Chemicals—Sediment-water chironomid toxicity test—Spiked water method
Some standard content:
ICs13.300
National Standard of the People's Republic of China
GB/T27858—2011
Chemicals
Sediment-water chironomid toxicity test-Spiked water method
Chemicals-Sediment-water chironomid toxicity test-Spiked water methodIssued on 2011-12-30
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaAdministration of Standardization of the People's Republic of China
Implementation on 2012-08-01
This standard was drafted according to the rules given in GB/11.1-2009. GB/T27858—2011
This standard has the same technical contents as the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide 219 Chemical Testing Guide - Chironomid Toxicity Test in Sediment-Water Systems - Spiked Water Method (April 2004) (English version). This standard has made the following structural and editorial changes: First, to be consistent with the existing series of national standards, the standard name is changed to "Chemical Sediment-Water System Chironomid Toxicity Test Spiked in Water Method":
The "Introduction" in OECD 219 is used as the "Introduction" of this standard; Annex 1 "Terms and Definitions" in OECD 219 is used as Chapter 3 "Terms and Definitions" of this standard; Annex 2, Annex 3, Annex 4 and Annex 5 correspond to Appendix A, Appendix B, Appendix C and Appendix D of this standard respectively. This standard is proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC 251). The drafting units of this standard are: Jiangsu Exit-Entry Inspection and Quarantine Bureau, China Chemical Economic and Technological Development Center. The main drafters of this standard are: Gan Hongsong, Tang Zhajun, Liu Junfeng, Wang Xiaobing, Dai Yucheng. I
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This standard is used to evaluate the effects of long-term exposure to chemicals on larvae of the genus Chironomus sp. in sediments. This standard is mainly based on the BBA standard, using a sediment-water system, including human soil and water column exposure scenarios11, and also draws on existing toxicity test methods for Chironamus riparius and Chiranomus tentans, which have been established and passed comparative tests in Europe and North America1. This standard may also use other well-documented chironomus species, such as Chironamus yoshimazsti-ia-i. The exposure scenario of this standard is the addition of the test substance to water. The appropriate exposure scenario should be selected based on the application purpose of the test. Water exposure scenarios (including the addition of the test substance to the water column) are intended to simulate the drift of pesticide spraying, while also taking into account the initial peak concentration in the interstitial water. Except when the duration of the accumulation process is longer than the test period, this standard is also useful for other types of exposure (including chemical splashes).
Test substances to be tested with organisms in sediments can usually exist in this system for a long time. Organisms in sediments can be exposed to the test substance through a variety of pathways. The relative importance of each exposure pathway, and the contribution of each exposure time to the overall toxic effect, depends on the relevant chemical. Physical and chemical properties of the chemical. For strongly adsorbed substances (e.g. substances with an octanol/water partition coefficient of 1gK.>5) or substances covalently bound to sediments, ingestion of food spiked with the test substance by the test organism may be an important exposure route. In order not to underestimate the toxicity of highly lipophilic substances, it may be considered to add a slurry to the sediment before using the test substance. In order to take all possible exposure routes into consideration, this standard will focus on long-term exposure. The test duration for C.riparius and C.yoshimatsui is 20d to 28d, and for C.tent4ns is 28d to 65d. If short-term data are required for special purposes, such as studying the toxic effects of unstable chemicals, additional parallel samples can be used for the test and discarded after 10 days. The final results of the test are the total number of adults that emerged and the time of emergence. If additional short-term data are required, it is recommended to only add additional parallel tests appropriately and measure the survival and growth of larvae after 10 days of the test. This standard recommends the use of artificial prepared sediments, which have several advantages over natural sediments: Because prepared sediments are renewable "standardized substrates", experimental uncertainties are reduced, and there is no need to find uncontaminated sources of clean sediments;
... The test can be started at any time without having to face seasonal changes in the test sediments; there is no need to pre-treat the sediments to remove native fauna; the use of prepared sediments also reduces the costs associated with collecting sufficient amounts of sediment in the field for standard tests;
: The use of prepared sediments allows toxicity data to be compared with each other, thereby classifying the toxicity of substances. TTTKAONYKACA
1 Scope
Test for Chironomid Toxicity in Chemical Sediment-Water System by Spiking Water
This standard specifies the test method for evaluating the toxicity of chironomids in sediment-water system by spike-in-water method. GB/T27858—2011
This standard is applicable to the evaluation of the effects of long-term exposure of chemicals on the larvae of the genus Chirooms SP. in sediment-water.
