ES2534955B1 - Environmental toxicity bioassay based on phycobionts isolated from lichen - Google Patents

Abstract

Environmental toxicity bioassay based on phycobionts isolated from lichens. # The present invention contemplates a method for assessing the toxicity of a sample based on a) obtaining a population of phycobionts in a sterile culture medium suitable for each type of assay. ; b) the preparation of a suspension with the population of phycobionts obtained in a) in a suitable culture medium; c) the exposure of the suspension obtained in b) to the sterilized sample whose toxicity is to be evaluated, d) the incubation of the suspension of phycobionts isolated from lichens (c) under conditions of temperature between 5 and 25 ° C, and a photoperiod of 8 at 12 h of light and 8 to 12 h of darkness of white light intensity between 5-200 {mi} mol {sup, m-2} s {sup, -1} for a suitable period (between 0 and 100 hours) , according to sample a); e) measurement of the optical dispersion of the culture obtained in step d); f) quantification of the DNA of the sample obtained in d); g) determination of the autofluorescence of the chlorophyll of the sample obtained in step d).

Description

BIOENSAY OF ENVIRONMENTAL TOXICITY BASED ON FICOBIONTES ISOLATED FROM LIQUES

FIELD OF THE INVENTION

The present invention relates to materials and methods for assessing the environmental toxicity of water, effluents, soils, leachates and even gaseous emissions, the invention being within the sector of biotechnology, biochemistry, cell physiology, environment and ecotoxicology. Specifically, it refers to environmental toxicity bioassays performed on phycobionts isolated from lichen.

BACKGROUND OF THE INVENTION

Environmental toxicology studies the impact of pollutants on the structure and function of biological systems. It is a relatively recent discipline that relies on the knowledge provided by different branches of knowledge such as Ecology, Organic Chemistry, Molecular Biology or Microbiology.

An extremely useful tool that uses environmental toxicology is bioassays. A bioassay can be defined as an experiment in which the biological activity of a substance is determined by observing its effect on intact organisms, organs, or different biological preparations. Such tests provide the possibility to evaluate the toxicity of a sample without knowing the complete chemical composition of the sample. Contaminated samples found in the environment may have a toxicity, not so much to a single pure compound, but to a complex matrix. This will lead to a series of interactions between these substances, which can cause different effects:

Additive effect: the combined effect of two substances is equal to the effect caused by each of them separately.

Synergistic effect: the effect of the combination of the two substances is much greater than that caused by each of them separately.

Empowerment: occurs when a substance that is not toxic increases the toxic effect of another substance that is.

Antagonism: occurs when two substances are counteracted or when one of them interferes with the action of the other.

Due to these possible interactions, the analysis of the quantity and composition of the contaminants in a sample is not sufficient to give complete information on the biological effects that it can cause. However, bioassays can provide us with that information (SA Mansour, MF Gad, Risk assessment of pesticides and heavy metal contaminants in vegetables: a novel bioassay method using Daphnia magna Slraus. Food and Chemical Toxicology, 2010, 48: 377-389; K. Wadhia, KC Thompson, Low-cost ecotoxicity testing of environmental samples using microbiotest for potential implementation of the Water Framework Directive. Trends in Analytical Chemistry, 2007, 26: 300-307).

There is a wide variety of toxicity bioassays that use different species, ranging from bacteria to birds. The species chosen for the performance of a bioassay must meet a series of criteria such as the possibility of being available in sufficient quantity, whether thanks to laboratory cultivation, breeding or collection in the field. It is also important that it supports the laboratory conditions well and that it allows obtaining a homogeneous population (K. Wadhia, K.C. Thompson,

Low-cost ecotoxicity testing of environmental samples using microbiotest for potential implementation of the Water Framework Directive. Trends in Analytical Chemistry, 2007, 26: 300-307).

Most bioassays are based on the evaluation of the mortality caused by the toxic sample in the organism chosen for the bioassay. Some examples of organisms used in bioassays are:

Daphnia: Daphnia magna, D. pulex, D. pulicaria. These organisms are widely used for toxicity bioassays in samples of Water. In this case the toxicity is evaluated by counting under the microscope the individuals that They die after coming into contact with the contaminated sample.

Bacteria: Vibrio fisheri. It is a marine bacterium used for both water samples and soil leachate. The bioassay is based on the bioluminescence emitted by the bacteria. That bioluminescence is directly affected proportionally

5 to the presence of toxic substances, so that the toxicity of the sample is evaluated by measuring the decrease in luminescence with spectrophotometric techniques.

