Some standard content:
ICS 13.300;13.020.40
National Standard of the People's Republic of China
GB/T 21805—2008
Chemicals
Alga growth inhibition test
Chemicals-Alga growth inhibition test2008-05-12Issued
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaStandardization Administration of the People's Republic of China
2008-09-01Implementation
GB/T21805—2008
This standard is equivalent to the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guidelines Na.201 (2006) Algae Growth Inhibition Test (English version).
This standard has made the following editorial changes:
Change the measurement units to the legal measurement units of my country. Adjust the original Appendix 1 terms and definitions to the main text. - Chloretta Vulgaris was added as a test organism. The content of the original Appendix 4 was adjusted to 6.1.2 Reserve culture of algae and 6.1.3 Pre-culture of algae in the main text. - The content related to "Source of algae" in the original Appendix 2 was deleted. Appendix A, Appendix B and Appendix C of this standard are informative appendices. This standard was proposed and coordinated by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC251). The responsible drafting unit of this standard: Chemical Registration Center of the Ministry of Environmental Protection. The participating drafting units of this standard: Safety Evaluation Center of Shenyang Research Institute of Chemical Industry, Shanghai Testing Center, Shanghai Research Institute of Environmental Sciences. The main drafters of this standard: Zhou Hong, Guan Xiaodong, Ma Xin, Cai Leiming, Zhao Yuyan, Rong Zhiyi, Shen Genxiang. T
1 Scope
Test for algal growth inhibition of chemicals
GB/T 21805—2008
This standard specifies the method overview, test preparation, test procedures, quality assurance and quality control, data and report of growth inhibition tests for chemicals.
This standard is applicable to the test of chemicals that are soluble in water under the test conditions. If volatile, strongly adsorbent, colored, insoluble or poorly soluble in water are to be tested, as well as chemicals that may affect the effective utilization of nutrients in the culture medium, the test procedures need to be modified (such as using a dense ball system, appropriate test containers). References [2], [3] and [4] provide some modification plans. 2 Terms and definitions
The following terms and definitions apply to this standard. 2.1
Biomass
The weight of living organisms per unit volume of test medium, for example grams per liter of test solution. Biomass is defined as mass, but in this standard, it is defined as mass per unit volume, and the determination of cell number or fluorescence per unit volume is used instead of biomass determination. 2.2
Coefficient of variation cnefficient of variation ratio of standard deviation to mean, usually expressed as a percentage, recorded as CV%. The coefficient of variation is a number without a metric, so it is easy to compare samples: the average coefficient of variation of the average specific growth rate of each parallel control group is calculated as follows: a) calculate the average specific growth rate of each parallel in each stage of the test, b) calculate the average coefficient of variation of the average specific growth rate of each parallel control group in each stage of the test, 2.3
Effect concentration EC
The liquid content of the test substance when the growth or growth rate of the test organism decreases by % (such as 50%) compared with the control. EC is divided into EC based on growth rate and EC based on growth amount.
Algae growth medium growtlimedium
Liquid or gelatinous substance containing specific nutrients. Algae are grown in a culture medium and exposed to the test substance. Usually the test substance is dissolved in the culture medium.
Growth rate
is the average specific growth rate, which is the exponential growth rate of algae biomass during exposure. 2.6
Lowest observed effect concentration; LoEC is the lowest test substance set concentration that has a significant (P 0.05) inhibitory effect on algal growth compared with the control during a certain period of exposure. All test concentrations above LOEC can observe the same or more severe toxic effects as at LOEC. If the above conditions are not met, the selection of LOEC (and NOEC) should be described in detail. 2.7
No observed effect concentration; NOEC is the test substance set concentration directly below the lowest observed effect concentration (LOEC). 1
GB/T 21805—2008
Response variableresponsc variable
Various parameters that can be used to evaluate the toxicity of the test substance to algae in place of biomass, such as growth rate and yield. 2.9
Specific growth rate
Daily growth of biomass during the test. The quotient of the difference between the natural logarithm of biomass and the difference in time. 2.10
Yield
The measured value of biomass growth during the test, the difference between the biomass of the species in the test container at the end of the test and the algae biomass in each test container at the beginning of the test.
3 Information on the test substance
Structural formula:
Vapor pressure;
Solubility in water
Shear coefficient of n-octanol-water (P.
