A system for extracting microplastics from water, sediment and tissue samples

Abstract

A system (100) for extracting microplastics from water, sediment and tissue samples, comprising: a sample collection unit (102) having means for collecting water, sediment and tissue samples from the collection point; a water sample processing unit (104) configured to extract microplastics from water samples; a sediment processing unit (106) configured to extract microplastics from sediment samples; and a biological sample processing unit (108) configured to extract microplastics from tissue samples.

Description

FIELD OF THE INVENTION

[0001] This disclosure relates to the extraction of microplastics from collected samples, in particular to a system for the extraction of microplastics from water, sediment and tissue samples. BACKGROUND OF THE INVENTION

[0002] Microplastic pollution has emerged as a significant environmental problem in aquatic ecosystems, including rivers, estuaries, and coastal waters. The detection, quantification, and characterization of microplastics in environmental samples pose several technical challenges that complicate accurate assessment and monitoring.

[0003] The existing challenges highlight the need for a standardized, efficient system that can accurately isolate, quantify, and characterize microplastics from various environmental matrices while minimizing sample contamination and maintaining the integrity of the microplastic particles. The present invention addresses these challenges by providing an integrated system for the extraction of microplastics from water, sediment, and tissue samples, followed by sample characterization. SUMMARY OF THE INVENTION

[0004] The present disclosure relates to a system for extracting microplastics from water, sediment, and tissue samples. The invention aims to isolate, quantify, and characterize microplastics from water, soil, and tissue samples collected from the Budhabalanga River estuary and the coast of Chandipur, Odisha. The water, sediment, and biological tissue samples are collected and then processed for microplastic isolation or extraction. The extracted microplastics are subjected to characterization using the Nile Red staining technique to detect hydrophobic particles and microscopy to examine different types of microplastics in the sample.

[0005] The present disclosure aims to provide a system for extracting microplastics from water, sediment, and tissue samples. The system comprises: a sample collection unit comprising means for collecting water, sediment, and tissue samples from the collection site; a water sample processing unit configured to extract microplastics from water samples; a sediment processing unit configured to extract microplastics from sediment samples; and a biological sample processing unit configured to extract microplastics from tissue samples.

[0006] In one embodiment, the sample collection unit comprises a Niskin water sampler and container for collecting water samples and a Van Veen grab for collecting sediment samples, and wherein the fish and shrimp are collected at the collection site from which the tissues are dissected.

[0007] In one embodiment, the water sample processing assembly comprises: a primary filtration unit configured to filter 1000 ml of water sample through a 1 µm sieve; a chemical treatment module configured to add 20 ml of 0.05M FeSO4 to the filtered sample.7H2O solution and, after 5 minutes, adding 20 ml of hydrogen peroxide (H2O2) to the filtered sample; a heating unit configured to maintain the temperature at 75 °C to boil the solution; a salt addition module configured to add and dissolve 6 g of NaCl; a stirring unit comprising a magnetic stirrer connected to the heating unit and the salt addition unit to stir the solution for thorough mixing; a multi-stage filtration unit with a stack of sieves from 5 mm to 1 µm to filter the cooled solution to collect microplastics in Petri dishes; a drying unit with an oven to dry the collected microplastics samples; and a closed storage container for storing the dried microplastics samples obtained from water samples.

[0008] In one embodiment, the sediment processing assembly comprises: a drying unit configured to dry sediment samples at 60°C for 96 hours; a grinding module configured to pulverize the dried sediments using a mortar and pestle; a salt mixing unit configured to mix 10 g of sediment sample with 100 ml of 4 M NaCl and perform three consecutive extractions of the supernatant; a chemical treatment module configured to add 20 ml of 0.05 M FeSO4 to the supernatant obtained from the salt solution.7H2O solution and 20 ml of hydrogen peroxide (H2O2) to the sieved supernatant obtained from the salt solution; a heating and stirring unit for boiling the solution to a specific temperature and stirring the solution using a magnetic stirrer; a salt addition unit for adding 3 g of NaCl to the solution, which is allowed to dissolve completely; and a filtration and collection unit configured to filter the solution through a stack of sieves to extract microplastics and collect the microplastic sample in a closed storage container.

