Chemicals—Sediment-water chironomid toxicity test—Spiked sediment method
Some standard content:
1cs 13.300
National Standard of the People's Republic of China
GB/T 27859—2011
Chemicals
Sediment-water chironomid toxicity test-Spiked sediment imethod
2011-12-30 Issued by
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China Standardization Administration of China
2012-08-01 Implementation
This standard was drafted in accordance with the rules given in GB/T1.1-2009 GB/T27859--2011
This standard has the same technical content as the Organization for Economic Cooperation and Development (OECD) Guidelines for Testing Chemicals 218 "Tests for Chironomid Toxicity in Sediment-Water Systems with the Sediment Method (English Version)". This standard has made the following structural and editorial revisions: - To be consistent with existing national standards, the name of the standard is changed to "Tests for Chironomid Toxicity in Sediment-Water Systems with the Sediment Method"; "Chironomid Toxicity in Sediment-Water Systems" in QECD 218 is used as the "Introduction" of this standard; "Terms and Definitions" in Annex 1 of QECD 218 is added as the third clause of this standard. This standard is proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC251). Drafting units of this standard: Jiangsu Entry-Exit Inspection and Quarantine Bureau, China Institute of Inspection and Quarantine. Who drafted this standard: Zhang Junxiang, Chen Huiming, Zhou Qiuhong, Xu Tan, Ding Hua, Lu Weiqun TTTKAONTKACA
GB/T27859-2011
This standard is used to evaluate the effects of long-term exposure of chemicals to Chironomus spp. in sediments. It is based on the existing toxicity test methods for Chironamus ripari and Chironamus entans, which have been established in Europe and North America and have passed comparative tests. This standard can also use other well-documented mosquito species, such as Chironomus yoshimatsziripi. The appropriate exposure scenario should be selected according to the application purpose of the test. The exposure scenario in this standard is to add a quantitative amount of the test substance to the sediment in a sediment-water system, that is, spike the sediment. This exposure scenario is used to simulate the accumulation level of chemicals present in sediment.
The test substance to be tested with organisms in sediment can usually exist in this system for a long time. Sediment organisms can be exposed to the test substance through various pathways. The relative importance of each exposure pathway, and the contribution of each exposure duration to the overall toxic effect, depends on the physicochemical properties of the relevant chemicals. For adsorbed substances (such as substances with a concentration of 1g>), or substances covalently bound to sediment, allowing the test organism to ingest food spiked with the test substance may be an important exposure pathway. In order not to underestimate the degradability of highly lipophilic substances, it may be considered to add feed to the sediment before using the test substance. In order to take all potential exposure pathways into consideration, this standard will focus on long-term exposure, with test durations of 20d-28d for C.riparius and C.yoshimatsui and 28d-65d for C.tentans. 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 abandoned 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 increase the 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. Compared with natural sediments, prepared sediments have several advantages: First, because prepared sediments are renewable "standardized matrix", the uncertainty of the experiment is reduced, and there is no need to find a source of clean sediment that is not contaminated. The
test can be started at any time without having to face seasonal changes in the test sediments, and 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 sediments in the field for routine testing:
The use of prepared sediments allows the data to be compared and the toxicity of substances to be classified.TTKANYKAcA
1 Scope
Chemicals
Test for Chironomid Toxicity in Sediment-Water Systems
Spiked Hand Sediment Method
This standard specifies the test method for evaluating the toxicity of chironomids in sediment-water systems by the spiked hand sediment method. GB/T 27859-—2011
This standard is applicable to the evaluation of the effects of long-term exposure to chemicals on the larvae of the genus Cironomid (Cironm sP.) in sediment-water.