Quantitative analysis method in aqueous solution
Photochemical process
White in water
Experimental
Light absorption
Water dissociation constant||tt ||4 Method Overview
4.1 Principle
Degradation and stability of the test:
The test concentration is not
, and the cyanobacteria are exposed to the aqueous solution containing the test green algae and the test algae in the best growth period. The test period is 72 hours and the following are recorded:
24 h, 48 h and 72 h. The biomass of the algae is determined and the inhibition rate (root ratio) is calculated. ECs and their 95% confidence intervals are obtained, and the lowest observable effect concentration (LOEC) and/or no observable effect (LOEC) are obtained statistically. The test period is obviously relatively short, but the effects can be evaluated after several generations of algae reproduction. The biomass of the algae at different times is measured to quantify the inhibition and growth inhibition of the algae. Since the dry weight of algae is difficult to measure, other parameters are often used as substitutes, such as cell concentration, carbon optical density, and optical density. The conversion factor between the replacement parameter used and biomass should be known. The determination endpoint is growth inhibition. It can be expressed as the average specific growth rate or the increase in biomass during the test. The concentration of the test substance that causes the growth rate or growth of the species to be inhibited by % (such as 50%) can be obtained from the average specific growth rate or growth amount under a series of test concentrations and expressed as E, C or EC (such as E, Csp or E, Co). 4.2 Reference substance
This standard recommends the use of 3,5-dichlorophenol as a reference substance. For green algae, potassium dichromate is also used as a reference substance. The effect of the reference substance on the growth of species should be tested regularly, at least twice a year. 5 Instruments and equipment
5.1 Test container
The test container and other instruments in direct contact with the test solution should be made entirely of glass or other chemically inert materials and should be thoroughly cleaned and sterilized before use in the test.
GB/T 218052008
The test container is a glass bottle with a certain volume, such as a triangular flask, to ensure that there is enough test liquid for testing during the test, and to ensure sufficient exchange of CO (there must be a certain surface area-volume ratio: the volume of the test liquid in a 125mL triangular flask should be 40mL~60mL, 250mL triangular flask should be 70mL~100mL, and 500mL triangular flask should be 100mL~150mL. Note that there must be enough liquid for analysis and determination.
In order to prevent organic or inorganic pollutants from affecting the growth of algae and the composition of the culture medium, the container should be sealed with cotton plugs, sponge plugs, filter paper, gauze (2-3 layers), tin foil, etc.
When testing volatile adjuvant chemicals, ground glass plugs should be used to seal completely. The containers of the same batch of tests should have the same specifications. 5.2 Culture equipment
Use a light incubator that can control the temperature within the range of ±2℃, 5.3 Illuminance meter
Instrument for measuring light intensity: Note that the method for measuring light intensity is very important, and the results obtained using different types of light receptors may be different. It is best to use a 4-element surface illuminance meter (which can accept direct or reflected light from all directions and angles) or a 2-element spherical illuminance meter (which can accept direct or reflected light from all angles above). 5.4 Instruments and equipment for measuring biomass
Cell counting is the most commonly used surrogate parameter, and the instruments and equipment used for counting mainly include electronic particle counters, microscopes, phytoplankton counting frames or blood cell counting plates, manual counters, etc. The conversion relationship between cell count and dry weight should be clarified. Other surrogate parameters can also be measured using spectrophotometers, colorimeters, fluorometers, flow cytometers, etc. In order to make effective measurements at low biomass, the light path of the absorption cell of the spectrophotometer must be at least 1 μm. 5.5 Other instruments and equipment
-pH meter;
Electronic analytical balance;
-Autoclave;
Mechanical oscillator, etc.
6 Test preparation
6. 1 Test organisms
6.1.1. Selection of test organisms
Select some green algae and blue algae that are not easy to adhere to the bottle wall as test organisms. a) Green vegetables
Pseudokirchneriella subcapitata: -Coconut (Desmodesmus subspicatus); Common Chlorella (Chiorella tulgaris) b) Diatoms
Naviculapelliculosa c) Blue algae
Anabaena flos-aguae: Synechococcus leopoliensis. Detailed information on the above algae can be found in Appendix A. Other algae may also be used, but their strains and (or) sources should be stated in the report, and it should be ensured that exponential growth is maintained under the corresponding test conditions during the test.