Lumbricides: Eisenia foetida. Very used in environmental impact studies of pesticides, herbicides, heavy metals and PAHs in soils. Toxicity is related to the number of individuals who die.

Unicellular algae: Chlamydomonas reinhardi, Se / enastfum capricornutum. These organisms are used in bioassays with water samples. In this case, the toxicity of the samples is evaluated by observing the growth of the algae.

Fish: Oncorhynchus mykiss (rainbow trout), Salvenilus fontinalis (brook trout), Carassius auratus (golden carp).

The death of individuals as a criterion for assessing toxicity is therefore of great interest and widely used in bioassays. However, it is sometimes difficult to record deaths and counts of a large number of samples should be done, which makes it a hard and slow job. It can also happen that the contaminants are in 25 non-lethal quantities or in non-bioavailable forms making immediate death unlikely (R Feito, Y. Valcárcel, M. Catalá, Biomarker assessment of toxicity with miniaturized bioassays: diclofenac as a case study, Ecotoxicology 2012, 21: 289-296; J. Rodríguez Pérez, S. Loureiro, S. Menezes, P. Palma, RR Frenandes, IR Barbosa, A.MV.M. Soares, Assessment of water quality in the Alqueva Reservoir (Portugal) using

30 bioassays, Environmental Science and Pollution Research, 2010, 17: 688-702). Another problem is the volume of environmental sample, which sometimes, as in bioassays with fish, is of the order of liters, which makes storage, maintenance and handling difficult.

There is a way to assess these non-lethal toxic effects, biomarkers, early. Biomarkers can be defined as any measurable and quantifiable biological parameter that serves as an index for the evaluation of physiological or health status. We can affirm that biomarkers are short-term indicators of long-term biological effects (M.P. Cajaraville, Science of the total environment, 2000, 247: 295-311).

Therefore, a correct use of biomarkers has the potential to anticipate changes in more complex levels of organization such as populations, communities and ecosystems and can be used in a predictive manner, allowing the application of corrective measures before environmental damage occurs irreversible. Among the biomarkers used are optical dispersion, autofluorescence of chlorophyll and DNA quantification.

Optical dispersion measurement is a method of indirect estimation of rapid and simple replication and growth. When a beam of light hits a suspended body, part of the light is reflected, part is scattered, part is absorbed and part is transmitted. Turbimetry is the technique that measures the light that is dispersed by hitting a particle as a decrease in the light transmitted through the medium. Thus, this method consists in measuring the turbidity, due to the scattering of light, of a liquid medium with cells in suspension. The scattered light will be greater as soon as the suspension contains more cellular material, so that the sample will be more cloudy. This turbidity is measured in absorbance units with a spectrophotometer or photocolorimeter. It should be noted that this method does not distinguish living cells from dead cells. (AM Manacorda, OP Cuadros, A. Alvarez, Practical Manual of Microbiology, National University of Comahue, 2007: 51; MK Chung, R. Hu, MH Wong, KC Cheung. Comparative toxicity of hydrophobic contaminants / 0 microalgae and higher plan / s. Ecotoxicology, 2007, 16: 393-402.).

Chlorophyll autofluorescence is a biomarker that in recent decades has become a key parameter when conducting studies with organisms of plant origin. Any alteration produced in the quantity or integrity of the chlorophyll molecule can affect the photosynthetic processes and, therefore, the physiological state of the organisms. This method is based on the phenomenon of remission (in the form of fluorescence) of the light absorbed by the chlorophyll molecules. The simplicity and sensitivity of the principle on which the method is based has allowed the development of easy fluorometers

management (P.B. Fai, A Grant, B. Reid, Chlorophyl a fluorescence as a biomarker for rapid loxicilyassessmenl, Enviornmental Toxicology and Chemistry, 2007, 26: 1520-1531).

Although the total amount of chlorophyll fluorescence is very small (only 1 or 2% of

5 the total absorbed light), the measurement is quite easy. The fluorescence spectrum emitted by chlorophyll is different from that of the absorbed light, the fluorescence emission peak being longer wavelength than that of absorption. Therefore, the fluorescence yield can be quantified by exposing a sample containing chlorophyll the light of defined wavelength and measuring the amount of light re-emitted in wavelengths

10 older (K. Maxwell, G.N. Johnson, Chlorophyll fluorescence -A practical guide, Journal of Experimental Botany, 2000, 51 (345): 665-668). This performance of chlorophyll fluorescence will depend on the conditions in which the organism is. Therefore, as conditions become more unfavorable for an organism that has chlorophyll, its fluorescence emission will be lower (R. Feito, Y. Valcárcel, M. Catalá,

15 Biomarker assessment of toxicity with miniaturized bioassays: diclofenac as a case study,

Ecotoxicology, 2012, 21: 289-296).

