Biological monitoring of water pollution - biological test method The method of assessing the pollution of a water body by using changes in the response or physiological function of the organism after being harmed or poisoned by the pollutant, and determining the safe concentration of the poison is called a biological test method. The method has two types: hydrostatic biological test and flow-through biological test. The former is to place the test organism in a non-flowing test solution, determine the relationship between the concentration of the pollutant and the biological poisoning reaction, thereby determining the toxicity of the pollutant; the latter put the test organism in a continuous or intermittent flow test In solution, determine the relationship between contaminant concentration and biological response. The test is divided into short-term ( not more than 96h) acute toxicity tests and long-term ( such as months or years ) chronic toxicity tests. In a test device, the test organisms may be one type or multiple types. Testing can be done in the laboratory or in contaminated water in the field. 1. Aquatic toxicity test Fish, mites, algae, etc. can be used for aquatic toxicity tests, and fish toxicity tests are widely used. Fish reacts very sensitively to changes in the water environment. When the pollutants in the water reach a certain concentration or intensity, they will cause a series of poisoning reactions. For example: abnormal behavior, physiological dysfunction, tissue cell disease, until death. The main purpose of the fish toxicity test is to find the semi-lethal concentration and safe concentration of certain poisons or industrial wastewater on fish, to provide a scientific basis for the development of water quality standards and wastewater discharge standards; to test the degree of pollution of water bodies and to check the effects of wastewater treatment. Sometimes fish toxicity tests are also used for special purposes, such as comparing the toxicity of different chemical substances, testing the relative susceptibility of different types of fish to poisons, and testing the effects of environmental factors on the toxicity of wastewater. The acute toxicity test of still water fish is described below. ( 1 ) Selection and domestication of test fish. Goldfish are easy to source and are often used for testing. It is necessary to select the same type and the same age of goldfish that are disease-free, lively, with complete fin extension, appetite and backwater resistance, and body length ( excluding the tail ) of about 3 cm . The selected fish must first be domesticated for 7 days or more under the living conditions ( temperature, water quality, etc. ) similar to the test conditions; the feeding should be stopped one day before the test; if the death occurs within 4 days before the test or the diseased fish is higher than 10 %, then Out of service. ( 2 ) Selection of test conditions. Each concentration of test solution is a group of at least 10 fish per group. The test vessel uses a glass cylinder with a volume of about 10L to ensure that the fish mass per liter of water does not exceed 2g . The temperature of the test solution should be suitable, 12 to 28 °C for cold water fish and 20 to 28 °C for warm water fish. In the same test, the temperature change is ± 2 °C; the test solution can not contain a large amount of oxygen-consuming substances, there must be enough dissolved oxygen, DO ≥ 5mg / L for cold water fish, DO ≥ 4mg / L for warm water fish; pH of test solution It should be 6.7 ~ 8.5 , the pH fluctuation range during the test should not exceed 0.4 pH units; hardness affects the toxicity of poisons. Generally speaking, hard water can reduce the toxicity of poisons, while soft water can enhance the toxicity of poisons. Therefore, it is necessary to pay attention to the hardness of test solutions. And noted in the report. Hardness should be 50 ~ 250mg / L ( calculated as CaCO 3 ) . The water used to prepare the test solution and domesticated fish should be uncontaminated river water or lake water. If tap water is used, it must be fully aerated before use. It is not advisable to use distilled water. ( 3 ) Test procedure. 1 Pre-test ( exploratory test ) : In order to ensure the smooth progress of the formal test, the concentration range of the test solution must be determined by exploratory test, that is, by observing the reaction and death of fish poisoning for 24h ( or 48h) , it is found that no death occurs. The concentration of all deaths and partial deaths. 2 Test solution concentration design: Reasonable design of test solution concentration is an important guarantee for the success of the test. Usually, 7 concentrations ( at least 5 ) are selected, and the concentration interval is equal to the logarithmic distance, for example: 10.0 , 5.6 , 3.2 , 1.8 , 1.0 ( Logarithmic spacing 0.25) or 10.0 , 7.9 , 6.3 , 5.0 , 4.0 , 3.6 , 2.5 , 2.0 , 1.6 , 1.26 , 1.0 ( log spacing 0.1) , unit available volume fraction ( eg wastewater ) or mass concentration (mg / L) ) said. Another control group was set up. The fishery mortality rate exceeded 10 % during the test period, and the whole test result could not be used. 3 Test: The test fish were placed in a glass jar containing different concentrations of solution and control water, and the time was recorded. The first 8 hours should be observed and recorded continuously. If it is normal, continue to observe and record the poisoning symptoms and death of the fish at 24h , 48h and 96h for the toxicity of the poison or industrial wastewater. 4 Toxicity determination: The median lethal dose (LD 50 ) or median lethal concentration (LC 50 ) is one of the main indicators for evaluating toxicity of toxicants. A simple method for LC 50 is to plot the data of more than half and less than half of the test fish deaths on the semi-logarithmic scale paper with the corresponding test solution poison ( or wastewater ) concentration ( logarithmic coordinates indicate poison concentration, arithmetic coordinates indicate mortality) ) , obtained by linear interpolation. The mortality values ​​of more than half of the deaths of the three test time test fish and the nearest half of the fish are marked with the coordinates of the corresponding wastewater concentration values, and are respectively connected, and then the vertical line of the abscissa is derived from the 50 % mortality rate. And intersect with the above three lines, the three intersection points are perpendicular to the ordinate, and the concentration at the intersection of the vertical line and the ordinate is LC 50. The LC50 of 24h , 48h and 96h are 5.2 %, 4.7 % and 4.4 %, respectively. 