GB/T 21805—2008
6.1.2 Reserve cultures of algae
After obtaining pure algae, they need to be preserved for use in the test. The species can be stored on the slant of solid culture medium in a test tube. Add 0.8% agar to the culture medium, pour it into the test tube after sterilization, cool it into a slant, and then inoculate the algae, seal it with cotton plugs, and it can be stored for a long time under low light and temperature conditions. Transfer it about every 2 months. If the test is carried out frequently. The reserve culture should be stored in a liquid culture medium. Add about 100mL of culture medium to a triangular flask, inoculate the algae, and culture it under the same temperature and light conditions required by the test. Transfer it once a week to keep the culture growing well and have sufficient quantity available for the test at any time. For fast-growing species, the inoculation amount is 1% of the cell concentration before transfer. Generally, it should be Transfer before the algae enter the growth stagnation period.
The growth of algae in the reserve culture should be checked regularly, including morphology and growth rate, as well as the presence of contamination by bacteria and other algae. Cultures with abnormal growth or contamination by other algae or fungi should be discarded or purified and rejuvenated. In order to avoid contamination of algae by bacteria and other algae, operations must be carried out in a sterile room. 6.1.3 Pre-culture of algae
A small amount of algae liquid is extracted from the reserve culture, mixed into fresh sterile culture medium, and cultured under the same conditions as required by the experiment. If the algae still show signs of growth (such as deformity, etc.) within 2 to 4 days, it should be discarded
6.2 Experimental conditions
6. 2. 1 Culture medium
Use OECD
Substances in different culture media
When the test substance is a compound or a gene of algae culture medium containing gold:
B. The composition of the modified culture medium should be stated in the culture medium, and
6.2.2 Other conditions 1
Temperature 21℃~2
4℃, in the test
. If a temperature other than that recommended by the manufacturer is used, the culture temperature should be kept constant.
Continuous uniform illumination, the range of cyanobacteria
400nm~~700nmbzxz.net
120 μE-m-3 8-1).
Recommended
reasonable
help||algae species, for
time for the test, if the species is contaminated or growth abnormal value and buffer volume are different, therefore, to a test when the degree of ionization is high.
time, can be appropriately modified, but in the report and fear of change every day in the culture of its position in the control requirements, may need to appropriately increase the culture
light intensity should be suitable for the test organisms. Spectrum - ~8 850 lx60 μE - m-2 . s-1 - he recommended algae species, especially Yonghua fish algae (Anabaeflasaquae) in the light intensity of
2 960 1x-~4 440 1x (40μE
±15% range.
-1 ～60 μE - ㎡-2· 8-) when the growth is good. The light intensity difference should be kept within
Mechanical oscillator: (100 ± 10) times/in or timed shaking 7 Test procedure
7.1 Preparation
7.1.1 Preparation of test solution
If the test substance is a chemical substance that is easily soluble in water, prepare a stock solution of the test substance with a sterilized fresh culture medium, and its concentration is twice the highest concentration required for the test. Use this stock solution to dilute and prepare a series of test solution with different concentrations, and its concentration is also twice the concentration required for the test.
If the test substance is a chemical substance that is poorly soluble in water, use an appropriate solvent, such as propylene glycol, butyl alcohol and dimethylformamide [2.[3, etc., to prepare a stock solution of the test substance, and its concentration should be 10 times the highest concentration required for the test. Use this stock solution to dilute and prepare a series of test solution with different concentrations, and its concentration is also 10 times the concentration required for the test. The solvent that has no effect on the growth of algae should be selected. The maximum allowable amount of solvent in the test solution is 100g/L. 1
GB/T21805—2008
7.1.2 Preparation of algae solution
Examine and count the pre-cultured algae under a microscope. If the algae grow well, they can be used for the test. The initial biomass of algae in the test solution (dry weight) should be less than 0.5 mg/L. Therefore, the initial cell concentrations of various algae in the test solution are as follows: a) Pseudakirchneriel&a subcapitata, 5×(1o°~1o*) cells/mL: b) Desmodesmus subspicatus, (2~5)×10° cells/mL c) Chiorella uiguris 1o* cells/mL d) Naiculapellicutosa, 10 cells/mL) Anabaenaftos-aquae, 10+ cells/mL: f) Synechococcus aquatiensis, 5×(1o*~105) cells/mL If the test substance is a chemical substance that is easily soluble in water, the algal cell concentration in the algal solution is twice the initial cell concentration; if the test substance is a chemical substance that is poorly soluble in water, the algal cell concentration in the algal solution is the initial cell concentration. 7.1.3 Preparation of test solution
If the test substance is a chemical substance that is easily soluble in water, mix the test substance solution and algae solution in a ratio of 1:1 to prepare the test solution. For the oral group, do not add the test substance solution but add the same volume of sterile culture medium. If the test substance is a chemical substance that is poorly soluble in water, add 10μL of the test substance solution to a certain volume of algae solution to prepare the test solution. Set up a bathing agent control group, in which 10μL of solvent is added. 7.2 Experimental operation
7.2.1 Preliminary test
Before the formal test, a preliminary test with a large range of concentration series is carried out to provide a basis for setting the concentration of the test substance for the formal test. The preliminary test is not parallel.