[0009] In one embodiment, the biological sample processing assembly comprises: a tissue preparation unit configured to process various biological tissues, including alimentary canal, muscle tissue, and digestive gland; a chemical digestion module configured to add 30 ml of 4M KOH per 5 g of tissue and 5 ml of hydrogen peroxide (H2O2); a chemical addition module configured to add 20 ml of 0.05 FeSO4, 7H2O, and 20 ml of H2O2 solutions; a heating and stirring unit configured to boil the solution and then stir it with a magnetic stirrer; a salt addition unit for adding 3 g of NaCl to the solution; and a filtration and drying unit configured to filter the solution through a stack of sieves to obtain a microplastic sample and dry the microplastic, which is then stored in a closed storage container.

[0010] An object of the present disclosure is to provide a system for extracting microplastics from water, sediment and tissue samples.

[0011] Another object of the present disclosure is to provide a system for assessing the occurrence, chemical extraction, Nile red staining, light and dark contrast microscopy for the isolation, quantification and characterization of microplastics from water, soil and tissue samples.

[0012] Another objective of this disclosure is the identification of microplastics by SEM analysis.

[0013] Another objective of the present disclosure is to characterize the extracted microplastics based on manual sorting after extraction to confirm the type of microplastics.

[0014] To further clarify the advantages and features of the present disclosure, a more detailed description of the invention will be given by reference to specific embodiments thereof illustrated in the accompanying drawings. These drawings are understood to represent only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings. BRIEF DESCRIPTION OF THE ILLUSTRATION

[0015] These and other features, aspects, and advantages of the present disclosure will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout the drawings, wherein: Fig. 1 shows a block diagram of a system for extracting microplastics from water, sediment, and tissue samples in accordance with an embodiment of the present disclosure.

[0016] Those skilled in the art will understand that the elements in the drawings are shown for convenience and are not necessarily drawn to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, one or more components of the device may be represented in the drawings by conventional symbols, and the drawing may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawing with details that would be readily apparent to those skilled in the art familiar with the present description. DETAILED DESCRIPTION:

[0017] To facilitate understanding of the invention, reference will now be made to the embodiment illustrated in the drawings and described in specific terms. It should be understood, however, that this is not intended to limit the scope of the invention, and such changes and further modifications to the illustrated system, and such further applications of the principles of the invention embodied therein, are contemplated as would normally occur to one skilled in the art to which the invention pertains.

[0018] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0019] When this specification refers to "one aspect," "another aspect," or the like, it means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the terms "in one embodiment," "in another embodiment," and similar expressions throughout this specification may or may not all refer to the same embodiment.

[0020] The terms "comprises," "including," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps not only comprises those steps, but may also include other steps not expressly listed or included in such process or method. Likewise, one or more devices or subsystems or elements or structures or components introduced with "comprises...a" do not preclude, without further limitation, the existence of other devices or other subsystems or other elements or other structures or other components or additional devices or additional subsystems or additional elements or additional structures or additional components.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and are not intended to be limiting.

[0022] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0023] Fig. 1 shows a block diagram of a system (100) for extracting microplastics from water, sediment and tissue samples in accordance with an embodiment of the present disclosure.

[0024] Referring to Fig.1, the system (100) comprises a sample collection unit (102) comprising means for collecting water, sediment, and tissue samples from the collection site; a water sample processing unit (104) configured to extract microplastics from water samples; a sediment processing unit (106) configured to extract microplastics from sediment samples; and a biological sample processing unit (108) configured to extract microplastics from tissue samples.

[0025] In one embodiment, the sample collection unit (102) comprises a Niskin water sampler and container for collecting water samples and a Van Veen grab for collecting sediment samples, and wherein the fish and shrimp are collected from the collection site from which the tissues are dissected.

