Method for determining acute toxicity and system using said method

Abstract

A method for determining the acute toxicity of an analyte comprising the steps of: a) exposing a first fish cell culture to a first reference solution; b) incubating the first fish cell culture in step a) with a fluorescent probe; c) obtaining a first fluorescence parameter from the stained first fish cell culture; d) establishing a first linear regression relation model between the first fluorescence parameter and the survival rate of the animal model; and e) determining the acute toxicity of the analyte by employing the first linear regression relationship model. A system for determining acute toxicity of an analyte using the method is also presented.

Description

Technical Field

[0001] The present invention relates to a method for determining the acute toxicity of an analyte, such as, in particular but not limited to, a sewage effluent sample; and a system for determining the acute toxicity of an analyte using the method. Background Art

[0002] Traditionally, acute toxicity tests require a large number of animals and use death as the top endpoint, which requires a large number of experimental animals and takes several days to obtain results. For example, in a test process, a large number of test animals (about 60-80) may be required, and two working days are required and death is used as an overall but rough endpoint. In addition, it is reported that in Europe alone, 2.56 million fish were used for research and testing purposes in 2019. It is understandable that such a large number of experimental fish not only bring additional costs, but also cause ethical issues. In addition to experimental animals, the required volume of test samples (about 500L) and sewage discharge (about 1500L) after the test is huge, which brings additional environmental problems.

[0003] Although there are reports of the development and / or use of alternative methods for determining acute toxicity, particularly the use of fish cell lines in place of conventional testing methods, it is believed that cell-based assays may show sensitivity that is several orders of magnitude lower than animal-based results.

[0004] The present invention seeks to obviate or at least mitigate these disadvantages, in particular by providing a new or otherwise improved cell-based assay having a combination of fluorescence techniques and bioimaging tools for assessing acute toxicity. Summary of the invention

[0005] In a first aspect of the present invention, a method for determining the acute toxicity of an analyte is provided, comprising the following steps: a) exposing a first fish cell culture to a first reference solution; b) incubating the first fish cell culture in step a) with a fluorescent probe; c) obtaining a first fluorescence parameter from the stained first fish cell culture; d) establishing a first linear regression relationship model between the first fluorescence parameter and the survival rate of an animal model; and e) determining the acute toxicity of the analyte by adopting the first linear regression relationship model.

[0006] In an optional embodiment, the first fish cell culture comprises a fin cell line isolated from Siganus fuscescens.

[0007] Optionally, step a) comprises the step of collecting a first reference solution from a reference location at a first point in time.

[0008] In an optional embodiment, the first reference solution comprises a 24-h flow-weighted composite effluent from a wastewater effluent treatment plant.

[0009] Optionally, the first reference solution has a pollutant volume concentration selected from any one of 0%, 6.5%, 12.5%, 25%, 50% and 100%.

[0010] Optionally, step b) comprises the step of incubating the mixture of the first fish cell culture and the fluorescent probe in the dark.

[0011] Optionally, the fluorescent probe is selected from the group consisting of: a lysosomal tracer, a mitochondrial tracer, (Z)-3-(4-(4-methylpiperazin-1-yl)phenyl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile (CSMPP), 7-ethoxyresorufin, H2DCFDA, Fluo-4, AM, ThiolTracker, and combinations thereof.

[0012] In an optional embodiment, step c) comprises the steps of: taking a confocal microscopy image of the stained first fish cell culture; and obtaining a first fluorescence parameter of the stained first fish cell culture from the confocal microscopy image.

[0013] Optionally, step c) further comprises step c1) obtaining the fluorescence intensity of the stained first fish cell culture by a fluorescence plate reader.

[0014] Optionally, the fluorescence parameter corresponds to the fluorescent probe and is selected from the group consisting of: lysosomal number, mitochondrial size, lysosomal pH, EROD activity, ROS production, Ca 2+ influx, GSH formation and their combination.

[0015] Optionally, step d) comprises the following steps: incubating the animal model with the first reference solution for at least 12 h; determining the survival rate of the animal model; and establishing a first linear regression equation between the first fluorescence parameter and the survival rate of the animal model, thereby obtaining an R square (R 2 )value.

[0016] In an optional embodiment, the animal model comprises any one of the following: an amphipod (Melita longidactyla), a barnacle larvae (Balanus amphitrite), and a shrimp (Metapenaeus ensis).

[0017] Optionally, the first reference solution has a pollutant volume concentration selected from any one of 0%, 6.5%, 12.5%, 25%, 50% and 100%.

[0018] Optionally, the R-squared value is at least about 0.70.

[0019] In an optional embodiment, the method further comprises the steps of: a') exposing the second fish cell culture to the analyte; b') incubating the second fish cell culture in step a') with a fluorescent probe; and c') obtaining a second fluorescence parameter from the stained second fish cell culture.

[0020] Optionally, step e) comprises the following steps: determining a reference value at 90% survival rate according to the first linear regression relationship model; and comparing the second fluorescence parameter with the reference value at 90% survival rate to determine the acute toxicity of the analyte.

[0021] Optionally, step e) further comprises the following steps: determining a reference value at 50% survival rate according to the first linear regression relationship model; and comparing the second fluorescence parameter with the reference value at 50% survival rate to determine the acute toxicity of the analyte.

[0022] In an optional embodiment, the analyte comprises sewage effluent.

[0023] In an optional embodiment, step a) is repeated at a second time point and a third time point using a second reference solution and a third reference solution from a reference location, respectively.

[0024] Optionally, the first time point, the second time point and the third time point are each separated by an interval of three months.

[0025] In a second aspect of the present invention, a system for determining the acute toxicity of an analyte according to the method of the first aspect is provided. The system comprises: a first fish cell culture stained with a fluorescent probe; a first reference solution, in which the first fish cell culture is incubated; and a first linear regression relationship model established based on a first fluorescence parameter obtained from the stained first fish cell culture and the survival rate of an animal model; wherein the first linear regression relationship model contains the acute toxicity of the analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention will now be described in more detail, by way of example only, with reference to the accompanying drawings, in which:

[0027] Figure 1The calibration curve of the ICP-MS intensity of the metal ions is shown. The CPS (counts per second) values ​​of the metal ions from the ICP-MS are compared with the concentration of the added metal ions. The equation of the linear phase and the R-squared value are also given;

[0028] Figure 2 is a table summarizing the fluorescent probes used for staining, the target organelles, the working concentration, the duration of staining, and the excitation / emission wavelengths of the probes;

[0029] Figure 3A The amount of metal ions in the sewage samples measured by ICP-MS is shown. The values ​​are expressed as the absolute concentration of each metal ion in the sewage samples (S1, S2, S3). Mean ± SD (n = 3);

[0030] Figure 3B The amounts of organic pollutants measured by ICP-MS are shown. The values ​​are expressed as the absolute concentration of each metal ion in the wastewater samples. Mean ± SD (n = 3);

[0031] Figure 4 Shows the LC values ​​of experimental animals exposed to different sewage 50 Values. These values ​​are expressed as sewage ratio (v / v). Mean ± SD (n = 4);

[0032] Figure 5A Cell viability of RFF cells exposed to different sewage contents for 24 hours and 48 hours is shown. Cell viability was measured by MTT assay. These values ​​are expressed as % of control compared to control readings (e.g. 100%, no sewage added). Mean ± SD (n = 6) *p < 0.05; **p < 0.01;

[0033] Figure 5B Cell viability of RFM cells exposed to different sewage contents for 24 hours and 48 hours is shown. Cell viability was measured by MTT assay. These values ​​are expressed as % of control compared to control readings (e.g. 100%, no sewage added). Mean ± SD (n = 6) *p < 0.05; **p < 0.01;

[0034] Figure 5C Cell viability of ZF4 cells exposed to different sewage contents for 24 hours and 48 hours is shown. Cell viability was measured by MTT assay. The values ​​are expressed as % of control compared to control readings (e.g. 100%, no sewage added). Mean ± SD (n = 6) *p < 0.05; **p < 0.01;

[0035] Fig. 6ARFF cells stained with lysosomal tracer (yellow) and mitochondrial tracer (red) after 24 hours of exposure are shown. Images were taken with a LSM900 confocal microscope and representative images are shown;