7.2. 2 Formal test
Perform the formal test based on the results of the preliminary test. It is best to set the test substance concentration series in geometric progression between 5% and 75% growth inhibition effect on algae. The formal test shall have at least 5 concentrations, with a concentration interval coefficient greater than or equal to 3.2, and 3 parallels for each concentration.
If the test does not need to obtain a no observable effect concentration (NOEC), the number of parallels can be appropriately reduced and the test concentration can be increased. A blank control group should also be set up. If a solvent is used, a solvent control group should also be added. The control group should have at least 3 parallels. If conditions permit, the number of parallels for the control group should be twice that of the treatment group. 7.2.3 Test cycle
The test cycle is 72h. However, if it is necessary to meet the requirements of quality assurance and quality control, the test cycle can be shortened or extended according to the actual situation.
7.2.4 Determination of algae growth
At the beginning of the test, every 24h, that is, at 24h, 48h, and 72h, samples are taken from each test container for microscopic examination and growth determination. A small amount of test liquid is aspirated for determination. After the determination is completed, it is strictly forbidden to put the taken test liquid back into the test container. The determination items include algae cell concentration, optical density or chlorophyll, etc. The determination method is as follows: a) Cell counting: Under a microscope, use a 0.1mL counting frame or a blood cell counting plate to count the number of algae cells. When using a counting frame, the field of view method can be used, that is, all cells in the field of view of the microscope are counted. The magnification is 40×10, and at least 10 fields are counted for each piece. If the density of algae cells is small, the counting field of view should be appropriately increased, and the number of algae is accumulated according to the field of view. Each count (samples from the same batch) should be counted using the same method (number of fields of view, magnification, etc.). Each sample should be counted at least twice. If the counting results differ by more than 15%, the count should be repeated. If the workload is too large, samples can be taken first, fixed with Digol's solution, and stored for later counting. The workload of microscopic counting is large, and an electronic particle counter can be used if conditions permit. b) Optical density: Take a certain amount of test liquid and measure its optical density on a spectrophotometer. The wavelength can be 650nm, 663nm or other wavelengths. It can also be measured with a fluorescence photometer. c) Chlorophyll: After centrifugation or filtration, the sample is extracted with acetone, ethanol or other solvents and spectrophotometrically determined. It can also be measured with a fluorescence photometer.
GB/T218052008
7.2.5 Analysis and detection
At the beginning and end of the test, the pH value of the test solution of the control group and each treatment group should be measured. The difference in pH value should be less than 1.5. If the test substance is a metal or mixture that is partially ionized at the test pH, pH excursions must be limited to ensure reproducible test results. Excursions less than 0.5 are theoretically possible and can be achieved by ensuring adequate CO exchange between air and the test solution, for example by increasing the frequency of shaking. Another approach is to reduce the initial biomass or shorten the test period to reduce the demand for CO.
Establish an analytical method for the concentration of the test substance in the test solution, and take samples at the beginning of the test and regularly during the test to verify the initial concentration of the test solution in each treatment group and the exposure concentration during the test. If the concentration of the test substance can be maintained within 20% of the set concentration (or the initial measured concentration) during the test, measure the concentration of the test substance in the test solution of a high concentration group, a concentration group and a concentration group with approximately 50% growth inhibition at the beginning and end of the test: if not, measure the concentration of the test substance in the test solution of each concentration group. If the test substance is volatile, unstable or strongly adsorbed, it should be measured every 24 hours. The medium used for the analysis of the concentration of the test substance should be treated in the same way as the medium used for the test, that is, it must be inoculated with green and cultured under the same tea conditions as the test. If the concentration of the dissolved test substance is to be analyzed, the algae must be separated from the culture medium. It is best to use centrifugation to separate, and the algae can be precipitated at a low speed. The result calculation is based on the measured concentration. If the measured concentration is 80% to 120% of the set concentration (or the initial measured concentration), the set concentration or the initial measured concentration can be used for calculation; if it exceeds this range, the geometric mean of the measured concentration or the model of the decrease in the concentration of the test substance is used for the calculation of the results [[.
