Biosensor device and method for assessing responses to pesticide applications in soils

Abstract

The invention refers to a biosensor device for assessing responses to pesticide applications in soils, comprising a vessel (1) with closed walls (2), a lid (3) with a first set of meshes (5), a cover (4) with a second set of meshes (6), a portion of a sample soil (7), and earthworms (8) held inside said biosensor device. The invention also refers to a method for assessing responses to pesticide applications in soils, which uses said biosensor device. The biosensor device for the field exposure of earthworms can be easily produced and become available to farmers for being applied in the field, before phytosanitary treatments and, collected afterward to be sent for the analysis of different molecular biomarkers referred to earthworms. The biosensor device assures that earthworms be exposed to pesticides while preventing the organisms from escaping.

Description

BIOSENSOR DEVICE AND METHOD FOR ASSESSING RESPONSES TO PESTICIDE APPLICATIONS IN SOILS

[0001] The present invention relates to the field of biosensors, and in particular to a biosensor comprising an earthworm as a model organism for assessing responses to pesticide applications in soils and a related assessing method.

[0002] Field exposures of organisms are of utmost importance, especially at higher tiers of a risk assessment, to measure the responses to potentially toxic contaminants, like pesticides, under more relevant and real scenarios of phytosanitary treatments. Non-target soil organisms undergo in-field and off-field pulse applications of mixtures of pesticides, with different modes of action and different spraying strategies, which may affect the magnitude and the frequency of the exposures. Therefore, such environmental exposures are different from single substances exposures conducted under laboratory conditions following ISO and OECD standard guidelines.

[0003] Earthworms are examples of model soil organisms, being the test species of standard protocols (e.g. ISO 17512-1:2008; ISO 11268-2:2012; OECD 222:2016), due to their relevant roles on soil functions and ecosystem services. They are also good soil health bioindicators as they may change their behavior when in contact with contaminated soil and they can uptake and accumulate the toxic substances from the soil, being possible to monitor their responses through different molecular biomarkers (Fruend et al., 2011), which can be measured by well-established protocols (e.g. Correia et al., 2017.; Lourenço et al., 2012).Technical Problem

[0004] The difficulties in conducting experimental field trials, are associated with different aspects, but one of these is the development of appropriate field chambers or cages for holding model soil organisms, to keep test organisms in physical conditions that do not affect their behavior and performance, and that are also not affected by the machinery and works ongoing in the fields.

[0005] Earthworms have been used for field exposures (Antunes et al., 2008; Lourenço et al., 2012), but those experiments did not consider the development of appropriate devices, that may guarantee real exposures, during phytosanitary treatment on different crop fields, while preventing organisms to escape.

[0006] In a study aimed at assessing the effect of soil contaminated with explosives, Robidoux et al. (2004) exposed earthworms (L. terrestris and E. andrei)in situusing 100 nylon mesh bags (25cm X 25 cm X 16 cm deep) containing 10 l of soil.

[0007] Antunes et al. (2008) and Lourenço et al. (2012) exposed earthworms of the speciesEisenia fetidato contaminated soil from a uranium mining area, and several molecular biomarkers were measured to assess the effects of metals and radionuclides. In both cases, any especial device was developed for such exposures, wherein plastic pots, commonly used for laboratory tests, were filled with the mine soil and buried in the soil. The earthworms were added to the pots and thus exposed to the contaminated soil. The objective was to maintain the earthworms under environmental climatic conditions. Kim et al. (2017) proposed a new test for assessing soil quality using tubes (height, 95 mm; diameter, 15 mm) that were filled with 5 g of reference soil containing 30% water. The tubes were open at both ends and buried in the soil. The objective was to assess if the earthworms added to the tubes (1 per tube), persisted in the reference soil within the tubes, escape to the top of the tubes, or disperse and burrow into the soil. According to the figures reported by this study, any special device was developed, and only graduated tubes, such as centrifuge tubes were used as test devices.

[0008] Moreover, the tests performed so far have not considered the leaching of the pesticide solution through the soil caused by possible precipitation after phytosanitary treatments, wherein this lack of assessment may result in false results referring to the impacts of phytosanitary treatments.

[0009] Still, there is a need to develop a biosensor device that is configured to properly address the interaction of earthworms, as model soil organisms, and pesticide formulations applied into the soil, providing better results that reflect the real impact of pesticides in soils when carrying out a phytosanitary treatment.Solution to Problem

[0010] The present invention solves the problems of the prior art by developing a biosensor device holding a plurality of earthworms as model soil organisms, wherein said biosensor device is configured to allow the pesticides entrance, as it is perforated on a top portion, as well as is configured to allow the pesticides leaching through a perforated bottom portion. Therefore, the earthworms are exposed to controlled environments that are closely related to real superior soil layers being exposed to phytosanitary treatments.Advantageous Effects of Invention

[0011] The biosensor device for the field exposure of earthworms can be easily produced and become available to farmers for being applied in the field, before phytosanitary treatments and, collected afterward to be sent for the analysis of different molecular biomarkers referred to the earthworms. The biosensor device can be filled with a standard artificial soil (OECD, 1984) or with field soil to better address the interaction of the components of the pesticide formulations with soil components, both mineral and organic, and thus better reflect their bioavailability in the soil of the site. The biosensor device can be made of a non-toxic material. The biosensor device assures that earthworms are exposed to pesticides while preventing the organisms from escaping, as well as allowing gas exchanges and water percolation.

