesticide by Nano-Gold Photocatalyst

Translational outcomes relevant to neurodevelopmental disorders following early life exposure of rats to chlorpyrifos

BackgroundNeurodevelopmental disorders (NDDs), including intellectual disability, attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD), are pervasive, lifelong disorders for which pharmacological interventions are not readily available. Substantial increases in the prevalence of NDDs over a relatively short period may not be attributed solely to genetic factors and/or improved diagnostic criteria. There is now a consensus that multiple genetic loci combined with environmental risk factors during critical periods of neurodevelopment influence NDD susceptibility and symptom severity. Organophosphorus (OP) pesticides have been identified as potential environmental risk factors. Epidemiological studies suggest that children exposed prenatally to the OP pesticide chlorpyrifos (CPF) have significant mental and motor delays and strong positive associations for the development of a clinical diagnosis of intellectual delay or disability, ADHD, or ASD.MethodsWe tested the hypothesis that developmental CPF exposure impairs behavior relevant to NDD phenotypes (i.e., deficits in social communication and repetitive, restricted behavior). Male and female rat pups were exposed to CPF at 0.1, 0.3, or 1.0 mg/kg (s.c.) from postnatal days 1-4.ResultsThese CPF doses did not significantly inhibit acetylcholinesterase activity in the blood or brain but significantly impaired pup ultrasonic vocalizations (USV) in both sexes. Social communication in juveniles via positive affiliative 50-kHz USV playback was absent in females exposed to CPF at 0.3 mg/kg and 1.0 mg/kg. In contrast, this CPF exposure paradigm had no significant effect on gross locomotor abilities or contextual and cued fear memory. Ex vivo magnetic resonance imaging largely found no differences between the CPF-exposed rats and the corresponding vehicle controls using strict false discovery correction; however, there were interesting trends in females in the 0.3 mg/kg dose group.ConclusionsThis work generated and characterized a rat model of developmental CPF exposure that exhibits adverse behavioral phenotypes resulting from perinatal exposures at levels that did not significantly inhibit acetylcholinesterase activity in the brain or blood. These data suggest that current regulations regarding safe levels of CPF need to be reconsidered.

Remediation of Wastewater from Chlorpyrifos Pesticide by Nano-Gold Photocatalyst

AbstractSuccessful disintegration of Chlorpyrifos pesticide by nearly monodisperse nano-gold photcatalyst (∼10 nm) for remediation of wastewater has been reported in current studies. Nano-Gold dispersion reaction with Chlorpyrifos solutions completes in 45 minutes at ambient temperature under the normal daylight and exhibit systematic variations in the solution color from wine red to pink to light sky blue and finally transforming into a transparent solution with fine precipitates. UV-Vis absorption studies correlates well with the systematic color changes as observed in the nano-gold treated Chlorpyrifos solutions with time. The characteristic localized surface plasmon resonance peak of nanogold dispersion observed at 529 nm red shifts to weak, medium and strong intensity peaks at ∼ 640 nm, 740 nm and 890 nm on increasing chlorpyrifos concentration owing to the aggregation of gold nanoparticles in small to bigger sized clusters. The fine turbidity in final transparent solution further confirms the aggregation of nanogold particles into stable bigger ensembles. IR transmission spectra of final transparent solutions showed disappearance of Chlorpyrifos νC-Cl and phosphorothioate functional group peaks indicating degradation of chlorpyrifos.

Literature Review: Food Processing in Minimizing Chemical Contamination of Rhodamin B in Shrimp Paste and Chlorpirifos Residue in Fresh Vegetables Garnish Which are Circulating in the Communities in Indonesia

Introduction: Food circulation among communities is prone to chemical contamination. The contamination is obtained from both food processing and food cultivation (planting, raw materials). One of the examples related to chemical contamination can be seen in the contamination of rhodamine B on Terasi. Although there is a prohibition on using Rhodamine B compounds in food add-ons, which is listed in Regulation of Ministry of Health of Republic Indonesia No.722/Menkes/Per/IX/1988, its usage is widely found. Similarly, chlorpyrifos pesticide residue in lalapan is the other example provided. The usage of it remains to continue even though the result of pesticide residue value is under BMR SNI 7313:2008. The article aims to study food processing efforts in minimizing the chemical contamination of Rhodamine B in shrimp terasi and chlorpyrifos residue in the lalapan vegetables in the communities in Indonesia. A literature review on scientific publications was undertaken through Google Scholar, ResearchGate, Neliti, ScienceDirect, LIPI, Directory Open Acces Journal, and Crossref. The keywords used were related to rhodamin B on terasi, synthetic dyes on terasi, natural dyes for terasi, vegetable pesticide residues, Chlorpyrifos residue on vegetables, and how to reduce chlorpyrifos residue in vegetables. All of them were published between the 2015 – 2020 period. Discussion: Chlorpyrifos residue in vegetables can be reduced by washing, soaking brine, and boiling. The usage of rhodamine B on Terasi can be replaced by the application of dragon fruit skin extract and angkak (red yeast rice) for natural dye alternatives. Conclusion: Washing food with running water and soaking it in hot water before food consumption can lower chlorpyrifos residue level in dark vegetables. However, it is more significant to lover the residue level through the boiling step. The use of natural pesticides can be used as the choice of alternative. Moreover, rhodamine B on terasi can be replaced with natural dye alternatives such as angkak (red yeast rice) and dragon fruit skin extract.