[0036] Figure 6B The calibration curve between the number of lysosomes and the content of sewage effluent is shown. The number of lysosomes after sewage effluent exposure was obtained from the confocal images by ImageJ analysis. Mean ± SD (n = 6). Statistical comparison with group 0 (no sewage effluent added); *p < 0.05; **p < 0.01. The equation of linear phase and R square value are also given;

[0037] Figure 6C The calibration curve between mitochondrial size and sewage effluent content is shown. Mitochondrial size after sewage effluent exposure was obtained from confocal images by ImageJ analysis. Mean ± SD (n = 6). Statistical comparison with group 0 (no sewage effluent added); *p < 0.05; **p < 0.01. The equation of linear phase and R square value are also given;

[0038] Fig. 7A RFF cells stained with lysosomal tracer (white) and CMSPP (green and red) after 24 hours of exposure are shown. Images were taken with a LSM900 confocal microscope and representative images are shown;

[0039] Figure 7B The changes in lysosomal pH under different concentrations of sewage effluent after 24 hours of exposure are shown. The results were obtained from the above confocal images and analyzed by ImageJ analysis. Mean ± SD (n = 6). Statistical comparison with group 0 (no sewage effluent added); *p < 0.05; **p < 0.01. The equation of linear phase and R square value are also given;

[0040] Fig. 8A The fluorescence intensity ratio of the two emission channels (615 nm (red), 503 nm (green)) obtained by Image J software is shown and named "R". The fluorescence intensity ratios at pH 2.6 and 6.8 are named R max and R min ;

[0041] Figure 8B The relationship between pH and log[(RR min ) / (R max -R)] and shows a linear relationship. The equations for the linear phase and R-squared value are also given;

[0042] Fig. 9A RFF cells stained with 7-ethoxyresorufin after 24 h of exposure are shown;

[0043] Fig. 9B The calibration curve between ERDO activity and sewage effluent content is shown. After sewage effluent exposure, the corresponding Fig. 9A ERDO activity was obtained in the confocal images of . Mean ± SD (n = 4). Statistical comparison with group 0 (no sewage effluent added); *p < 0.05; **p < 0.01. The equation of linear phase and R square value are also given;

[0044] Fig. 9C RFF cells stained with DC-FHDA after 24 h exposure are shown;

[0045] Fig.9D The calibration curve between ROS formation and sewage effluent content is shown. After sewage effluent exposure, the corresponding Fig. 9C ROS formation was obtained in the confocal images of . Mean ± SD (n = 4). Statistical comparison with group 0 (no sewage effluent added); *p < 0.05; **p < 0.01. The equation of linear phase and R square value are also given;

[0046] Fig.9E RFF cells stained with Fluo-4 after 24 h of exposure are shown;

[0047] Fig.9F Shows Ca 2+ Calibration curve between influent and effluent content. After effluent exposure, the corresponding Fig.9E The confocal images of Ca 2+ Inflow. Mean ± SD (n = 4). Statistical comparison with group 0 (no sewage effluent added); *p < 0.05; **p < 0.01. The equation of linear phase and R square value are also given;

[0048] Figure 9G ThiolTracker was used after 24 hours of exposure. TM Violet-stained RFF cells;

[0049] Figure 9H The calibration curve between GSH content and sewage effluent content is shown. After sewage effluent exposure, the corresponding Figure 9G GSH content was obtained from the confocal images of . Mean ± SD (n = 4). Statistical comparison with group 0 (no sewage effluent added); *p < 0.05; **p < 0.01. The equation of linear phase and R square value are also given;

[0050] Fig. 10ACalibration curves between cellular parameters (lysosome number) obtained from confocal microscopy and fluorescent plate reader are shown. Mean ± SD (n = 3). The equation of linear phase and R square value are also given;

[0051] Fig. 10B Calibration curves between cellular parameters (mitochondrial size) obtained from confocal microscopy and fluorescent plate reader are shown. Mean ± SD (n = 3). The equation for the linear phase and R square value are also given;

[0052] Fig. 10C Calibration curves between cell parameters (EROD activity) obtained from confocal microscopy and fluorescent plate reader are shown. Mean ± SD (n = 3). The equation of linear phase and R square value are also given;

[0053] Fig. 10D Calibration curves between cellular parameters (ROS formation) obtained from confocal microscopy and fluorescent plate reader are shown. Mean ± SD (n = 3). The equation for the linear phase and R square value are also given;

[0054] Fig. 10E Cell parameters (Ca) obtained from confocal microscopy and fluorescence plate reader are shown. 2+ Calibration curve between 1:1 and 2:1. Mean ± SD (n = 3). The equation of the linear phase and R-squared value are also given;

[0055] Fig.10F Calibration curves between cellular parameters (GSH content) obtained from confocal microscopy and fluorescent plate reader are shown. Mean ± SD (n = 3). The equation of linear phase and R square value are also given;

[0056] Fig.11A The changes in cell parameters (mitochondrial size) at different exposure durations are shown. RFF cells were exposed to different concentrations of sewage effluent for 6 hours, 12 hours and 24 hours. The cells were then harvested by trypsin and stained with the above-mentioned fluorescent probes, and the fluorescence intensity of various parameters was obtained by a plate reader. Mitochondrial size was measured separately and normalized by cell number. The values ​​are expressed as fold change compared to the basal reading (such as 1, no sewage effluent added). Mean ± SD (n = 4);

[0057] Fig. 11BThe changes in cell parameters (EROD activity) at different exposure durations are shown. RFF cells were exposed to different concentrations of sewage effluent for 6 hours, 12 hours and 24 hours. The cells were then harvested by trypsin and stained with the above-mentioned fluorescent probes, and the fluorescence intensity of various parameters was obtained by a plate reader. Mitochondrial size was measured separately and normalized by cell number. The values ​​are expressed as fold change compared to the basal reading (such as 1, no sewage effluent added). Mean ± SD (n = 4);

[0058] Fig. 11C The changes in cellular parameters (ROS formation) at different exposure durations are shown. RFF cells were exposed to different concentrations of sewage effluent for 6 hours, 12 hours and 24 hours. The cells were then harvested by trypsin and stained with the above-mentioned fluorescent probes, and the fluorescence intensity of various parameters was obtained by a plate reader. Mitochondrial size was measured separately and normalized by cell number. The values ​​are expressed as fold change compared to the basal reading (such as 1, no sewage effluent added). Mean ± SD (n = 4);

[0059] Fig.11D Cell parameters (Ca 2+ Influx). RFF cells were exposed to different concentrations of sewage effluent for 6 hours, 12 hours and 24 hours. The cells were then harvested by trypsin and stained with the above fluorescent probes, and the fluorescence intensity of various parameters was obtained by a plate reader. Mitochondrial size was measured separately and normalized by cell number. The values ​​are expressed as fold change compared to the basal reading (such as 1, no sewage effluent added). Mean ± SD (n = 4);

[0060] Fig.11E The changes in cell parameters (lysosomal pH) at different exposure durations are shown. RFF cells were exposed to different concentrations of sewage effluent for 6 hours, 12 hours and 24 hours. The cells were then harvested by trypsin and stained with the above fluorescent probes, and the fluorescence intensity of various parameters was obtained by a plate reader. Lysosomal pH was calculated based on the reported equation. Mean ± SD (n = 4);

[0061] Fig. 12A Calibration curves between mitochondrial size and survival of test animals (amphipods, barnacles and shrimp) at 24 h exposure are shown. Cellular parameters (obtained by confocal microscopy) were compared with the survival of animals at Fig.13A and Fig. 13B The survival rates at the same sewage concentrations (90% and 50%) are compared as shown in . The equation of linear phase and R-squared value are also given;

[0062] Fig. 12BThe calibration curves between the number of lysosomes and the survival of the tested animals (amphipods, barnacles and shrimp) at 24 h exposure are shown. Cellular parameters (obtained by confocal microscopy) were compared with the survival of the animals at Fig.13A and Fig. 13B The survival rates at the same sewage concentrations (90% and 50%) are compared as shown in . The equation of linear phase and R-squared value are also given;

[0063] Fig. 12C The calibration curves between the number of lysosomes and the survival of the tested animals (amphipods, barnacles and shrimp) at 24 h exposure are shown. Cellular parameters (obtained by confocal microscopy) were compared with the survival of the animals at Fig.13A and Fig. 13B The survival rates at the same sewage concentrations (90% and 50%) are compared as shown in . The equation of linear phase and R-squared value are also given;

