USE OF A CNIDARIAN-DINOFLAGELLA SYMBIOTIC ORGANISM AS A MARKER FOR EVALUATING THE ECOTOXICITY OF A PRODUCT, AND METHOD FOR EVALUATING THE ECOTOXICITY OF A PRODUCT

Abstract

The present invention relates to the use of a cnidarian-dinoflagellate symbiotic organism as a marker for assessing the ecotoxicity of a product. The present invention also relates to a method for assessing the ecotoxicity of a product, comprising the following steps: 1) culturing at least one cnidarian-dinoflagellate symbiotic organism in a suitable medium, 2) adding at least one product to the medium, 3) measuring, in the medium and in the cnidarian, the dissociation of the dinoflagellates from the symbiosis and / or the growth rate of cnidarian cuttings, particularly hard corals. Figure 1

Description

Title of the invention: USE OF A CNIDARIAN-DINOFLAGELLA SYMBIOTIC ORGANISM AS A MARKER FOR EVALUATING THE ECOTOXICITY OF A PRODUCT, AND METHOD FOR EVALUATING THE ECOTOXICITY OF A PRODUCT technical field

[0001] The present invention relates to the use of a cnidarian-dinoflagellate symbiotic organism, as well as to a method for evaluating the ecotoxicity of a product.

[0002] The present invention finds applications particularly in the field of toxicology, especially ecotoxicology.

[0003] In the description below, references in brackets ([ ]) refer to the list of references presented at the end of the text. State of the art

[0004] It has been demonstrated in recent years that the marine environment is particularly impacted by the toxicity of certain ingredients in cosmetic products, especially sunscreens. Indeed, some products can cause coral bleaching, or even their death. These organisms are already severely affected by climate change. In this context, the cosmetics industry is increasingly subject to strict regulations aimed in particular at ensuring the safety of its products with respect to the marine environment and more specifically cnidarians, the zoological group to which tropical corals belong.

[0005] Cnidarian-dinoflagellate associations, such as those that form reef-building corals, are mutualistic symbioses of fundamental importance in the marine environment. These partnerships constitute the trophic and structural basis of coral reef ecosystems. This intracellular association is centered on nutrient exchange and is essential for both partners to thrive in nutrient-poor tropical seas. Cnidarian hosts, such as corals and anemones, harbor photosynthetic dinoflagellate endosymbionts of the genus Symbiodinium within gastrodermal cells in phagosomally derived vacuoles called symbiosomes. Initial colonization most often occurs when the host's gastrodermal cells lining the gastric cavity phagocytose the symbionts ingested orally during feeding.

[0006] Although cnidarian-dinoflagellate symbioses are stable under non- Stressed by various environmental stressors, including the high temperatures caused by climate change, symbiotic relationships can destabilize these relationships and lead to the loss of host tissue symbionts. This phenomenon, known as cnidarian or coral bleaching, results in a significant reduction in the host's physical condition, slowed growth, and even death, potentially leading to the destruction of reefs. More recently, it has been shown that certain components in cosmetic products can have disastrous environmental repercussions, including the bleaching of tropical corals. The democratization of low-cost air travel in recent years has exacerbated this phenomenon, as increasing numbers of people flock to tropical beaches each year, bordering coral reefs already weakened by climate change.Cosmetic products, and more specifically sunscreens, come into contact with corals in high concentrations during swimming.

[0007] When the cnidarian bleaches, it is deprived of the food source provided by dinoflagellates. The cnidarian dies within a few weeks if it has not been recolonized by symbionts. Even low concentrations over the long term can have deleterious effects, notably slowing coral growth. Generally, a coral species living on the upper parts of the reef (e.g., Acropora spp., Pocilopora spp., Seriatorpora spp., etc.), under stable physiological conditions, will gain up to 1% of its mass per day. This growth rate can decrease significantly if the species in question is subjected to any kind of stress, such as chronic exposure to a sublethal toxic substance. During these acute or chronic impacts, an entire ecosystem is weakened, with very significant socio-economic consequences.Indeed, these ecosystems, beyond the economic benefits they bring to tourism, provide local populations with food and coastal protection. It is estimated that one billion people worldwide depend directly on coral reefs as their primary food source. It is therefore crucial to urgently implement a comprehensive policy for these ecosystems and to better manage the negative impact of large-scale tourism, which carries a whole range of chemicals with harmful ecological consequences.

