Pesticides are recognized as environmental pollutants, particularly in the ground and water 1. The presence of pesticides has also been noted in the air by scientists and addressed by regulatory authorities 23. However, in contrast with water, no obligatory monitoring or limitation of pesticide levels in air exists. Moreover, little is known about pesticide airway penetration and their impact on the respiratory system. Recently, public institutions developed pesticide air monitoring programs in several regions of France to characterize the level of exposure and to identify the principal compounds 4. Theses studies collected PM₁₀ (particulate matter collected with a 50% efficiency for particles with an aerodynamic diameter of 10 μm) and reported that in vine-growing regions during spring and summer (the treatment periods for vines) one of the main air-polluting pesticides was folpet 5678910. In rural settlements, folpet was detected in the air at a large range of concentrations; mean levels were between 0.16–1.2 μg/m³ depending on meteorological conditions, sprayed quantity and duration of treatment 58. Folpet was also found in urban areas, the average concentration was around 0.01 μg/m³ 67910 excepted for the city of Reims (0.05–0.15 μg/m³) 5678. Such data indicates that the general population of these regions could be exposed. However, the most exposed populations are the workers who manipulate the product in the course of their work. The average concentration of folpet in the air during its spraying on crops was found to be 40.13 μg/m³(interquartile range: 1.7–14.95 μg/m³; maximum value: 857 μg/m³; Baldi, unpublished data).

Folpet (N-[(trichloromethyl)thio)phthalimide]) is a contact fungicide belonging to the dicarboximide family. It has been used for the past 50 years and is still widely employed in Europe as a preventive or curative treatment against mildew, gray mold, spoilage fungi and wood rot fungi 11. Folpet is able to inhibit spore germination 12, its mode of action is centred on its reaction with thiol groups 13. The folpet degradation pathway consists of hydrolysis with cleavage of the sulfur-nitrogen bond to give thiophosgene and phthalimide 14. Phthalimide is hydrolyzed to phthalamic acid and then to phthalic acid (Fig. 1). Thiophosgene is a highly reactive short-lived compound, rapidly degraded to form HCl and SH₂ in water.

Folpet is classified as a harmful substance, noxious by inhalation and with possible risks of irreversible effects 1115. Furthermore, studies performed in vitro on mammalian cells have shown folpet to induce cell-cycle deregulation 16, enzyme inhibition 17, clastogenic effects 18 and to have mutagenic effects)19.

Nevertheless, many publications on folpet are relatively old and studies were not really performed using commercial forms of this fungicide. For agricultural treatment, folpet is generally available associated with one or two other fungicides and more rarely alone. It is formulated with vehicles as wettable powders, wettable granules (for example Folpan 80WG^®) or suspension concentrates (for example Myco 500^®) 11. For use, these concentrated forms are diluted in water at a final concentration of 1 g/l and sprayed on vines or other crops such as apple trees 11. At this manufacturer recommended working concentration, folpet is slightly water-soluble 13, remains on the surface of treated plants as particle form and acts as a contact fungicide. Depending on meteorological conditions and spray methods, air contamination can occur during application and, later on, by resuspension of folpet particles residing on treated areas. The general population can be exposed to folpet by this pathway, which might be particularly damaging to vulnerable persons.

The objective of this study was to determine the physicochemical characteristics of folpet particles of two commercial forms of folpet under their typical application conditions and to assess the cytotoxic effect in vitro on human cells. First, the granulometry and morphometry of the two commercial forms tested (Folpan 80WG^® and Myco 500^®) were determined using analytical scanning electron microscopy and laser light diffraction methods. The stability of folpet particles and their degradation products were then evaluated using an analytical method specially developed for this study. These physicochemical characteristics led us to test the impact of these particles and/or degradation products on the respiratory tract using an in vitro model. Cultured human bronchial epithelial cells (16HBE14o-) were exposed to these particles; their cytotoxic effect was assessed using the neutral red release assay, the reactive oxygen species (ROS) detection was assessed using DCFH-DA stain.

Pesticides have only recently been considered as air pollutants in France 4 which has led folpet, a contact fungicide that has been widely used for the past 50 years, to be detected in rural and urban ambient air of several regions of France 20. In light of this and in the absence of studies performed on commercial forms, we determined the physicochemical characteristics and cytotoxicity of two commercial forms of this fungicide (Folpan 80WG^® and Myco 500^®). Here we report that these forms under their typical application conditions are composed of particles and that most of them are smaller than 5 μm, slightly water-soluble, stable over time and thus are potentially inhalable by the general population. Furthermore, these particles were found to be cytotoxic in vitro to human bronchial epithelial cells (IC₅₀ = 2.9–5.1 μg/cm²) and to generate ROS production.