[0012] To promote an understanding of the principles of the embodiments of the present invention, reference will be made to the embodiments illustrated in the figures and to the language used to describe the same. Anyway, it must be understood that there is no intention of limiting the scope of the present invention to the contents of the figures. Any alterations or later changes of the inventive features illustrated herein, and any additional application of the principles and embodiments of the invention shown, which would occur normally for one skilled in the art when reading this description, are considered as being within the scope of the claimed invention.Fig.1

[0013] illustrates a cross-sectional view of the biosensor device;Fig.2

[0014] illustrates an embodiment of a biosensor device;Fig.3

[0015] illustrates experimental results regarding the percolation of water through a biosensor device;Fig.4

[0016] illustrates experimental results regarding the stress of earthworms held in a biosensor device;Fig.5

[0017] illustrates the average acetylcholinesterase activity in earthworms exposed to the application of the pesticide chlorpyrifos in a first type of soil;Fig.6

[0018] illustrates the average acetylcholinesterase activity in earthworms exposed to the application of the pesticide chlorpyrifos in a second type of soil;Fig.7

[0019] illustrates the average acetylcholinesterase activity in earthworms exposed to the application of different pesticides and mixtures thereof;Fig.8

[0020] illustrates the average glutathione S-transferases activity in earthworms exposed to the application of different pesticides and mixtures thereof;Fig.9

[0021] illustrates the average lactate dehydrogenase activity in earthworms exposed to the application of different pesticides and mixtures thereof;Fig.10

[0022] illustrates the average concentration of thiobarbituric acid reactive substances in earthworms exposed to the application of different pesticides and mixtures thereof.

[0023] The present invention refers, in the first aspect, to a biosensor device for assessing responses to pesticide applications in soils, wherein said biosensor device comprises:

[0024] a vessel (1), which comprises at least one closed wall (2); and

[0025] a lid (3), which is removably positioned on an end of said vessel (1); and

[0026] a cover (4), which is connected to an opposite end of said vessel (1); wherein

[0027] said lid (3) comprises a first set of meshes (5); and

[0028] said cover (4) comprises a second set of meshes (6); and

[0029] a portion of a sample soil (7) is held within a zone formed by an interior surface of at least one closed wall (2), the first set of meshes (5), and the second set of meshes (6); and

[0030] at least an earthworm (8) is held in said portion of the sample soil (7).

[0031]

[0032] The wall (2) or walls (2) of the vessel (1) are closed, avoiding the soil sample, its water content, and the earthworms leaving the biosensor device through said walls.

[0033] In the preferred embodiments of the present invention, the vessel (1) has a horizontal cross-sectional shape that is polygonal, preferably in the form of a polygon selected from the group consisting of a triangle, a quadrilateral, a pentagon, a hexagon, a heptagon, or an octagon. Alternatively, said vessel (1) has a horizontal cross-sectional shape of a circle or an ellipse. Even more preferably, said vessel (1) has a cylindrical shape. An example of proper dimensions for a cylindrical vessel (1) is a diameter in the range of 4 to 16 cm and a height in the range of 3 to 12 cm. More preferably, the diameter is in the range of 7 to 10 cm and the height is in the range of 4 to 6 cm.

[0034] In the preferred embodiments, the vessel (1), the lid (3), and the cover (4) are made of a rigid material, for instance, a polymer, a copolymer, a metal, or an alloy. Examples of suitable polymers for preparing a vessel (1), a lid (3), or a cover (4) comprise polypropylene, polypropylene, polyethylene, polyethylene terephthalate, polystyrene, polyvinyl chloride, copolymers thereof. Examples of suitable metals or alloys for preparing a vessel (1), a lid (3), or a cover (4) comprise aluminum or stainless steel.

[0035] In the preferred embodiments, said first set of meshes (5) and said second set of meshes (6) have a mesh size in the range of 1.5 mm to 2.5 mm, more preferably in the range of 1.6 mm to 2.0 mm, and even more preferably 1.8 mm, wherein these mesh sizes are proper for allowing the exposure of the sample soil (7) in the interior of the biosensor device to the sprayed pesticides, besides avoiding earthworms (8) to escape.

[0036] In the preferred embodiments, the lid (3) is removably positioned on an end of said vessel (1) and is connected to the vessel (1) by means of at least a pair of male and female connectors or through a threaded connection. Examples of male connectors are screws or bolts. Examples of female connectors are nuts or threaded holes. An illustrative embodiment is presented in, wherein the perimeter of an end of the said vessel comprises a plurality of channels having a first longitudinal section and a second transversal section arranged in an angle, such as 90º, wherein said channels are female connectors. The lid (3) comprises a plurality of male projections, wherein each male projection matches a first longitudinal section, wherein the connection is made through sliding the male projection along the first longitudinal section, and further sliding of the male connector along the second transversal section, locking the lid (3), through its rotation, to the vessel (1).

