KS I ISO 23893-2-2009 Water quality－Biochemical and physiological measurements on fish－Part 2：Determination of ethoxyresorufin-O-deethylase(EROD)《水质 鱼的生物化学和生理测量 第2部分 乙氧基异吩唑酮 脱乙基酶(E.pdf

1、 KSKSKSKS KSKSKSK KSKSKS KSKSK KSKS KSK KS KS I ISO 238932 2：- O-(EROD) KS I ISO 238932 :2009 2009 12 18 http:/www.kats.go.krKS I ISO 238932:2009 : ( ) ( ) ( ) : (ISO/TC147) () ( ) () : (http:/www.standard.go.kr) ： ：2009 12 18 2009-0831 ： ：(ISO/TC147) ( 02-509-7266) (http:/www.kats.go.kr). 10 5 , .

2、KS I ISO 238932:2009 2：-O- (EROD) Water qualityBiochemical and physiological measurements on fish Part 2：Determination of ethoxyresorufin-O-deethylase(EROD) 2007 1 ISO/TS 238932, Water qualityBiochemical and physiological measurements on fishPart 2：Determination of ethoxyresorufin-O-deethylase(EROD)

3、 , . 1 (liver) ( EROD .) -O-(Ethoxyresorufin-O-deethylase, EROD) . ( ) . EROD 1 pmol/(minmg) . () . 2 . . ( ) . KS I ISO 238931, 1： , 3 KS I ISO 238931 . () , S9 . S9 EROD 7 -(7-ethoxyresorufin) (resorufin) . ( B ) . EROD S9 KS I ISO 238932:2009 2 . 4 20 2 , S9 4 ( , ). 5 , . 5.1 , 1 S/cm . 5.2 , 15

4、0 mmol/L 1 L (5.1) 11.2 g KCl( 74.6) . 4 3 6 . 5.3 , 100 mmol/L; pH 7.80.1 pH EROD , (species) . EROD . 2 A B . A ：1 L (5.1) 17.4 g K 2 HPO 4 (, 174.2) . B ：1 L (5.1) 13.6 g KH 2 PO 4 (, 136.1) . B A pH 7.80.1 . 4 3 6 . 5.4 (20 %) (5.3) 20 % (C 3 H 8 O 3 ; 92.1) . 20 % 100 mmol/L . 5.5 (108 mg/L) ,

5、100 mL (DMSO) 10.8 mg ( C 12 H 6 NNaO 3 ； 235.2) 2 . 572 nm . (1) c(mmol/L) . l D c (1) D ： (cm 1 ) ： , 572 nm 73.2(mmol/L) 1 cm 1 ( 17) KS I ISO 238932:2009 3 54.01.1(mmol/L) 1 cm 1 ( 36) . l ： (cm) , 20 , . 6 . . 5.6 11.5 mol/L 10 mL DMSO (5.5) . . 5.7 B B(Rhodamine B, C 28 H 30 N 2 O 3 ： 442.55)

6、25 mg 250 mL , 0.1 mol/L . 4 3 6 . 5.8 NADPH(Nicotinamide adenine dinucleotide phosphate) , -NADPH(C 21 H 26 N 7 Na 4 NO 17 P 3 ； 833.35) 19.2 mg 2 mL (5.1) 10 mmol/L . , . () , 41.7 mg NADPH 1 mL (5.1) 50 mmol/L . 5.9 7- (7-Ethoxyresorufin：C 12 H 6 NaO 3 ； 235.17) (：DMSO 5 mg/mL). 1 . 5.10 7- (46 m

7、ol/L) 482 nm (1) (5.9) . 482 nm 2.2510 4 (mol/L) 1 cm 1 . (5.9) DMSO 46 mol/L . . () 400 mol/L . 5.11 - -( -Naphthoflavone, C 19 H 12 O 2 ： 272.3) 5 mg/L . - 50 mg/kg(/) ( 1 g KS I ISO 238932:2009 4 10 L ). 45 5 . EROD 50 mg/kg 10 . . 6 , . 6.1 6.2 , 70 6.3 , 96-well . 6.4 6.5 , Potter Elvehjem 6.6

8、pH 6.7 7 7.1 , KS I ISO 238931 . 10 , , . , . EROD . . a) b) (, , , , , ) KS I ISO 238931 . ( ) . KS I ISO 238932:2009 5 . 7.2 , , KS I ISO 238931 . 1 g . (bile) . 7.3 , 70 . 70 (6.2) 3 . EROD 1 (8.1) , 4 . 8 8.1 (fractions) (5.2) , (6.5) 4 mL (5.3) . , ： 1 g：4 mL . 4 . 0.5 2 , . 5 10(strokes) . 4 3

9、 10 20 10 000 g( 90 000 m/s 2 ) . (S9 ) 4 3 . , 1 . (7.3) , 7.3 (6.1) 70 . , (5.4) . 8.2 S9 22 2 . (kits) . (in-house standards) , BSA(Bovine serum albumin) . 8.3 EROD 8.3.1 8.3.1.1 KS I ISO 238932:2009 6 (5.6) B (5.7) . B 1.2 . 8.3.1.2 . . . . DMSO ：1; 5; 10; 50; 100 (5.6) . 7- S9 (BSA ) 8.3.2.1 .