Compared to most other short-term aquatic toxicity tests, the algal growth inhibition test is a dynamic test system. The actual exposure concentration in the test is difficult to determine, especially for strongly adsorbed substances at low concentrations. Therefore, due to adsorption on algae in the biomass tower, the disappearance of the test substance in the test solution does not mean that it disappears from the test system. When calculating the results, it is necessary to check whether the decrease in the concentration of the test substance is accompanied by a decrease in the growth inhibition effect. If this happens, it is recommended to use an appropriate model of the decrease in the concentration of the test substance. If this does not happen, it is best to use the analytical results of the initial concentration (set or measured) for calculation. 7.2.6 Experimental observation
At the end of the test, examine the algal cells in the test solution to see if they are growing normally and healthily, and record and explain any abnormal conditions of the cells, such as deformities (caused by exposure to the test solution).
7.3 Limit test
If the results of the preliminary test show that the test substance has no observable effect on the test algae at a concentration of 100 mg/L (or at the maximum solubility in the test solution, which is greater than 100 mg/L), the formal test can use a limit test with a concentration of 100 mg/L. If the maximum solubility of the test substance in the test solution is greater than 100 mg/L, the maximum solubility is taken as the concentration of the limit test.
The limit test is repeated 6 times, and the control group and the treatment group should be carried out at the same time. All the above-mentioned contents about test conditions and quality assurance and quality control are applicable to the limit test. The response variables measured in the control group and the treatment group need to be compared and analyzed by statistical methods, such as the Student t test: if the variables obtained from the two groups are irregular, the t test method should be adjusted. 8 Quality Assurance and Quality Control
The test is valid only when the following indicators are met:
a) Within 72 hours after the start of the test, the algal cell concentration in the control group should increase by at least 16 times, that is, the specific growth rate should not be less than 0.92d-1. For commonly used test algae species, the growth rate is much higher than this (see Appendix A). If other slower growing algae species are used in the test, this indicator may not be met. If this happens, the test period should be extended until the algal cells in the control group grow exponentially and the concentration increases by 16 times: at the same time, the test period can also be shortened, but it should be at least 48 hours and the supply is sufficient, and the algal cell concentration in the control group should also increase exponentially and increase by 16 times.
b) At each stage of the test, such as 0d～1d, 1d~2d and 2d-3d, the average value of the coefficient of variation of the specific growth rate of the control group is less than 35%.
GB/T21805—2008
Throughout the test period, the coefficient of variation of the average specific growth rate of each parallel control group is not more than 7% for Pseudokirchneriella subcapitata and Desnadesmus subspicatus, and not more than 10% for other recommended algae. 9 Data and Reporting
9.1 Data Processing
9.1.1 Plotting Growth Curves
Use the measured alternative parameters (such as algal cell concentration, fluorescence) to express the biomass of algae in the test container. List the algal biomass in the test container of the control group and each treatment group at 24h, 48h and 72h in tabular form. With the algae biomass as the horizontal axis, the algae biomass as the vertical axis, and the average value of each parallel, the algae growth curves of the control group and each treatment group are drawn. Both logarithmic coordinate axes and linear coordinate axes can be used, but the growth curve drawn with the logarithmic coordinate axis is a straight line, and its slope is the specific growth rate of the algae, which can better express the growth pattern of the algae. Observe the growth curve and check whether the exponential growth of the control group during the test reaches the growth rate of the period. Carefully check all data points and related charts, check the original data and the links that may cause errors. Carefully check all data points that may deviate from the systematic error. If an obvious procedural error is found or the possibility of the error is very high, mark the relevant data points as outliers and exclude these data when performing the next statistical analysis (if the concentration of one of the parallels is zero, it may be that the parallel test solution is not inoculated with algae or the washing method of the blood is incorrect). The test report should state the reason for the data being excluded as abnormal values. Acceptable reasons are limited to procedural errors and precision differences. The statistical procedure for determining outliers is only applicable to this type of problem and cannot replace expert review. It is best to retain outliers in subsequent graphs and tables. 9.1.2 Parameter calculation
This standard should calculate the following parameters to evaluate the effects of the test substance on the species: a) Specific growth rate: the daily increase in biomass during the test period, see formula (1): Fi
_ InX, -lnX:
Where:
i-——specific growth rate from time to time, in units of days (d\\); biomass at time Xi;
X, —i biomass.