[0037] In other embodiments, the cover (4) can also be connected to an opposite end of the said vessel (1) by means of at least a pair of male and female connectors or through a threaded connection, as well as the lid (3) is connected to the said vessel (1).

[0038] In the preferred embodiments of the invention, a ratio of 2 to 5 adult earthworms (8) are held in a portion of about 250 cm3of sample soil (7). In the preferred embodiments of the invention, the mass of an adult earthworm (8) is in the range of 300 to 900 mg.

[0039] Preferably, said sample soil (7) said sample soil (7) is an artificial soil, a natural soil, or mixtures thereof. Moreover, preferably, said sample soil (7) is moistened to a level between 30 to 60% of its maximum water holding capacity, for instance, about 50% of its maximum water holding capacity (WHC), which is measured according to the standard ISO 11268-2:2012.

[0040] The present invention refers, in the second aspect, to a method for assessing responses to pesticide applications in soils, wherein said method utilizes the biosensor device, as defined in the first aspect of the invention, and said method comprises the following steps:

[0041] a) holding a portion of a sample soil (7) within a zone delimited by the interior surface of at least one closed wall (2) of said vessel (1), the first set of meshes (5), and the second set of meshes (6); and

[0042] b) holding at least one earthworm (8) within said portion of the sample soil (7); and

[0043] c) Burying said biosensor device in the soil, while ensuring that said first set of meshes (5) remains substantially aligned with the surface of said soil; and

[0044] d)Exposing the confined at least one earthworm (8) to a pesticide application; and

[0045] e) Removing said biosensor device from the soil; and

[0046] f) Removing said at least one earthworm (8) from said biosensor device; and

[0047] g) Analyzing at least one biomarker comprised in at least one earthworm (8) to assess responses to pesticide applications in soils.

[0048]

[0049] The exposures of earthworms (8) inside the biosensor device to the pesticide applications last for a proper period, for instance, seven days in the field, wherein the evaluation period must be coincident with phytosanitary treatments. After the evaluation period, the biosensors devices are removed from the soil and sent for analysis. The transport of the samples is preferably made in moistened and controlled temperature conditions to prevent the stress of the organisms.

[0050] Preferably, said method further comprises a step of moistening said sample soil (7) to 30 to 60% of its maximum water holding capacity, wherein this further step is executed before the step d) of exposing the confined at least one earthworm (8) to a pesticide application.

[0051] Preferably, said method further comprises an acclimatization step of at least one earthworm (8) to said sample soil (7), which is executed before the step d) of exposing the confined at least one earthworm (8) to a pesticide application.

[0052] Preferably, said method further comprises in the step g) a comparison with results of referred to a second set of earthworms exposed to a pesticide application, wherein said second set of earthworms is held in the soil outside said biosensor device.Examples

[0053] A biosensor device made of a plastic comprising a cylindrical vessel (1) with a diameter of 4.7 cm and a height of 8.4 cm was used. The first set of meshes (5) and the second set of meshes (6) have a mesh of 1.8 mm.

[0054] a) Evaluation of penetration of water and aqueous solutions into the biosensor device

[0055] An agricultural soil was collected at Campus de Vairão and dried at 105ºC overnight. A plastic box was filled with dried soil, up to 30 cm in height. Inside the box were placed three biosensor devices, which were filled with the same dried soil, being the upper surface of the device aligned with the surface of the soil. The whole box was homogeneously sprayed with the volume of water required to attain between 50 to 60% of its maximum water-holding capacity. The box was left at room temperature for 4h to let water percolate. Water was used as an equivalent to an aqueous solution, such as a soluble solution of a plant protection product (PPP) during spray applications.

[0056] Afterward, three soil samples outside the biosensor devices were collected, immediately at the surface (0-10mm) and at the maximum depth for which the presence of water was evident (10-30 mm). The soil inside the biosensor devices was gently removed and soil from two different depths was separated (0-10mm; 10-30mm). The presence of water inside the biosensor devices was also not evident for depths above 30mm.

[0057] The soil samples were weighted up to 0.01 g, placed in porcelain crucibles, and left to dry at 105ºC overnight. The dry weight was registered, and the moisture percentage of the different samples was calculated.displays the average soil moisture percentages and the respective standard deviations of the different soil samples collected at different depths inside (I) and outside (O) the biosensor devices. Although a slight reduction in soil moisture is recorded between 10-30 mm inside the biosensor device, no significant statistical differences (ANOVA: F=1.938; df: 12, 8; p=0.202) were recorded between the samples collected inside and outside.

[0058] The water was slightly intercepted by the biosensor device lid with the studied mesh size, however, this effect was not statistically significant.

[0059] b) Evaluation of stress on the earthworms in the biosensor device and the subsequent results

[0060] The same agricultural soil used in experiment 1 was moistened with the volume of water required to adjust its maximum water-holding capacity to 50%. A plastic box was filled with moistened soil, up to 30 cm in height. Three biosensor devices were filled with the same moistened soil and three earthworms were added to each one and the devices were closed. The biosensor devices were then buried into the soil, being the upper surface of the device aligned with the surface of the soil. Nine earthworms were randomly selected and added to the soil in the box (i.e. outside de devices). All the earthworms have the same eight (400-600mg) and were obtained from synchronized LABRISK cultures maintained under controlled conditions of substrate, photoperiod, and temperature. The earthworms were previously acclimated to the soil used in the experiment for 24h. The organisms were not fed during the experiment.