2 Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) shall apply to this document. GB/T 21809 Acute toxicity test for chemicals 3 Terms and definitions
The following terms and definitions apply to this document. 3.1
formuated sediaent
prepared sediment
mixture of the physical composition of natural streams, which may be called regenerated sediment, artificial sediment or synthetic sediment. 3.2
overlying water
water above the sediment in the test container. 3.3
interstitialwater orporewaterwater between sediment and soil particles.
spiked sediment
test sediment to which the test substance has been added. 4 Principle
The first instar mosquito is exposed to a series of sediment-water systems containing different concentrations of the test substance for the test. First, add the test substance to the sediment. After the sediment and water in the beaker have aged, introduce a chironomid larvae into the beaker. At the end of the test, measure the number of chironomids emerging and the development rate. If necessary, the number and mass of surviving larvae can also be measured after 10 days (using appropriate additional parallel samples). The measured experimental data can be analyzed by regression model to estimate the concentration that causes the emergence rate or larval survival rate or growth rate to decrease by <% (such as 15% effective concentration EC15, half effective concentration EC, etc.), or use 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 Test substance information
The solubility, vapor pressure, distribution in sediment obtained by measuring or calculation, and stability in water and sediment of the test substance should be known. A reliable analytical method is also required for the quantitative determination of the test substance in overlying water, interstitial water and sediment, with known and reported accuracy and detection limits. Useful information also includes the structural formula, purity, chemical fate (such as attenuation, biological and non-biological degradation, etc.) of the test substance. If the physicochemical properties of the test substance make it difficult to conduct this test, refer to the relevant literature-, 6 Reference substances
Tests of reference substances should be 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, trifluralin, pentafluorophenol, sawyl chloride and potassium chloride [1-2.5-,a7]. 7 Quality Assurance and Quality Control
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 70% [1.; b) C. riparius and C. yashimatsui in the control container should emerge as adults within 12 to 23 days after introduction; C. tentans should take 20 to 65 days; c) At the end of the test, the pH value and dissolved oxygen concentration in each test container should be measured. The dissolved oxygen concentration should not be less than 60% of the air saturation value (ASV) at that temperature, and the pH value of the overlying water in all test containers should be between 6.0 and 9.0; d) The water temperature does not change by more than ± 1.D. The water temperature can be controlled in a constant temperature chamber and recorded at appropriate time intervals. B. Description of test method
8.1 Test container
The test is carried out in a 600mL glass beaker with a diameter of 8cm. Other containers may also be used, but appropriate depths of overlying water and sediment should be ensured. The sediment surface should be sufficient to provide an area of 2cm to 3cm for each larva. The ratio of the sediment depth to the overlying water depth should be 1.4. The test container and other utensils in direct contact with the test system should all be made of glass or other chemically inert materials (such as vinyl).
, B.2 Selection of test species
The appropriate species selected for the test is C. irius. C. tentans can also be used, but the test operation is more difficult and the test period is longer. Cyoshimatsui can also be used. Appendix A provides detailed cultivation methods for C. riparius. Cultivation conditions for other species are also available, such as C, tentans and C. yoshimatsuicn). The test species should be confirmed before the test, but if indoor-cultured organisms are used, such confirmation 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 characterized (at least pH value and organic carbon content should be measured; other parameters such as carbon-nitrogen ratio and particle size are also recommended), and the natural sediment should be uncontaminated and free of other organisms that compete with or prey on the midges. Before using it in the chironomid toxicity test, it is recommended to age the natural sediment for several days under conditions similar to those of the subsequent test. The following prepared sediment is based on the artificial soil used in GB/T21809. It is recommended to use t.412
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GB/T 27859—2011
—4%～5% (dry weight) peat in this test: pH value should be between 5.5 and 6.0 as much as possible; it is important to use powdered peat, ground into powder (particle size ≤1 mm), and air-dried. - 20% (by weight) aged clay (preferably with a kaolinite content greater than 30%). - 75%~76% (by weight) quartz sand (mainly fine sand, more than 50% of the quartz sand particles should be between 50μm and 200μm). - Add deionized water to make the moisture content of the final mixture 30%~50%. - Add chemically pure magnetic acid (CaCO,) to adjust the pH value of the final mixture to 7.D ± 0.5. - The organic carbon content of the final mixture should be 2.0% ± 0.5%, which can be adjusted with appropriate amounts of high-speed peat and quartz sand respectively. 8.3.2 The sources of peat, aged clay and quartz sand should be clear. The sediment components should be checked for chemical pollution (such as heavy metals, organic gas compounds, organic phosphorus compounds, etc.). Appendix B describes the preparation of the prepared sediment. If it can be demonstrated that no separation of sediment components (e.g., floating of peat particles) occurs after the addition of overlying water, and if the peat or sediment has been sufficiently aged, the preparation may also be made by direct mixing of the dry components.