For each treatment group and control group, calculate the average value of each parallel for toxicity evaluation. 13
The average specific growth rate of the entire test period (usually 0d~3d) calculated using the set value of the initial inoculated biomass is more accurate than the calculation using the measured value. Especially when the biomass concentration is low, if a very precise instrument (such as a flow cytometer) is used to determine the biomass, the measured value of the initial biomass concentration can be used for calculation. Calculate and evaluate the specific growth rate of each day (0d~1d, 1d~2d and 2d~3d) during the test, and check whether the growth rate of the control group meets the requirements of quality assurance and quality control. If the specific growth rate on the first day is quite low compared with the total average specific growth rate, it means that the algae are in a growth stagnation period for one day. The growth stagnation period in the control group is eliminated or minimized by pre-culture. The growth stagnation in the exposure group indicates that the algae may recover after the test substance or that the exposure is reduced due to the loss of the test substance (including adsorption to the algae). Therefore, in order to evaluate the effect of the test substance on the growth of algae during the exposure period, the growth rate of each stage of the test should be evaluated. If there is a significant difference between the average specific growth rate and the growth rate of each stage, it means that the algae have not maintained continuous exponential growth. Therefore, the growth curve should be carefully checked. b) The inhibition rate based on the specific growth rate is shown in formula (2): I, = two × 100
In the formula:
I.-Inhibition rate based on specific growth rate, %; (2)
GB/T21805-2008
uc-the average value of the specific growth rate of each parallel control group: T--the specific growth rate of each parallel treatment group. Growth: During the test period, the growth of biomass is shown in formula (9): e=Y2×100
Wherein;
I, inhibition rate based on growth, %, Yc-average growth of each parallel group in the control group; Y-growth of each parallel group in the treatment group.
If a solvent control group is set up in the test, the solvent control group should be used as the standard for inhibition rate calculation. 3
The toxicity data obtained from the specific growth rate and growth are not comparable, and they should be distinguished when applying the test results. Due to different calculation methods, the EC. (i.e. E, C) obtained from the average specific growth rate is generally higher than the EC (i.e. E, C) obtained from the growth volume. Since only the calculation methods used are different, the difference in sensitivity of the two variables does not need to be specifically explained. The definition of the average specific growth rate is based on the exponential growth pattern of algae under nutrient-sufficient conditions. Toxicity evaluation is based on the effect of the test substance on the growth rate, rather than the absolute level of the control group specific growth rate, the slope of the dose-effect curve or the same period of the test. In contrast, the results based on growth are based on all these other variables. Due to the differences in species and strains, the ECs derived from the average specific growth rate and the maximum specific growth rate of the species in each test are also different. This response variable cannot be used to compare the sensitivity of species and strains to each substance. It is more scientific to use the average specific growth rate for toxicity evaluation.
9. 1.3 Draw a dose-effect curve
Use the logarithm of the test substance concentration as the horizontal axis and the inhibition rate of the specific growth rate or the inhibition rate of growth as the vertical axis to draw a regression curve, which is the dose-effect curve. When drawing the regression curve, the data that have been identified as abnormal values in the previous stage should be ignored. Use linear interpolation or other computer statistical software to obtain the key values of ECsp, ECI and (or) EC. However, this method may sometimes not be applicable:
a) The computer statistical software is not suitable for the obtained data. Expert review is more conducive to obtaining more reliable results. In this case, some software cannot even give a reliable solution (iteration is not concentrated, etc.). b) Standard computer statistical software is not suitable for handling data on stimulatory effects. 9.1.4 Statistical Analysis
Quantitative dose-effect relationships were obtained by regression analysis. Linear transformation of the data, such as probit, logarithmic, or Weibull units, may allow statistical analysis using weighted linear regression, but nonlinear regression is the preferred method that better handles irregular data and data that deviate from the curve but cannot be ignored. Irregular data that are difficult to analyze, such as those close to 0% inhibition or 100% inhibition, may be exaggerated by transformation. Note that standard methods for transforming to probit, logarithmic, or Weibull units are designed for quantal data (e.g., mortality or survival) and must be modified to make them compatible with growth and biomass data. Specific procedures for determining the EC from continuous data are described in Refs. [9] [10] and [11]. Appendix C details the use of nonlinear regression analysis.