[0061] This experiment lasted for seven days. After this period, the earthworms outside the devices were removed from the soil and randomly grouped in three pools of three earthworms each. All the earthworms were gently washed, and the pools or earthworms were added to Falcon tubes, which were immediately frozen in liquid nitrogen.

[0062] The concentration of thiobarbituric acid reactive substances was measured following the methodology described by Buege and Aust (1978). Data were normalized for the total protein content of each earthworm’s sample, following the method described by Bradford (1976) adapted to a microplate reader. Thiobarbituric acid reactive substances, as the malondialdehyde (MDA) measured spectrophotometrically, are the byproduct of lipid peroxidation, caused by cellular oxidative stress.

[0063] displays the average concentration of MDA recorded in the earthworms kept both inside the biosensor devices (I1, I2, I3) and outside the biosensor devices (O1, O2, O3). Highly significant differences were recorded between the earthworms inside the devices and those maintained outside the devices (F=10.536; d.f.: 22,16, p<0.01). A significantly lower concentration of MDA was recorded in earthworms kept inside the biosensor devices for seven days (Tukey: p<0.5). These results demonstrate that the physical structure of the biosensor device did not cause stress on the organisms exposed, during the exposure period analyzed. The highest concentrations of MDA recorded on earthworms that were kept outside the device cannot be interpreted as the highest level of stress, since the values recorded may be within earthworms’ baseline values.

[0064] The biosensor device does not cause stress to the test organism, considering the proper earthworm density for the volume of the biosensor device and for the exposure period considered.

[0065] c) Evaluation of the capacity of the biosensor device to allow exposition of earthworms to plant protection products; evaluation of the type of soils in the results; evaluation of the avoidance as a sensitive endpoint to be assessed in the sensors

[0066] Two agricultural soils were used in this experiment, namely an agricultural soil collected at Campus de Vairão (VA) and a vineyard soil collected at Quinta do Azevedo (QA) (Lama, Barcelos). The physical and chemical characterization of both soils (pH, soil organic matter content – OM, and water holding capacity - WHC) is provided in Table 1. Both soils were dried at room temperature and stored for all the experiments.

[0067] Sampling sitepH (H2O)OM (%)WHC (%)VA6.066.6039.5QA5.801.6245.3

[0068] For each soil type (VA and QA) one box was prepared with three biosensor devices for the pesticide application and another box with three biosensor devices for the control (CTL). Each biosensor device had 8.4 cm eight and 4.7 cm of diameter. The pesticide used in this experiment was the commercial formulation of chlorpyrifos, CICLONE® 48EC from SAPEC AGRO Portugal. The soils were moistened to 50% of their WHC and then used to fill each plastic box. The biosensor devices were filled with the same moistened soil, three earthworms were added to each one and the devices closed. The biosensor devices were then buried into the soil of each box, being the upper surface of the device aligned with the surface of the soil. Nine earthworms were also added to the soil outside the biosensor devices to test for possible physical effects of the devices on the organisms. All the earthworms (Eisenia fetida) have the same weight (about 600mg) and were obtained from synchronized cultures that are maintained under controlled conditions of substrate, photoperiod, and temperature. The earthworms were previously acclimated for 24h to the soils used in the experiment. The organisms were not fed during the experiment.

[0069] The pesticide solution was prepared to respect the application dose recommended by the vendor (2L / ha of the commercial formulation diluted to 200mL / hL) and adjusted to the surface area of the soil in the box. Respecting the application doses an exposure of 0.192 mg chlorpyriphos / m2of soil surface was tested. One liter of the pesticide solution was prepared with the required concentration. Afterward, all the pesticide solution was sprayed randomly to cover the soil surface. The box from the CTL was sprayed with the same volume of tap water.

[0070] After seven days of exposure, earthworms were removed from the soil and from the biosensor devices. The earthworms were placed in a moistened paper to remove soil particles from the body surface and then immediately frozen in liquid nitrogen and stored at -80ºC before analysis. The tissues were homogenized in an ice-cold phosphate buffer (50 mM, pH 7.0 with 0.1 % Triton X-100) using a tissue homogenizer (T10 basic ULTRA-TURRAX®), at 40ºC. Homogenized samples were centrifuged (15,000xg for 10 min at 40ºC), and the supernatants were divided into aliquots and frozen in Eppendorf tubes at -80ºC, according to the methodology described by Correia et al. (2017).

[0071] Acetylcholinesterase (AChE) was determined according to Ellman et al. (1961). The procedure was done with a reaction solution composed of phosphate buffer (0.1 M; pH=7.2), acetylcholine 0.075 M and DTNB (Ellman’s reagent) 10 mM. The product of AChE activity was measured at a wavelength of 414 nm and presented in nmol min-1 mg-1 protein.