[0064] Fig.12D Calibration curves between ROS formation and survival of test animals (amphipods, barnacles and shrimp) at 24 h exposure are shown. Cellular parameters (obtained by confocal microscopy) were compared with the survival of animals at Fig.13A and Fig. 13B The survival rates at the same sewage concentrations (90% and 50%) are compared as shown in . The equation of linear phase and R-squared value are also given;

[0065] Fig.12E Shows the Ca content of the samples after 24 h exposure. 2+ Calibration curves between influx and survival of test animals (amphipods, barnacles and shrimp). Cellular parameters (obtained by confocal microscopy) were compared with the survival of animals at Fig.13A and Fig. 13B The survival rates at the same sewage concentrations (90% and 50%) are compared as shown in . The equation of linear phase and R-squared value are also given;

[0066] Fig.12F The calibration curves between GSH content and survival of the test animals (amphipods, barnacles and shrimp) at 24 h exposure are shown. Cellular parameters (obtained by confocal microscopy) were compared with the survival of the animals at Fig.13A and Fig. 13B The survival rates at the same sewage concentrations (90% and 50%) are compared as shown in . The equation of linear phase and R-squared value are also given;

[0067] Figure 12G Calibration curves between lysosomal pH and survival of test animals (amphipods, barnacles and shrimp) at 24 h exposure are shown. Cellular parameters (obtained by confocal microscopy) were compared with the survival of animals at Fig.13A and Fig. 13BThe survival rates at the same sewage concentrations (90% and 50%) are compared as shown in . The equation of linear phase and R-squared value are also given;

[0068] Fig.12H Calibration curves between cell survival and survival of test animals (amphipod, barnacle, and shrimp) at 24 h exposure are shown;

[0069] Fig.13A is a table summarizing the cell parameters calculated when the survival rate of the test animals is 90%;

[0070] Fig. 13B is a table summarizing the cell parameters calculated when the survival rate of the test animals is 50%;

[0071] Fig.14A Calibration curves between cell viability and sewage effluent content of RFF at 24 and 48 h exposure are shown. Mean ± SD (n = 6);

[0072] Fig. 14B Calibration curves between cell viability and animal survival for RFF at 24 and 48 h exposure are shown. The equation for the linear phase and R-squared value are also given;

[0073] Fig.15A Shows the corresponding Fig. 14B The survival rates of different animals (amphipods, barnacles and shrimps) exposed to different concentrations of sewage sample S1 for 48 hours;

[0074] Fig. 15B Shows the corresponding Fig. 14B The survival rates of different animals (amphipods, barnacles and shrimps) exposed to different concentrations of sewage sample S2 for 48 hours;

[0075] Fig. 15C Shows the corresponding Fig. 14B The survival rates of different animals (amphipods, barnacles and shrimps) exposed to different concentrations of sewage sample S3 for 48 h;

[0076] Fig.15D is the summary corresponding to Fig.15A Table of animal survival and fish cell survival after 48 hours of exposure to sewage sample S1. The values ​​are expressed as the survival rate of animals or fish cells after exposure. Mean ± SD (n = 4);

[0077] Fig.15E is the summary corresponding to Fig. 15B Table of animal survival and fish cell survival after 48 hours of exposure to sewage sample S2. The values ​​are expressed as the survival rate of animals or fish cells after exposure. Mean ± SD (n = 4);

[0078] Fig.15F is the summary corresponding to Fig. 15C Table of animal survival and fish cell survival after 48 hours of exposure to sewage sample S3. The values ​​are expressed as the survival rate of animals or fish cells after exposure. Mean ± SD (n = 4);

[0079] Fig.16A The calibration curves between the cellular parameters (mitochondrial size, obtained by fluorescence plate reader) and the animal survival rate of the tested animals (amphipods, barnacles, shrimps) at 12 hours of exposure are shown. Fig.17A and Fig. 17B The survival rates (90% and 50%) at the same sewage content are compared as shown in . The equation of linear phase and R square value are also given;

[0080] Fig. 16B The calibration curves between the cell parameters (EROD activity, obtained by fluorescence plate reader) and the animal survival rate of the tested animals (amphipods, barnacles, shrimps) at 12 hours of exposure are shown. Fig.17A and Fig. 17B The survival rates (90% and 50%) at the same sewage content are compared as shown in . The equation of linear phase and R square value are also given;

[0081] Fig. 16C The calibration curves between the cell parameters (ROS formation, obtained by fluorescence plate reader) and the animal survival of the tested animals (amphipods, barnacles, shrimps) at 12 hours of exposure are shown. Fig.17A and Fig. 17B The survival rates (90% and 50%) at the same sewage content are compared as shown in . The equation of linear phase and R square value are also given;

[0082] Fig.16D Cell parameters (Ca 2+ Calibration curves were plotted between cell parameters (obtained by fluorescence plate reader) and animal survival of the test animals (amphipods, barnacles, shrimp). Fig.17A and Fig. 17B The survival rates (90% and 50%) at the same sewage content are compared as shown in . The equation of linear phase and R square value are also given;

[0083] Fig.16EThe calibration curves between the cell parameters (lysosomal pH, obtained by fluorescence plate reader) and the animal survival rate of the tested animals (amphipods, barnacles, shrimps) at 12 hours of exposure are shown. Fig.17A and Fig. 17B The survival rates (90% and 50%) at the same sewage content are compared as shown in . The equation of linear phase and R square value are also given;

[0084] Fig.17A is a table summarizing the cellular parameters calculated when the survival rate of the test animals is 90%; and

[0085] Fig. 17B is a table summarizing the cell parameters calculated when the survival rate of the test animals was 50%. DETAILED DESCRIPTION

[0086] As used herein, the forms “a,” “an,” and “the” are intended to include both the singular and the plural, unless the context clearly indicates otherwise.

[0087] The words "example / embodiment" or "exemplary" used in the present invention are intended to be used as examples, instances or illustrations. Any aspect or design described as "exemplary" in this disclosure is not necessarily to be construed as being more preferred or advantageous than other aspects or designs. On the contrary, the use of the words "example" or "exemplary" is intended to present concepts in a concrete way. As used in this application, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or". That is, unless otherwise specified or clear from the context, "X employs A or B" is intended to mean any natural inclusive arrangement in the natural inclusive arrangement. That is, if X employs A; X employs B; or X employs both A and B, then "X employs A or B" is satisfied under any of the aforementioned examples.

[0088] As used herein, the phrase "about" is intended to refer to values ​​that slightly deviate from the values ​​stated herein. For example, "about 0.70" can refer to any value among 0.68, 0.685, 0.69, 0.692 ... 0.7, 0.705 ... 0.71 ... 0.718 ... 0.72.

[0089] Without wishing to be bound by theory, the inventors have designed a cell-based assay by their own research, test and experiment, which combines fish cell lines with fluorescence techniques to reveal the subcellular toxicity of analytes (particularly sewage effluents). Particularly, by means of a confocal microscope, the changes in organelles and enzymatic activity can be visualized and quantitative. In an exemplary embodiment, it is found that various cell indexes such as lysosome number, mitochondrial size, lysosomal pH can sensitively respond to sewage effluent exposure. By comparing cell index and animal survival rate, it is shown that the present invention can more accurately represent sewage content and / or toxicity. In addition, by means of this cell-based assay, only 10mL of exposure medium will be enough to be used for the alternative assessment of the quality of the whole sewage effluent and / or toxicity determination, and similar results can be achieved in a much shorter time. It is believed that the present invention can potentially be expanded to other applications, such as providing preliminary toxicity results of medicines, cosmetics and additives etc. It is also believed that the present invention can provide a replacement, reduction or improvement approach to chronic animal testing, which is characterized by being easy to follow, less time-consuming and cheaper.

[0090] In a first aspect of the present invention, a method for determining the acute toxicity of an analyte is provided, comprising the following steps: a) exposing a first fish cell culture to a first reference solution; b) incubating the first fish cell culture in step a) with a fluorescent probe; c) obtaining a first fluorescence parameter from the stained first fish cell culture; d) establishing a first linear regression relationship model between the first fluorescence parameter and the survival rate of an animal model; and e) determining the acute toxicity of the analyte by adopting the first linear regression relationship model.