[0008] Thus, bioassays have been developed with the aim of evaluating the ecotoxicity of a product.

[0009] Some bioassays are commercially available as "ready-to-use" kits based on miniaturization and standardization of conventional toxicity tests (https: / / www.microbiotests.com / toxkit / ). These kits contain, in particular, test organisms such as daphnia, rotifers, microaglues, etc., which For example, they can be in the form of dormant eggs in the case of daphnia (sporocysts). This form of commercialization has the advantage of freeing the user from the constraints of breeding and cultivation. It also allows for easy access to a variety of bioassays that can be used to create a minimum battery of tests to characterize the ecotoxicity of a sample. However, these bioassays are not very representative of reef ecology.

[0010] Other tests have been developed to assess the ecotoxicology of cosmetic products on corals, such as the "ReefTox" (SGS, France). This test aims to evaluate the toxicological impact of a cosmetic ingredient or product on a coral population representative of tropical / Indo-Pacific swimming areas (known as the "Coral Triangle"). Thus, this test assesses the toxicity of products on 12 or 50 species / specimens of hard and soft corals in order to obtain a true representation of the coral population. Although these bioassays represent a real advance in reef ecotoxicology, they have the drawback of being extremely expensive, time-consuming, and cumbersome to implement. Furthermore, the physicochemical or biological (coral survival) methods used for water analysis are only indirect measures of the stress caused.It is therefore difficult to determine whether any mortality observed in the quarantine tank is due to exposure to the tested chemical or to the fact that the cnidarian was moved to a different tank during the experiment. These organisms are indeed very sensitive, and any change in environment can lead to bleaching or even death. Furthermore, bleaching of a cnidarian exposed to a toxic substance can spread to other neighboring organisms not exposed to the same chemical compounds, resulting in a difficult, or even impossible, interpretation of the results.

[0011] Finally, other tests have been developed using cell cultures. For example, the VIRIS test (INNOV&SEA) is a cytotoxicity test using cell cultures from the anemone Anemonia viridis. This test is time-consuming (14 days). Furthermore, this anemone lives in the cold waters of the Mediterranean and the Atlantic, making it an organism that is not very representative of the animals inhabiting coral reefs.

[0012] There is therefore a real need for testing to evaluate the toxicity, and in particular the ecotoxicity, of a product, especially on coral reefs. Description of the invention

[0013] The present invention is specifically designed to address these needs and drawbacks of the prior art.

[0014] Following extensive research, the Applicant has succeeded in developing a standardized method for assessing the health status of cnidarians by measuring bleaching and / or growth rate.

[0015] The invention results in a simple, quick to implement, and little process costly, allowing us to highlight the suffering of cnidarians subjected to exposure to chemicals such as those found in cosmetic products.

[0016] Advantageously, this process can be carried out by a laboratory technician, and requires only a small amount of weekly maintenance time, which can be as little as about 1 hour per week.

[0017] Furthermore, the method of the invention has high sensitivity. Advantageously, it can detect sublethal signs of animal suffering such as coral bleaching or a decrease in growth rate.

[0018] Moreover, the invention is particularly representative of the impact of chemical pollution on a coral reef.

[0019] Advantageously, the invention can be used on a large scale to assist in decision-making in order to combine economic development, product development, particularly cosmetics, and preservation of the marine environment.

[0020] Finally, the invention can make it possible to assess the health status of corals and to know if they are subjected to acute and / or chronic stress.

[0021] Thus, a first object of the invention relates to the use of a cnidarian-dinoflagellate symbiotic organism as a marker for the evaluation of the ecotoxicity of a product.

[0022] A second object of the invention relates to a method for evaluating the ecotoxicity of a product, comprising the following steps: 1) culture of at least one cnidarian-dinoflagellate symbiotic organism in a suitable medium, 2) addition, in the medium, of at least one product, 3) measurement, in the environment and the cnidarian, of the dissociation of dinoflagellates from the symbiosis and / or of the growth rate of cnidarian cuttings, particularly hard corals.