[0072] The concentration of thiobarbituric acid reactive substances (TBARs) was measured following the methodology described by Buege and Aust (1978). Data were normalized for the total protein content of each earthworm sample, following the method described by Bradford (1976) adapted to a microplate reader. Thiobarbituric acid reactive substances, as the malondialdehyde (MDA) measured spectrophotometrically, are the byproduct of lipid peroxidation, caused by cellular oxidative stress. The amount of TBARS was measured at a wavelength of 535 nm and presented as nmol of MDA equivalents / mg of protein.

[0073] The total concentration of protein of the earthworms’ homogenates was determined according to Bradford’s protocol (Bradford 1976), adapted to a microplate, and measured spectrophotometrically at 595 nm.

[0074] TBARS determination points once again for the inexistence of oxidative stress caused by the physical structure of the devices in both experiments, as was already shown in the previous experiments.

[0075] anddisplay the average activity of AChE on earthworms exposed in VA and QA soils to the recommended application dose of Ciclone®. Figure 5a illustrates the average AChE activity in earthworms exposed to CTL (D1-D3) and chlorpyrifos sprayed devices in Vairão (VA) soil (D4-D5). Figure 5b illustrates average values for the three devices of each treatment. Error bars stand for standard deviation. Figure 6a illustrates the average AChE activity in earthworms exposed to CTL (D1-D3) and chlorpyrifos sprayed devices in Quinta do Azevedo (QA) soil (D4-D5). Figure 6b illustrates the average values for the three devices of each treatment. Error bars stand for standard deviation.

[0076] No significant differences (VA: p=0.557, QA: p=0.889) were found between the control and the sprayed biosensor devices regarding AChE activity, for both experiments with different soils.

[0077] All the earthworms were found in the bottom of the devices, both in the CTL and in the chlorpyriphos treatment.

[0078] Chlorpyriphos is a neurotoxic insecticide, wherein its mode of action includes the inhibition of the acetylcholinesterase activity preventing this enzyme to clean the synaptic clefts, affecting neuromuscular junctions, and ultimately causing the death of the organisms. Thus, a significant inhibition of AChE activity in earthworms when compared to the control was expected in our experiment, however, it was not recorded. The height of the devices (8 cm) allows the avoidance of the earthworms to the bottom preventing their exposure and subsequently the neurotoxic effects. Rodríguez-Seijo et al. (2018) recorded the ability of earthworms to avoid soils contaminated with the same pesticide. Although this behavior was already known, it was considered that keeping this possibility within the devices helps to maintain their ecological relevance, as the same behavior happens in the field allowing the organisms to protect themselves against exposure to abiotic stressors. Nevertheless, although the soil was not sieved its structure was altered with sampling. Under field conditions, the soil is more compacted, and epigeic organisms (as the species tested) which leave more in the litter and on the soil surface may not have the ability to burrow to such a depth, being more exposed during pesticide applications. Therefore, reducing the height of the biosensor devices is a preferred approach.

[0079] Rao et al (2003) recorded a LC50value of 0.37 mg / m2(concentration that causes 50% mortality in exposed organisms) in earthworms of the speciesEisenia fetidain a direct contact test and for an exposure of 48h. The same authors start to record a 60% inhibition of AChE after 12h of exposure. Following the application doses recommended by the vendor we had an application dose of 0.192 mg / m2, which was about 1.9 times lower, Further, in the exposure of Rao and collaborators (2003) the earthworms were forced to be in contact with the filter paper, moistened with the solution of the pesticide for 48h. In our case the earthworms also have the possibility to bury in the soil, avoiding the exposure to the pesticide. Although this may suggest that the sensitivity of the biosensor is low, in fact, we consider that it is reliable for laboratory and field exposures, as low concentrations will achieve the soils after spraying, which is done directly on the crop species or on a soil covered with weeds.

[0080] The avoidance behavior was not a sensitive parameter to pesticide exposure, as earthworms were found at the bottom of the biosensor devices in all the treatments. Avoidance behavior has been proven to be a sensitive response of oligochaetes to chemical stressors, to chlorpyriphos (Rodríguez-Seijo et al. (2018)) and it was the basis to set up a standard ecotoxicological assay (ISO 17512-1:2008). However, in this standard protocol, the whole soil is contaminated with pesticides, and the height of the soil layer in the test chamber is limited to a few centimeters (about 5 cm). The avoidance behavior is tested horizontally, as the contaminated and the non-contaminated soil are placed side-by-side. In the tested devices the earthworms are allowed to naturally move to the highest depth in the soil to escape from the pesticide that persists on the surface (maximum 3 to 4 cm, according to the first experiment but also to look for a soil-less vulnerable to water loss.

[0081] Therefore, the soil had no effect on the response of earthworms, despite the differences in its physical and chemical parameters.

[0082] d) Assessing the sensitivity of the biosensor device to more realistic phytosanitary treatments, that frequently include more than one active ingredient in the same application

[0083] A vineyard soil collected at Quinta do Azevedo (QA) (Lama, Barcelos) was used in this experiment. The soil was dried at room temperature and stored until use. The physical and chemical characterization of QA soil (pH, soil organic matter content (OM), and water holding capacity (WHC)) are presented in Table 2.