[0091] In one embodiment, step a) may include incubating the first fish culture with the first reference solution for at least 12 h, such as 12 h to 48 h, 12 h to 40 h, 12 h to 36 h, 12 h to 30 h, 12 h to 28 h, and in particular 12 h to 24 h. It is believed that incubation times varying between 12 h and 24 h do not differ significantly in the fluorescence parameters determined in subsequent steps. Therefore, it is believed that the incubation time can be shortened from 24 h to 12 h to enable the present invention to achieve faster toxicity determination.

[0092] The first fish culture may be seeded with a plurality of fish cells in, for example, a confocal dish, a culture well / plate / dish, etc. The fish cells may have a size of about 1×10 6In one embodiment, the first fish culture may include any one of a fin cell line (RFF) isolated from brown basket fish (grey rabbitfish), a macrophage-like cell line (RFM) isolated from brown basket fish, and a fibroblast cell line (ZF4) isolated from zebrafish. In a preferred embodiment, the first fish culture may include RFF isolated from brown basket fish.

[0093] In one embodiment, step a) may also include the step of collecting a first reference solution from a reference location at a first time point. In particular, different (first) reference solutions may be used, depending on the nature of the analyte. In an exemplary embodiment, the analyte may include sewage effluent, and in this case, the first reference solution may be a composite effluent, such as a 24-h flow-weighted composite effluent collected from a sewage effluent treatment plant / factory. It is understood that the effluent may be collected by suitable technical means.

[0094] Optionally or additionally, step a) can be repeated using a second reference solution and a third reference solution from a reference location (such as a wastewater treatment plant as described herein) at a second time point and a third time point, respectively. In particular, the first time point, the second time point, and the third time point can each differ by an interval of three months. For example, if the first reference solution is collected from the reference location at a first time point in October 2022, the second reference solution can be collected from the reference location at a second time point in January 2023, and the third reference solution can be collected from the reference location at a third time point in April 2023.

[0095] The first fish culture may be exposed to a first reference solution having a pollutant volume concentration selected from any one of 0%, 6.5%, 12.5%, 25%, 50% and 100%. In some embodiments, the pollutants may include heavy metals, organic pollutants and combinations thereof. In some embodiments, the heavy metals may include any one of arsenic, cadmium, cobalt, chromium, copper, manganese, lead, antimony and zinc. In some embodiments, the organic pollutants may include any one of residual chlorine, bromoform, bromodichloromethane, chloroform, dibromochloromethane, dichloromethane, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, tetrachloroethylene, bromoacetic acid, chloroacetic acid, dibromoacetic acid, dichloroacetic acid, trichloroacetic acid and 1,2,4-trichlorobenzene.

[0096] After exposing the first fish culture to the first reference solution in step a), in step b), the first fish culture can be incubated with a fluorescent probe. In particular, the fluorescent probe can be added to the first fish culture in step a) to form a mixture, and then the mixture is incubated in the dark. In some embodiments, the fluorescent probe can be selected from the group consisting of: lysosomal tracers, mitochondrial tracers, (Z)-3-(4-(4-methylpiperazine-1-yl)phenyl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile (CSMPP), 7-ethoxyresorufin, H2DCFDA, Fluo-4, AM, ThiolTracker, and combinations thereof. Based on the fluorescent probe used, the concentration and incubation time can be adjusted according to actual needs and / or protocols. For example, in the case where the fluorescent probe can be a mitochondrial tracer (such as MitoTracker TM In some embodiments of Deep Red M22426), the first fish culture in step a) can be incubated with the mitochondrial tracer at a concentration of about 0.1 μM for about 15 min.

[0097] Step c) may include the step of taking a confocal microscopy image of the first fish cell culture of the stain; and obtaining a first fluorescence parameter of the first fish cell culture of the stain from the confocal microscopy image. In some embodiments, before taking the confocal image, the first fish culture of the stain may be washed with, for example, 1X PBS. The confocal image may be taken by a confocal microscope having a specific excitation and emission corresponding to the fluorescent probe used in step b). Thereafter, the confocal image may be analyzed and quantified by specific software (such as ImageJ software) to obtain the first fluorescence parameter.

[0098] In some other embodiments, the first fluorescence parameter can be determined by a fluorescence plate reader instead of confocal microscopy. In these embodiments, step c) may also include step c1) obtaining the fluorescence intensity of the stained first fish cell culture by a fluorescence plate reader. After obtaining the fluorescence intensity, it can be converted into a standardized fluorescence intensity by dividing the fluorescence intensity by the number of cells. By comparing with the fluorescence intensity of the first fish culture incubated with a first reference solution having a 0% contaminant concentration, the standardized fluorescence intensity can be further converted into a multiple of the change in fluorescence intensity. In short, in these embodiments, the first fluorescence parameter can be a multiple of the change in fluorescence intensity as described herein.

[0099] In some embodiments, the first fluorescence parameter can correspond to a fluorescent probe, such as the fluorescence intensity of a fluorescent probe determined from the confocal images described herein, and is selected from the group consisting of: lysosome number, mitochondrial size, lysosomal pH, EROD activity, ROS production, Ca 2+influx, GSH formation and their combination.

[0100] In some embodiments where the first fluorescence parameter can be determined by a fluorescence plate reader, the first fluorescence parameter can be a fold change in fluorescence intensity corresponding to the fluorescent probe and is selected from the group consisting of: lysosome number, mitochondrial size, lysosomal pH, EROD activity, ROS production, Ca 2+ influx, GSH formation and their combination.

[0101] In step d), it may include the following steps: incubating the animal model with the first reference solution for at least 12 hours; determining the survival rate of the animal model; and establishing a first linear regression equation between the first fluorescence parameter and the survival rate of the animal model, and thereby obtaining R square (R 2 )value.

[0102] In some embodiments, the animal model may include any one of the following: amphipods (long-fingered Maltese hook shrimp), barnacle larvae (striped barnacles) and shrimps (neopeneraeus). One or more of these animal models can be incubated with a first reference solution having a pollutant volume concentration selected from any one of 0%, 6.5%, 12.5%, 25%, 50% and 100% for at least 12 hours, 12 hours to 48 hours, 18 hours to 48 hours, 20 hours to 48 hours, and particularly 24 hours to 48 hours. Thereafter, the survival rate of the animal model can be determined by calculating the number of animal models that survived the number of animal models exposed to the first reference solution having a pollutant volume concentration of 0%. The survival rate of the animal model obtained can then be used to construct a first linear regression model relationship with the first fluorescence parameter by plotting the survival rate of the animal model relative to the first fluorescence parameter. By doing so, a first linear regression equation between the first fluorescence parameter and the survival rate of the animal model and the R square (R 2 ) value. In particular, R 2 The value may be at least about 0.70, such as about 0.68 to about 0.98, about 0.69 to about 0.98, about 0.69 to about 0.97, about 0.70 to about 0.97, and the like.

[0103] In some embodiments, step d) can be performed after completing steps a) to c). In some other embodiments, step d) can be performed in parallel with any one of steps a) to c).

[0104] The method of the present invention may also include the following steps: a') exposing a second fish cell culture to an analyte, such as the sewage effluent described herein; b') incubating the second fish cell culture in step a') with a fluorescent probe; and c') obtaining a second fluorescence parameter from the stained second fish cell culture.

[0105] In some embodiments, the second fish cell culture can be any one of a fin cell line (RFF) isolated from brown basket fish, a macrophage-like cell line (RFM) isolated from brown basket fish, and a fibroblast cell line (ZF4) isolated from zebrafish. Preferably, the second fish cell culture can be the same as the first fish culture used to obtain the first fluorescence parameter, which can contain RFF isolated from brown basket fish.

[0106] In particular, step a') can be performed in a similar manner to step a), wherein the second fish cell culture can be incubated with the analyte for at least 12 h, followed by incubation of the second fish culture with a fluorescent probe as described herein. Thereafter, the stained second fish culture can be arranged to obtain a second fluorescence parameter by confocal microscopy or a fluorescent plate reader, as described in step c).

[0107] In some embodiments, steps a') to c') may be performed after completing steps a) to d). In some other embodiments, steps a') to c') may be performed in parallel with any one of steps a) to d).