2 Normative References
The following documents are essential for the application of this document. For all dated references, only the dated version applies to this standard. For all undated references, the latest version (including all amendments) applies to this document. GB/T 21809 Chemicals Test for Acute Toxicity to Earthworms and Shrimp 3 Terms and Definitions
The following terms and definitions apply to this document
Forinulated sediment is a mixture used to simulate the physical composition of natural sediments. It may also be called regenerated sediment, artificial sediment or synthetic sediment. 3.2
Overlying water
Water above the sediment in the test container. 3.3
Interstitlal water or pore water Water between sediment and soil particles.
Spiked water
Test water to which the test substance has been added. 4 Principle of the Test
The first-instar Chironomid larvae are exposed to a series of sediment-water systems containing different concentrations of the test substance for the test. At the beginning of the test, the first-instar Chironomid larvae are introduced into the beaker containing the sediment-water system, and then the test substance is added to the water. At the end of the test, the number of chironomids emerging and the rate of development are measured. If necessary, the number and mass of surviving larvae can also be measured after 10 days (using appropriate additional replicate samples). The experimental data can be analyzed by regression model to estimate the concentration that causes a 15% decrease in the emergence rate or larval survival rate or growth rate (such as 15% effective concentration ECis, half effective concentration ECs, etc.), or by statistical hypothesis testing to determine the no observable effect concentration (NOEC) or the lowest observable effect concentration (LOEC). The latter requires a statistical test method to compare the effect value with the control value. 1
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5 Information on the test substance
The solubility, vapor pressure, measured or calculated distribution in sediment, and stability in water and sediment of the test substance should be known. There is also a need for a reliable analytical method for the quantitative determination of the test substance in overlying water, interstitial water and sediment, and it has a known confirmed accuracy and detection limit. Useful information also includes the structure, purity, and chemical fate of the test substance (such as decay, biological and non-biological degradation, etc.). If the physicochemical properties of the test substance make it difficult to conduct this test, please refer to the literature [12]. 6 Reference substances
Tests on reference substances are carried out regularly to verify the reliability of the test plan and test conditions. Reference poisons that have been successfully used in comparative tests and effectiveness studies include lindane, fluralin, pentachlorophenol, radium chloride and potassium chloride [12; 1.5-13]. 7 Quality assurance and quality control
In order for the test to be effective, the following conditions should be met: a) At the end of the test, the emergence rate of the control group should not be less than 0%1.6; h) C.riparius and C.yoshinatsui in the control container should emerge as adults within 12d23d after introduction; C. tentans requires 20 d~65 dt
c) At the end of the test,The pH and dissolved oxygen concentration should be measured in each test container. The dissolved oxygen concentration should not be less than 60% of the air saturation value (ASV) at that temperature. The pH of the overlying water in all test containers should be between 6.0 and 9.0. The water temperature should not vary by more than ±1.0°C. The water temperature may be controlled by a constant temperature chamber and recorded at appropriate intervals. 8 Description of the Test Method
8.1 Test Containers
The test is conducted in a 600 mL glass beaker with a diameter of 8 cm. Other containers may be used, but appropriate depths of overlying water and sediment should be ensured. The sediment surface should be sufficient to provide an area of 2 cm* to 3 cm for each larva. The ratio of the sediment layer depth to the overlying water depth should be 1:4. The test containers and other equipment in direct contact with the test system should be made of glass or other chemically inert materials (such as tetrafluoroethylene).
8.2 Selection of Test Species
The appropriate species selected for the test is C. riparius. C. tentans may also be used, but the test is more difficult and the test period is longer. C. yoshimatsui may also be used. Appendix A provides detailed culture methods for C. ribarius. Cultivation conditions for other species are also available, such as C. tentansl4I and C. yasi matsui [1]. The species to be tested should be confirmed before the test, but if indoor cultured organisms are used, this is not necessary before each test.
8.3 Sediment
8.3.1 Prepared sediment is preferred. If natural sediment is used, its properties should be identified (at least pH and organic carbon content should be measured, and other parameters such as carbon-nitrogen ratio and degree are also recommended), and the natural sediment should be uncontaminated and free of other organisms that compete with or prey on the midges. It is recommended that the natural sediment be aged for several days under the same conditions as in subsequent tests before use in mosquito toxicity tests. The following prepared sediment is based on the artificial soil used in GB/T 21809 and is recommended for use in this test = 1.4.15. 1. 4%~5% (dry weight) charcoal: pH value should be between 5.5~6.0 as much as possible; it is important to use powdered peat, ground into powder (particle size ≤1mm), only air drying.
