《DD-ISO-TS-23893-2-2007.pdf》由会员分享，可在线阅读，更多相关《DD-ISO-TS-23893-2-2007.pdf（26页珍藏版）》请在三一文库上搜索。
1、DRAFT FOR DEVELOPMENT DD ISO/TS 23893-2:2007 Water quality Biochemical and physiological measurements on fish Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) ICS 13.060.70 ? Licensed CopyChinese University of Hong Kong, 20/10/2008 08:32, Uncontrolled Copy, (c) BSI DD ISO/TS 23893-2:2007
2、 This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 31 March 2008 BSI 2008 ISBN 978 0 580 53706 6 National foreword This Draft for Development is the UK implementation of ISO/TS 23893-2:2007. This publication is not to be regarded as a Brit
3、ish Standard. It is being issued in the Draft for Development series of publications and is of a provisional nature. It should be applied on this provisional basis, so that information and experience of its practical application can be obtained. Comments arising from the use of this Draft for Develo
4、pment are requested so that UK experience can be reported to the international organization responsible for its conversion to an international standard. A review of this publication will be initiated not later than three years after its publication by the international organization so that a decisio
5、n can be taken on its status. Notification of the start of the review period will be made in an announcement in the appropriate issue of Update Standards. According to the replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion in
6、to an international Standard, to extend the life of the Technical Specification or to withdraw it. Comments should be sent to the Secretary of the responsible BSI Technical Committee at British Standards House, 389 Chiswick High Road, London W4 4AL. The UK participation in its preparation was entrus
7、ted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/5, Biological methods. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsibl
8、e for its correct application. Amendments/corrigenda issued since publication DateComments Licensed CopyChinese University of Hong Kong, 20/10/2008 08:32, Uncontrolled Copy, (c) BSI Reference number ISO/TS 23893-2:2007(E) TECHNICAL SPECIFICATION ISO/TS 23893-2 First edition 2007-11-15 Water quality
9、Biochemical and physiological measurements on fish Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) Qualit de leau Mesurages biochimiques et physiologiques sur poisson Partie 2: Dosage de lthoxyrsorufine-O-dthylase (EROD) DD ISO/TS 23893-2:2007 Licensed CopyChinese University of Hong Kon
10、g, 20/10/2008 08:32, Uncontrolled Copy, (c) BSI ii Licensed CopyChinese University of Hong Kong, 20/10/2008 08:32, Uncontrolled Copy, (c) BSI iii Contents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references. 1 3 Principle. 1 4 Test environment 1 5 Reagents 2 6 Apparatus 3 7 Sampling a
11、nd preparation of samples 4 7.1 Sampling of fish 4 7.2 Killing of fish and dissection. 4 7.3 Storage. 4 8 Procedure 4 8.1 Preparation of the fractions. 4 8.2 Determination of protein 5 8.3 Determination of the EROD activity 5 8.4 Checking the sensitivity of the biological reagent and compliance with
12、 the operating method. 6 9 Expression of results . 7 9.1 Calculation of the EROD activity. 7 9.2 Statistical processing of the data . 7 10 Test report . 8 Annex A (informative) Examples of results. 9 Bibliography. 14 DD ISO/TS 23893-2:2007 Licensed CopyChinese University of Hong Kong, 20/10/2008 08:
13、32, Uncontrolled Copy, (c) BSI iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body int
14、erested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnic
15、al Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the
16、technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. In other circumstances, particularly when there is an urgent market requirement for such documents, a technical commit
17、tee may decide to publish other types of normative document: an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting
18、a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether
19、it will be confirmed for a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn. Attention is
20、 drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO/TS 23893-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological metho
