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GB/T 35515-2017: Chemicals -- Fish assay for oestrogenic and androgenic activity, and aromatase inhibition
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Chemicals -- Fish assay for oestrogenic and androgenic activity, and aromatase inhibition
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GB/T 35515-2017
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Basic data | Standard ID | GB/T 35515-2017 (GB/T35515-2017) | | Description (Translated English) | Chemicals -- Fish assay for oestrogenic and androgenic activity, and aromatase inhibition | | Sector / Industry | National Standard (Recommended) | | Classification of Chinese Standard | A80 | | Classification of International Standard | 13.300; 13.020.40 | | Word Count Estimation | 38,321 | | 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 35515-2017: Chemicals -- Fish assay for oestrogenic and androgenic activity, and aromatase inhibition
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Chemicals-Fish assay for oestrogenic and androgenic activity, and aromatase inhibition
ICS 13.300; 13.020.40
A80
National Standards of People's Republic of China
Chemical fish estrogens, androgens and aromatase
Inhibitory activity test method
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 Fish euthanasia 4
8.4.4 Observing Secondary Characteristics 4
8.4.5 Determination of vitellogenin 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 230 (2009) "21d Fish Test. Short-Term Screening Test for Estrogen, Androgen and Aromatase Inhibition Activity" (English)
Consistent.
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, Guangdong Province Microbial Analysis and Detection Center, Shanghai Testing Center, Ningbo
Entry Inspection and Quarantine Bureau.
The main drafters of this standard are. Cui Yuan, Chen Huiming, Zeng Guofu, Li Haishan, Xie Wenping, Mei Chengfang, Yin Haowen, Liang Yihuai, and Chen Xiaoqing.
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 21d exposure period, the end points of the biomarkers for male and female fish were determined and used as test chemicals for estrogen stimulation.
Prime, aromatase inhibition or androgenic activity indicators. These test endpoints include vitellogenin and secondary sexual characteristics. For black-headed octopus, Japanese green pheasant
And zebrafish can be assayed for vitellogenin, while secondary sexuality can only be determined on blackheaded and Japanese barley.
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.
Chemical fish estrogens, androgens and aromatase
Inhibitory activity test method
1 Scope
This standard specifies the methodological overview, quality assurance, and quality of test methods for the inhibition of estrogenic, androgen and aromatase activities of chemicals in fish
Controls, equipment, test preparation, test procedures, test procedures, data, and reports.
This standard applies to the test of inhibitory activity of estrogens, androgens and aromatase enzymes in fish.
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.
ELISA. enzyme-linked immunosorbent assay
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. For other biological test indicators, refer to Appendix C, Table C.1. Set 3
Concentration of test substance and a blank control, if necessary, solvent control. For barley and zebrafish, at least 2 per test concentration
Test containers (5 male fish and 5 female fish per test container); for blackheads, at least 4 test concentrations per test concentration
(2 male fish and 4 female fish are included in each test vessel). 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
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