[0108] Now turning to step e), which may include the following steps: determining a reference value at 90% survival rate according to the first linear regression relationship model; and comparing the second fluorescence parameter with the reference value at 90% survival rate to determine the acute toxicity of the analyte. In particular, by using the first linear regression equation obtained in step d), the animal model survival rate can be fitted into the equation to obtain the corresponding reference value.

[0109] In one embodiment, in a graph of animal model survival (y-axis) versus a first fluorescence parameter (such as mitochondrial size) (x-axis) using a first linear regression equation (such as y=2.36x-167.24), by placing a 90% animal model survival rate into the equation, a first reference value (x-value) will be determined to be 71.25. Then, by comparing the second fluorescence parameter (such as mitochondrial size) determined in step c') to this reference value, and if it is lower than this reference value, it can be expected that the analyte (sewage effluent) can cause 10% mortality in the animal model and can therefore be considered toxic.

[0110] In another embodiment, the animal model survival rate (y-axis) is plotted against a first fluorescence parameter (such as Ca) using a first linear regression equation (such as y=-0.69x+127.37). 2+ In the graph of (x-axis), by putting 90% animal model survival rate into the equation, the first reference value (x value) will be determined to be 183.3. Then, by adding the second fluorescence parameter (such as Ca) determined in step c') 2+Influent) is compared to this reference value, and if it is above this reference value, it can be expected that the analyte (wastewater effluent) may cause 10% mortality in the animal model and may therefore be considered toxic.

[0111] In an optional or additional embodiment, step e) may further include the following steps: determining a reference value at 50% survival rate according to the first linear regression relationship model; and comparing the second fluorescence parameter with the reference value at 50% survival rate to determine the acute toxicity of the analyte. Similarly, in this embodiment, by using the first linear regression equation obtained in step d), the animal model survival rate (in this case, 50% survival rate) can be fitted to the equation to obtain the corresponding reference value. For example, again referring to the first linear regression equation, such as y=2.36x-167.24, at 50% animal model survival rate, the corresponding first reference value (x value) will be determined to be 71.08, and by comparing the second fluorescence parameter (such as mitochondrial size) determined in step c') with this reference value, and if it is lower than this reference value, it can be expected that the analyte (sewage effluent) can cause 50% death in the animal model and can therefore be considered toxic.

[0112] Similarly, referring again to the first linear regression equation, such as y=-0.69x+127.37, at a 50% survival rate of the animal model, the corresponding first reference value (x value) will be determined to be 183.87, and by comparing the second fluorescence parameter (such as mitochondrial size) determined in step c') with this reference value, and if it is higher than this reference value, it can be expected that the analyte (sewage effluent) can cause 50% mortality in the animal model and can therefore be considered toxic.

[0113] In a second aspect of the present invention, a system for determining the acute toxicity of an analyte according to the method described herein is provided. The system may include: a first fish cell culture stained with a fluorescent probe as described herein; a first reference solution as described herein, in which the first fish cell culture is incubated; and a first linear regression relationship model established based on a first fluorescence parameter obtained from the stained first fish cell culture and the survival rate of an animal model as described herein; wherein the first linear regression relationship model contains the acute toxicity of the analyte.

[0114] In some embodiments, the system can further include a second fish cell culture stained with a fluorescent probe having a second fluorescence parameter as described herein.

[0115] Hereinafter, the present invention is described in more detail by way of Examples, but the present invention is not limited thereto.

[0116] Example

[0117] Materials and methods

[0118] Cell culture

[0119] Keratinocyte cell lines and macrophage cell lines were isolated from brown basket fish (S. fuscescens) and hepatocyte cell lines were isolated from zebrafish (Danio rerio). Fish fin (RFF) cells and macrophages (RFM) were cultured as reported. The complete growth medium for RFF and RFM cells was Dulbecco's modified Eagles medium (DMEM) supplemented with 100 IU / mL penicillin, 100 μg / mL streptomycin, 2 μg / mL amphotericin-B, and 20% heat-inactivated fetal bovine serum. The complete growth medium for ZF4 cells was Dulbecco's modified Eagles medium F-12 (DMEM F-12) supplemented with 100 IU / mL penicillin, 100 μg / mL streptomycin, 2 μg / mL amphotericin-B, and 10% heat-inactivated fetal bovine serum. All these cell lines were incubated at 28°C in a water-saturated 5% CO2 incubator. After 70% confluence, cells were harvested with 1 mL of 0.25% trypsin for 3 minutes, and trypsinization was terminated by adding 5 mL of complete growth medium.

[0120] Sewage collection

[0121] Three 24-hour flow-weighted composite effluent samples were collected from sewage treatment plants in Hong Kong, China (October 2022, January 2023, and April 2023) and named S1, S2, and S3, respectively. The effluent samples were transported to the laboratory immediately on the day of collection. Salinity (‰), dissolved oxygen (mg / L), and pH were determined. The concentrations of metal pollutants were determined by inductively coupled plasma mass spectrometry (ICP-MS). Briefly, 200 μL of sewage effluent was added to 200 μL of 69% HNO3 for digestion and then diluted 100 times for ICP-MS (N8150060, Perkin Elmer) analysis. The standard curves of metals are as shown in Figure 1As shown in . For organic pollutant testing, total residual chlorine was tested based on APHA 4500Cl G method; bromoform, bromodichloromethane, chloroform, dibromochloromethane, dichloromethane, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, tetrachloroethylene were tested by GC-MS based on USEPA 8260B method; bromoacetic acid, chloroacetic acid, dibromoacetic acid, dichloroacetic acid, trichloroacetic acid were tested by GC-ECD based on TG-ENV-WW-79; 1,2,4-trichlorobenzene was tested by GC-MSD based on TG-ENV-WW-86 method. Details of GC-MS experiments, TG-ENV-WW-79 and TG-ENV-WW-86 are as follows:

[0122] TG-ENV-WW-79 based on U.S. Environmental Protection Agency's METHOD 8121

[0123] The dual column / dual detector method involves the use of two 30m x 0.53mm ID fused silica open tubular columns with different polarities and therefore different selectivities for the target compounds. The columns are connected to an injection tee and two identical detectors.

[0124] Column 1 - 30m x 0.53mm ID fused silica open tubular column, cross-linked and chemically bonded using 95% dimethyl and 5% diphenyl polysiloxane (DB-5, RTx-5, SPB-5 or equivalent), with a film thickness of 0.83μm or 1.5μm.

[0125] Column 2 - 30 m x 0.53 mm ID fused silica open tubular column, cross-linked and chemically bonded using 14% cyanopropylphenyl and 86% dimethylpolysiloxane (DB-1701, RTx-1701 or equivalent), film thickness 1.0 μm.

[0126] Splitter: If the splitter method for dual column injection is selected, the following are three recommended splitters. Equivalent splitters are acceptable. See Section 7.5.1 for considerations on splitter use.

[0127] Splitter 1 - J&W Scientific press fit Y-glass 3-way connector splitter (J&W Scientific, catalog number 705-0733).

[0128] Splitter 2 - Supelco 8 inch glass injection tee, deactivated (Supelco, cat. no. 2-3665M).

[0129] Splitter 3 - Restek Y-type fused silica connector (Restek, catalog number 20405).

[0130] Column flushing kit (optional): Bonded phase column flushing kit (J&W Scientific,

[0131] Catalog No. 430-3000 or equivalent).

[0132] Microinjectors - 100 μL, 50 μL, 10 μL (Hamilton 701N or equivalent) and 50 μL (blunt tip Hamilton 705SNR or equivalent). Balance - Analytical, 0.0001 g.

[0133] Volumetric Flasks, Class A - 10mL to 1000mL.

[0134] TG-ENV-WW-86 based on ISO 11423-1 test method

[0135] ISQ evaluation at a scan speed of 2,650u / sec TM GC-MS, which means 10 full scan mass spectra (scanning m / z 35 to 300 in 0.1 sec) acquired per second. An OI Eclipse equipped with a sample heater and a 4551A autosampler was used. TM 4660 purge and trap sample concentrator delivers 5mL sample for analysis. Internal standard and surrogate are added by Standard Addition Module (SAM) unit. The calibration curve range in sample is 0.4μg / L to 40μg / L. Due to 40mL / min split flow during injection, the amount on column of each compound is .05ng to 5ng.

[0136] GC-MS test process and parameters

[0137]

[0138]

[0139] Determination of benzene and some derivatives in water samples was performed according to Part 1: Headspace gas chromatography. ISO 11423-1, 1997.