Regarding the quantification of DNA, it should be mentioned that it is a method based on the amount of DNA present in a sample. It is a method that provides high reliability and repeatability. The determination of the amount of a sample is carried out by spectrofluorimetry by adding to the sample of intercalating fluorochromes. (P.K.S. Lam, Use of biomarkers in environmental monitoring, Ocean & Coastal Management, 2009,

52: 348-354).

25 Among these fluorochromes is Hoechst which is a specific and stoichiometric intercalator of DNA. It acts by preferentially binding to the AT base pairs exposed in the minor groove of the double strand of DNA, emitting fluorescence at wavelengths of "excitation = 360 nm," emission = 465 nm (B. Welsblum, E. Haenssler, Fluorometric properties of the bisbenzimidazole derivative Hoechst 33258, a fluorescent

30 probe specific for AT concentration in chromosomal DNA. Chromosoma, 1974, 46: 255-260) to be collected by the fluorimeter detector. Any change that affects the amount of DNA present in a sample will be detected by the fluorimeter, thus indicating increases in cell number decreases (KA Haque, RM Pfeiffer, MB Beerman, JP Struewing, SJ Chanock, AW Bergen, Performance 01 high- Ihroughput ONA quanlilicalion

35 melhods, BMC Biotechnology, 2003, 28 (3): 20).

Now, the authors of the present invention have developed a bioassay that evaluates the toxicity of a sample by indirect methods, determining the optical dispersion of a suspension, the autofluorescence of chlorophyll and / or the amount of DNA present in a sample, using as Phycobiontic model organisms isolated from lichens.

When carrying out toxicity microbioassays, a key component in them is the species involved, since they must be properly selected so that the global toxicity in the ecosystem can be evaluated and relevant results can be obtained. Ecological (K. Wadhia, KG. Thompson, Low-cost ecotoxicity testing of environmental samples using microbiotest for potential implementation of the Water Framework Directive, Trends in Analytical Chemistr, 2007, 26: 300-307 .; M. Catalá, M. Esteban , JL Rodríguez-Gil, LG Quintanilla, Deve / opment of a naturally miniaturized testing method based on the mitochondrial activity of fern spores: A new higher plant bioassay, Chemosphere, 2009, 77, 983-988).

Among the requirements that these species must meet are, their sensitivity to the environmental or material factors in question, have a wide distribution and availability in sufficient quantities and throughout the year, be a taxon with importance from an ecological point of view and / or economical, it must be able to be easily maintained in the laboratory, it must be in good condition (free of parasites or diseases) and above all, it must be compatible with bioassay techniques (MA Capó Martí, Principles of Ecotoxicology: Diagnosis, Treatment and Environmental Management; entity, McGraw-HiII, 2002; K. Wadhia, KC Thompson, Low-cost ecotoxicity testing of environmental samples using microbiotest for potential implementation of the Water Framework Directive, Trends in Analytical Chemistry, 2007, 26: 300- 307).

Species that meet these characteristics are selected, among other things, to assess the environmental toxicity of discharges to surface waters. Among them are aquatic invertebrates (for example, Ceriodaphnia dubia, Daphnia magna), fish (as is the case with Pimephales promelas), bacteria (such as Pseudomonas polished), microalgae (for example, PseudokirchnerieJ / a subcapitata), and vascular plants (for example, Lemna minar) (SM Paixao, L. Silva, A. Fernandes, K. Q'Rourke, E. Mendoc; a, A. Picado, Performance of a miniaturized algal bioassay in phytotoxicity screening, Ecotoxicology, 2008, 17 : 165-171.). In particular, microalgae have been widely used for toxicity tests due to their high sensitivity and reproducibility. Toxicity bioassays carried out with microalgae use free-living algae, without

However, a good option is the use of algae associated with lichen, environmental biomarkers par excellence (1. Shitanda, S. Takamatsu, K. Watanabe, M. Itagaki,

Amperometric screen-printed algal biosensor with flow ¡njection analysis system for detection of environmentaJ toxic compounds, Electrochimica Acta, 2009, 54: 4933-4936).

Lichen talus has a part composed of heterotrophic fungi (mycobiont) and another part of photosynthetic algae or cyanobacteria (photobiont or in the specific case of algae, also called phycobiont). Studies with lichen show that, generally, the most sensitive part to contaminants is the phycobiont (M. Backor, P.