[0084] LocalpH (H2O)OM (%)WHC (%)QA5.806.045.3

[0085] In this experiment two commercial herbicides were tested, glyphosate (RoundUp® UltraMax from Bayer©) and flazasulfuron (Zagaia from Ascenza®), tested individually and combined. Four treatments were tested:

[0086] 1) the biosensor devices were sprayed with only water (CTL);

[0087] 2) the biosensor devices were sprayed with glyphosate (Gly);

[0088] 3) the biosensor devices were sprayed with flazasulfuron (Flaz);

[0089] 4) the biosensor devices were sprayed with a mixture of glyphosate and flazasulfuron (Mix).

[0090] For each treatment, three plastic pots, each one with a biosensor device (4.1 height and 7.9 diameter), were prepared. The soil was moistened to 50% of its water-holding capacity and then used to fill each plastic pot. The biosensor devices were filled with the same moistened soil, three earthworms were added to each one and the sensor devices closed. The devices were then buried into the soil of each pot, being the upper surface of the device aligned with the surface of the soil. All the earthworms (Eisenia fetida) have the same weight (about 600mg) and were obtained from synchronized cultures from LABRISK, at the Faculty of Sciences of the University of Porto. The pots were maintained under controlled conditions, photoperiod (16hL:8hD) and temperature (202ºC). The earthworms were previously acclimated for 24 h to the soil used in the experiment. The organisms were not fed during the experiment.

[0091] The concentrations used for each herbicide were prepared following the indications given by the supplier for the recommended application dose (10 L / ha for Gly and 200 g / ha for Flaz) and adjusted for the m2used in the experiment. For the mixture treatment, the same application dose of each herbicide was used and mixed in the same solution. One liter of each solution was prepared and sprayed randomly on a 1 m2surface area where the pots were distributed. In the case of the CTL, only tap water was sprayed.

[0092] After seven days of exposure the earthworms were removed from the biosensor devices and placed in moistened paper to remove soil particles from the body surface and then immediately frozen in liquid nitrogen and stored at -80ºC before analysis. The tissues were homogenized in an ice-cold phosphate buffer (50 mM, pH 7.0 with 0.1 % Triton X-100) using a tissue homogenizer (T10 basic ULTRA-TURRAX®), at 4oC. Homogenized samples were centrifuged (15,000xg for 10 min at 4oC), and the supernatants were divided into aliquots and frozen in Eppendorf tubes at -80oC, according to the methodology described by Correia et al. (2017).

[0093] Acetylcholinesterase (AChE) was determined according to Ellman et al. (1961). The procedure was done with a reaction solution composed of phosphate buffer (0.1 M; pH=7.2), acetylcholine 0.075 M and DTNB (Ellman’s reagent) 10 mM. The product of AChE activity was measured at a wavelength of 414 nm and presented in nmol min-1mg-1protein.

[0094] Glutathione S-Transferases (GST) are a group of enzymes that catalyze the conjugation reaction of the substrate (CDNB - 1-chloro-2,4-dinitrobenzene) with reduced glutathione (GSH), which led to the formation of a thioether (Ɛ=9.6mM⁻¹cm⁻¹) that can be monitored by the increase in absorbance at 340 nm (method described by Habig et al. (1974). GST activity is expressed as nmol min-1mg-1protein.

[0095] The Lactate Dehydrogenase (LDH) activity was measured by monitoring the decrease in absorbance at 339 nm due to the oxidation of β-NADH in the presence of LDH and pyruvate (method described by Vassault, 1983). LDH activity is expressed in nmol min-1mg-1protein.

[0096] The concentration of thiobarbituric acid reactive substances (TBARs) was measured following the methodology described by Buege and Aust (1978). Thiobarbituric acid reactive substances, as the malondialdehyde (MDA) measured spectrophotometrically, are the byproduct of lipid peroxidation, caused by cellular oxidative stress. The amount of TBARS was measured at a wavelength of 535 nm and presented as nmol of MDA equivalents per mg of protein.

[0097] The total concentration of protein of the earthworms’ homogenates was determined according to Bradford’s protocol (Bradford 1976), adapted to a microplate, and measured spectrophotometrically at 595 nm.

[0098] As showed in, no significant differences (p>0.05) were found between the control and the sprayed biosensor devices regarding AChE activity for all the treatments. Error bars stand for standard deviation. Other authors have already reported the insensitivity of AChE in earthworms exposed to herbicides such as glyphosate (e.g. Owagboriaye et al., 2020). This agrees with the mode of action of both herbicides which were designed to interfere with the metabolism of aromatic aminoacids (Franz et al., 1997), and not with neurotransmission.

[0099] However, significant differences were recorded for Glutathione S-Transferases (GST) (F3,29=7.43; p=0.0008), with an increase in the activity after spraying the biosensor device with a mixture of glyphosate and flazasulfuron (). In, error bars stand for standard deviation and asterisks indicate statistical differences from the CTL (p<0.05). This may indicate that both herbicides reach a level inside the biosensor device able to activate the antioxidant system of earthworms.