-20% (dry weight) kaolin clay (kaolinite content is preferably greater than 30%). GB/T27858——2011
---75%~76% (weight) quartz sand (mainly fine sand, more than 50% of the quartz sand particles should be 50μm~~200μm). . Add deionized water to make the moisture content in the final mud compound 30%~50%. 2. Add chemical absolute calcium carbonate (CaCO.) and adjust the pH value of the mixture to 7.0±0.5. The organic carbon content in the final mixture should be 2.0% ± 0.5%, which can be adjusted with appropriate amounts of the above-mentioned peat and quartz sand. 8.3.2 The sources of peat, kaolin clay and quartz sand should be clear. Sediment components should be checked for chemical pollution (such as heavy metals, organic chlorides, organic phosphorus compounds, etc.). Appendix B describes the preparation of prepared sediments. If it can be confirmed that no separation of sediment components will occur after the addition of overlying water (such as the floating of peat particles) and the peat or sediment has been fully aged, it can also be prepared by directly mixing the dry components. bzxz.net
Appendix A and Appendix C list the chemical indicators of the dilution water that can be used. Any water that meets these indicators can be used as test water. During the entire cultivation and test process, if the midges can survive in it without showing any discomfort, any suitable water, including natural water (surface water), prepared water (see Appendix A) and deoxygenated tap water, can be used as cultivation water and test water. At the beginning of the test, the pH of the test water should be between 6.0 and 9.0 and the total hardness should not exceed 400 mg/L (calculated as CaCO). However, if there is a suspicion of a reaction between the hardness ions and the test substance, water with a lower hardness should be used (thus, in this case, Elendt M4 medium cannot be used). The same type of water should be used throughout the test. The quality characteristics listed in the appendix should be determined at least twice a year and should also be tested when these characteristics may change significantly. 8.5 Stock solution - spiked water
The test concentration is calculated based on the concentration of the water column (the water body located in the sediment). The test solution of the selected concentration is usually prepared by diluting the stock solution. The test substance is dissolved in the test medium to prepare a stock solution. Sometimes, some solvents or dispersants are used to prepare a stock solution of appropriate concentration. The solvents that can be selected are: acetone, ethanol, methanol, ethylene glycol-ethyl ether acetate, ethylene glycol-dimethyl ether, dimethylformamide and triethylene glycol. The dispersants that can be used are: polyoxyethylene ether (10) hydrogenated sesame oil, Tween 80, methylcellulose 0.01 and HCO-40. In the final test medium, the concentration of the cosolvent should be as low as possible (e.g. not more than 0.1 II I/L) and the same in all treatment groups. If a cosolvent is used, a control test with only the cosolvent should be conducted to confirm that the cosolvent does not affect the survival of chironomid larvae or produce other two observed adverse effects. Therefore, the cosolvent should be used as little as possible. 9 Experimental Design
9.1 General
The experimental design involves the selection of the number of test concentrations and the interval between concentrations (group interval), the number of test containers for each concentration and the number of larvae in each test container. The estimation of the EC point, the estimation of the NOEC, and the design of the limit test should be described. 9.2 Design of regression analysis
9.2.1 The effect concentration (e.g. ECr5, ECs>) and the effect concentration range of the test substance of interest should be included in the concentration range used in the test. Generally speaking, the accuracy and especially the effectiveness of the effect concentration (EC_) can be improved when the effect concentration is within the test range. The situation that it is much lower than the lowest positive concentration and much higher than the highest concentration should be avoided. Preliminary experiments to explore the concentration range will help to select the concentration range to be used.
9.2.2 If the EC is to be estimated, at least 5 groups of test concentrations should be set, with 3 replicates for each concentration. In order to make the evaluation model more accurate, the test scale should be sufficient, and the concentration ratio factor should not be greater than 2 (unless the slope of the measurement is very small, when the test of different effects is 3
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When the number of test concentrations increases, the number of replicates for each concentration can be reduced. Increasing the number of replicates or reducing the interval between test concentrations can reduce the confidence interval of the test results. If it is necessary to evaluate the survival and growth of larvae at 10 days, an additional half-row of samples should be added.