[0140] Cytotoxicity studies

[0141] Cell viability was determined by MTT assay to quantify cytotoxicity. Cells were plated at 2 × 10 4The density of 1 cell / well was inoculated in 96-well plates and cultivated for 12h. The sewage effluent samples were mixed with the culture medium (DMEM) of various concentrations (6.5%, 12.5%, 25%, 50%, 100% v / v). After treatment, 0.5mg / mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added, and dimethyl sulfoxide (DMSO) was added to dissolve the purple crystals produced. Absorbance was measured at 570nm by a microplate reader.

[0142] Visualization of organelle and lysosomal pH

[0143] RFF cells were cultured at 1x10 6 The cells were seeded at a density of 10 cells / dish in confocal culture dishes and cultured for 12 h. The cells were then exposed to culture medium with different sewage effluent samples at final concentrations of 6.5%, 12.5%, 25%, 50% and 100% (v / v) for 24 h. The organelles lysosomes and mitochondria were stained with 0.5 μM lysosomal tracer (LysoTracker Deep Red DND-99, L7528, Thermo Fisher) and 0.1 μM mitochondrial tracer (MitoTrackerTM DeepRed M22426) for 30 min ( Figure 2 ).

[0144] To visualize lysosomal pH, the cyanostyrene derivative (Z)-3-(4-(4-methylpiperazin-1-yl)phenyl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile (CSMPP) was added to the culture medium at a final concentration of 2.5 μM and stained for 45 min ( Figure 2 ). After staining, 1X PBS was used to wash the residual probe, and confocal images were collected by confocal microscope LSM900 (CarlZeiss). The fluorescence intensity of lysosomes and mitochondria was measured by a microplate reader (FlexStation multi-mode microplate reader) with such excitation and emission wavelengths as described above. For lysosomal pH calculation, the fluorescence intensity ratio of two emission channels (615nm (red), 503nm (green)) was obtained by Image J software and named "R". When pH was 2.6 and 6.8, the fluorescence intensity ratio was named R max and R min pH value and log[(RR min ) / (R max -R)] showed a linear relationship. Lysosomal pH was calculated according to the following equation:

[0145] pH = -log [(RR min ) / (R max-R)]-2.908 / (-0.6189)

[0146] Cellular enzyme and protein measurements

[0147] In brief, RFF cells were seeded in confocal culture dishes. After overnight culture, the cells were exposed to culture media with different sewage effluent concentrations. For the ethoxyresorufin-O-deethylase (EROD) activity assay, 7-ethoxyresorufin was added to the cell culture medium at a final concentration of 0.5 mg / L after exposure and incubated for 40 min (Table 1). The formation of reactive oxygen species (ROS) in RFF cells was visualized by H2DCFDA (D399, Thermo Fisher). In brief, the exposed RFF cells were incubated with 1 μM H2DCFDA reagent for 10 min. The influx of calcium ions (Ca) in the cells was measured by Fluo-4, AM (F14201, Thermo Fisher). In brief, the exposed RFF cells were incubated with 0.1 μM Fluo-4, AM reagent for 30 min. By ThiolTracker TM Violet (T10095, ThermoFisher) was used to measure the glutathione (GSH) content in cells. TM Violet reagent for 30 min. After staining, wash the cells twice with 1X PBS to remove the solution. 2+ Confocal images were collected by confocal microscope LSM900 (Carl Zeiss) with λex=492 / λem=520nm for GSH and λex=405 / λem=590nm for GSH, and quantified by ImageJ software thereafter. The fluorescence intensity of the cell solution was measured by a microplate reader (FlexStation multimode microplate reader) with the above-mentioned specific excitation and emission wavelengths. Specific EROD activity, ROS formation, Ca ions were calculated based on relative fluorescence units (RFU) as the product of resorufin. 2+ Influx and GSH content were normalized to cell number. A summary of the different probes used for staining purposes is given in Figure 2 Given in.

[0148] In vivo toxicity of sewage effluents

[0149] Animals used for sewage effluent toxicity testing included amphipods (Long-fingered Maltese hook shrimp), barnacle larvae (Barnacles striata) and shrimp (Penaeus striata). Amphipods were collected from local coastal waters of Sai Kung, with a body length of about 0.5-0.7 cm. The experimental amphipods were acclimatized in fully aerated seawater (temperature: 22 ± 1 ° C, salinity: 30‰) in the laboratory before testing. Barnacle larvae of stage II were released from adult barnacles collected from Sai Kung. The experimental larvae were acclimatized in fully aerated seawater (temperature: 22 ± 1 ° C, salinity: 30‰) maintained in a 500 mL glass beaker and fed with Chaetoceros gracilis for 24 h in the laboratory before testing. After acclimatization, the experimental animals were exposed to sewage effluents at final concentrations of 6.5%, 12.5%, 25%, 50% and 100% (v / v) for 48 h. The number of surviving animals was counted after exposure.

[0150] Statistical analysis

[0151] All data were analyzed using SPSS software. The results are expressed as mean ± SD. The differences between the control group and each experimental group were evaluated using a one-way T test (p < 0.05).

[0152] Example 1

[0153] Pollutant indicators and overall animal toxicity of wastewater effluents

[0154] The salinity of all three wastewater effluent samples was 20‰, the dissolved oxygen was 6.8-7.0 mg / L, and the pH was 7.8-7.9. The metal concentrations detected in the wastewater effluent samples are shown in Figure 3A In all three wastewater effluent samples, Mn was the most abundant metal, followed by Cu and Zn. Other pollutants in the wastewater samples were quantified and displayed in Figure 3B Among the pollutants tested, dichloromethane was the most abundant (10-13 μg / L), followed by residual chloride (about 6 μg / L). The levels of all organic pollutants in the sewage samples were below the limit values ​​in Hong Kong, China.

[0155] The salinity of the sewage was adjusted to 30‰ before testing. The survival rates of different animals after 48 hours of exposure to the three sewage effluent samples were as follows: Figure 4As shown in . Among the tested animals, amphipods were the most sensitive, followed by barnacle larvae and shrimp. Similarly, previous studies have also shown that amphipods are generally sensitive to sewage effluents. The survival rate of all animals was slightly lower under S2 and S3 exposures, which may be due to the higher concentrations of metals in these two sewage effluent samples. The LC50 of the effluents determined was 25.0-28.8%, 29.0-54.6% and 68.4-83.3% for amphipods, barnacle larvae and shrimp, respectively ( Figure 4 ).

[0156] Example 2

[0157] Cell survival under emission exposure

[0158] The cytotoxicity of sewage effluent S1 on RFF cells and two other fish cell lines, including macrophages from brown basket fish (RFM) and a hepatocyte line from zebrafish (ZF4), was tested and shown in FIG. 5A to FIG. 5C Here, different concentrations of sewage effluent (6.5%, 12.5%, 25%, 50% and 100% v / v) were added to the culture medium and cultured for 24h and 48h, respectively. Saline with the same salinity as the sewage effluent samples was used as a negative control. Meanwhile, when sewage effluent was added at a concentration of 6.5%, a significant decrease in cell viability occurred. Only when the sewage effluent content reached about 100% did the cell viability of RFM and ZF4 cells show a significant decrease, indicating that RFM showed the most sensitive response to sewage effluent exposure ( Figure 5A ). The results showed that the RFF cell line could accurately and sensitively reflect the content of sewage effluent under exposure.

[0159] Example 3

[0160] Cellular responses to sewage effluent exposure

[0161] To further investigate the 'structure' of the organelles, several probes were applied to visualize subcellular changes. Among the organelles, mitochondria are believed to act as the powerhouse of the cell regulating the tricarboxylic acid cycle, oxidative phosphorylation (OXPHOS) and ATP production, thus providing energy for cell survival and function. This organelle hosts important biosynthetic and bioenergetic processes in the cell, not only controlling cell proliferation, differentiation and death, but also contributing to the body's immune response. This organelle is also responsible for the transport of different metal cations, including Ca 2+ , K + 、Na + Mg 2+ 、Zn 2+ , which is essential for maintaining metal homeostasis in cells. Impairment of metal homeostasis leads to mitochondrial dysfunction.