Testing of commercial forms of pesticides better resembles the real-life situation of their use. The two commercial forms of folpet tested here (Folpan 80WG^® and Myco 500^®) were chosen because they contain only folpet as the active ingredient and because they differ in their initial formulations: the first is a wettable granulate (80% folpet w/w) and the second, a highly concentrated liquid suspension (500 g/l folpet). Studies performed on commercial forms of folpet and published in peer-review journals deal with the levels of residues on plants 212223 not with the toxicity of these formulations on mammals. Yet several studies have been published using the technical grade molecule (for review see Gordon 24). Here we report the first morphology and granulometry results of commercial forms of folpet fungicide formulations. The initial forms of Folpan 80WG^® and Myco 500^® differed morphologically. Yet once diluted in water to the manufacturer recommended working concentration of 1 g/l folpet and sprayed on grape vine leaves, both were found to be small polygonal particles, the majority of which less than 5 μm in size. Laser light diffraction, recognised as the most robust method for particles size determination was employed for the 1 g/l aqueous suspension. However, scanning electron microscopy was used for particles deposited on grape vine leaves after spraying owing to their solid state. The two methods gave similar granulometry results of either commercial form. These results were in close agreement with the measurement of folpet in air of French vine-growing regions as these studies collected PM₁₀ 678.

We next tested the stability of these particles in an aqueous environment and found that even after 30 days, in a suspension of 1 g/l folpet of both Folpan 80WG^® and Myco 500^®, more than 75% of the folpet remained. Folpet was found to be slightly water-soluble in accordance with previously published solubility data 13 and once dissolved, it degraded rapidly into different compounds. The loss of folpet in the total fraction was due to the dissolution of folpet particles in water and its subsequent hydrolysis. This was confirmed by a low level of folpet in the dissolved fraction and by an increased amount of degradation products over time. After 30 days, although the same quantity of folpet was degraded and all known degradation products were found 24, Folpan 80WG^® and Myco 500^® differed in their profile of degradation products. More phthalimide and less phthalic acid was found for Myco 500^® than for Folpan 80WG^®. This may be explained by a more rapid folpet degradation in Folpan 80WG^® than in Myco 500^® during the first 21 days of our degradation study which itself may be due to a difference in vehicle composition. After 21 days, in the total fraction, there was no statistical difference in folpet concentration between Folpan 80WG^® and Myco 500^® and the rate of degradation was reduced to an inconsiderable level. This could be because the aqueous hydrolysis folpet is pH-dependent, the rate of folpet degradation is decreased at acidic pH 2425. The acidification found during our experiment could be a result of HCl released from the degradation of thiophosgene 26. However, after 30 days the folpet in the dissolved fraction did not accumulate which could be due to an additional negative effect of the pH on the solubility folpet particles in water.

Taken together, we report here that the commercial forms of folpet, Folpan 80WG^® and Myco 500^®, persist as particles in their typical application conditions and are stable in aqueous suspension. This could explain the presence of folpet (<1–50 ng/ml) in the aquatic environment 27 and in rural and urban air, during and after the period of treatment 28. The risk of inhalation of particles by humans is determined by their size: particles under 10 μm are inhalable and those less than 5 μm are known to reach the deep lung and alveolar area but principally deposited on the bronchial area 29. In their initial commercial forms, both Folpan 80WG^® and Myco 500^® could not be inhaled by humans because of the high size of Folpan 80WG^® granulates and the high concentrated suspension (500 g/l) with a doughy aspect for Myco 500^®. However, at their recommended dilution and under their typical application conditions, 75% of the particles of both forms are less than 5 μm in size and could be inhaled.

It is suggested that folpet expresses its primary toxicity locally rather than by systemic effects 26. However, few studies have been published on the cytological effects of folpet on mammalian cells 24. Furthermore, to our knowledge, no study has specifically investigated the human respiratory system, which we addressed by using an in vitro model of human bronchial epithelial cells (16HBE14o-cells). These cells were chosen in accordance with the granulometric results for the particles.

The toxic effect of folpet was first investigated using an in vitro cytotoxicity test. This test allowed to study direct effects of particles on target cells following acute exposure. It is the starting point to provide mechanistic information and it is useful to define basal cytotoxicity giving the non-cytotoxic concentrations to be used for ROS detection. The estimated IC₅₀ values for folpet on human bronchial epithelial cells (2.89 μg/cm² or 16 μM for Folpan 80WG^® and 5.11 μg/cm² or 30 μM Myco 500^®) was similar to that reported for an other halogenated fungicide on human lung fibroblasts (chloropicrin, IC₅₀ of 30 μM) 30. Phthalimide, phthalamic acid and phthalic acid, the degradation products of folpet were found not to have a cytotoxic effect on 16HBE14o-cells, as well as the vehicles of Folpan 80WG^®. These results support the hypothesis that thiophosgene contributed at least in part to the cytotoxic effect of folpet 3132. Further supporting this, we found that Folpan 80WG^® was more cytotoxic than Myco 500^® over 24 h which could be attributable to the differential kinetics of folpet degradation where more thiophosgene would have been released by Folpan 80WG^®.

