1、DRAFT FOR DEVELOPMENTDD ISO/TS 23893-2:2007Water quality Biochemical and physiological measurements on fish Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) ICS 13.060.70g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g
2、36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58DD ISO/TS 23893-2:2007This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 31 March 2008 BSI 2008ISBN 978 0 580 53706 6National forewordThis Draft for Developme
3、nt is the UK implementation of ISO/TS 23893-2:2007.This publication is not to be regarded as a British 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
4、 its practical application can be obtained.Comments arising from the use of this Draft for Development 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
5、 later than three years after its publication by the international organization so that a decision 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 t
6、he review period, the responsible BSI Committee will decide whether to support the conversion into 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
7、House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted 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
8、 does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Amendments/corrigenda issued since publicationDate CommentsReference numberISO/TS 23893-2:2007(E)TECHNICAL SPECIFICATION ISO/TS23893-2First edition2007-11-15Water quality Bioche
9、mical 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 23893-2:2007ii iiiContents Page Foreword iv Introduction v 1 S
10、cope . 1 2 Normative references . 1 3 Principle. 1 4 Test environment 1 5 Reagents 2 6 Apparatus 3 7 Sampling and 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
11、Determination of the EROD activity 5 8.4 Checking the sensitivity of the biological reagent and compliance with 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
12、 results . 9 Bibliography . 14 DD ISO/TS 23893-2:2007iv 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
13、. Each member body interested 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 Intern
14、ational Electrotechnical 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 Sta
15、ndards adopted by the 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 documen
16、ts, a technical committee 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 par
17、ent committee casting 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 or
18、der to decide whether 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 w
19、ithdrawn. Attention is 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
20、SC 5, Biological methods. 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 Specifi
21、cation DD ISO/TS 23893-2:2007vIntroduction The measurement of pollution biomarkers in fish, such as the measurement of biotransformation enzyme activities, is likely to provide information about exposure levels, bioavailability and the early biological effects of substances present in aquatic ecosys
22、tems. 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 dioxins. A large amount of research work bears witness to the extent of
23、 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 exposure of the fish to organic contaminants, account being taken of the i
24、nhibition 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 monitoring programme. The intercalibration exercises on the measurement of the
25、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 become familiar with the analysis of enzyme activities and the possibl
26、e error factors. DD ISO/TS 23893-2:2007blank1Water 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
27、-mitochondrial fraction of fish liver homogenate (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 la
28、boratory. This method is applicable for EROD 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 d
29、ated references, only the edition cited applies. 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 Pr
30、inciple Samples of fish are collected and dissected 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 me
31、asurement of the increase in fluorescence due 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
32、proteins in the S9 fraction. 4 Test environment 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 23893-2:20072 5 Reagents Unless
33、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). This solution is stable for 6 months at a tempera
34、ture 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 EROD activity for most species of fish. Prepare t
35、he 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 (5.1). Adjust solution A to pH 7,8 0,1 with solution
36、 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 concentrations are then 20 % glycerol and 100 mmol/l phospha
37、te. 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 spectrophotometer. Calculate the exact resorufin con
38、centration, c, in millimoles per litre, using Equation (1): Dcl= (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)1cm1at 572 nm (Reference 17) and 54,0 1,1 (mmol/l)1cm1(Reference 36) ha
39、ve 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 Resorufin is very unstable under daylight conditions. 5.6
40、 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 23893-2:200735.7 Rhodamine B standard solution. Dissolve 25 mg of rhodamine B (C28H30N2O3; relative molec
41、ular mass 442,55) in 250 ml of ethylene glycol monomethyl ether. Dilute this solution with ethylene glycol monomethyl ether, so as to obtain a standard 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
42、 adenine dinucleotide phosphate (NADPH). For the microplate method, dissolve 19,2 mg of -NADPH (C21H26N7Na4O17P3; relative molecular mass 833,35) in 2 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 i
43、ce 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-Ethoxyresorufin stock solution Prepare a stock solution of concentrated 7-ethoxyresorufin (C12H6NaO3; relative molecular mass 235,17), e.g. 5 mg/ml, in D
44、MSO. 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 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(
45、mol/l)1cm1. Dilute the stock solution (5.9) with DMSO in order to obtain a 46 mol/l working solution for the microplate method. Prepare this solution 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
46、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 (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 s
47、olution 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 little importance. 6 Apparatus Usua
48、l laboratory and dissection equipment and in particular the following. 6.1 Cryogenic tubes. 6.2 Liquid nitrogen container or freezer, set at a temperature below 70 C. 6.3 Cell or microplate spectrofluorimeter, for 96-well microplates. NOTE The use of white opaque microplates allows a significant red
49、uction of the fluorimetric background noise. 6.4 Centrifuge. 6.5 Homogeniser, of Potter Elvehjem or equivalent type. DD ISO/TS 23893-2:20074 6.6 pH meter. 6.7 Spectrophotometer. 7 Sampling and preparation of samples 7.1 Sampling of fish Sampling should be carried out in the natural environment by fishing or in a laboratory on fish exposed to substances or effluents as specified in ISO 238931. Sample at least 10 fish of the same species and sex and of uniform size from each group to be ex
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