[0026] In one embodiment, the water sample processing assembly (104) comprises: a primary filtration unit configured to filter 1000 ml of water sample through a 1 µm sieve; a chemical treatment module configured to add 20 ml of 0.05M FeSO4 to the filtered sample.7H2O solution and, after 5 minutes, adding 20 ml of hydrogen peroxide (H2O2) to the filtered sample; a heating unit configured to maintain the temperature at 75 °C to boil the solution; a salt addition module configured to add and dissolve 6 g of NaCl; a stirring unit comprising a magnetic stirrer connected to the heating unit and the salt addition unit to stir the solution for thorough mixing; a multi-stage filtration unit comprising a stack of sieves ranging in size from 5 mm to 1 µm to filter the cooled solution and collect microplastics in Petri dishes; a drying unit comprising an oven for drying the collected microplastic samples; and a closed storage container for storing the dried microplastic samples obtained from water samples.

[0027] In one embodiment, the sediment processing assembly (106) comprises: a drying unit configured to dry sediment samples at 60°C for 96 hours; a grinding module configured to pulverize the dried sediments using a mortar and pestle; a salt mixing unit configured to mix 10 g of sediment sample with 100 ml of 4 M NaCl and perform three consecutive extractions of the supernatant; a chemical treatment module configured to add 20 ml of 0.05 M FeSO4 to the supernatant obtained from the salt solution.7H2O solution and 20 ml of hydrogen peroxide (H2O2) to the sieved supernatant obtained from the salt solution; a heating and stirring unit for boiling the solution to a specific temperature and stirring the solution using a magnetic stirrer; a salt addition unit for adding 3 g of NaCl to the solution, which is allowed to dissolve completely; and a filtration and collection unit configured to filter the solution through a stack of sieves to extract microplastics and collect the microplastic sample in a closed storage container.

[0028] In one embodiment, the biological sample processing assembly (108) comprises: a tissue preparation unit configured to process various biological tissues, including alimentary canal, muscle tissue, and digestive gland; a chemical digestion module configured to add 30 ml of 4M KOH per 5 g of tissue and 5 ml of hydrogen peroxide (H2O2); a chemical addition module configured to add 20 ml of 0.05 FeSO4, 7H2O, and 20 ml of H2O2 solutions; a heating and stirring unit configured to boil the solution and then stir it with a magnetic stirrer; a salt addition unit for adding 3 g of NaCl to the solution; and a filtration and drying unit configured to filter the solution through a stack of sieves to obtain a microplastic sample and dry the microplastic, which is then stored in a closed storage container.

[0029] The present invention relates to a system for extracting microplastic samples from water, sediment, and tissue samples, with the resulting samples subsequently being characterized. The water, soil, and tissue samples were collected from the Budhabalanga River estuary and the coast of Chandipur, Odisha.

[0030] Water samples were collected in duplicate from the surface in the estuary and coastal regions using a Niskin water sampler and stored in a 1-liter amber Tarson container for analysis. Sediment samples were collected using a VanVeen grab, and biological samples were obtained from local fish catches. Both sediment and fresh tissue samples were transferred to a zip-lock bag, stored in an ice box, and subjected to the proposed system.

[0031] The water sample treatment system extracts the microplastics from the collected water samples. The microplastic extraction process begins with the filtration of a 1000 ml water sample through a 1 µm sieve, followed by the collection of the residue in a sterilized, labeled glass beaker containing distilled water. During the chemical treatment, the reagents are added sequentially: First, 20 ml of a 0.05M FeSO4 7H2O solution is added and kept at room temperature for 5 minutes. Then, 20 ml of H2O2 is added, mixed thoroughly, and incubated for another 5 minutes at room temperature. The mixture is thermally treated at 75 °C while continuously stirring with a magnetic stirrer at 350 rpm for 30 minutes. Then, 6 g of NaCl is added with thorough mixing, and the solution is cooled to room temperature.During the final separation, the cooled solution is passed through a cascade of sieves (5 mm to 1 µm), with the retained microplastics being collected in separate Petri dishes. These samples are oven-dried, and the dried microplastic particles are stored in sealed vials for further analysis.