[0023] Within the scope of the invention, any cnidarian-dinoflagellate symbiotic organism known to a person skilled in the art may be used.

[0024] In particular, the cnidarian may be a "hard" coral (with a calcareous skeleton), a "soft" coral (without a calcareous skeleton), or an anemone. It may be a whole organism, a fragment (for example, a cutting) of a whole organism, and / or cultured cells. For example, the cnidarian may be at least one cnidarian selected from Aiptasia spp., for example, Aiptasia pallida, Zoanthus spp., Xenia spp., Clavilaria spp., Acropora spp., Pocilopora spp., Seriatorpora spp., Stylophora spp., Turbinaria spp., and a scleractinian. The "hard" coral may be a scleractinian. The dinoflagellate may be, in particular, of the genus Symbiodinium, also known as zooxanthellae.

[0025] Preferably, the cnidarian used for the measurement step, in the medium and the Cnidarian, resulting from the dissociation of dinoflagellates from symbiosis, can be Aiptasia spp., and in particular Aiptasia pallida. It possesses characteristics that make it an excellent model organism, including rapid growth, easy maintenance requirements, and its ability to be bleached and reinfected. It is possible to maintain large cultures of symbiotic and aposymbiotic A. pallida, and numerous molecular and cellular biology techniques are available for Aiptasia (Detournay et al., Dev Comp Immunol, 2012 ([1]), including a database of 12,000 ESTs of A. pallida sequences. A. pallida can range in size from less than 2 mm to 4–5 cm in height. It reproduces asexually via pedal laceration, in which motile polyps leave behind small pieces of the pedal disc that develop into tiny new polyps less than 1 mm in size.Large colonies (i.e., hundreds to thousands of individuals) of genetically identical animals can therefore be generated and maintained in culture indefinitely. A. pallida can be "bleached" by exposing the individuals to a product to be tested for ecotoxicity. Within a few days or weeks, the polyps are free of algae (aposymbiotic), and if kept in the dark, they will remain aposymbiotic indefinitely.

[0026] Preferably, the cnidarian used for the growth rate measurement step can be the scleractinian hard coral Seriatopora hystrix. It possesses particularly interesting characteristics for measuring growth rate. It is a fast-growing species that can be easily propagated by simple fragmentation, preferably into fragments longer than 1 cm. These small fragments will heal quickly and can therefore be used in growth rate assessment tests when exposed to potentially toxic substances.

[0027] The product may be any substance or mixture of substances whose potential toxicity to marine reef fauna is to be verified. In particular, it may be at least one product selected from among a chemical and a biological product, for example, a cosmetic product, a petrochemical product, a detergent product, an agri-food product, or a plant protection product, generally speaking. Specifically, it may be a product used in the composition of a sunscreen.

[0028] Within the framework of the invention, the cnidarian-dinoflagellate symbiotic organism is used as a marker in that the measurement of the dissociation of the dinoflagellates from the symbiosis indicates suffering of the latter, and therefore ecotoxicity, due to the presence of the product to be tested in the environment.

[0029] Step 1) of culture can be carried out by any method known to those skilled in the art that allows the symbiotic organism to multiply without time limitation. The culture conditions, in particular temperature, oxygen level, salinity, Food and light are controlled and can be adjusted by a person skilled in the art depending on the symbiotic organism used and its quantity in the medium. The culture medium can be any medium commonly used for a bioassay, for example, an aquarium, a microplate, or a jar; this list is not exhaustive. Advantageously, the culture can be carried out in a standardized manner in a multi-well plate.

[0030] For the purposes of this invention, "suitable medium" means any medium that allows the multiplication of the cnidarian, without any time limitation. This could be, for example, seawater or artificial seawater.

[0031] Step 2) of adding at least one product to the medium can be carried out by adding the at least one product at one or more known concentrations. The culture time can be adjusted by a person skilled in the art so that the possible effects of the product(s) on the symbiotic organism can be observed and / or measured.