9.3 Design for evaluating NOEC/LOEC
If NOEC/LOEC needs to be evaluated, 5 groups of test concentrations should be set, with at least 4 replicates for each concentration, and the concentration interval scaling factor should not be greater than 2. In order to ensure sufficient statistical power, at the 5% significance level (power = 0. 05) Detect a 20% difference from the control group. The number of parallel samples should be sufficient. If you need to evaluate the growth rate, you can use analysis of variance (ANOVA), such as Dunnett's test and Wiliatns test [ia-1e. When evaluating the emergence rate, you can use the Cochran-Armitage test, Fisher's exact test (Ponfcrroni positive) or Mantel-Haentzal test: 9. 4 Limit tests
If any effect is observed in the pilot study to explore the concentration range, a limit test (a test concentration and a control concentration) should be conducted. The purpose of a limit test is to test at a concentration high enough to enable the decision maker to exclude the possibility of a quasi-effect of the substance. The limit value is set at a concentration that is not expected to occur under any circumstances. The recommended concentration is 1000 mg/kg (dry weight). At least 6 replicates are required for each test and control. It should be shown that the test has sufficient statistical power to detect a difference of 20% from the control at a 5% significance level (p=0.05). For the measured effect outcomes (growth rate and mass), if the data meet the requirements of the test (normality, homogeneous variance), the test is an appropriate statistical method. If the data do not meet these requirements, the t-test with unequal variances or a non-parametric test such as the Wilcoxon-Mann-Whithey test can be used. For irradiation rate, the Fish's exact test can be used.
10 Test Procedure
10.1 Required Conditions
10.1.1 Preparation of Spiked Water-Sediment System 10.1.1.1 Add an appropriate amount of prepared sediment to the test container to form a thickness of at least 1.A layer of 5 cm should be added, followed by water of 6 cm depth. The ratio of sediment thickness to water depth should not exceed 1:4, and the thickness of the sediment layer should not exceed 3 cm. Before adding the test organism, the sediment-water system should be gently aerated for 7 days (see Appendix B). When adding the test solution to the water column, in order to avoid sediment stratification and re-stirring up fine particles suspended in the water, a plastic sheet can be placed over the sediment, the solution can be poured onto the plastic sheet and then the plastic sheet can be quickly removed. Other suitable containers can also be used. 10.1.1.2 A glass tray or other container should be placed over the test container. If necessary, water can be added during the test to make the water depth exceed the initial volume to compensate for the evaporation of water. Only distilled water or electric water should be added to avoid increasing the salt concentration. 10.1.2 Addition of test organisms
10.1.2.1 Four to five days before the test organism is introduced into the test container, the egg mass should be removed from the incubator and transferred to a small container with culture medium. You can use the ready-made aged medium in the incubator or the newly prepared medium. If the latter is used, add green algae to the culture medium, and/or filter out a small drop of the suspended liquid after grinding sliced fish food. Only eggs that have just been laid can be used. Under normal circumstances, the larvae begin to hatch 2d~3d after the eggs are born (C.riparius, 2d~3d at 20℃; C.Lentans 1d~4d at 23℃, C.yoshimatsui, 1d--4d at 25℃>), and the larvae grow to four instars from larvae to adults, and each instar is 4d~8d. The first-instar larvae (2d~3d or 1d~4d after hatching) were used for the experiment. d). The age of the larvae can be determined by measuring the width of their head shell. 10.1.2.2 Use a pipette to randomly add 20 first-instar larvae to each test container that has been added with standard water and sediment. Stop ventilation when adding larvae to the test container and keep it there for 24 hours after adding. The number of larvae added to each concentration depends on the different test methods used. When using the EC point estimation method, at least 60 larvae are required for each concentration, and when using the NOEC determination method, at least 80 larvae are required. 10.1.2 .3 24 hours after the addition of the larvae, add the test substance to the upper water column (use a pipette to pour a small amount of the test substance solution into the water surface), and again give slight aeration to carefully mix the upper water column, taking care not to stir up the sediment. 10.1.3 Test concentrations
10.1.3.1 In order to select the concentration range for the formal test, a preliminary test of the concentration range can be carried out; for this purpose, a series of widely spaced test substance concentrations are required. The chironomids are exposed to each test substance concentration for a period of time under the same chironomid surface density as in the formal test. Appropriate test concentrations can be screened out by estimation. No parallel samples are required for exploratory tests of a concentration range. 10.1.3.2 The test concentrations for the formal test are determined based on the results of the preliminary tests of the exploratory range, and at least five groups of concentrations should be selected. The concentrations selected should be consistent with the requirements of 9.2 and 9.3. 10.1.4 Control
