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GB/T 35517-2017: Chemicals -- Fish short term reproduction toxicity assay
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Chemicals -- Fish short term reproduction toxicity assay
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GB/T 35517-2017
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Basic data | Standard ID | GB/T 35517-2017 (GB/T35517-2017) | | Description (Translated English) | Chemicals -- Fish short term reproduction toxicity assay | | Sector / Industry | National Standard (Recommended) | | Classification of Chinese Standard | A80 | | Classification of International Standard | 13.300; 13.020.40 | | Word Count Estimation | 38,392 | | Date of Issue | 2017-12-29 | | Date of Implementation | 2018-07-01 | | Issuing agency(ies) | General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, Standardization Administration of the People's Republic of China | GB/T 35517-2017: Chemicals -- Fish short term reproduction toxicity assay---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Chemicals-Fish short term archetype toxicity assay
ICS 13.300; 13.020.40
A80
National Standards of People's Republic of China
Short-term test methods for chemical fish reproductive toxicity
Published on.2017-12-29
2018-07-01 Implementation
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China
China National Standardization Administration released
Directory
Preface III
Introduction IV
1 Scope 1
2 Terms, definitions and abbreviations 1
3 Method Overview 1
4 Quality Assurance and Quality Control 2
5 Equipment 2
6 Test preparation 2
6.1 Test Water 2
6.2 Domestication of fish 2
6.3 Pre-exposure and fish selection 3
7 Test procedure 3
7.1 General instructions 3
7.2 Selection of test concentration 3
8 Test procedure 3
8.1 Selection and weighing of test fish 3
8.2 Exposure conditions 3
8.2.1 Time 3
8.2.2 Domestication 3
8.2.3 Lights and Temperature 4
8.3 Test frequency 4
8.4 Observation 4
8.4.1 Mortality 4
8.4.2 Behavior and appearance 4
8.4.3 Fertility 4
8.4.4 Euthanasia of fish 4
8.4.5 Observing Secondary Characteristics 4
8.4.6 Determination of vitellogenin 5
8.4.7 Gonadal Histopathology Evaluation 5
9 Data and Reports 5
9.1 Evaluation of Biomarker Response Using ANOVA
9.2 Test Result Report 5
Appendix A (informative) Sample collection procedure for vitellogenin analysis 7
Appendix B (informative annex) Assessment of the detection of secondary sexual characteristics of blackheadhead and barley with specific endocrine-active substances16
Appendix C (Informative Appendix) Experimental Conditions for Fish Endocrine Screening Test 21
Appendix D (informative) Spawning substrates for zebrafish and blackheaded fish23
Appendix E (Informative) Some Chemical Characteristics of Acceptable Dilution Water 25
Appendix F (Informative Appendix) Analysis of Yolk Protein Addition and Inter-Platform Standards 26
Appendix G (Informative) Statistical Analysis Process 27
Appendix H (Normative Appendix) Standards for Interpretation and Acceptability of Test Results 28
References 29
Figure A.1 Cut along the pectoral fins with scissors 8
Figure A.2 Using scissors along the abdomen line into the abdomen about 2mm from the head of the anus
Figure A.3 Using surgical forceps to open the abdominal wall, exposing the liver and other internal organs (or securing the abdominal wall laterally) 9
Figure A.4 Slowly separating and resecting the liver with forceps 10
Figure A.5 Slowly removing the intestine with surgical forceps 10
Figure A.6 End of the bowel and any mesentery appendages cut with scissors 11
Figure A.7 (female) Same for the female fish process 11
Figure A.8 Complete the entire process 12
Figure A.9 Segmentation of zebrafish fish head 15
Figure B.1 Number and size of black-headed fish beads 17
Figure B.2 Gender Differences in Anal Fin Shape and Size 19
Figure B.3 Protuberance process on the joint plate of the hip fin line 20
Figure B.4 Fish with cutting points Photo 20
Figure D.1 Zebrafish Spawning Substrates 23
Figure D.2 Blackhead spawning substrate 24
Figure G.1 Statistical analysis flow chart 27
Table B.1 Blackhead Fish Star Template 18
Table C.1 Experimental conditions for the fish endocrine screening test 21
Table E.1 Some Chemical Characteristics of Acceptable Dilution Water 25
Foreword
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
The technical content of this standard and the Organization for Economic Cooperation and Development (OrganizationforEconomicCooperationandDevelopment,
OECD) Test Guideline 229 (2012) "Fish Short Term Reproductive Toxicity Test (2012)" (in English) Consistent technical content.
This standard is proposed and managed by the National Standardization Technical Committee for Hazardous Chemicals Management (SAC/TC251).
This standard was drafted by. China Academy of Inspection and Quarantine, China National Accreditation Service for Conformity Assessment, Ningbo Entry-Exit Inspection and Quarantine Bureau,
Guangdong Province Microbial Analysis and Testing Center, China Chemical Industry Economic and Technological Development Center, and Shanghai Testing Center.
The main drafters of this standard. Cheng Yan, Cui Yuan, Tang Wei, Sun Yun, Chen Huiming, Li Haishan, Xie Wenping, Cao Mengran, Yin Haowen, Zhang Jingyi, Zeng Guoji,
Mei Chengfang.