[0032] Sediment samples were collected from the estuary and the seabed using a Van Veen grab and stored in labeled zip-lock bags until further processing. These sediments were transferred to clean, labeled Petri dishes and dried at 60°C for 96 hours. After drying, they were finely ground using a mortar and pestle. A 10-g portion of the sediment was placed in a clean 500-mL beaker and 100 mL of 4M NaCl was added. The mixture was stirred vigorously to ensure thorough mixing. The supernatant was filtered through a 1-µm sieve and collected in a clean beaker; this process was repeated three times. Then, 20 mL of 0.05 M FeSO4-7H2O solution was added, and the mixture was incubated at room temperature for 5 minutes. Then 20 ml of hydrogen peroxide (H2O2) was added and incubation continued for another 5 minutes under the same conditions.The solution was heated to 75 °C and stirred with a magnetic stirrer at 350 rpm for 30 minutes. 3 g of NaCl was then added to the beaker and allowed to dissolve completely. The solution was then cooled in a cold water bath. It was sieved through a series of sieves ranging in size from 5 mm to 1 µm, and the extracted microplastics were collected in separate, labeled Petri dishes. These Petri dishes were oven-dried, and the dried samples were stored in sealed vials for further analysis.

[0033] The biological sample preparation facility isolates the microplastics from the tissue samples. Crab muscle tissue and mussel digestive glands were dissected, cleaned, and separately transferred into a 100 ml borosil beaker. 30 ml of 4M KOH was then added for digestion, with 30 ml of 4M KOH added to the sample for every 5 g of tissue. After digestion, 5 mL of hydrogen peroxide (H2O2) was added to the samples, which were then allowed to rest overnight. The next day, 20 ml of a 0.05 M FeSO4-7H2O solution was added to the mixture and incubated for 5 minutes at room temperature. A further 20 ml of hydrogen peroxide was then added, and the solution was thoroughly mixed before incubating for another 5 minutes at room temperature. The mixture was then heated to 75 °C and stirred with a magnetic stirrer at 350 rpm for 30 minutes.After this step, 3 g of sodium chloride (NaCl) were added, which dissolved completely. The solution was then cooled in a cold water bath. After cooling, it was sieved through a series of sieves ranging in size from 5 mm to 1 µm, allowing the extracted microplastics to be collected in separate Petri dishes. Finally, the Petri plates were oven-dried, and the dried samples were transferred to sealed vials for storage.

[0034] The extracted microplastics are subjected to characterization using a staining technique, whereby the microplastics are stained with Nile Red, a lipophilic dye (9-diethylamino-5H-benzo[α]phenoxazin-5-one) commonly used to detect intracellular lipid droplets, hydrophobic particles, and in cytofluorometry. A stock solution was prepared by dissolving 1 mg of Nile Red in 1 mL of acetone. For the working solution, the stock solution was diluted with acetone to achieve a concentration of 10- 5mg / ml. A 1:1 ratio was maintained between the Nile Red stain and the extracted microplastic solution. To stain the microplastic, three drops of the extracted solution were placed on a clean slide and allowed to dry completely. The Nile Red working solution was then added dropwise to the dried area using a micropipette and dried in the dark. The stained slide was viewed under a fluorescence microscope at 20x magnification.

[0035] The dried slides prepared with the microplastic sample were examined under a compound microscope at 10x magnification to determine the type and quantity of microplastics present in the sample. A Nikon microscope equipped with a camera was used to capture images of the microplastics. For scanning electron microscopy (SEM), the extracted microplastic solution was thoroughly dried to remove moisture. Double-sided tape was applied to the sample holder, and the processed microplastic sample was carefully placed onto the tape using a toothpick. The sample was evenly distributed in a single layer to improve visibility. The slide holder was then coated with a layer of gold particles and held under the electron beam for imaging. SEM analysis was performed using a Gemini SEM 300.