21、ds. ISO 23893 consists of the following parts, under the general title Water quality Biochemical and physiological measurements on fish: Part 1: Sampling of fish, handling and preservation of samples Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) Technical Specification DD ISO/TS 23893
22、-2:2007 Licensed CopyChinese University of Hong Kong, 20/10/2008 08:32, Uncontrolled Copy, (c) BSI v Introduction The measurement of pollution biomarkers in fish, such as the measurement of biotransformation enzyme activities, is likely to provide information about exposure levels, bioavailability a
23、nd the early biological effects of substances present in aquatic ecosystems. The measurement of the EROD enzyme activity allows the diagnosis of the exposure of fish to inducers of the P450 1A cytochrome, such as certain polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and
24、dioxins. A large amount of research work bears witness to the extent of the studies conducted (see Bibliography). An induction of EROD activity reflects the presence of inducers such as those mentioned above. On the other hand, the absence of induction does not necessarily reflect the absence of exp
25、osure of the fish to organic contaminants, account being taken of the inhibition phenomena of the EROD induction of possible modification of the bioavailability of the inducers or of low exposure concentrations. The application of a standardised reference method is strongly advised within a monitori
26、ng programme. The intercalibration exercises on the measurement of the EROD enzyme activity undertaken since 1991 have revealed multiple sources of errors, which are very easy to avoid (dilution of resorufin, determination of proteins, calculation of the enzyme activity, etc.) once laboratories have
27、 become familiar with the analysis of enzyme activities and the possible error factors. DD ISO/TS 23893-2:2007 Licensed CopyChinese University of Hong Kong, 20/10/2008 08:32, Uncontrolled Copy, (c) BSI blank Licensed CopyChinese University of Hong Kong, 20/10/2008 08:32, Uncontrolled Copy, (c) BSI 1
28、 Water quality Biochemical and physiological measurements on fish Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) 1 Scope This part of ISO 23893 specifies a method for measuring the ethoxyresorufin-O-deethylase (EROD) enzyme activity on a post-mitochondrial fraction of fish liver homoge
29、nate (subcellular fraction in which the EROD activity is located) employing a cell or microplate fluorimetric method. It applies to fish that are sampled in their natural environment (fresh water or salt water) or exposed to substances or effluents in a laboratory. This method is applicable for EROD
30、 values greater than or equal to 1 pmol/(minmg) of proteins. A higher sensitivity may be achieved by using a cell (test tube) procedure. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited appl
31、ies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 23893-1, Water quality Biochemical and physiological measurements on fish Part 1: Sampling of fish, handling and preservation of samples 3 Principle Samples of fish are collected and di
32、ssected as described in ISO 23893-1 to obtain pieces of liver. Homogenates of fish liver are prepared by crushing (homogenisation) and the supernatant S9 fraction recovered by centrifugation. The EROD enzyme activity in the S9 fractions is determined by measurement of the increase in fluorescence du
33、e to the transformation of the 7-ethoxyresorufin into resorufin. The fluorescence is reported in quantities of resorufin by means of a calibration range (external calibration of resorufin or of rhodamine B). The EROD activity is related to the quantity of proteins in the S9 fraction. 4 Test environm
34、ent All of the tests and handling operations with the S9 fraction shall be carried out at a temperature close to 4 C (e.g. handling in crushed ice), except the enzyme reaction which shall be performed at 20 C 2 C. DD ISO/TS 23893-2:2007 Licensed CopyChinese University of Hong Kong, 20/10/2008 08:32,
35、 Uncontrolled Copy, (c) BSI 2 5 Reagents Unless otherwise specified, use only reagents of recognised analytical grade. 5.1 Ultra pure water, having a conductivity below 1 S/cm. 5.2 Potassium chloride, 150 mmol/l solution. Dissolve 11,2 g of KCl (relative molecular mass 74,6) in 1 l of water (5.1). T
