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Suscripción a IA
Sorry I couldn't contact the ChatGPT think tank 🙁Abstract
Methylisothiazolinone (MIT) and chloroxylenol (PCMX) are popular disinfectants often used in personal care products (PCPs). The unregulated discharge of these micropollutants into the environment, as well as the use of sewage sludge as fertilizer and reclaimed water in agriculture, poses a serious threat to ecosystems. However, research into their ecotoxicity towards nontarget organisms is very limited. In the present study, for the first time, the ecotoxicity of biocides to Pseudomonas putida, Pseudomonas moorei, Sphingomonas mali, and Bacillus subtilis was examined. The toxicity of MIT and PCMX was evaluated using the microdilution method, and their influence on the viability of bacterial cells was investigated by the AlamarBlue® test. The ability of the tested bacteria to form biofilms was examined by a microtiter plate assay. Intracellular reactive oxygen species (ROS) production was measured with CM-H2DCFDA. The effect of MIT and PCMX on phytohormone indole-3-acetic acid (IAA) production was determined by spectrophotometry and LC‒MS/MS techniques. The permeability of bacterial cell membranes was studied using the SYTOX Green assay. Changes in the phospholipid profile were analysed using LC‒MS/MS. The minimal inhibitory concentrations (MICs) values ranged from 3.907 to 15.625 mg L−1 for MIT and 62.5 to 250 mg L−1 for PCMX, indicating that MIT was more toxic. With increasing concentrations of MIT and PCMX, the cell viability, biofilm formation ability and phytohormone synthesis were maximally inhibited. Moreover, the growth of bacterial cell membrane permeability and a significantly increased content of ROS were observed, indicating that the exposure caused serious oxidative stress and homeostasis disorders. Additionally, modifications in the phospholipid profile were observed in response to the presence of sublethal concentrations of the chemicals. These results prove that the environmental threat posed by MIT and PCMX must be carefully monitored, especially as their use in PCPs is still growing.
Introduction
Currently, much attention is given to the pollution of the natural environment with toxic compounds of anthropogenic origin. Mass production and application of synthetic chemicals increase this threat. One of the groups of pollutants is micropollutants, which are not covered by existing statutes due to their low accumulation in the environment (i.e., ng L−1 up to µg L−1). These contaminants have been introduced into ecosystems for many years, but current advances in analytical processes have allowed the detection of their presence (despite their low quantities in the environment)1. Among these micropollutants, personal care products (PCPs) and household chemicals are important emerging contaminants. PCPs form an integral part of the daily lives of humans and include skincare products, hair care formulations, toothpastes, soaps, sunscreens and perfumes. These chemicals are pervasive in the environment due to their incomplete elimination by conventional biological wastewater treatment systems. Although most PCPs are considered readily biodegradable in environmental matrices, their threat to the environment is not due to persistency but rather to their biological activity together with their continuous emission, which characterizes them as “emerging contaminants” or “prospective pollutants”2,3.
Chloroxylenol (PCMX, 4-chloro-3,5-dimethylphenol, p-chloro-m-xylenol) and methylisothiazolinone (MIT, 2-methyl-4-isothiazolin-3-one) are broad-spectrum antimicrobial agents that are used extensively in industrial, consumer and health care products, including cosmetics, household chemicals and disinfection products such as preservatives or disinfectant agents4,5. PCMX is a to strong antimicrobial capable of reducing populations of bacteria, such as Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, and Salmonella typhi, and fungi, such as Aspergillus niger, Aspergillus flavus, Candida albicans and Candida parapsilosis. Moreover, the virucidal activity of PCMX towards Ebola virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been confirmed, making it widely used for disinfection during the SARS-CoV-2 pandemic6–11. Generally, it is assumed that the mechanism of action of chloroxylenol is similar to that of other phenolic and halophenolic antibacterial agents, particularly those perturbing cell membranes and causing cell leakage4,12. MIT, which is the best representative of preservatives from the isothiazolinones group, is a powerful biocide and has been described to be able to diffuse across the bacterial cell membrane and the cell wall of fungi. The main mechanism of the inhibitory effect is based on the presence of a reducing sulfur in the MIT molecule. Sulfur is able to react with nucleophilic groups in cellular components and inactivate thiols in cellular proteins to form disulfide bonds (-S-S-). By blocking several specific enzymes, isothiazolinones effectively stop respiration and inhibit the synthesis and utilization of adenosine triphosphate (ATP), which inhibits cellular activity and ultimately causes bacterial and fungal cell death. Moreover, ROS accumulate because the vital pathways in cellular metabolism are disrupted; this accumulation leads to cellular death13–17.