5 Application of fish toxicity test: An important purpose of the fish toxicity test is to estimate the safe concentration of poison based on the test data, and provide a basis for setting the maximum allowable concentration of toxic substances in water. The last application is more common. For chemical substances that are easy to decompose and accumulate less, the coefficient is generally 0.05 to 0.1 . For stable chemical substances that can accumulate in fish, the coefficient is generally 0.01 to 0.05 . After calculating the safe concentration according to the formula, further verification tests, especially poisons or waste water with volatility and instability, should be tested with a constant current device for a long time ( such as one month or several months ) . The control group is compared. If symptoms of poisoning are found, the concentration of the poison or wastewater should be reduced and the test should be carried out until a certain concentration is safe for the fish, which can be determined as a safe concentration. In addition, bait must be fed during the verification test. 2 , luminescent bacteria method ( 1 ) Method principle Luminescent bacteria are a class of non-pathogenic Gram-negative microorganisms that emit visible blue-green light ( 450-490 nm ) under appropriate conditions. When the toxic component of the sample is in contact with the luminescent bacteria, it may affect or interfere with the metabolism of the bacteria, so that the luminescence intensity of the bacteria is decreased or not. In a certain range of toxic concentrations, the concentration of toxic substances is inversely related to the luminescence intensity, so the relative luminescence intensity of the water sample can be measured using a bioluminescence luminometer to monitor the concentration of toxic substances. In the national standard ( GB/T 15441 - 1995 ), mercury chloride is used as a reference toxicity to characterize the toxicity of wastewater or soluble chemicals. Half effective concentration ( EC 50 ) can also be used, that is, when the luminous intensity is half of the maximum luminous intensity. or wastewater the concentration of soluble chemical species is characterized; selection Photobacterium phosphoreum T 3 subspecies luminescent bacteria. Since the bacteria is a marine bacterium, the water sample and the reference toxic solution should contain a certain concentration of sodium chloride. At present, fresh luminescent bacteria culture method and freeze-drying luminescent powder powder preparation method are often used. (2) Measurement points 1 Preparation of test materials: special biological toxicity tester, luminescent bacteria agar culture solution, liquid medium, 0.02~0.24mg/L series HgCl 2 standard solution, fresh bright photobacterium T 3 subspecies or bright luminescent freeze-dried powder, Chemical poisons or integrated wastewater. 2 Preparation of fresh luminescent bacterial suspension: Pick a loop of bacteria from the oblique tube surface of the luminescent bacterium, inoculate the new luminescent bacteria agar slant, and add the appropriate amount of diluent when the slant is covered with the lawn and obviously emits light. Prepare the bacterial suspension; inoculate 0.1mL bacterial suspension in 50mL liquid medium, shake culture at 22 °C to the middle of logarithmic growth ( 12~14h ), dilute the bacterial suspension to 5 × 10 with diluent 7 (cells) / [mL (bacterial suspension) ] , stored at 4 °C for later use. 3 sample determination: the water sample to be removed by filtering and removing particulate matter impurities is added to the NaCl which accounts for 3 % of the water sample mass, and the dilution liquid and the water sample to be tested are sequentially added, and the same amount of luminescent bacteria suspension is added after constant temperature ( 20 ± 0.5 ) °C. The luminous intensity is measured in turn. 4 Test result analysis: Calculate the relative refractive index according to the measured luminous intensity of the water sample to be tested. 3. Mutagenicity and carcinogen detection Also known mutagenic carcinogens and mutagens, which detection methods micronucleus assay, Ames (Ames) test, chromosome aberration test method. The principle of micronucleus assay is based on the fact that chromosomes in biological cells often undergo some breakage during the process of replication. Under normal circumstances, most of these breaks can heal by themselves, but if they are subjected to external mutagens, some freeness will occur. Chromosome fragments form envelopes and become small spheres ( micronuclei ) of varying sizes, which are proportional to the intensity of external mutagens and can be used to assess environmental pollution levels and damage to organisms. The biological material used in the method can be a plant or animal tissue or cell. Plants widely use the tip of purple grass and broad bean. The purple sage uses the chromosome of the pollen mother cell as a mutagen in the meiosis process, and the micronucleus number formed in the tetrad is used as an indicator of chromosome damage, and the degree of damage is evaluated. Vicia faba root tip cells have large chromosomes and high DNA content, which are sensitive to mutagens. The Ames test detects the presence or absence of mutagenicity of a test substance by the property of a histidine auxotrophic strain of Salmonella typhimurium . This strain contains a gene that controls histidine synthesis and cannot grow in a histidine-free medium. However, if a mutagen is present, it acts on the DNA of the strain, causing a mutation in a specific part to respond. It is a wild-type strain that can grow in histidine-free medium. Considering that many substances show mutagenicity after metabolic activation in vivo, Ames et al. adopted a method of adding a mammalian liver microsomal enzyme system ( s - 9 mixed solution ) to activate the test substance in vitro. Improve the reliability of the test. The chromatic aberration test is based on the change of chromosome number and structure of biological cells under the action of mutagens, such as chromatid cleavage, chromatid exchange, etc., to detect mutagens and their strength. For more information, please pay attention: Yingnuo Instrument (Shanghai) Co., Ltd. Website: http:// Related instruments: BOD analyzer , hand-held oil detector , solid waste toxicity leaching equipment , BOD online analyzer , handheld chlorophyll analyzer Ningbo Staneex Imp. & Exp. Co., Ltd. , https://www.staneex.com