10 Váczi, Copper toleranee in lhe lichen photobiont Trebouxia erici (Chlorophyta), Environmental and Experimental Botany, 2002, 48: 11-20).

It has been observed that phycobionts have characteristics such as tolerance to desiccation or anhydrobiosis (characteristic that algae do not have free life), tolerance to a wide range of working temperatures, growth capacity in liquid and solid medium (F. Gasulla Vidal, Insights on desiccation tolerance of lhe fichen photobiont TR9 pi. In bolh Ihalline and isolaled ones, Doctoral thesis, 2009; A. Del Hoyo, R. Álvarez,

E.M. Del Campo, F. Gasulla, E. Barreno, L.M. Casan 0, Oxidative stress induces distinct physiological responses in the two Trebouxia phycobionts of the fichen Ramalina farinacea,

20 Annals 01 Botany, 2010, 1-10; L.M. Casano, E. Del Campo, F. Garcia-Breijo, J. ReigArmiñana, .F Gasulla, A. Del Hoyo, A. Guéra, E. Barreno, Two Trebouxia algae with different physiological performances are ever present in fichen thalli of Ramafina farinacea . Coexislence vs. Competence Environmental Microbiology, 2010, 13: 806-818). These qualities are very useful in the development of miniaturized bioassays, more adaptable,

25 versatile and low cost allowing easy storage, handling and use.

Thus, the present invention has allowed the development of a new bioassay for the evaluation of the toxicity of a sample, fast, simple, economical and reliable, which has important advantages over those currently employed in the state of the art.

OBJECT OF THE TNVENTION

First, a method of assessing the toxicity of a sample based on the determination of the optical dispersion, the autofluorescence of chlorophyll and / or the amount of DNA present in a sample of phycobionts isolated from lichen

The object of the invention is also the use of optical dispersion, of the chlorophyll autofluorescence and of the quantification of DNA in phycobionts isolated from lichen as biomarkers in methods for assessing toxicity in a sample.

Finally, a kit for evaluating the toxicity of a sample by the method mentioned above, as well as the fundamentals of an environmental biosensor, is also the subject of the invention.

DESCRIPTION OF THE INVENTION

The present invention reveals a family of bioassays capable of determining the environmental toxicity of water, soil and air samples, and the methods for carrying them out with lichen phycobionts from the measurement of optical dispersion, chlorophyll autoflowering and DNA quantification.

The biology of phycobionts can be altered by the presence of toxic substances, or mixtures of them present in the environment, resulting in a variation in a series of parameters such as optical dispersion, autofluorescence of chlorophyll and / or DNA . Thus, contacting the isolated phycobionts of lichens with field samples will produce, or not, a variation in the optical dispersion, autofluorescence of chlorophyll or amount of DNA depending on the toxicity of the samples,

Thus, in a main aspect of the invention, a method for assessing the toxicity of a sample based on a) obtaining a phycobionate population in a sterile culture medium suitable for each type of assay is contemplated; b) the preparation of a suspension with the population of phycobionts obtained in a) in a suitable culture medium; c) the exposure of the suspension obtained in b) to the sterilized sample whose toxicity is to be evaluated; d) the incubation of the suspension of phycobionts isolated from lichen (c) under temperature conditions between 5 and 25 oC, and a photoperiod from 8 to 12 h with a white light intensity between 5-200 ~ mol m, 2 S, 1 for a suitable period (between O and 100 hours),

according to the sample: e) measurement of the optical dispersion of the culture obtained in step d) at a wavelength of 650 nm; f) quantification of the DNA of the sample obtained in d); g) determination of the autofluorescence of the chlorophyll of the sample obtained in step d).

5 The characteristics of this test allow the analysis of samples of very different nature:

aqueous samples: including, but not limited to, groundwater, surface water, drinking water, soil leachate, waste leachate, 10 liquid waste, etc.

solid or semi-solid samples: including, but not limited to, soil, sludge, compost, granular aggregates, etc.

15 gaseous samples: including, but not limited to, gaseous effluents, either directly or through their aqueous absorption.

In a particular embodiment of the invention the axenic population of phycobionts in a) is obtained from an established collection, such as the UTEX collection of the

20 University of Texas, or through its isolation and purification from a liquid thallus (for example with the method of F. Gasulla, A. Guéra, E. Barreno, A simple and rapid method for isolating lichen photobionts, Symbiosis 2010 51 : 175-179).