[0162] In addition to mitochondria, lysosomes have been studied as the most important organelles. The main structure of lysosomes is an acidic cavity, which contains a large number of hydrolases, including nucleases, proteases, phosphatases, lipases, and sulfatases. This organelle constitutes the main site for the sequestration and detoxification of toxic metals and organic pollutants. The response of this organelle has been shown to be a sensitive stress indicator in response to metal pollution. According to these characteristics, lysosomal changes have received widespread attention as a potential 'early warning tool' for estimating the biological effects of pollutants. Therefore, changes in lysosomes, including their size and pH, are monitored.

[0163] RFF cells were exposed to different concentrations of sewage effluent for 24 h. The cells were then stained with fluorescent probes to visualize the structure of lysosomes and mitochondria. Confocal images ( Fig. 6A ), and the number of lysosomes and mitochondrial size in each cell were quantified based on confocal images. The number of lysosomes per cell increased from about 20 to about 60 ( Figure 6B ). At the same time, the number of lysosomes increased significantly at a sewage effluent content of 12.5%. When comparing the number of lysosomes with the sewage effluent concentration, the number of lysosomes was linearly correlated with the sewage effluent content (R 2 =0.93)( Figure 6B ). The increased number of lysosomes is a cellular response to the digestion of pollutants, which is consistent with muscle cells exposed to Cd and Hg.

[0164] Due to their susceptibility to damage, mitochondria are highly sensitive to environmental toxins. Upon exposure to sewage effluent, the size of mitochondria in each cell decreases from approximately 125 to approximately 73 μm. 2 A significant decrease was observed when the sewage effluent content reached 12.5%. In addition, the decrease in mitochondrial size was linearly correlated with the sewage effluent concentration ( Figure 6C ). This subcellular response is similar to that of hepatocytes under small particle exposure. The reduction in mitochondrial size is associated with dysfunction of this organelle, which leads to subsequent cell death. Thus, under sewage effluent exposure, more lysosomes are formed and mitochondria shrink in RFF cells. According to these results, the number of lysosomes and the size of mitochondria can be used as two parameters representing the subcellular effects of sewage effluents.

[0165] To further investigate changes in lysosomal function under sewage effluent exposure, lysosomal pH was also stained and measured ( Fig. 7A and Figure 7B Here, the specific probe CSMPP was used to stain the cells after exposure to sewage effluent. The fluorescence intensity of the green and red channels was used to calculate the pH value ( Fig. 8A and Figure 8B). Under sewage effluent exposure, lysosomes were acidified and the pH decreased from 6.8 to 6.5. In addition, the pH decreased linearly with increasing sewage effluent concentration ( Figure 7B ). Digestive enzymes within lysosomes require low pH to be active. The reduced pH further confirmed the digestive process of lysosomes under sewage effluent exposure. This result indicates that lysosomal pH can be used to represent the impact of sewage effluent on RFF cells.

[0166] In addition to studying changes in organelles, several enzymes related to cellular function and detoxification were visualized and measured, including EROD activity ( Fig. 9A and Fig. 9B )、ROS production( Fig. 9C and Fig.9D ), calcium (Ca 2+ )Inflow( Fig.9E and Fig.9F ), glutathione (GSH) formation ( Figure 9G and Figure 9H ). The induction of 7-ethoxyresorufin-O-deethylase (EROD) activity is believed to be a valuable biomarker in ecotoxicological studies. EROD activity is highly correlated with the concentration of organic pollutants. In cell-based assays, EROD activity increases with the concentration of sewage effluent ( Fig.9D ). One possible explanation is that the increase in EROD is mainly related to organic pollutants in sewage effluent. The increase in cellular EROD under wastewater exposure is consistent with rainbow trout and mirror carp.

[0167] It is understood that ROS is a double-edged sword in cellular metabolism. At low to moderate levels, it acts as a signal transducer to activate cell proliferation, migration, invasion, and angiogenesis. In contrast, high levels of ROS will cause damage and lead to cell death. The generation of ROS decreases with the concentration of sewage effluent ( Fig. 9C and Fig.9D The reduction in ROS formation may be caused by the shrinkage of mitochondria ( Figure 6C ), mainly because about 90% of ROS are generated from mitochondria.

[0168] Intracellular Ca 2+ Concentration plays a major role in cell cycle progression, cell growth, proliferation and division. It is believed that Ca 2+ Signaling is associated with persistent organic pollutants and metal pollutants. To this end, the Ca 2+ Inflow( Fig.9E ). It was found that intracellular Ca 2+ The concentration of sewage effluent increased in a dose-dependent manner ( Fig.9F ). It is believed that Ca 2+The increase was mainly related to the dysfunction of cells under the exposure of sewage effluent.

[0169] GSH plays a key role in regulating redox homeostasis and metal homeostasis. The formation of GSH increases with the concentration of sewage effluent ( Figure 9H ), which may be related to metal contaminants in sewage effluent. This response is similar to that of catfish under oxidative stress and metals and European carp under metal exposure.

[0170] All four indicators (EROD activity, ROS production, Ca 2+ Influx and GSH formation) were linearly correlated with the effluent concentration, indicating the potential for representing the toxicity of the effluent. However, significant differences only occurred when the effluent content reached about 50%. The sensitivity of these four cellular indicators was not as high as that of lysosomes and mitochondria.

[0171] To further simplify the facilities required for cell-based testing, a fluorescence plate reader was applied to replace the use of confocal microscopy in fluorescence intensity quantification. Here, the fluorescence intensity of the previous cell parameters in RFF cells was measured and normalized by the number of cells after sewage effluent exposure. The normalized fluorescence values ​​were then compared with the values ​​without the addition of sewage effluent to obtain the fold change of the cell parameters under sewage effluent exposure.

[0172] To confirm the accuracy of the plate reader results, each cell parameter was compared with the fold change in fluorescence intensity ( FIG. 10A to FIG. 10F ). Based on the calibration curve, mitochondrial size, EROD activity, ROS formation and Ca 2+ The linear relationship between the flow chart results and the plate reader results (R 2 >0.8)( FIG. 10B to FIG. 10E ), while the number of lysosomes and GSH content obtained from confocal microscopy were inconsistent with those of the plate reader (R 2 =0.64 and 0.45)( Fig. 10A and Fig.10F ).

[0173] These results indicate that the plate reader can partially replace the use of confocal microscopy in this cell-based test. In addition, attempts were made to shorten the exposure duration. RFF cells were exposed to three sewage effluent samples (S1, S2, S3) for different exposure periods (6h, 12h, 24h) ( FIG. 11A to FIG. 11E). Then, the fluorescence values ​​of the cell parameters were tested by plate reader as described above. Similar trends were observed in the fold change of fluorescence intensity. Except for lysosomal pH, the cell parameters did not show changes at 6 hours of exposure. When compared with saline at the same salinity, significant changes were found in the sewage effluent content of 12.5% ​​at 12 hours of exposure ( FIG. 11A to FIG. 11E ). At the same time, the fold changes of cellular indicators did not show significant differences between 12-h and 24-h exposure treatments. This helps to shorten the time and simplify the facilities required for toxicity determination.

[0174] Example 4

[0175] Relationship between cell markers and animal survival rate

[0176] To further confirm that subcellular indicators can represent acute toxicity to animals, cellular parameters obtained by confocal microscopy and fluorescence plate reader were compared with toxicity in test animals (amphipods, barnacles, and shrimp) at the same sewage effluent levels.

[0177] Except for GSH content, all cellular indices showed linear relationships with the survival rates of amphipods and barnacles (R 2 >0.8)( Fig.12F ). Since GSH may not directly affect animal survival, GSH content was not linearly correlated with survival of all three tested animals and was not used for further toxicity determination. 2 Only mitochondrial size, EROD activity, and lysosomal pH showed a good linear relationship with shrimp survival ( Fig. 12A , Fig. 12C and Figure 12G ). This indicates that acute toxicity results for amphipods, barnacles, and shrimp can be expressed by these cellular indicators. Amphipods and barnacle larvae are considered sensitive model animals in toxicity determination and water quality assessment. In contrast to subcellular parameters, cell viability did not show a good linear relationship with the level of sewage effluent (R 2 <0.8), which further demonstrates the inaccuracy of using cell viability alone to determine toxicity ( Fig.14A ). By plotting cell survival relative to animal survival ( Fig.12H , Fig. 14B and FIG. 15A to FIG. 15F ), the cell survival rate of RFF cells showed a poor linear relationship with the animal survival rate at the same exposure concentration (R 2 This result indicates that cell survival is not representative of animal survival under sewage effluent exposure.