[0032] Advantageously, at least one product can be introduced into the medium at different concentrations to obtain at least one threshold value chosen from among the NOC (no observed effects concentration), the NOC (minimum observed effects concentration), and the EC50 (effective concentration). If different product concentrations are tested, the medium containing the symbiotic organism is divided in step 1) into as many samples as necessary. A negative control (i.e., without product) can also be prepared to validate and / or interpret the results and establish one or more reference values ​​that characterize the ecotoxicity level of a product or mixture of products. Several replicates of each of the tested concentrations can be prepared to ensure the accuracy and reliability of the results.Although not mandatory, it is also possible to introduce a positive control which can provide information on the maximum observed biological effect. The results of the samples should then be expressed in relation to this maximum observed effect.

[0033] As indicated above, the reference values ​​may be the following: - the no-observed-effect concentration (NOEC): this is the highest concentration of the substance / sample tested for which no effect significantly different from the control was observed; - the minimum concentration with observed effects CMEO (in English: LOEC): this corresponds to the lowest concentration for which a significantly different effect from the control was observed; - The effective concentration EC50 (in English: EC50): this corresponds to the concentration of the substance / sample that produces an effect 50% different from the control. The EC50 is determined statistically based on a model.

[0034] Step 3) of measuring, in the medium and the cnidarian, the dissociation of the dino- The measurement of dinoflagellate dissociation from the symbiosis can be carried out by any method known to those skilled in the art. Advantageously, the measurement of dinoflagellate dissociation from the symbiosis can include measuring the concentration of dissociated dinoflagellates in the medium, and / or measuring the disappearance, for example by digestion, of the dinoflagellate present within a cnidarian tissue, for example by microscopic analysis of the cnidarian tissue, or counting tissue dinoflagellates after grinding and directly deducing the rate of dinoflagellate expulsion and / or digestion. It could also involve, for example, measuring the concentration of symbiotic dinoflagellate microalgae in the culture supernatant. This concentration can be determined, for example, using a microscope and counting chamber, a plankton counter, or a spectrophotometer.

[0035] The measurement can be carried out at a frequency allowing the detection of aigii stress, in particular measurable from one day to the next.

[0036] Advantageously, the assessment of acute stress can be carried out by measuring, in the medium and the cnidarian, the dissociation of the dinoflagellates from the symbiosis, optionally followed by a measurement of the expression of at least one gene known as a stress indicator, for example HSP (heat shock protein) and / or TGFbeta. Advantageously, the culture supernatant can then be discarded and the cnidarians digested in order to determine the amount of biomass by measuring total protein or the amount of microalgae residing in the animal. An advantage is that it is then possible to standardize the rate of microalgae expelled in the culture supernatant, in particular by relating this result to the animal biomass (for example by measuring total protein) or to the total number of microalgae, corresponding to the amount of dissociated microalgae plus the amount of microalgae in the animal.It is also possible to deduce from these results the no-observed-effect concentration (NOEC), the minimum observed-effect concentration (MOEC), and the effective concentration EC50.

[0037] For the investigation of chronic stress, it is possible to measure the growth rate of fragments, for example, fragments of approximately 1 cm, of hard corals, for example, scleractinian corals, by measuring their submerged mass, optionally followed by a measurement of the expression of at least one gene known as a stress indicator, for example, HSP (heat shock protein) and / or TGFbeta. This submerged mass measurement can be carried out by attaching a submerged fragment to a thread, for example, fishing line, which is itself placed on the hook of an analytical balance. By taking into account the density of seawater, the density of the coral calcareous skeleton, and the buoyant force, the mass of the coral skeleton can be determined very precisely.

[0038] An increase in the dissociation of dinoflagellates from the symbiosis, compared to the value measured at the beginning of step 2) or compared to the control, or a growth rate lower than that of the control, implies the existence of ecotoxicity of the product.

[0039] Optionally, the method of the invention may further include an analysis of the expression of at least one genetic stress marker by the cnidarian, for example by QRT-PCR, and / or an analysis of the growth rate, in particular of hard corals, such as scleractinians, by measuring the submerged weight, in particular to detect chronic stress evolving over several weeks, or even months.