Once the aliquots are prepared, the exposure (stage e) and incubation of

25 these with the sample of water, soil or air whose toxicity is to be evaluated (step d). The incubation temperature range of stage d) ranges between 5 and 25 oC depending on the species of phycobiont used, and whatever its optimum temperature range. Likewise, depending on the species to be used, the photoperiod of light and dark hours will vary between 8 and 12 h. After incubation, you will extract an aliquot from each sample to measure the dispersion

30 optics (stage e). In a particular embodiment of the invention, the average optical dispersion is measured in a 650 mm plate reader. In the same way an aliquot will be extracted to measure the autofluorescence of chlorophyll (step d). In a particular embodiment of the invention, a fluorimeter with a 485 nm excitation filter, and a 635 nm emission filter is used to measure the autofluorescence of chlorophyll. The rest

35 of the sample is intended for quantification of DNA (step f). In a particular embodiment

of the invention, the sample is processed by the method of Feito el aL, with a series of modifications adapted for these microorganisms (R. Feite, Y. Valcárcel, M. Catalá, Biomarker assessment of toxicity with miniaturized bioassays: diclofenac as a case study, Ecotoxicology, 2012, 21: 289-296). According to this method, the cells are subjected to 5% 70% ethanol and incubated for 24 hours at room temperature. After the incubation time, the samples settle, for example by centrifuging at 13,000 g. They are resuspended in a TNE-lysis medium (1: 1), composed of lysis solution (0.2% Triton X-100 and 1% NaOH 1) and TNE (100 mM TRIS, 10 mM EDTA and 1 M NaCl ). Subsequently, one aliquot of each sample will be diluted in TNE and another aliquot of the same volume will be diluted in TNE 10 bisbenzimide Heechst 33258 (Sigma-Aldrich ehemie Gmbh, Steinheim, Germany) 0.2 ~ g / ml. In this way, a target is created that eliminates the autofluorescence that cells can emit, to obtain only the fluorescence of the DNA bound to the Hoechst. Both should be stirred for 1 min and incubated in the dark for 5 minutes. In a particular embodiment of the invention, Hoechst fluorescence is measured in a plate reader.

15 fluorimeter with an excitation filter of 360 nm, and an emission filter of 465 nm. The fluorescence data should be interpolated in a straight standard of purified DNA in the appropriate concentration range of O -10 f.lg / ml, for example.

In a particular embodiment, racks of 96 tubes of 1.2 ml capacity with 20 sterilized caps are used, adding 500 J..II of a suspension with 106 cells mr 'to each tube.

In a preferred embodiment, isolated phycobionts of lichen Ramalina farinacea are chosen, hereinafter referred to as Trebouxia sp. whose suspension requires the following incubation conditions (step a): 12 hour light photoperiod, at a temperature of 15

25 oC, for 15 days. Under these conditions, step c) is carried out at a temperature of 15 oC with a 12-hour light photoperiod for a period of time between 24 and 96 hours in axenic conditions.

In another preferred embodiment, Asterochloris erici {Ahmadjian) Skaloud phycobionts are chosen

30 et Peksa (formerly known as Trebouxia eriel) whose suspension requires the following incubation conditions (stage a): a 12-hour light photoperiod, at a temperature of 20 oC, for 15 days. Under these conditions, stage c) is carried out at a temperature of 20 oC with a 12-hour light photoperiod for a period of time between 24 and 96 hours in axenic conditions, and on racks of 96 tubes of 1, 2

35 ml

The use of lichen-isolated phycobionts provides the bioassay object of the present invention with a series of characteristics that confer advantages over other bioassays existing in the state of the art:

1) Phycobionts isolated from lichens are easy to reproduce in the laboratory. Sufficient amount can be obtained economically to process the samples that are needed, since they reproduce at low temperatures and low light intensities.

2) They tolerate storage at 4 oC directly under growing conditions, and freezing in the appropriate medium. They are also adapted to anhydrobiosis, recovering their physiological activity quickly after rehydration, which greatly facilitates their transport, storage and conservation.

3) The phycobionts isolated from lichen grow easily in semi-solid medium. So they do not need the constant maintenance of liquid cultures.

4) This bioassay provides greater biological relevance than other existing bioassays especially for the terrestrial environment. Because lichens are known as bioindicators par excellence, the use of phycobionts isolated from lichens will maintain this particularity. Phycobionts reproduce by meiosis and mitosis, so that the cells used in the bioassay are going to be, in part, genetically different individuals and also the formation of subpopulations has been observed. Other bioassays use clones of individuals (algae, bacteria, cell lines or primary cultures), which although they have a lower dispersion in the results, give the bioassay less biological relevance.