Introduction
This test method describes an in vivo screening test that exposes sexually mature male fish to females in the oviposition phase to chemistry
21d in the product. At the end of the 21-day exposure period, the end points of the biomarkers for male and female fish were determined as test chemical substances
Secretion activity index. These test endpoints include vitellogenin and secondary characteristics for yolks of black-headed sturgeon, Japanese clams, and zebrafish
Protoplasma assays, while secondary sexuality can only be determined on blackheaded and Japanese barley. Monitor fertility every day throughout the testing process;
Retention of gonads can assess the reproductive health of the test animals by histopathology and also provide stronger evidence for other test endpoints.
Vitellogenin is usually stimulated by circulating endogenous estrogens and is produced by the liver of female oviparous vertebrates. Vitellogenin is
The precursor of vitellin, once produced in the liver, enters the ovary from the blood stream and is absorbed and modified by the developing egg. The vitellogenin is incomplete
The plasma of the cooked females and males was barely detectable because they lacked enough circulating estrogen but were stung by exogenous estrogens.
When stimulated, the liver can synthesize and secrete vitellogenin.
The determination of vitellogenin can detect different estrogenic modes of action for chemicals, and the detection of estrogens may pass
The production of vitellogenin in males was measured. Reduction of estrogen circulating levels in females, eg by inhibiting the conversion of endogenous males
An aromatase that is a natural estrogen 17β-estradiol causes a decrease in vitellogenin levels, which is used to detect aromatase
Inhibiting properties of chemicals.
Use standardized tests that have been standardized. Use of immunochemical, species-specific enzyme-linked immunosorbent assay (ELISA) assays
In the method, the blood of blackheaded fish, the homogenate of the blood or head/tail of the zebrafish, and the liver of the barley were collected as a sample for the measurement of VTG. for
Barley, VTG measured in the blood has a good correlation with the liver. Appendix A provides analysis of vitellogenin
Sample collection recommended procedure. The vitellogenin can be tested by different methods, but all are based on validated species-specific ELISA.
method.
The secondary sexual characteristics of males of specific species are observable and quantitatively responsive to endogenous androgen circulating levels. These characteristics are in blackheads.
Fish and barley can be reflected, but the zebrafish is not reflected because the zebrafish does not have a quantifiable secondary sexuality.
For blackheaded fish, the main indicator of exposure to exogenous substances is the number of bead stars located on the female's lips. For barley, the number of mastoids
The major markers of exogenous exposure to androgens in females were composed. Appendix B illustrates the use of blackheaded fish and barley
The process of evaluating sexual characteristics.
The 21-day fish test included quantitative fecundity and gonadal histopathology. Additional end points may be required in the experiment
A more complete assessment of the reproductive health of the subject was conducted, or to prevent the vitellogenin and secondary sexual characteristics from responding to chemical exposure. although
Some of the endpoints are clear conclusions (eg male induces vitellogenin formation, female induces formation of bead) but not all of the tests
Endpoints (eg, fecundity and gonadal histopathology) directly indicate the mechanism of toxicity. In addition, endocrine disruption
To speculate. Although not a specific endocrine effect, due to its sensitivity to endocrine-disrupting active substances, fertility is considered
Consider the important end. Because when it and other endpoints are not affected, it is more certain that this compound is likely not to have endocrine dryness.
Disturb activity. When fertility is affected, it will provide strong evidence for inference. This test provides further interpretation of data results
Guidance on the acceptability of test results.
Short-term test methods for chemical fish reproductive toxicity
1 Scope
This standard specifies the methodological overview, quality assurance and quality control, instrumentation and equipment for short-term test methods for chemical fish reproductive toxicity.
Preparations, test procedures, test procedures, data and reports.
This standard applies to chemical fish reproductive toxicity test.
2 Terms, definitions and abbreviations
2.1 Terms and Definitions
The following terms and definitions apply to this document.
2.1.1
Loading rate loadingrate
The wet weight of a fish in a unit volume of water.
2.1.2
Carrying density stockingdensity
The number of fish per unit volume of water.
2.1.3
Vitellogenin vitelogenin;VTG
A phospholipid glycoprotein, a precursor of yolk protein, which is commonly found in sexually mature female individuals of all oviparous animals.
2.1.4
Maximum tolerated concentration maximumtoleratedconcentration;MTC
The test article causes the highest test concentration of less than 10% mortality.
2.2 Abbreviations
The following abbreviations apply to this document.
CV. coefficient of variation
ELISA. enzyme-linked immunosorbent assay
HPG axis. hypothalamic-pituitary-gonadal axis
3 Method Overview
Females and males in the breeding period were co-exposed to the test substance. According to the selected test fish species its relevant biomarker endpoint
Indicators are measured. Among them, sexual characteristics can be visually confirmed after dissection. See Appendix C for other biological test indicators. Set 3 test substances
As well as a blank control, a solvent control can be used if necessary. For barley and zebrafish, at least 2 test vessels per test concentration
(5 male fish and 5 female fish per test container); for blackhead fish, at least 4 test containers per test concentration (per
The test vessel included 2 tail males and 4 females). Exposure was conducted for 21 days and fish were sampled on Day 21 exposure.
On the 21st day, all animals were euthanized. For the detection of secondary sexual characteristics of black-headed finfish and barley, refer to Appendix B;
Blood samples of zebrafish and blackheaded fish, or zebrafish head/tail tissue homogenate samples for detection of vitellogenin, see Appendix A; for blue
Alas, collect the liver for the detection of vitellogenin (see Appendix A). After dissection, the whole gland is fixed for pathological evaluation.
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