[0040] Advantageously, the method of the invention may include a step of comparing the value obtained in step 3) with a predicted or measured value in a natural environment, making it possible to assess the ecotoxicity associated with the product. In this case, it is also possible to compare any threshold values ​​obtained with the predicted or measured concentrations in the natural environment to assess the ecotoxicity associated with the product under study.

[0041] Other advantages may become apparent to a person skilled in the art upon reading the examples below, illustrated by the accompanying figures, which are given by way of illustration. Brief description of the drawings

[0042] Figure 1 shows a curve showing the fit to experimental results, of the percentage of bleaching as a function of the product tested (pg / L, log scale). EXAMPLES OR METHODS OF IMPLEMENTATION

[0043] Example 1: Implementation of the process of the invention with A. pallida

[0044] Culture of A. pallida

[0045] A. pallida (symbiotic) mother cultures are maintained in aquariums at room temperature (approximately 24°C) and at 60 pmoles / m² / sec on a 12-hour light / dark cycle. The mother cultures are fed twice weekly with Artemia nauplii. Before the start of an experiment, the cnidarians are removed from the aquariums and placed in individual wells of multi-well plates in artificial seawater (ASW). The plates are placed in an incubator at 24°C, 60 pmoles / m² / sec on a 12-hour light / dark cycle, and allowed to acclimate for 1 to 2 days before the experiment. The anemones are then subjected to several treatments.

[0046] Performing the tests

[0047] The tests are carried out in 6 replicates. Each column of 6 wells (6 replicates) is subjected to a specific concentration of the product to be tested.

[0048] The first column contains no substance and therefore serves as a negative control (T-), while the second column serves as a positive control (T+) in which the cnidarians are subjected to a precise concentration of a substance known to induce the bleaching of the cnidarian, of Copper sulfate (CuSO4, 5H2O) at 50-100 pg / L). Then, from column 3 to 8, the cnidarians are subjected to decreasing concentrations of the product to be tested.

[0049] The plate is placed in a climatic chamber for 18 h (lighting 200 PAR; temperature 28°C).

[0050] At the end of this incubation period, the concentration of symbiotic dinoflagellate microalgae in the culture supernatant is determined using either a microscope and a counting chamber, or the HydraL (a planktonic counter developed by PLANKTOVIE sas), or a spectrophotometer.

[0051] Next, the supernatant from the wells is removed. The cnidarians from each well are then digested in order to determine the amount of biomass by measuring total protein (e.g., Bradford Assay) or the amount of resident microalgae in the animal.

[0052] Thanks to this, it is therefore possible to standardize the rate of microalgae expelled in the culture supernatant per unit of animal protein, or per total quantity of microalgae per well (dissociated microalgae + microalgae in the animal). From these results, we deduce: • the no-observed-effect concentration (NOEC), • the minimum concentration with observed effects (MCEO), • the effective concentration EC50

[0053] Comparing these threshold values ​​with predicted or measured concentrations in the natural environment makes it possible to assess the environmental risk associated with the product studied.

[0054] Additional biomolecular tests can be carried out on animals subjected to these threshold concentrations such as measurements of the expression of certain genes known as indicators of stress, for example HSP (heat shock protein) and / or TGFbeta.

[0055]

[0056] Example 2: Implementation of the method of the invention with Seriatopora hystrix

[0057] Evaluation of the mass gain of hard corals, type Seriatopora hystrix (scleractinian).

[0058] Cuttings of approximately 1 cm in length of S. hystrix (symbiotics) are taken from mother colonies. These cuttings are then attached to a short fishing line (10 cm long) and suspended in the water column of aquariums at room temperature (approximately 24°C) and at 60 pmoles quanta / m² / sec on a 12-hour light / dark cycle. These cuttings must be completely healed before they can be used in subsequent bioassays. This is generally the case after 1–2 weeks. Before the start of an experiment, the cnidarians are removed from the aquariums and placed in individual wells of multi-well plates in artificial seawater (ASW). The plates are placed in an incubator at 24°C, 60 pmoles quanta / m2 / sec on a 12-hour light / dark cycle and allowed to acclimate for 1 to 2 days before experimentation. The cuttings are then subjected to several treatments.