During the test, control containers should be prepared. These containers do not contain the test substance, but contain sediment. An appropriate number of parallel samples should also be prepared for the control containers (see 9.2 and 9.3). If a certain solvent is used in 8.5, the sediment solvent should be added. 10.1.5 Test System
A static system should be used. In some special cases, such as when the water quality indicators become unsuitable for the test organisms or affect the chemical balance (for example: the dissolved oxygen content in the water is too low, the excreta content is too high, or the minerals precipitated from the sediment affect the pH value or hardness of the water), semi-static or flowing systems can also be used: intermittent or continuous renewal of the upper water column. However, it is usually better to use other methods to improve the quality of the upper water column, such as aeration, and avoid the use of semi-static or flowing systems. 10. 1.6 Feed
Larvae should be fed regularly, preferably once a day or at least three times a week. For small larvae during the first 10 days, 0.25 mg/d to 0.5 mg/d (0.35 mg to 0.5 mg for C. ynshzmatui) of fish feed (a water-based suspension or ground fine feed such as Tetra-Min or Tetra-Phyll, see Appendix A) per larva is sufficient. For larger larvae, slightly more feed should be used: 0.5 mg/d to 1.0 mg/d per larva should be sufficient for the remainder of the experiment. If fungal growth is found or mortality is observed in the control, the feed supply should be reduced in all experimental and control groups. If fungal growth cannot be stopped, the experiment should be repeated. When testing strongly adsorbed substances (such as K5 substances) or substances that covalently bind to the coagulant, a diet sufficient for human use in sediment should be prepared before the aging period to ensure survival and natural growth of the larvae. In this case, a vegetable diet may be used instead of a fish diet. For example, 0.5% (dry weight) of ground leaves from Uitica dioeca, Morusatba, Trifolium repens broth (Spinacia aleracea), or other vegetable materials (Cerophyl or cellulose). 10.1.7 Incubation conditions
10.1.7.1 Gently aerate the upper water in the test vessel 24 h after the addition of the larvae and maintain this condition until the end of the test (care should be taken that the dissolved oxygen concentration does not fall below 60% of the ASV). Aeration is carried out by means of a glass Pasteur tube fixed 2 to 3 cm above the sediment layer (i.e. 1 or more bubbles per second). When testing volatile chemicals, the sediment-water system should not be aerated. 10.1.7.2 The test should be carried out at a constant temperature of 20 °C ± 2 °C. For C. tentans and C. oshimatti, the recommended temperatures are 23 °C and 25 °C ± 2 °C respectively. A 16 h photoperiod is usually used and the light intensity should be 5001x~1000[x, 10.1.8 Exposure time
Exposure starts when the larvae are in the spiked containers and control containers. The maximum exposure time for C. riparius and C. yoshimatu is 28 days, and for C. tentans it is 65 days. If the mosquitoes emerge early, the test can be ended at least 5 days after the last adult emerges in the control container.
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10.2 Observation
10.2.1 Emergence
10.2.1.1 Determine the development time and the total number of fully emerged male and female mosquitoes. Male mosquitoes can be easily identified by their feathery antennae. 10.2.1.2 At least three observations per week should be made to assess any unusual behaviour of larvae in the spiked containers (e.g. leaving the sediment, unusual swimming) compared with larvae in the control containers. During the expected period of emergence, the number of emerging mosquitoes should be counted daily and the sex and number of fully emerged mosquitoes recorded at each stage. After identification, mosquitoes should be removed from the containers. All egg masses laid should be recorded before the end of the test and then removed to prevent reintroduction of larvae into the sediment. The number of observed failures to emerge should be recorded. The method for measuring emergence is given in Appendix D. 10.2, 2 Development and Survival
If data on the survival and development of larvae to 10 days are required, additional test containers should be prepared at the beginning of the test so that they can be used in subsequent tests. The sediment removed from these additional test containers should be filtered through a 250 μm filter to remove the larvae. Mortality is defined as dead or unresponsive to mechanical stimulation. Larvae that are not recovered should also be counted as dead (those that died early in the test may have been decomposed by microorganisms). Determine the dry weight of the surviving larvae in each test container (which should be free of foreign matter) and calculate the average dry weight of the individual larvae in each test container. It is useful to determine the age of the surviving larvae and, for this purpose, measure the width of the head capsule of each larva.