Considering the large presence of MIT and PCMX in everyday products, their entry into water or soil systems is inevitable, and their presence may lead to alterations in the edaphic environment. Moreover, because popular antimicrobial agents such as triclosan and triclocarban have been banned by the Food and Drug Administration of the United States, it is anticipated that alternative chemicals, such as MIT and PCMX, which are commonly perceived as less harmful, will be used in PCPs in higher quantities. Therefore, the concentrations of MIT and PCMX in the natural environment should be continually surveyed18. The occurrence and fate of MIT and PCMX in wastewater treatment plant (WWTP) systems have frequently been detected at the level of nano- and micrograms per litre19–25. Discharge of wastewater after incomplete removal of preservatives from WWTPs leads to the accumulation of residue in the receiving environment. PCMX was detected in the range of 20–1200 ng L−1 in surface waters in Jakarta city26,27. There are few data in the literature about the occurrence of MIT in surface water. Paijens et al.28 describes the contamination of Paris wastewater by MIT at a concentration of 14 ng L−1.
Because preservatives, such as MIT and PCMX, are designed to elicit biological effects, the potential exists for these compounds to affect nontarget organisms. The toxic effects of MIT and PCMX have been explored previously, mainly on aquatic organisms. In zebrafish and rainbow trout, prolonged PCMX and MIT exposure cause hatching delays or inhibition, altered gene expression, embryonic mortality, morphological abnormalities, neurotoxicity and DNA damage to erythrocytes29,30. Moreover, Lee et al.31 revealed that exposure to MIT caused modulation of genes involved in thyroid hormone regulation. PCMX also modulated antioxidant enzymatic activity, produced changes in swimming speed and caused neurotoxic and mitochondrial malfunction in the estuarine rotifer Brachionuskoreanus32.
The possible influence of PCPs on terrestrial fauna, especially soil-dwelling organisms, has not been adequately examined. Soil is frequently the final reservoir for most pollutants that penetrate the environment. The application of sewage sludge or the use of reclaimed water as an important water resource in the field is becoming more controversial. A great benefit of this practice is the conversion of precious components such as plant nutrients and organic matter. However, biosolids and recycled water, which are sources of harmful xenobiotics, can reach agricultural lands, and the chemicals can persist in soil for prolonged periods of time33,34. Moreover, new data in the literature suggest that, due to their biocidal activity, preservatives from the isothiazolinone group could be used as a new class of insecticides for the control of pests, which makes them a new source of environmental contamination35. Literature data show that MIT is present in soil in Poland at concentrations ranging from 1.04 to 10.8 µg kg−124. Soils work as a large bioreactor for degrading pollutants and simplifying nutrient transformation. Moreover, soils play a vital role in ecosystems as a habitat for organisms and plants. Microorganisms are one type of living form that are abundant in soil and are referred to as soil microbiomes. Soil contains a wide variety of microorganisms, including bacteria, archaea, fungi, algae, and nematodes, that can be beneficial or pathogenic. Beneficial microbes in soil are essential for maintaining soil ecosystem health by breaking down organic matter, recycling elements, and promoting plant growth36. Among the variably distributed heterotrophic microflora, populations of bacteria belonging to different species make up approximately 15% of the overall microbial populations37. Undoubtedly, soil is a notable sink antimicrobial agents, which has a negative impact on indigenous microbes by killing specific groups of soil microbial flora38,39. Since it is known that MIT and PCMX have antibacterial activity, they could be expected to exert deleterious effects on bacteria in the soil. The antibacterial effects of MIT and PCMX have been primarily studied on pure cultures of human pathogenic bacteria40–43. Similar to other xenobiotics, the negative impact of these preservatives on environmentally significant bacteria is gradually increasing, but it is still poorly investigated.
The present study, for the first time, investigated the ecotoxicity of these two preservatives on species belonging to the three different genera Pseudomonas, Sphingomonas and Bacillus, which are representative genera of beneficial soil bacteria. The species belonging to these genera are well known for their multifaceted functions ranging from producing highly beneficial phytohormones, such as indole acetic acid, gibberellins and sphingan, to remediating many types of environmental contamination. These microbes have also been noted to reduce stress factors, such as salinity, heavy metals, and drougth, leading to improved plant growth in agricultural soil37,44,45.