Appendix and Appendix C list the chemical parameters of water release that may be used. Any water that meets these parameters may be used as test water. Any suitable water, including natural water (surface water), prepared water (see Appendix A), and dechlorinated tap water, may be used as culture water and test water if the chironomids can survive in it without discomfort throughout the entire incubation and test process. At the beginning of the test, the pH value of the test water should be between 6.0 and -9.0, and the total hardness should not exceed 400 mg/L (calculated as CaCO3). However, if there is a reaction between the cyclic hardness ions and the test substance, water with a lower hardness should be used (thus, EledtMt medium cannot be used in this case, see Appendix B). The same type of water should be used throughout the entire test process. The water quality characteristics listed in Appendix C should be measured at least twice a year and should also be tested when there is a concern that these characteristics may change significantly. 8.5 Reserve Standard - Spiked Sediment
The selected concentration of spiked sediment is usually prepared by adding the test substance solution directly to the sediment. The test substance is dissolved in deionized water to prepare a stock solution, and then the stock solution is mixed with the prepared sediment using an oscillator, agitator or manual mixing. If the test substance is poorly soluble in water, it can be dissolved in as little as possible of a suitable organic solvent (such as n-hexane, propane or chloroform); this solution is mixed with 10g of fine quartz sand, and every 10g of quartz sand is suitable for one test container. The organic solvent is evaporated until it is completely removed from the quartz sand. The quartz sand is then mixed with an appropriate amount of sediment in a beaker. Only volatile reagents can be used to dissolve, disperse or emulsify the test substance. When preparing the sediment, the amount of quartz carried by the mixture of the test substance and quartz sand should be considered (i.e., the amount of quartz sand used in the sediment is reduced by the relative pressure). Care should be taken to ensure that the test substance added to the sediment is completely and evenly distributed in the sediment. If necessary, duplicate samples can be analyzed to determine its uniformity.
9 Design of the experiment
9.1 General
The design of the experiment involves the selection of the number of groups and the interval between concentrations (group intervals) in the test volume, the number of test containers for each concentration, and the number of larvae in each test container. The estimation of EC points, the estimation of NOECs, and the design of limit tests should be described. 9.2 Design of regression analysis
9.2.1 The effect concentration (e.g., EC15, ECs) and the effect concentration range of the test substance of interest should be contained within the range of liquids used in the test. In general, the accuracy and especially the validity of the estimated effect concentration (E center) can be improved when the effect concentration is within the range of test concentrations. The situation of being much lower than the lowest positive concentration and much higher than the highest concentration should be avoided. Preliminary tests to explore the concentration range are helpful in selecting the concentration range to be used.
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9.2.2 If it is necessary to estimate EC, 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 concentrations tested should be sufficient. The proportional factor between concentrations should not be greater than 2 (unless the slope of the dose-response curve is very small). When the number of groups of test concentrations with different responses increases, the number of parallel samples for each concentration can be reduced. Increasing the number of parallel sample groups 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 10, additional parallel samples are required.