[0178] To confirm the accuracy of the results based on 12 hours of exposure, the changes in cell indices (as obtained by fluorescent plate reader) were compared with the animal test results (Figure 16). These cell indices similarly showed good linear relationships with the amphipod and barnacle results, but poor relationships with shrimp. These results indicate that cell-based tests can be operated by fluorescent plate readers and that the 12-hour exposure duration can reflect the toxicity of sewage effluents. In general, subcellular changes can partially represent the acute toxicity of sewage effluents in vivo, especially animals that are very sensitive to pollutants.

[0179] Example 5

[0180] Toxicity determination using calibrated cell parameters and animal survival

[0181] After calibrating the cell parameters with the animal survival rate (i.e., establishing a linear regression relationship model between the cell parameters and the animal survival rate), the linear regression equation of the model can be used to calculate the values ​​of the cell parameters when the animal mortality rate reaches 10% (LC10) and 50% (LC50). For example, when the cell parameters are obtained by confocal microscopy, the following can be established: FIG. 12A to FIG. 12G A set of linear regression relationship models are shown in . Then, we can use FIG. 12A to FIG. 12G The linear regression equation shown in was used to calculate the values ​​of the cell parameters when the animal mortality reached 10% (LC10) and 50% (LC50). The values ​​of each cell parameter for each animal are listed in the table, as shown in Fig.13A and Fig. 13B Therefore, it is believed that Table ( Fig.13A and Fig. 13B ) are the criteria for determining the (acute) toxicity of wastewater samples (LC10 or LC50).

[0182] To determine the acute toxicity of an unknown sewage effluent sample, first, the RFF cells stained with the fluorescent probe may be incubated with the sewage effluent sample. Given that it is apparent from the above examples that the incubation times of 12 hours and 24 hours have an insignificant effect on the obtained cell parameters, the stained RFF cells may be incubated for 12 hours or, optionally, 24 hours, depending on actual needs. In this example, the stained RFF cells may be incubated with the sewage effluent sample for 24 hours. Then, one or more cell parameters from the RFF cells exposed to the sewage effluent are recorded and quantified by confocal microscopy. Thereafter, this cell parameter may be compared with the Fig.13A and Fig. 13B The cellular parameters in the samples were compared for acute toxicity determination.

[0183] like Fig.13A and Fig. 13BAs shown in the figure, if the test value is higher than the value in the orange area or lower than the value in the green area, it indicates that this sewage effluent sample may cause 10% or 50% of certain animals to die and this sample is considered to be toxic. It also provides such an indication that such samples need to be treated or diluted to meet the disposal standards. For example, assuming that by Fig. 13B By comparing the mitochondrial size and EROD data in the effluent samples, it was determined that the mitochondrial size of the RFF cells after exposure to the sewage effluent sample was 50 and the EROD value was 30, which can be determined that this sewage effluent sample can cause 50% mortality of amphipods, barnacles and shrimps. And the sewage effluent sample can be considered toxic.

[0184] Alternatively, when cell parameters are obtained by fluorescence plate reader, the following may be used: FIG. 16A to FIG. 16E The linear regression relationship model and linear regression equation shown in were used to calculate the values ​​of cell parameters when the animal mortality reached 10% (LC10) and 50% (LC50), and these values ​​are shown in Fig.17A and Fig. 17B To determine the acute toxicity of an unknown sewage effluent sample, similarly, the stained RFF cells can be incubated with the sewage effluent sample for, for example, 12 hours, and then one or more cell parameters of the RFF cells exposed to the sewage effluent can be recorded using a fluorescent plate reader, and the recorded cell parameters can be compared with the Fig.17A and Fig. 17B For acute toxicity determinations, the values ​​shown in Fig.17A Neutralization Fig. 17B As shown, if the test value is higher than the value in the orange area or lower than the value in the green area, it indicates that this sewage effluent sample may cause 10% or 50% death of certain animals and this sample is considered to be toxic.

[0185] The present invention is presented by way of example only, and various other modifications and / or alterations to the described embodiments may be made by those skilled in the art without departing from the scope of the present invention as specified in the appended claims.

Claims

1. A method for determining the acute toxicity of an analyte, comprising the following steps: a) exposing a first fish cell culture to a first reference solution; b) incubating the first fish cell culture in step a) with a fluorescent probe; c) obtaining a first fluorescence parameter from the stained first fish cell culture; d) establishing a first linear regression relationship model between the first fluorescence parameter and the survival rate of the animal model; and e) determining the acute toxicity of the analyte by using the first linear regression relationship model.

2. The method of claim 1, wherein the first fish cell culture comprises a fin cell line isolated from brown basket fish.

3. The method of claim 1, wherein step a) comprises the step of collecting the first reference solution from a reference location at a first point in time.

4. The method of claim 3, wherein the first reference solution comprises a 24-hour flow-weighted composite effluent from a wastewater effluent treatment plant.

5. The method of claim 1, wherein the first reference solution has a pollutant volume concentration selected from any one of 0%, 6.5%, 12.5%, 25%, 50% and 100%.

6. The method of claim 1, wherein step b) comprises the step of incubating the mixture of the first fish cell culture and the fluorescent probe in the dark.

7. The method of claim 1, wherein the fluorescent probe is selected from the group consisting of: lysosomal tracers, mitochondrial tracers, (Z)-3-(4-(4-methylpiperazin-1-yl)phenyl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile (CSMPP), 7-ethoxyresorufin, H2DCFDA, Fluo-4, AM, ThiolTracker, and combinations thereof.

8. The method of claim 1, wherein step c) comprises the following steps: taking a confocal microscopy image of the stained first fish cell culture; and The first fluorescence parameter of the stained first fish cell culture is obtained from the confocal microscopy image.

9. The method of claim 1, wherein step c) further comprises step c1) obtaining the fluorescence intensity of the stained first fish cell culture by a fluorescence plate reader.

10. The method of claim 1, wherein the fluorescence parameter corresponds to the fluorescent probe and is selected from the group consisting of: lysosome number, mitochondrial size, lysosomal pH, EROD activity, ROS production, Ca 2+ influx, GSH formation and their combination.

11. The method of claim 1, wherein step d) comprises the following steps: Incubating the animal model with the first reference solution for at least 12 hours; determining the survival rate of the animal model; as well as Establish a first linear regression equation between the first fluorescence parameter and the survival rate of the animal model, and thereby obtain R 2 value.

12. The method of claim 1, wherein the animal model comprises any one of the following: Maltese gammarus, barnacles, and shrimp Metapenaeus lanceolatus.

13. The method of claim 11, wherein the first reference solution has a pollutant volume concentration selected from any one of 0%, 6.5%, 12.5%, 25%, 50% and 100%.

14. The method of claim 11, wherein said R 2 The value is at least 0.

70.

15. The method of claim 1, further comprising the steps of: a') exposing a second fish cell culture to said analyte; b') incubating the second fish cell culture in step a') with a fluorescent probe; and c') Obtain a second fluorescence parameter from a second stained fish cell culture.

16. The method of claim 15, wherein step e) comprises the following steps: Determine the reference value at 90% survival rate according to the first linear regression relationship model; as well as The second fluorescence parameter is compared to the reference value at the 90% viability to determine the acute toxicity of the analyte.

17. The method of claim 15, wherein step e) further comprises the following steps: Determine the reference value at 50% survival rate according to the first linear regression relationship model; as well as The second fluorescence parameter is compared to the reference value at the 50% viability to determine the acute toxicity of the analyte.

18. The method of claim 1, wherein the analyte comprises sewage effluent.

19. The method of claim 3, wherein step a) is repeated using a second reference solution and a third reference solution from the reference location at a second time point and a third time point, respectively.

20. The method of claim 19, wherein the first time point, the second time point, and the third time point are each separated by an interval of three months.

21. A system for determining the acute toxicity of an analyte according to the method of claim 1, comprising: First fish cell cultures stained with fluorescent probes; a first reference solution, incubating the first fish cell culture in the first reference solution; as well as a first linear regression relationship model established based on the first fluorescence parameter obtained from the stained first fish cell culture and the survival rate of the animal model; The first linear regression relationship model includes the acute toxicity of the analyte.

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