5) The bioassay described provides greater ecological relevance than other bioassays currently used. There is a wide variety of lichen species and some of them are characteristic of very specific habitats or ecosystems. This means that certain species of phycobionts can be useful tools in the monitoring and environmental monitoring of specific habitats.

6) The bioassay described allows to process a large number of samples with a minimum cost. Both the biological material and the reagents used in this bioassay

Are cheap. The necessary laboratory equipment for the bioassay can be found in any basic microbiology laboratory.

7) The characteristics of the bioassay make it possible to adapt to automatic systems of optical techniques such as multiwell plates and continuous systems.

8) The bioassay object of the present invention employs small volumes of sample. The bioassay can be carried out with sample volumes of the order of microliters. This feature makes the bioassay especially useful in cases where the sample quantity is limited. It also reduces the need to store high amounts of sample, which in many cases need refrigeration, which causes space problems.

Finally, another main aspect of the invention contemplates the possibility of developing a kit to evaluate the toxicity of a sample, which comprises a population of phycobionts isolated from lichens in suspension, solid or semi-solid substrate, and the suitable means for their use. Likewise, a protocol of use should be included to determine the toxic effect on the optical dispersion and the autofluorrescence of chlorophyll of water, soil or air samples. On the other hand, a fluorescent probe can also be included for the quantification of the DNA of the phycobionts and the appropriate means for its use to determine the toxic effect of water, soil or air samples.

BRIEF DESCRIPTION OF THE FIGURES

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to a preferred example of practical realization thereof, a set of figures in the accompanying part is accompanied as an integral part of said description. where, for illustrative and non-limiting purposes, the following has been represented:

Figure 1. Optical dispersion, autofluorescence of chlorophyll and quantity of ONA with respect to control after 24, 48, 72 and 96 hours with 5 concentrations of the herbicide Atrazine in Trebouxia sp. The columns represent the averages and the bars show the typical error of the average. At the base of each column the value of n appears at each point. (*) indicates statistically significant difference from the control (ANOVA, p <0.05).

Figure 2. Optical dispersion, autofluorescence of chlorophyll and amount of ONA with respect to control after 24, 48, 72 and 96 hours with 6 concentrations of the anti-inflammatory Diclofenac in Trebouxia sp. The columns show the average and the bars show the typical error of the average. At the base of each column the value of n appears at each point. (*) indicates statistically significant difference from the control (ANOVA, p <Q, 05).

Figure 3. Optical dispersion, autofluorescence of chlorophyll and amount of DNA with respect to the control after 24, 48, 72 and 96 hours with the concentrations of the herbicide Atrazine in Asterochloris erici. The columns show the average and the bars show the typical error of the average. At the base of each column the value of n appears at each point. (*) indicates statistically significant difference from the control (ANOVA, p <O, 05).

Figure 4. Optical dispersion, autofluorescence of chlorophyll and amount of DNA with respect to the control after 24, 48, 72 and 96 hours with 6 concentrations of the anti-inflammatory Diclofenac in Asterochloris erici. The bars show the typical error of the average. At the base of each column the value of n appears at each point. (*) indicates a statistically significant difference from the control (ANOVA, p <0.05).

Exposed heretofore the distinctive features of the present invention, one of the possible ways of carrying it out is described.

PREFERRED EMBODIMENT OF THE INVENTION

The present invention is further illustrated by the following example which is not intended to be limiting of the scope of the invention.

A bioassay was carried out with phycobionts isolated from Ramalina farinacea, which from now on we will call Trebouxia sp., And Asterochloris erici, against the presence of phytosanitary products and drugs found in river and drinking waters, such as the herbicide Atrazine and the anti-inflammatory Diclofenac (Ma, J., Zheng, R., Xu, L., Wang, S., 2002.

Differential Sensilivily 01 Two Green Algae, Scenedesmus obliqnus and Chlorella pyrenoidosa, to 12 Pesticides. Ecotoxicology and Environmental Safety 52, 57-61; Altenburger, R., Bodeker, W., Faust, M., Horst Grimme, L., 1990; EvaJuation of the isobologram method for the assessment of mixtures of chemicals: Combination effect studies