9.3 Design of NOEC/LOEC Evaluation
If NOEC/LOEC needs to be evaluated, 5 test concentrations should be set, with at least 4 parallel samples for each concentration, and the concentration interval ratio factor should not be greater than 2. In order to ensure sufficient statistical power to detect a difference of 20% from the control group at a significant level of 5% (power = 0.05>), the number of parallel sample groups should be sufficient. For evaluating growth rate, analysis of variance (ANOVA) can be used, such as Dunnett's test and Wil-lieans test = -1p. When evaluating eclosion rate, Cochra-Armitagc test, Fishcr Exact test (Bonferroni correction) or Mantel-Hacntzal test. 9.4 Limit test
If no effect is observed in the preliminary test to explore the concentration range, a limit test (one test concentration and one control concentration) can be carried out. The purpose of the limit test is to test at a concentration high enough to exclude possible toxic effects of the substance. The limit value is set at a concentration that is not expected to be toxic under any circumstances. The recommended concentration is 1000ng/kg (dry weight). At least 6 parallel samples are required for the test and control. . It should be demonstrated that the trial has sufficient statistical power to detect a 20% difference from the control group at a significance level of 5% (yield minus 0.05). For the measured effect outcomes (growth rate and mass), if the data meet the requirements of the 1 test (normality, homogeneity), then the test is an appropriate statistical method. If the data do not meet these requirements, a test for unequal variances can be applied, or a nonparametric test such as the Wilcoxon-Mann-Whithcy test, or for eclosion rate, the Fishcr exact test can be used. 10
Test Steps
10. 1 Exposure conditions
10.1.1 Preparation of the spiked sediment-water system 10.1.1.1 Addition of the test substance It is recommended to refer to the spiked procedure described in GB/T 21809. Place the spiked sediment in the test container and add overlying water to form a sediment-water system with a volume ratio of 1:4 (see 8.1 and 8.4). The thickness of the sediment layer should be 1.5 cm~3 cm. When adding the test solution to the water body, in order to avoid sediment stratification and re-suspending small particles in the water, a round plastic sheet can be placed on the sediment, and the condensate can be poured on the plastic sheet and then immediately removed. Other suitable containers can be used. 10.1.1.2 A glass cap or similar cover should be placed on the test container. If necessary, water may be added during the test to make up the initial volume to compensate for the evaporation of water. Only distilled or deionized water should be added to avoid the addition of salt. 10.1.2 Aging
After the standard sediment-water system is prepared: the test substance should be allowed to redistribute between the water phase and the sediment [4..13]. Aging should be carried out under the same temperature and ventilation conditions as the test. The appropriate equilibrium time depends on the characteristics of the sediment and the chemical, and may be from hours to days, and in rare cases up to several weeks (4 weeks to 5 weeks). Since long periods of time will cause degradation of many chemicals, it is not necessary to wait for equilibrium to occur, and it is recommended to set the equilibrium time to 48. At the end of the equilibrium period, the concentration of the test substance in the upper water, interstitial water and sediment should be tested. At least the distribution of the test substance in the test system with the highest test concentration and a lower concentration should be tested. These analytical determination results of the test substance can be used to calculate the mass balance and express the test results related to the measured concentration. 10.1.3 Addition of test organisms 10.1.3.1 Four to five days before the addition of the test organisms to the test container, the egg masses should be removed from the incubator and transferred to a small container filled with the selection medium (GB/T 27859-2011). The aged medium in the incubator or the newly prepared medium can be used. If the latter is used, green and/or a few drops of filtrate from a suspension of finely ground sliced fish food (see Appendix A) should be added to the culture medium. Only freshly laid egg masses can be used. Under normal circumstances, the larvae begin to hatch 2 to 3 days after the eggs are laid (C.riparius, 2 to 3 days at 20°C; C.tentans, 1 to 4 days at 23°C; D.osmatui, 1 to 4 days at 25°C). The larvae grow to adults in four instars, with each instar lasting 4 to 4 days. The test is conducted on first instar larvae (23 or 14 days after hatching). The first instar of the larvae can be determined by filling the test container with 10.1.3.2 Using a blunt-ended pipette, 20 first instar larvae are randomly placed in each test container containing the spiked sediment and water. When the larvae are added to the test container, ventilation must be stopped and the container must be kept for 24 h. The number of larvae added to each concentration depends on the different experimental designs used. When using the EC point estimation method, at least 60 larvae should be added to each concentration, and when using the NOEC determination method, at least 80 larvae should be added. 10.1.4 Test concentrations