10.3 Analytical tests
10.3.1 Concentrations of the test substance
10.3.1.1 At the beginning (preferably 1 h after the addition of the test substance) and at the end of the test, samples of the overlying water, the intermediate water and the sediment should be taken from at least the highest concentration group and one of the lower concentration groups; these concentrations of the test substance provide an indication of the behaviour and distribution of the test chemical in the water-sediment system. Sampling of the sediment at the beginning of the test may affect the test (e.g. by removing the test larvae); therefore, if appropriate, separate test containers should be used for sampling at the beginning and during the test (see 10.3.1.2). If, under comparable conditions (e.g. flow-to-water ratio, application type, organic carbon content of sediment), the distribution of the test substance in water and sediment can be clearly determined in water-sediment studies, it is not necessary to measure the concentration of the test substance in the flow. 10.3.1.2 When measurements are to be made during the experiment (e.g. 7th day) and a large number of samples need to be analyzed, the removal of samples will inevitably affect the entire test system. In this case, additional test containers should be used; these additional test containers are treated under the same conditions as the formal test containers (including the introduction of the test organisms), but are not used for biological observation, but only for sampling and analysis. 10.3.1.3 It is recommended to separate the interstitial water by centrifugation under the following conditions: 10 000 g (g is the value of the free fall acceleration) and 1°C for 30 min. However, filtration can also be used if it can be demonstrated that the test substance is not adsorbed on the filter. In some cases, it is almost impossible to analyze the intermittent concentration because the sample size is too small.
10.3.2 Physical and chemical parameters
Measure the pH and temperature in the test container by appropriate methods (see Chapter 7). At the beginning and end of the test, the hardness and nitrogen content of the test container with the highest concentration and the control container should be measured. 11 Data and reporting
11.1 Results processing
11.1.1 The purpose of this test is to determine the effect of the test substance on the development rate of chironomid mosquitoes and the total number of fully emerged male and female mosquitoes, or to determine the effect of the test substance on the number and mass of surviving larvae in a 10-day test. If there is no indication that there is a difference in statistical sensitivity between the sexes of chironomids, the results of males and females can be combined. The existence of a difference in sensitivity can be determined by statistical methods, such as the X\-×2 table test. During the 10-day test, the number of larvae present and the mean dry weight of the larvae in each container should be determined. 11.1.2 The effect concentration should be calculated based on the concentration of the test substance in the sediment at the beginning of the test. The effect concentration should be based on the dry mass. 11.1.3 For the calculation of EC or other EC values, the statistical value of each test container can be regarded as the true replicate value. When calculating any confidence interval, the deviation between these values should be taken into account or it should be shown that the deviation is small enough to be ignored. When the least squares model is used, the statistical value of each test container should be transformed to improve the homogeneity of the deviation. However, when calculating the EC value, the response data should be transformed back to the original value.
11.1.4 For the determination of NOEC/LOEC, when the hypothesis test method is used for the analysis of the variance, the deviation of the statistical value of each test container should be taken into account. In this case, the nested ANOVA method can be used; or, when the standard ANOVA setting is not met, more and more sufficient experiments are required [2].
11.2 Emergence rate
11.2.1 Emergence rate is a discrete data. When the dose-effect relationship is expected to be unidirectional and the data are consistent with this expectation, the Cachran-Armitage test can be performed using the method of regression. Otherwise, the Fisher's exact test or the Mantel-Haentzal test with the Bonfeeroni-Halm test should be used. When the deviation of parallel experiments at the same concentration is greater than the binomial distribution (usually called "extra-binomial" deviation), the full Cochran-Armitage test or Fisher's exact test should be used. 1I.2.2 The total number of mosquitoes that emerged in each test container, n. + divided by the number of larvae added, n, is the emergence rate, see formula (1): ER-\
Wu:
ER——Emergence rate
Yuan·The number of mosquitoes that emerged in each container, n-the number of larvae added to each container.