36、his solution is stable for 6 months at a temperature of 4 C 3 C. 5.3 Phosphate buffer, 100 mmol/l; pH 7,8 0,1. The ionic composition and pH can affect the EROD activity, and optimal conditions may vary between species. The following buffer is expected to provide measurable and comparable values of E
37、ROD activity for most species of fish. Prepare the following two solutions, A and B: Solution A: dissolve 17,4 g of K2HPO4 (relative molecular mass 174,2 as the anhydrate) in 1 l of water (5.1); Solution B: dissolve 13,6 g of KH2PO4 (relative molecular mass 136,1 as the anhydrate) in 1 l of water (5
38、.1). Adjust solution A to pH 7,8 0,1 with solution B. This solution is stable for 6 months at a temperature of 4 C 3 C. 5.4 Glycerol (20 %) in phosphate buffer solution. Add a mass fraction of 20 % of glycerol (C3H8O3; relative molecular mass 92,1) to the phosphate buffer (5.3). The final concentrat
39、ions are then 20 % glycerol and 100 mmol/l phosphate. 5.5 Resorufin (108 mg/l) stock solution. Dissolve in the dark, shaking for 2 h, about 10,8 mg of resorufin (sodium salt C12H6NNaO3; relative molecular mass 235,2) in 100 ml of dimethylsulfoxide (DMSO). Read the optical density at 572 nm on the sp
40、ectrophotometer. Calculate the exact resorufin concentration, c, in millimoles per litre, using Equation (1): D c l = (1) where D is the optical density (corresponding to the absorbance wavenumber in cm1); is the molar extinction coefficient for resorufin, values of = 73,2 (mmol/l)1 cm1 at 572 nm (R
41、eference 17) and 54,0 1,1 (mmol/l)1 cm1 (Reference 36) have been reported; l is the optical pathlength, in centimetres. Prepare this solution at the time of determination and store aliquots of this solution frozen at 20 C and sheltered from the light. These aliquots can be stored for 6 months. NOTE
42、Resorufin is very unstable under daylight conditions. 5.6 Resorufin working solution. Dilute the stock solution (5.5) with DMSO to obtain approximately 10 ml of 11,5 mol/l working solution. Prepare this solution at the time of determination. DD ISO/TS 23893-2:2007 Licensed CopyChinese University of
43、Hong Kong, 20/10/2008 08:32, Uncontrolled Copy, (c) BSI 3 5.7 Rhodamine B standard solution. Dissolve 25 mg of rhodamine B (C28H30N2O3; relative molecular mass 442,55) in 250 ml of ethylene glycol monomethyl ether. Dilute this solution with ethylene glycol monomethyl ether, so as to obtain a standar
44、d 0,1 mol/l solution to be stored in aliquots. This solution remains stable for 6 months in the dark and at a temperature of 4 C 3 C. 5.8 Nicotinamide adenine dinucleotide phosphate (NADPH). For the microplate method, dissolve 19,2 mg of -NADPH (C21H26N7Na4O17P3; relative molecular mass 833,35) in 2
45、 ml of water (5.1) to obtain a concentration of 10 mmol/l. Prepare this solution at the time of determination and keep it sheltered from light in an ice bath. NOTE For the cell (test tube) method, 41,7 mg of NADPH are dissolved in 1 ml of water (5.1) to obtain a concentration of 50 mmol/l. 5.9 7-Eth
46、oxyresorufin stock solution Prepare a stock solution of concentrated 7-ethoxyresorufin (C12H6NaO3; relative molecular mass 235,17), e.g. 5 mg/ml, in DMSO. Store this solution in the dark at room temperature for a maximum of 1 year. 5.10 7-Ethoxyresorufin (46 mol/l) working solution Measure the exact
47、 concentration of the stock solution (5.9) by spectrophotometry at 482 nm using Equation (1). At 482 nm, the molar absorption coefficient is 2,25 104 (mol/l)1 cm1. Dilute the stock solution (5.9) with DMSO in order to obtain a 46 mol/l working solution for the microplate method. Prepare this solutio
48、n at the time of determination. NOTE A 400 mol/l working solution is used for the cell (test tube) method. 5.11 -Naphthoflavone dissolved in peanut oil For the injection of a dose of -naphthoflavone (C19H12O2, relative molecular mass 272,3) into the fish at a mass per body mass fraction of 50 mg/kg
49、(injection of 10 l of solution into the oil per gram of fish), prepare a solution of -naphthoflavone in peanut oil at a concentration of 5 mg/ml. This solution is shaken and brought up to a temperature of 45 C 5 C using a water bath in order to improve the homogeneity. NOTE A dose even 10-fold lower than 50 mg/kg can be sufficient for EROD induction. It is possible, therefore, that the exact dose to be used is of l