[0036] The drawings and the foregoing description provide examples of embodiments. Those skilled in the art will understand that one or more of the described elements may well be combined to form a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Furthermore, the actions of a flowchart need not be performed in the order shown; nor do all actions necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of the embodiments is by no means limited by these specific examples.Numerous variations are possible, whether explicitly stated in the description or not, such as differences in structure, dimensions, and use of materials. The scope of the embodiments is at least as broad as indicated in the following claims.

[0037] Advantages, other benefits, and solutions to problems have been described above with respect to specific embodiments. However, the advantages, benefits, solutions to problems, and components that may cause an advantage, benefit, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all of the claims. References 102 sampling unit 104 Water sample processing assembly 106 Sediment processing assembly 108 Biological sample processing assembly

Claims

[1] A system (100) for extracting microplastics from water, sediment and tissue samples, comprising: a sample collection unit (102) having means for collecting water, sediment and tissue samples from the collection point; a water sample processing unit (104) configured to extract microplastics from water samples; a sediment processing unit (106) configured to extract microplastics from sediment samples; and a biological sample processing unit (108) configured to extract microplastics from tissue samples. [2] The system of claim 1, wherein the sample collection unit (102) comprises a Niskin water sampler and container for collecting water samples and a Van Veen grab for collecting sediment samples, and wherein the fish and shrimp are collected from the collection site from which the tissues are dissected. [3] The system of claim 1, wherein the water sample processing assembly (104) comprises: a primary filtration unit configured to filter 1000 ml of water sample through a 1 µm sieve; a chemical treatment module configured to: - add 20 ml of a 0.05M FeSO4.7H2O solution to the filtered sample; - after 5 minutes add 20 ml of hydrogen peroxide (H2O2); a heating unit configured to maintain the temperature at 75°C to boil the solution; a salt addition module configured to add and dissolve 6 g of NaCl; a stirring unit comprising a magnetic stirrer connected to the heating unit and the salt addition unit to stir the solution to mix it thoroughly a multi-stage filtration unit with a stack of sieves ranging in size from 5 mm to 1 µm for filtering the cooled solution to collect microplastics in Petri dishes; a drying unit with an oven for drying the collected microplastic samples; and a closed storage container for storing the dried microplastic samples obtained from water samples. [4] The system of claim 1, wherein the sediment processing unit (106) comprises: a drying unit configured to dry sediment samples at 60 °C for 96 hours; a grinding module configured to pulverize the dried sediments using a mortar and pestle; a salt mixing unit configured to: - Mix 10 g of sediment sample with 100 ml of 4M NaCl; - perform three consecutive extractions of the supernatant; a chemical treatment module configured to add 20 ml of a 0.05 M FeSO4.7H2O solution and 20 ml of hydrogen peroxide (H2O2) to the sieved supernatant from the saline solution; a heating and stirring unit for boiling the solution to a certain temperature and stirring the solution using a magnetic stirrer; a salt addition unit for adding 3 g of NaCl to the solution, which can dissolve completely; and a filtration and collection unit configured to filter the solution through a stack of sieves to extract microplastics and collect the microplastic sample in a closed storage container. [5] The system of claim 1, wherein the biological sample processing arrangement (108) comprises: a tissue processing unit configured to process various biological tissues including the alimentary canal, muscle tissue, and digestive gland; a chemical digestion module configured to: - Add 30 ml of 4M KOH per 5 g of tissue; - Addition of 5 ml of hydrogen peroxide (H2O2); a chemical addition module configured to add 20 ml of 0.05 FeSO4.7H2O and 20 ml of H2O2 solutions; a heating and stirring unit configured to boil the solution and then stir it with a magnetic stirrer; a salt addition unit for adding 3 g of NaCl to the solution; and a filtration and drying unit configured to filter the solution through a stack of sieves to obtain a microplastic sample and to dry the microplastic, which is then stored in a closed storage container.

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