with pesticides in a / gal biotests. Ecotoxicology and Environmental Safety 20, 98-114; Gonzalez Alonso, S .; Catala, M .; Romo Maroto, R .; Rodriguez Gil, J. L; Gil de Miguel, A .; Valcarcel, Y., 2010. PoJ / ution by psychoactive pharmaceutica / s in the Rivers of Madrid metropolitan area (Spain). Enviroment International 36: 195-201.; Feito, R; Valcárcel, Y .; Catalá, M .; Biomarker assessment of toxicity with miniaturized bioassays: diclofenac as a case study. Lawrence, J.R., Swerhone, G.DW., Topp, E., Korber, DR., Neu, T.R., Wassenaar, LI., 2007. Structural and functional responses of river biofilm communities to the nonsteroidal anti-inflammatory diclofenac. Environmental Toxicology and Chemistry 26, 573582). The microbioassays try to determine the toxicity of these compounds in the organisms mentioned above, after subjecting them to different concentrations of each compound and facing different exposure times in a preferred embodiment (24, 48, 72 and 96 hours). The biomarkers studied for the determination of toxicity were: measurement of optical dispersion (turbidity), autofluorescence of chlorophyll and quantification of DNA by fluorescent probes.

In a first stage a preculture of a suspension of phycobionts of Trebouxia sp. and Aslerochloris erici, with the aim of facilitating acclimatization and obtaining known suspensions in later stages.

For all microbioassays, cultures in liquid medium, prepared 15 days before, of each of the selected species were used. This is because the maximum growth rate of these phycobionts is between 7 and 17 days of cultivation (Gasulla, 2009). 1.2 ml tubes were taken and 500.JI of phycobionts were inoculated in suspension with a concentration of 106 cells / ml for each tube. To each of them 500¡JI of 3NBBM medium and contaminant were added in order to identify its effect. Each of these 1.2 ml tubes was stored in racks with capacity for 96 tubes, to determine what influence the phycobionts had on both the concentration of the compound and the exposure time to which it was subjected. 8 replicates were performed per concentration of the compound, including controls. In the case of Atrazine, the control was composed of sterile 3NBBM culture, acetone and phycobiontes in suspension, since Atrazine is an apolar compound and acetone was used for its dissolution. For the drug Diclofenac, the control was composed of sterile 3NBBM culture medium and suspended phycobionts. All cultures were kept in a culture chamber at 15 oC for the case of Trebouxia sp., And at 21 ° C for the case of Aslerochloris erici, under a photoperiod 12 h of white light with an intensity of 30 ~ mol m-2 s -'for both strains. At the end of the exposure time, 200¡JI were taken for optical dispersion measurement, 200¡JI was used to determine the

autofluorescence of chlorophyll and 600 1-11 for quantification of DNA by fluorescent probes.

In the case of measurement of the optical dispersion, 200 µI of sample were taken and 100 1-11 were deposited per well of 96-well plate, so that two plates were obtained for duplicate absorbance measurements. These were obtained using the multi-well plate reader (spectrophotometer) model 17-550 Anthos 2010 at a wavelength "= 650 nm (Chung el aL, 2007). The results show that both the herbicide atrazine and the anti-inflammatory diclofenac at very low concentrations induce significant alterations in the optical properties of the suspensions of both phycobionts, especially Aslerochloris erici, more sensitive.The variation of the optical properties of the suspensions is linked both to the number of cells and to the change in the pattern of aggregation linking the effects at the level individual with the population level This parameter reports alterations in the population structure and is a clear biomarker of exposure without prejudice to other applications.

In the case of the chlorophyll autofluorescence measurement, 200 IJI samples were also taken and 100 [JI] were deposited per well of 96-well Greiner plate, so that two plates were obtained for duplicate fluorescence measurements. These were obtained by the SPECTRAFluor Plus plate reader (Tecan Group Ud., Mannedorf, Switzerland) with filters of A eKcitation = 485 nm and "emission = 635 nm. The results presented in the figures show that both substances induce significant alterations in Chlorophyll autofluorescence is especially striking the significant decrease induced by Aslerochloris erici attrazine, a herbicide that inhibits photosynthesis.In this sense, Trebouxia sp. seems more tolerant, which is consistent with the classification as toxitolerant of its lichen Ramalina farinacea origin.

For the quantification of DNA, the Hoechst fluorometric probe was used, which is a specific and stoichiometric intercalator of DNA. It acts by preferentially binding to the A-T base pairs exposed in the minor groove of the double strand of DNA, emitting fluorescence at wavelengths of A eKcitation = 360 nm, A emission = 465 nm. Previously, it was necessary to make a chemical treatment of the samples in order to break or generate pores in the cell membranes and facilitate the entry of the Hoechst probe. For this, the samples were sedimented, at 1300 g for 10 minutes in the Digicen 20 centrifuge, arranged with a floating rotor capable of centrifuging the racks. Subsequently, the supernatant was removed and the sample was subjected to 70% ethanol exposure. It was stirred for a few seconds in the vortex and incubated at room temperature for 24 hours. After this period of time, the samples were centrifuged again under the same conditions mentioned above and the supernatant was removed. Next, the 1: 1 TNE-lysis solution, composed of

5 lysis solution (0.2% Trilon X-1 OO and 1% NaOH 1N) and TNE (100 mM TRIS, 10 mM EDTA and 1 M NaCI) (Cesarone et aL, 1979). Finally, the samples were sonicated for 5 minutes and placed in the bath for 1 hour at 37 oC, being ready for the application of the probe and the corresponding measurement.