[0100] Glutathione transferases are enzymes that catalyze the conjugation of glutathione conjugation to electrophilic compounds, primarily produced from exogenous xenobiotics by biotransformation. The activation of this enzyme has been described for earthworms exposed to glyphosate and other herbicides, as a response to deal with organic xenobiotic molecules.

[0101] The most sensitive endpoint to both herbicides, plus the mixture, was the activity of LDH, which was inhibited in all the treatments when compared to the control (). Significant lower values were recorded for the earthworms in the biodevices devices sprayed with the herbicides (F3,29=12.02; p<0.0001). This result demonstrates that the anaerobic glycolysis was inhibited, which may have accounted for the accumulation of pyruvate. Pyruvate was reported as one of Kreb’s cycle intermediates (KCI) with a protective capacity against oxidative stress in cells. Therefore, this may explain the significant reduction (F3,28=21.65; p<0.0001) () in the concentration of malonaldehyde, a thiobarbituric acid reactive substance (TBAR) measured to assess lipid peroxidation, resulting from oxidative stress.

[0102] It is known that LDH is reversibly inactivated by natural peptides. Considering that both herbicides interfere with the metabolism of aminoacids, the inhibition of LDH recorded could be explained by both a direct interaction of the herbicides with the enzyme, or indirectly through the interaction with by-products resulting from the interference of the enzyme on aminoacids metabolism. Nevertheless, the inhibition of LDH has been a response reported for earthworms exposed to herbicides such as glyphosate (Owagboriaye et al., 2020).

[0103] The results recorded indicated that the biosensor devices do not allow earthworms to escape to a greater depth, preventing the assessment of responses to more real exposures to pesticides, and its subsequent risks to biota.

[0104] As used in this description, the expressions “about” and “approximately” refer to a range in values of roughly 10% of the specified number.

[0105] As used in this description, the expression. “substantially” means that the real value is within an interval of about 10% of the desired value, variable or related limit, particularly within about 5% of the desired value, variable or related limit or particularly within about 1% of the desired value, variable or related limit.

[0106] Moreover, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is unless specified otherwise, or clear from the context, the phrase “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, the phrase “X employs A or B” is satisfied by any of the following instances: X employs A; X employs B; or X employs both A and B.

[0107] In addition, the articles “a” and “an” as used in this application and the appended claims should generally be construed to mean “one or more” unless specified otherwise or clear from the context to be directed to a singular form.

[0108] Further, as used herein, the term “exemplary” is intended to mean serving as an illustration or example of something and is not intended to indicate a preference.

[0109] The subject matter described above is provided as an illustration of the present invention and must not be interpreted to limit it. The terminology used with the purpose of describing specific embodiments, according to the present invention, must not be interpreted to limit the invention. As used in this description, the definite and indefinite articles, in their singular form, aim to include in the interpretation the plural forms, unless the context of the description explicitly indicates the contrary. It will be understood that the expressions “comprise” and “include”, when used in this description, specify the presence of the characteristics, the elements, the components, the steps, and the related operations, but do not exclude the possibility of other characteristics, elements, components, steps, and operations from being also contemplated.

[0110] All modifications, providing that they do not modify the essential features of the following claims, must be considered within the scope of protection of the present invention.

[0111] 1. A vessel;

[0112] 2. A closed wall;

[0113] 3. A lid;

[0114] 4. A cover;

[0115] 5. A first set of meshes;

[0116] 6. A second set of meshes;

[0117] 6. A second set of meshes;

[0118] 8. An earthworm;

[0119] 9. A pair of male and female connectors.Non Patent Literature

[0120] Antunes, S.C., Castro, B.B., Nunes, B., Pereira, R., Gonçalves, F., 2008. In situ bioassay with Eisenia andrei to assess soil toxicity in an abandoned uranium mine. Ecotoxicology and Environmental Safety 71 (2008) 620–631.

[0121] Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.

[0122] Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods in Enzymology 52, 302–310.

[0123] Correia, B., Lourenço, J., Marques, S., Nogueira, V., Gavina, A., Rasteiro., M.G., Antunes, F., Mendo, S., Pereira, R., 2017 Oxidative stress and genotoxicity of an organic and an inorganic nanomaterial to Eisenia andrei: SDS / DDAB nano-vesicles and titanium silicon oxide. Ecotoxicology and Environmental Safety 140: 198-205.

[0124] Ellman, G.L., Courtney, K.D., Andres, V.Jr., Feather-Stone RM, 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88–95.

[0125] Franz, J.E., Mao, M.K., Sikorski, J.A., 1997. Glyphosate: a Unique Global Herbicide. American Chemical Society.

[0126] Fruend, H.C., Graefe, U., Tischer, S., 2011 Earthworms as biodinicadorts of soil quality. In Biology of Earthworms, Soil Biology 24, 261. Springer-Verlag Berlin Heidelberg.

[0127] Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases – the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130–7139.

[0128] Kim, S.W., Kim, D., Moon, J., Chae, Y., Kwak, J.I.,Park, Y., Jeong, S.-W., An, Y.-J., 2017. Earthworm dispersal assay for rapidly evaluating soil quality. Ecotoxicology and Environmental Chemistry 36(10): 2766-2772.