---+( 1
11.2.3 When there is an exobinomial bias, the alternative method that is most suitable for large sample sizes is to treat the emergence rate as a continuous effect value. When the dose-effect relationship is expected to be unidirectional and the ER value also meets this expectation, a procedure such as the Williams test should be used; when the dose-effect relationship does not remain unidirectional, the Dunnett test is applicable. This single defines a large sample size as: the number of emergences and non-emergence in one experiment (one container) exceeds 5. 11.2.4 When using the ANOVA method, the ER value should be transformed by square root-arcsine or Turkey-Ferccman to obtain an approximate normal distribution and homogeneity of variance. When using absolute numbers, the Cochran-ArmitaBe test, Fisher's fine (Bonferroni) test, or Mantel-Haentzal test can be used. Square root-arcsine transformation is a test for ER. The square root of the inverse sine (sina-l) of the value.
11.2.5 Calculate the emergence rate EC using regression analysis (e.g., probit-31, logit, Weibull, or suitable commercial software). If regression analysis is not possible (e.g., there are fewer than two partial effect sizes), use other nonparametric methods such as moving averages or simple interpolation.
11.3 Eclosion rate
11.3.1 Mean developmental time represents the average time from the introduction of larvae (or experimental day 0) to the emergence of a large group of experimental adults (in order to correctly calculate the developmental time, the age of the larvae at the time of introduction should be considered). The developmental rate (unit: I) is the reciprocal of the developmental time and represents the rate of larvae emerging per day. For the evaluation of sediment toxicity studies, the developmental rate is more important because it is less biased, more uniform, and closer to a normal distribution than the developmental time, so more valid parametric testing procedures can be used to calculate the developmental rate rather than the developmental time. Because developmental rate is a continuous effect value, EC, values can be estimated using regression analysis. L2 = -23]. 11.3.2 For the following statistical tests, the number of chironomids observed on the observation day is assumed to be those that have emerged from the rth to the Lth day (the length of the observation interval, usually d), and the average developmental rate per container [) is calculated according to formula (2): 7
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to—average developmental rate per container, observation interval index;
maximum value of observation
f number of chironomids that have emerged during the observation interval; 1
to the total number of midges that have emerged from the last stage of the experiment (-b); developmental rate of midges that have emerged during the observation interval. 11.3.3 The development rate of the larvae during the observation interval 1 is calculated according to formula (3): r: -1/(d, -
: number of days of observation (calculated from the date of addition of larvae); tt - length of the observation interval (the date band is 1 day). 11.4 Test report
11.4.1 Test substance:
. (3)
Characteristics, relevant physico-chemical properties [water solubility, vapor pressure, distribution coefficient in soil (or sediment), water stability, etc.]; chemical identification information (common name, chemical name, structural formula and AS number, etc.), including purity and quantitative analysis method. 11.4.2 Test organisms:
--The species, scientific name, source and breeding conditions of the organisms used in the test; - Information on the method for handling egg masses and larvae; - The age of the test organisms when added to the test container. 11.4.3 Test conditions:
Sediments used: natural or artificially prepared sediments. For natural sediments, the location of the sediment sampling area should be recorded and described. If possible, the pollution history and characteristics of the natural sediments should also be included: pH value, organic carbon content, carbon-nitrogen ratio and particle size (if applicable). Preparation of prepared sediments: composition and characteristics (pH value before the start of the test , organic carbon content, pH value, moisture, etc.: preparation of test water (such as using prepared water) and its characteristics (oxygen content, pH value, conductivity, hardness, etc. before the start of the test); - thickness of sediment and overlying water;
.... - volume of soil overlying water and interstitial water; the mass of sediment with and without interstitial water; test container (material and size)
preparation method of stock solution and test concentration! Use of test substance: test concentration of test substance used, number of parallel samples and solvent used (if used): incubation conditions: temperature, photoperiod and intensity, ventilation (frequency and intensity); specific information on feeding including type of feed, preparation, feeding quantity and slow feeding plan. 11.4.4 Results:
theoretical test concentration, actual measured test concentration and all analytical results of test substance in quick test container; - quality of water in test container, such as: pH value, temperature, dissolved oxygen content,Hardness and ammonia content - if compensation for evaporation of the test water is made during the test, the number of male and female midges grown in each container each day should be recorded; the number of larvae that failed to grow into chironomids in each container; the average dry mass of individual larvae in each container; the average dry mass of each instar, if appropriate; the percentage of emergence for each replicate and each concentration tested (male and female mosquitoes combined); the average development rate and treatment rate of fully emerged adults in each test container (male and female mosquitoes combined); the estimated toxic effect value, such as EC (and its associated confidence interval), N (EC and/or LC) EC, and the statistical method used; discussion of the results, including the impact of any deviation from this standard on the conclusions of the test,
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