Statistical analysis was performed for each response variable and the EC- and its 95% confidence interval were calculated based on the dose-effect relationship. (If possible. It is best to plot or statistically analyze whether the response data is consistent with the regression model. Use the data obtained from each parallel instead of the average of each parallel for regression analysis. If the table data is too scattered and it is difficult or impossible to use a nonlinear curve, the average of each parallel should be used for regression, which can reduce the influence of possible outliers. If you choose to do so, it should be explained and verified in the report as a deviation from the routine procedure, because it is not a good result for the curve to be consistent with individual parallels. If the data cannot be used to calculate the EC5 and its 95% confidence interval through the available regression model/method, linear interpolation can be used.
The average values of the effects of the test substance on algal growth in each treatment group are compared by single-solid analysis of variance (ANOVA) to obtain LOEC and NOEC. Appropriate multiple comparison methods must be used to compare whether there is a significant difference between the average values of each treatment group and the average value of the group. CB/T 21805—2008
If the data are different, it is recommended to use Dunnett's method or Williams' method for multiple comparisons, see references [12], [14], [15][16]. [17]. It is necessary to evaluate whether the assumption of homogeneity of variables in ANOVA is valid. This can be evaluated by drawing or testing [1. It is recommended to use Levcne's method or Bartlett's method to test whether the variables are homogeneous. If the result is negative, it should be corrected by logarithmic transformation. If the non-homogeneity is abnormally significant and difficult to correct by data transformation, it is recommended to use decreasing regression. The Jonkheere trend test was used for analysis. Other inferences on determining NOEC can be found in reference [11]. Recent scientific developments have advocated abandoning the concept of NOEC and replacing it with EC_. For algae tests, the most appropriate value has not yet been determined. Values in the range of 10% to 20% are relatively appropriate (depending on the response variable). It is best to give both EC1 and EC9.1.5 Growth stimulation
tests may observe a low growth stimulation effect (negative inhibition rate). This effect may be caused by hormesis (hormesis effect) or by the addition of growth stimulating factors in the test substance to the minimum amount of culture medium. Note that the addition of inorganic nutrients does not have a direct effect because the nutrients in the culture medium are kept in excess during the test. When calculating EC, if a significant stimulatory effect is observed or the median of the EC to be solved is low, it is recommended to use the hormesis model. Otherwise, it can be ignored. When calculating, try to avoid removing the hormesis effect from the original data. If the statistical software used does not allow the occurrence of stimulatory effects (secondary status), use linear interpolation.
9.1.6 Algal growth inhibition caused by non-test substance fungi Since shading will reduce effective light, the growth inhibition phenomenon is aggravated for test substances with light absorption. The effects on algal growth caused by physical and chemical properties should be distinguished by modifying the test conditions and test methods. For relevant difficulties, see references [2] and [3]. 9.2 Test report
The test report should include the following:
) Test substance
Physical properties and relevant physical and chemical properties, including solubility in water: Chemical characteristics (such as CAS number),Including purity: b) The species, source or provider of the tested organisms
algae, and the culture conditions. ) Step conditions
Test start date and duration:
Test design: Test container, culture volume and biomass density at the start of the test; culture medium composition;
Test concentration and parallel (such as the number of parallels, the number of test concentrations and their common ratios); test solution preparation, including the use of solvents; culture equipment;
light intensity and light quality (source, whether it is uniform); temperature:
concentration determination: set concentration and all measured concentrations, the method of adding recovery test and the minimum detection limit of the instrument should be stated; all deviations from this standard;
the determination method of biological exhaustion, and the relationship between the measured parameters and dry weight. d) Results
pH value of each treatment group at the beginning and end of the test - Method of biomass determination, and biomass in each test container at each measurement point: Growth curve (biomass-time):
- Calculated values of each parameter for each parallel, and its mean value and coefficient of variation; - Dose-effect curve:
- Evaluation of the toxicity of the test substance to algae, such as ECs, ECoECa and their confidence intervals. LOEC and NOEC and their statistical 9
GB/T 21805—2008
Method (optional);
If one-way analysis of variance (ANOVA) is used for statistics, indicate the least significant difference: Any results that have a stimulatory effect on algal growth in each group; Other effects observed, such as morphological changes in algae; Discussion of results, including explanations for deviations. 10
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