10.1.4.1 In order to select the concentration range for the formal test, a pilot test of the concentration range can be carried out: for this purpose, a series of widely spaced concentrations of the test substance are used. The appropriate test concentration can be estimated by exposing the mosquitoes to each concentration of the test substance for a period of time under the same mosquito surface density as in the formal test. Replicate samples are not required for pilot tests of the concentration range. 10.1.4.2 The concentration of the test substance to be used in the formal test shall be determined based on the results of preliminary tests to explore the concentration range. At least five concentrations shall be selected. The concentrations shall be selected in accordance with the requirements of 9.2 and 9.3. 10.1.5 Controls
Control containers shall be prepared for the test. These containers do not contain the test substance, but contain sediment. An appropriate number of parallel samples shall be prepared for the control containers (see 9.2 and 9.3). If a solvent is used in 8.5, a sediment solvent control shall be added. 10.1.6 Test system
Static systems should be used. In 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 precipitation of minerals from the sediment affects the permanent pH or hardness), semi-static or flowing systems can also be used to renew the upper water column intermittently or continuously. However, it is generally better to use other methods of improving water quality, such as aeration, and avoid using semi-static or flowing systems. 10.1.7 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, a fish diet (an aqueous suspension or a ground fine feed, such as Tetra-Min or Tetra-Phyll, see Appendix A) containing 0.25 mg/d to 0.5 mg/d (0.35 mg to 0.5 mg for C. yoshimatti) per larva should be sufficient. For larger larvae, the diet should be slightly more: 0.5 mg/d to 1.0 tg/d per larva should be sufficient for the remainder of the test. If fungal growth is found or mortality is observed in the control, the amount of feed provided should be reduced in all samples and controls. If fungal growth cannot be stopped, the test should be repeated. When testing strongly adsorbed substances (e.g. substances with a K of > 5 per 1 g) or substances covalently bound to the sediment, sufficient feed should be added to the prepared sediment before the aging period to ensure the survival and natural growth of the larvae. For this purpose, a plant-based feed should be used instead of fish feed. For example, 0.5% (by weight) of ground plant leaves from Uitzica dioeca, Morusalba, Trifolium repens, Spinacia oleracea, or other plant materials (Cerophyl or cellulose) should be added. 10.1.8 Incubation conditions
10.1.8.1 Gently aerate the upper water in the test container 24 h after the addition of the larvae and maintain this condition until the end of the test (care should be taken that the oxygen content does not fall below 60% of the ASV). Aeration is carried out by means of a glass Pasteur pipette fixed 2 to 3 cm above the sediment (i.e. 1 or more bubbles per second). When testing volatile chemicals, the sediment-water system should not be ventilated. 10.1.8.2 The test should be carried out at a constant temperature of 20 ℃ ± 2 ℃. For C. tentans and C. yashimatui, the recommended temperature is 23 ± 2 ℃ and 25 ± 2 ℃, respectively. A 16-h photoperiod is usually used, and the illuminance should be 500 lux to 1000 lux. 5
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10.1,9 Exposure time
The maximum exposure starts when the larvae are added to the spiked container and the control container. The maximum exposure time for C. riparius and C. yoshimatui 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.
10.2 Observation
10.2.1 Emergence
10.2.1.1 Determine the development time and total number of fully emerged females. Males can be easily identified by the fusion of their feathers. 10.2.1.2 At least three observations per week, visually assess any abnormal behavior of larvae in spiked containers (e.g., leaving the sediment, abnormal swimming) compared with larvae in control containers. During the expected emergence period, count the number of emerging mosquitoes daily and record the sex and number of fully emerged mosquitoes daily. After identification, remove mosquitoes from containers. All egg masses laid before the termination of the test should be recorded and then removed to prevent larvae from reintroducing into the sediment. The number of pupae that fail to emerge that can be observed should be recorded. The method for measuring emergence is shown in Appendix D. 10.2.2 Development and Survival
If data on the survival and development of larvae up to 10 days are required, additional test containers should be prepared at the beginning of the test for use in subsequent tests. The sediment from these additional test containers should be filtered through a 250 mesh filter to remove larvae. Death is defined as dead or unresponsive to mechanical stimulation. Larvae that are no longer found should also be counted as dead (larvae that died early in the test may have been decomposed by microorganisms). Determine the dry weight of surviving larvae in each test container (which should be free of foreign matter) and calculate the average dry weight of each larva in each test container. It is useful to determine the age of the surviving larvae, and for this purpose the head shell thickness of each larva should be measured. 10.3 Analytical Tests
10.3.1 Concentration of Test Substance wwW.bzxz.Net
10.3.1.1 Before the start of the test (i.e., the addition of larvae), the sediment from at least one of the test containers should be removed for the concentration of the test substance for each test concentration. At the beginning and end of the test, at least the overlying water, intermediate water and sediment samples in the highest concentration group and one lower concentration group should be measured. The test results of the test substance concentration can show the behavior or distribution of the test substance in the water-sediment.