In vivo and in vitro studies have shown that the mechanism of toxicity of folpet could be due to a gluthation depletion 33 and enzyme alteration 1734 by folpet reaction with thiol compounds 35. In our study, folpet was found to generate ROS by increasing DCFH-DA fluorescence. This result supports the hypothesis that folpet may mediate its toxicity directly or indirectly through ROS-mediated alterations and cause an oxidative stress. This biological effect has been reported in vivo on an aquatic plants with a commercial form of folpet (Folpan 500^®) 36. This probable mechanism of toxicity has also been reported for several ambient air particulate matter pollutants like PM_2.5or diesel exhaust particles (DEP) on respiratory cells 3738394041424344. Indeed, on 16HBE14o^- cells, from 10–30 μg/cm², DEP and PM_2.5 have been found to be potent inducers of oxidative stress with ROS production 4244. There is good evidence that these ambient PM can induce acute asthma exacerbation due to their ability to induce oxidative stress. ROS have been found to play an active role in the genesis of pulmonary inflammation and contribute to antigen-induced airway hyperreactivity 4546. Although concentrations of folpet particles in air are lower than those of PM_2.5, folpet could have an additive effect or synergic effect with PM_2.5 on oxydative stress production and then on respiratory toxicity. Moreover, folpet particles measured at concentrations 100-fold lower than PM_2.5 in urban area (e.g. the city of Reims 47) and only 10-fold lower in rural area, induce relatively high cytotoxicity on 16HBE14o-cells compared to other air pollutant particles. Indeed, from 10 to 30 μg/cm², DEP and PM_2.5 have been reported to have no effect on the viability of these cells 4248 while, at these concentrations folpet particles have been found to induce more than 90% lethality. More studies should be performed to know whether the presence of folpet particles in air and particularly in rural area could represent a risk for the heath of this population.

The concentrations tested in our experiment were in the same range than other particles air pollutants like PM_2.5 or DEP concentration (0.2–20 μg/cm²) tested on bronchial epithelial cells or macrophages 4249. Using calculations to relate in vitro DEP dose-response effects to in vivo PM dosimetry, Li and al. 49 reported that it is possible to achieve the in vitro dose range of 0.2–20 μg/cm². When further corrections were made for the individual variations (e.g. deposition at airway bifurcation points, nasal breathing), PM_2.5 were calculated to deposit in the tracheobronchial region at 2.3 μg/cm² for human subjects with uneven airflow (e.g. asthma) and at 1.13 μg/cm² for normal breathing individuals, exposed to an ambient total particulate matter average level of 79 μg/m³. Although, these calculations are based on 24 h exposure, workers exposed to an average concentration of folpet in air at 40.13 μg/m³ during its manipulation could be probably exposed in the tracheobronchial region to folpet concentration closed to those inducing biological effects.

Using a personal air sampling pump, total suspended particles were collected to determine the potential inhalation exposure rate during folpet mixing and application. The time weighed average rate was evaluated to be 68.2 μg/h of work (IQR: 2.9–25.4 μg/h; maximum value: 1457 μg/h) during a working time average of 4.8 h (± 2.2) (Baldi, unpublished data). More research should focus on the workers, the most exposed population with an acute exposure to high concentration level during their work possibly in the range of concentration having biological effects.

On the other hand, the rural population in vine-growing regions less exposed than workers, but 10 to 100 fold more exposed than urban population should be monitored because little is known on the effect of a chronic exposure to these lower concentrations in humans. It is an important concern because the general population and particularly children could be exposed.

The data presented here constitute the first step towards a risk assessment of folpet particles by inhalation for human health. Further studies are required, particularly to identify the mechanism of cytotoxicity and the in vivo impact on respiratory cells. Our data support the hypothesis that folpet found in the environment can persist, is inhalable by humans and is cytotoxic in vitro on human bronchial epithelial cells. The mechanism of toxicity of folpet could have been approached: folpet particles may mediate its toxicity directly or indirectly through ROS-mediated alterations. This work confirms the need for further studies on the effect of environmental pesticides on the respiratory system especially as no obligatory monitoring or limitation of pesticides in air exists as they do for water or food.

The author(s) declare that they have no competing interests.

MCR carried out the SEM analysis, folpet particles stability study and in vitro cytotoxicity studies, analyzed the data and drafted the manuscript. BL assisted in in vitro cytotoxicity studies design and coordination. BM assisted in folpet particles stability study. ES helped to prepare all specimens for electron microscopy and perform SEM analyses. FF assisted in granulometric study. PR participated in the interpretation of results and drafting of the manuscript. OC, JC, IB, MM, NM participated in the interpretation of the results. PB assisted in study design, supervised the experimental work and coordination and helped in manuscript preparation. All authors read and approved the final manuscript.