10 An aliquot of 100 1-11 of each sample was diluted in TNE to generate the targets (measurement of the autofluorescence of phycobionts) and another aliquot of the same volume was diluted in Hoechst 0.1 I-Ig / ml -TNE ( measurement of autofluorescence from phycobionts and DNA) in black multi-well Greiner plates. On the other hand, it was carried out the realization of a straight DNA pattern that will later serve

15 to interpolate the DNA results of the samples and thus know their DNA concentration. Each plate was stirred for one minute and subsequently incubated in the dark for 5 minutes and at a temperature of 37 ° C. After this period of time, they were stirred again for 1 minute to ensure the homogeneity of the sample and the measurement was carried out by SPECTRAFluor Plus plate reader (Tecan Group Ud.,

20 Mannedorf, Switzerland) at wavelengths of A excitation = 360 nm and A emission = 465 nm (Cesarone et al, 1979). The results of the figures show significant alterations in the number of cells induced by various concentrations of the pollutants at various times, although the interpretation of this parameter is complex since at the time tested, quite long, there could be subpopulation selection.

25 tolerant with higher proliferation rates and replacement of the original population. The exposure period should be studied for each case.

Claims (10)

1. Method for assessing the toxicity of a sample from lichen phycobionts comprising:

to.

obtaining a population of phycobionts;

b.

preparation of a suspension with the population of phycobionts obtained in a) in a suitable culture medium,

C.

exposure of the suspension obtained in b) to the sterilized sample whose toxicity is to be evaluated;

d.

incubation of the suspension of phycobionts of lichens obtained in e) under temperature conditions between 5 oC and 25 oC, photoperiod of 8 or 12 h of light and 8 or 12 h of darkness of white light intensity between 5-200 Ilmol m- 2s · 1 for a suitable period (between O and 100 hours), depending on the sample to be analyzed;

and.

measurement of the optical dispersion of the sample obtained in d); I

F.

quantification of the DNA of the sample obtained in di; I

g.

determination of the autofluorescence of the chlorophyll of the sample obtained in di.

h.

Analysis of the data obtained in e) to g) to assess the toxicity of the sample.

2.

Method according to the preceding claim, characterized in that the suspension obtained in step d) can be frozen at a maximum temperature of -18 ° C.

3.

Method, according to the preceding claims, whereby the amount of lichen phycobiont DNA is determined, where a specific DNA probe and suitable excitation and emission wavelengths are used.

Four.

Method, according to the preceding claims, by which the autofluorescence of lichen phycobiont chlorophyll is measured where an excitation filter with a wavelength between 450 nm and 500 nm and with an emission filter with a wavelength is used between 600 nm and 650 nm.

5.

Method, according to the preceding claims, by which the optical dispersion of the phycobiont suspension is measured using a device capable of measuring the absorbance between 550 and 700 nm.

Method according to any of the preceding claims, wherein the microassay is based on phycobionts isolated from lichen Ramalina farinacea. 7. Method according to Gl: the elldisro of claims 1 to 5, wherein the microassay is based on the liquid phycobiont Asterochloris erici. 10 8. Use of optical dispersion in lichen phycobionts as a biomarker in methods to assess the toxicity of a sample. 9. Use of the amount of ONA in lichen phycobionts as a biomarker in 15 methods to assess the toxicity of a sample. 10. Use of chlorophyll autofluorescence in lichen phycobionts as a biomarker in methods to assess the toxicity of a sample. 20 11. Kit for assessing the toxicity of a sample using alterations in the optical activity and autofluorescence of chlorophyll as biomarkers, according to the method described in the preceding claims, comprising, at least, a vial with a suspension of phycobionts in culture medium , a bottle with sterile culture medium and a protocol with instructions to perform the bioassay. 12. Kit for assessing the toxicity of a sample using the amount of DNA as a biomarker, according to the method described in the preceding claims, which comprises, in addition to the material described in claim 11, a vial with concentrated reagent, dilution buffer and a protocol with instructions to perform the 30 bioassay and DNA quantification.