10.3.1.2 When measurements are required during the sensitive period (e.g., the 7th day) and when more 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 observations, but only for sampling and analysis. 10.3.1.3 It is recommended to separate the source water by centrifugation under the following conditions: 10,000g (g is the value of the acceleration of free fall), 4℃, and 30min. However, if it can be proved that the test substance is not adsorbed on the filter, filtration can also be used. In some cases, it is almost impossible to analyze the concentration of interstitial water because the sample volume is too small.
1.3.2 Physical and chemical parameters
Measure the pH and temperature of the basic test container by appropriate methods (see Chapter 7). At the beginning and end of the test, the hardness and nitrogen content of the highest filled test container and the control container should be measured. 11 Data and reporting
11.1 Handling of results
11.1.1 The purpose of this test is to determine the effect of the test substance on the development rate of chironomids 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 test lasting 6
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10 days. If there is no evidence of differences in statistical sensitivity between the sexes of mosquitoes, the results of males and females can be combined and the presence or absence of differences in sensitivity can be determined by statistical methods, such as the X\-r×2 table test. During the 10-day test, the number of surviving larvae and the average dry weight of individuals in each container should be determined. 11.1,2 The effect concentration is calculated based on the concentration of the test substance in the sediment measured at the beginning of the test. The effect concentration should be based on dry mass. 11.1,3 For the calculation of ECsc or other EC values, the statistical data values of each test container can be regarded as the true parallel test values. When calculating the confidence interval for any EC, the deviations between these values should be taken into account or shown to be negligible. When the least squares model is used, the statistical data for each test container should be transformed to make them homogeneous. However, when calculating the EC, the response data should be transformed back to their original values.
11.1.4 When the statistical analysis is performed by hypothesis testing for the determination of NOEC/LOEC, the deviations between the statistical data for each test container should be taken into account. In this case, a set of ANOVA methods can be used: or, when the usual ANOVA settings are not met, more adequate studies are needed [2].
11.2 Eradication rate
11.2.1 Eradication rate is a discrete data. When the dose-response relationship is expected to be unidirectional and the data are consistent with this expectation, the Cochren-Armitage test can be used by the method of regression. Otherwise, Fisher's exact test or the Mantel-Haentzal test with Bonfeeroni-Holm correction should be used. When the deviation of parallel tests at the same concentration is larger than the binomial distribution (often referred to as "extra-binomial deviation"), the robust Cochran-Armitage test or Fisher's exact test should be used. 11.2.2 The total number of mosquitoes that emerged in each test container, n, is divided by the number of larvae added, na, to obtain the emergence rate, as shown in formula (1): ER = n
Where:
ER—·-emergence rate;
n. - number of mosquitoes that emerged in each container;, number of larvae added to each container.