[0059] Performing the tests

[0060] The tests are carried out in 3-6 replicates. Each column of 6 wells (6 replicates) is subjected to a specific concentration of the product to be tested.

[0061] The first column contains no substance and therefore serves as a negative control (T-), while the second column serves as a positive control (T+) in which the cnidarians are subjected to a precise concentration of a substance known to decrease coral growth, Copper sulfate (CuSO4, 5H2O) at 5-10 pg / L. Then, from column 3 to 8, the cnidarians are subjected to decreasing concentrations of the product to be tested.

[0062] The plate is placed in a climate chamber for the entire duration of the chronic stress assessment, typically over a period of 1-2 months (200 PAR lighting; 28°C temperature). Each week, the culture medium in each well is replaced with fresh medium containing the substance to be tested.

[0063] Also every week, an immersed mass measurement is carried out using an analytical balance equipped with a suspension hook.

[0064] The fishing line to which the cutting is attached is placed on the hook of the analytical balance, and the mass of the cutting is recorded.

[0065] From these results, we deduce: • the no-observed-effect concentration (NOEC), • the minimum concentration with observed effects (MCEO), • the effective concentration EC50

[0066] Comparing these threshold values ​​with predicted or measured concentrations in the natural environment makes it possible to assess the environmental risk associated with the product studied.

[0067] Additional biomolecular tests can be carried out on animals subjected to these threshold concentrations such as measurements of the expression of certain genes known as indicators of stress, for example HSP (heat shock protein) and / or TGFbeta. REFERENCES

[0068] 1. Detournay et al.: “Regulation of cnidarian-dinoflagellate mutualisms: Evidence that activation of a host TGF[3 innate immune pathway promotes tolerance of the symbiont”, Dev Comp Immunol. 2012 Dec;38(4):525-37.

Claims

Demands

1. Use of a cnidarian-dinoflagellate symbiotic organism as a marker for the ecotoxicity assessment of a product.

2. Method for evaluating the ecotoxicity of a product, comprising the following steps: 1) culture of at least one cnidarian-dinoflagellate symbiotic organism in a suitable medium, 2) addition, in the medium, of at least one product, 3) measurement, in the medium and the cnidarian, of the dissociation of dino-flagellates from the symbiosis and / or the growth rate of cnidarian cuttings, in particular of hard corals.

3. A method according to claim 2, wherein said at least one product is introduced into the medium at different concentrations to obtain at least one threshold value selected from the NOC (no observed effect concentration), the NOC (minimum observed effect concentration) and the EC50 (effective concentration).

4. A method according to claim 2 or 3, comprising a step of comparing the value obtained in step 3) with a predicted or measured value in a natural environment, allowing the ecotoxicity associated with the product to be assessed.

5. A method according to any one of claims 2 to 4, wherein the culture is carried out in a standardized manner in a multi-well plate.

6. A method according to any one of claims 2 to 5, wherein the measurement of the dissociation of dinoflagellates from the symbiosis includes measuring the concentration of dinoflagellates dissociated from the symbiosis in the medium and / or measuring the disappearance, for example by digestion, of the dinoflagellate present within a tissue of the cnidarian, for example by microscopic analysis of the tissue of the cnidarian, or counting of tissue dinoflagellates after grinding, and direct deduction of the rate of expulsion and / or digestion of the dinoflagellate.

7. A method according to any one of claims 2 to 6, further comprising an analysis of the expression of at least one genetic stress marker by the cnidarian, for example by QRT-PCR, and / or an analysis of the growth rate of the cnidarian by measurement of submerged mass.

8. Use according to claim 1, or method according to any one of claims 2 to 7, characterized in that the cnidarian is at least one cnidarian selected from Aiptasia spp, for example Aiptasia pallida, Zoanthus spp,

9. Use or method according to claim 8, characterized in that the cnidarian is a whole organism, a fragment of a whole organism, or cells in culture.

10. Use or process according to claim 8 or 9, characterized in that the product is at least one product selected from a chemical and a biological product, for example a cosmetic product, a petrochemical product, a detergent product, an agri-food product or a plant protection product.

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