This study was designed to investigate the tolerance of the bacterial species Pseudomonas putida (DSM 291), Pseudomonas moorei (DSM 12647), Sphingomonas mali (DSM 10565), and Bacillus subtilis (DSM 3657) to MIT and PCMX, reactive oxygen species production, changes in the phospholipid profile, biofilm formation and indole-3 acetic acid production. The objectives of the present work were to gain insight into the toxic effects of MIT and PCMX on beneficial soil bacteria and to evaluate the ecological risks they pose to soil environments.
Results and discussion
Determination of bacterial sensitivity and viability towards MIT and PCMX
The rich diversity and abundance of soil microflora and their activity determine the good quality and fertility of the soil. Although chemicals such as antibiotics, personal care products and plant protection products are an integral part of human life, their impact on nontarget organisms poses a serious threat to the functioning of the entire environment, including soil ecosystems. Disrupting the number or ratio of microbes in the soil can potentially inhibit the recycling and transformation processes of elements or pollutants. Studies on biocide toxicity on soil bacteria and other microorganisms are limited and generally focused on biodegradation of these pollutants or antibacterial activity in the context of using them in the final consumer products46–48. In the present study, for the first time, the ecotoxicology potential of MIT and PCMX has been evaluated towards gram-negative P. putida, P. moorei, and S. mali and gram-positive B. subtilis bacterial strains by determining their growth after treatment with various concentrations of MIT and PCMX. The results are presented as the percent of biotic control growth (control of bacterial growth without the addition of xenobiotics). Moreover, based on the level of bacterial growth, the MIC was measured. The effects of MIT and PCMX concentrations on bacterial growth and MIC values are illustrated in Fig. 1. The tested preservatives showed differential antimicrobial activity. For the first time, it was observed that the MIC values of preservatives towards soil microorganisms ranged from 3.907 to 15.625 mg L−1 for MIT and from 62.5 to 250 mg L−1 for PCMX, which clearly indicated that MIT was the most ecotoxic compound. The incubation of P. putida with MIT or PCMX at concentrations of 0.12225 mg L−1 and 0.977 mg L−1, respectively, caused statistically significant stimulation of growth. A similar effect was observed for P. moorei after incubation with MIT (concentration range of 0.0305625–0.2445 mg L−1) and B. subtilis after incubation with PCMX (concentration range of 1.954–15.625 mg L−1) with p < 0.05. It is known that some beneficial soil bacteria, such as Pseudomonas sp. and Bacillus sp., which are characterized as plant growth-promoting rhizobacteria (PGPR), can degrade various contaminants in soil and use them as a source of energy that stimulates bacterial growth49,50. Thus, it could not be excluded that MIT and PCMX, in low concentration ranges, are used as sources of carbon and energy.
Both strains of Pseudomonas were similarly susceptible to the action of MIT and PCMX. The first significant inhibition of growth was observed at an MIT concentration of 0.977 mg L−1, which caused approximately 80% growth inhibition of these bacteria. A further increase in the concentration led to a complete inhibition of growth. In the case of PCMX, an increased sensitivity of Pseudomonas was observed at concentrations above 31.25 mg L−1, where the xenobiotic reduced bacterial growth by more than 50% and 60% for P. putida and P. moorei, respectively. S. mali and B. subtilis were the most resistant microorganisms to the tested concentrations of MIT. In both cases, the concentration of 0.977 mg L−1 inhibited bacterial growth by approximately 23%. However, in contrast to that of B. subtilis, S. mali growth was highly reduced at the next tested concentration (1.954 mg L−1).
The toxicity of the investigated compounds, at five significant concentrations, towards bacterial species was also examined using an AlamarBlue® (AB) assay. The AB assay is widely applied in studies for monitoring microbial viability based on measuring the difference in the fluorescence intensity of the nonfluorescent, blue dye resazurin and the highly fluorescent reduced form resofurin. This redox reaction is a result of a metabolic pathway and cell respiration of metabolically active bacteria51,52. The results are presented as the percent of biotic control. The results presented in Fig. 2 indicate that the inhibition of AB dye reduction could be highly correlated with the inhibition of bacterial species growth, which might suggest that the inhibition of cell growth is caused by the inhibition of metabolic activity by xenobiotics.