11.2.3 When there is an extra-binomial deviation, the most suitable alternative method for large sample sizes is to treat the emergence rate as a continuous effect value. When the dose- When the response relationship is expected to be unidirectional and the ER value is consistent with this, a procedure such as the Williamson test should be used. When the dose-response relationship does not remain unidirectional, the Duanett test is appropriate. This defines a large sample size as: the number of emergences and non-emergence in a parallel experiment (one container) is greater than 5. 11.2.4 When using the ANOVA method, the ER value should be transformed by square root-arcsine or Turkcy-Freeman transformation to obtain an approximate normal distribution and homogeneity of variance. When using absolute frequencies, the Cachran-Artnitagc test, Fisher's exact (Bonferrari) test or Mant's test can be used. El-Haentzal test. The square root-arcsine transformation is the arcsine of the square root of ER (sine-value
11.2.5 Use regression analysis to calculate the emergence rate EC, (for example, you can use probit[\], logit, Weibull, or suitable commercial software, etc.). If regression analysis cannot be used (for example, some effect values are less than two), use other non-parametric methods such as moving average or simple interpolation. 11.3 Development rate
11.3.1 The average development time represents the average time from the introduction of insects (experimental day 0) to the emergence of a large group of experimental adults (in order to correctly calculate the development time, the age of the larvae when introduced should be considered). The development rate (unit: 1/d) is the reciprocal of the development time and represents the rate of larvae that emerge per day. For the evaluation of sediment sex studies, the development rate is more important because it is less biased, more uniform, and closer to a normal distribution than development time; thus more valid parametric testing procedures can be used to calculate the development rate rather than development time. Because the development rate is a continuous effect value, EC values can be estimated using regression analysis (32-z3). 11.3.2 For the following statistical tests, the number of mosquitoes observed on the observation day x is assumed to have emerged from the day -L to the day -L (L = the length of the observation interval, usually 1d). The average development rate () for each container is calculated according to formula (2): GB/T 27859—2011
In the formula;
Average development rate of each container;
Observation interval index:
Observation interval maximum value:
Number of chironomids that emerged during the observation interval: f
Total number of chironomids that emerged until the end of the experiment (= f) Development rate of chironomids that emerged during the observation interval 1. 11.3.3#
Development rate of chironomids that emerged during the observation interval r: Calculated according to formula (3): r -1/(d; --
In the formula:
d:——number of days of observation (calculated from the date of addition of larvae), 1:——length of the observation interval (date, usually 1d). 11.4 Test report
11.4. 1 Test substance:
(2)
- Physical properties, relevant physical-chemical properties [solubility, vapor pressure, distribution coefficient in soil spots (or sediments), water stability, etc.];
Chemical identification information (common name, chemical name, structural formula and CAS number, etc.), including purity and quantitative analysis methods. 11.4.2 Test organisms:
- Species, scientific name, source and breeding conditions of the organisms used in the test; - Information on the method of handling eggs and larvae; - Age of the test organisms when added to the test container. 11.4.3 Test conditions:
Sediment used, such as natural or artificially prepared sediment! 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 =Disk, carbon-nitrogen ratio and particle size (if appropriate); Preparation of prepared sediment: composition and characteristics (organic carbon content before the start of the test, H value, moisture, etc.); Preparation of test water (if prepared water is used) and its characteristics (oxygen concentration, pH value, conductivity, hardness, etc. before the start of the test); Thickness of sediment and overlying water;
Volume of overlying water and interstitial water, mass of sediment with and without interstitial water; Test container (material and size);
Method of spiking sediment: test concentration, number of parallel sample groups and solvent used (use) Stable equilibrium phase of the spiked sediment-water system: duration and state: incubation conditions: temperature, photoperiod and intensity, ventilation (frequency and intensity): Specific information on feeding including type of feed, preparation, feeding quantity and feeding plan. 11.4.4 Results, theoretical test concentration, actual measured test concentration and all analytical results of the test substance content in the quick test container: water quality in the test container, such as pH, temperature, dissolved oxygen, hardness and ammonia content: if compensation for evaporated test water is made during the test, it should be recorded; the number of male and female mosquitoes grown in each container each day; the number of larvae that can grow into chironomids in each container GB/T27859—2011
- the average dry mass of individual mosquitoes in each container; if appropriate, measure the average dry mass of each instar; the percentage of emergence (male and female mosquitoes combined) of each parallel sample and each test concentration; the average development rate and treatment rate of fully emerged adult mosquitoes in each test container (male and female mosquitoes combined); estimated toxic effect values, such as EC (and its related confidence interval), NOEC and/or LOEC, and the statistical methods used
discussion of the results, including the effect of any deviation from this standard on the conclusions of the test. 9
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