[0129] Lourenço, J., Pereira, R., Silva, A., Carvalho, F., Oliveira, J., Malta, M., Paiva, A., Gonçalves, F., Mendo, S.Evaluation of the sensitivity of genotoxicity and cytotoxicity endpoints in earthworms exposed in situ to uranium mining wastes, 2012. Ecotoxicology and Environmental Safety 75: 46–54.

[0130] Owagboriaye,F., Dedeke, G., Bamidele, J., Aladesida, A., Isibor, P., Feyisola, R., Adeleke, M., 2020. Biochemical response and vermiremediation assessment of three earthworm species (Alma millsoni, Eudrilus eugeniae and Libyodrilus violaceus) in soil contaminated with a glyphosate-based herbicide. Ecological Indicators 108:105678.

[0131] Rao, J.V., Yenamandra, S.P., Madhavendra, S., 2003. Toxicity of chlorpyrifos on the morphology and acetylcholinesterase activity in the earthworm Eisenia fetida. Ecotoxicology and Environmental Safety 54(3):296-301.

[0132] Robidoux, P.Y., Dubois, C., Hawari, J., Sunahara, G.I., 2004. Assessment of Soil Toxicity from an Antitank Firing Range Using Lumbricus terrestris and Eisenia andrei in Mesocosms and Laboratory Studies. Ecotoxicology, 13, 603–614.

[0133] Rodríguez-Seijo, A., Santos, B., Ferreira da Silva, E., Cachada, A., Pereira, R., 2018. Low-density polyethylene microplastics as a source and carriers of agrochemicals to soil and earthworms. Environmental Chemistry 16(1) 8-17.

[0134] Vassault, A., 1983. Lactate dehydrogenase: UV-method with pyruvate and NADH. In: Bergmeyer HU (ed) Methods of enzymatic analysis, 3rd edn. Verlag Chemie, Weinheim, pp 118–126.

Claims

A biosensor device for assessing responses to pesticide applications in soils, wherein said biosensor device comprises:a vessel (1), which comprises at least one closed wall (2); anda lid (3), which is removably positioned on an end of said vessel (1); anda cover (4), which is connected to an opposite end of said vessel (1); characterized in thatsaid lid (3) comprises a first set of meshes (5); andsaid cover (4) comprises a second set of meshes (6); anda portion of a sample soil (7) is held within a zone formed by an interior surface of at least one closed wall (2), the first set of meshes (5), and the second set of meshes (6); andat least an earthworm (8) is held in said portion of the sample soil (7).The biosensor device, according to the previous claim,whereina ratio of 2 to 5 adult earthworms (8) are held in a portion of about 250 cm3of sample soil (7).The biosensor device, according to the previous claim,whereinthe mass of an adult earthworm (8) is in the range of 300 to 900 mg.The biosensor device, according to any of the previous claims,whereinsaidsample soil (7) is moistened to a level between 30 to 60% of its maximum water holding capacity.The biosensor device, according to any of the previous claims,whereinsaid vessel (1) is made of a rigid material.The biosensor device, according to the previous claim,whereinsaid vessel (1) has a cylindrical shape.The biosensor device, according to any of the previous claims,whereinsaid first set of meshes (5) and said second set of meshes (6) have a mesh size in the range of 1.5 mm to 2.5 mm.The biosensor device, according to any of the previous claims,whereinsaidsample soil (7) is an artificial soil, a natural soil, or mixtures thereof.A method for assessing responses to pesticide applications in soils,characterized byutilizing the biosensor device, as defined in any of the previous claims,whereinsaid method comprises the following steps:a) holding a portion of a sample soil (7) within a zone delimited by the interior surface of at least one closed wall (2) of said vessel (1), the first set of meshes (5), and the second set of meshes (6); andb) holding at least one earthworm (8) within said portion of the sample soil (7); andc) Burying said biosensor device in the soil, while ensuring that said first set of meshes (5) remains substantially aligned with the surface of said soil; andd)Exposing the confined at least one earthworm (8) to a pesticide application; ande) Removing said biosensor device from the soil; andf) Removing said at least one earthworm (8) from said biosensor device; andg) Analyzing at least one biomarker comprised in at least one earthworm (8) to assess responses to pesticide applications in soils.The method for assessing responses to pesticide applications in soils, according to the previous claim, wherein said method further comprises a step of moistening said sample soil (7) to 30 to 60% of its maximum water holding capacity, which is executed before the step d) of exposing the confined at least one earthworm (8) to a pesticide application.The method for assessing responses to pesticide applications in soils, according to any of claims 9 or 10, wherein said method further comprises an acclimatization step of at least one earthworm (8) to said sample soil (7), which is executed before the step d) of exposing the confined at least one earthworm (8) to a pesticide application.The method for assessing responses to pesticide applications in soils, according to any of claims 9 to 11, wherein said method further comprises in the step g) a comparison with results of referred to a second set of earthworms exposed to a pesticide application, wherein said second set of earthworms is held in the soil outside said biosensor device.

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