10、S9 . 8.3.2.2 . . . 8.3.1.3 B (6.3) B (5.7) 3 well . B/ ( ). B , . 8.3.2.2 . 1,2 0.1 mol/L . 8.3.2 8.3.2.1 S9 , 1 . NADPH . 3 , 3 . 1 (5.3) 1.96 mL 200 L 100 mmol/L 100 mmol/L 100 mmol/L 7-(5.10) 10 L 10 L 400 mol/L 46 mol/L 2 mol/L S9 (8.1) 20 L 10 L 5 mg10 mg protein/mL 1.1 mg2.3 mg protein/mL 50 g

11、100 g protein/mL NADPH(5.8) 20 L 11.5 L 50 mmol/L 10 mmol/L 0.5 mmol/L KS I ISO 238932:2009 7 NADPH (5.8) . , 1 2 . 2 . (lag phase) . 7 - (2 mol/L) . (8.3.1.2) . 8.3.2.2 (6.3) , . , (6.3) , 530 nm535 nm (extinction) 580 nm 590 nm (emission) . . , . 8.4 . -( -naphthoflavone)(5.11) ( 10). (：96 ). , -

12、(： 10 g 100 L ). ：(zebrafish), (DMSO - ) . 48 . 50 mg/kg , ( ) ( ) EROD , . , ( - ) , 10( 3 ) , , 100( 30 ) ( 11). 9 9.1 EROD S9 , . (8.3.1.2) B (8.3.1.3) . S9 EROD (A EROD , pmoles/min) (2) . r S9 EROD V RV A (2) KS I ISO 238932:2009 8 R ： (pmoles/ Lmin nmoles/ Lmin ) V r： ( L) ：S9 (mg/ L) V S9： S9

13、 ( L) 9.2 ( , , ) (：Levene test). (variable transformation) ( 1, 12). , (non-parameter) (：Mann and Whitney U-test). (batch) EROD (ANOVA, Student t-test). () . ( 95 % ). A . 10 . a) (KS I ISO 238932) b) , , ( , ), (KS I ISO 238931 .) c) , , , , (KS I ISO 238931 .) d) 8.1 EROD e) ( ) (EROD ) f) KS I I

14、SO 238932:2009 9 A () A.1 (Rainbow trout, Oncorhynchus mykiss) 96 . 10 - naphthoflavone 50 mg/kg . A.1 , A.2 (batch) . A.1 (Batch) mm g EROD pmol/(minmg) proteins Trout001 86 7.9 11 Trout002 93 9.8 8 Trout003 90 9.4 7 Trout004 90 10.4 13 Trout005 102 12.8 9 Trout006 100 11.6 10 Trout007 100 12.7 9 T

15、rout008 90 9.8 6 Trout009 91 8.6 5 Trout010 84 7.5 10 Trout011 98 12.1 205 Trout012 110 17 779 Trout013 92 9.7 285 Trout014 112 15.3 820 Trout015 105 13.7 730 Trout016 92 9.2 530 Trout017 98 10.8 616 Trout018 98 11.5 542 Trout019 93 11.7 347 Trout020 104 15.3 412 Trout021 2 % 100 11.7 8 Trout022 2 %

16、 96 10.2 7 Trout023 2 % 102 12.3 10 Trout024 2 % 90 8.2 15 Trout025 2 % 82 6.7 15 KS I ISO 238932:2009 10 A.1 () (Batch) mm g EROD pmol/(minmg) proteins Trout026 2 % 92 9.7 14 Trout027 2 % 96 10.5 7 Trout028 2 % 92 9.8 6 Trout029 2 % 78 6.2 9 Trout030 2 % 92 10.4 9 Trout031 10 % 100 12 39 Trout032 1

17、0 % 92 9.7 26 Trout033 10 % 84 7.8 37 Trout034 10 % 100 12.5 26 Trout035 10 % 87 8.1 27 Trout036 10 % 110 14 40 Trout037 10 % 94 9.8 27 Trout038 10 % 92 9.5 22 Trout039 10 % 95 9.7 77 Trout040 10 % 102 12.7 25 Trout041 33 % 98 11.1 77 Trout042 33 % 86 7.9 68 Trout043 33 % 98 10.1 42 Trout044 33 % 92

18、 8.7 83 Trout045 33 % 87 7.3 51 Trout046 33 % 94 9.5 80 Trout047 33 % 102 11.7 103 Trout048 33 % 94 9.6 46 Trout049 33 % 82 6.3 51 Trout050 33 % 87 7.3 68 Trout051 67 % 95 10.2 77 Trout052 67 % 87 8.4 59 Trout053 67 % 86 7.9 65 Trout054 67 % 105 14.6 102 Trout055 67 % 95 10 72 Trout056 67 % 102 12.9

19、 49 Trout057 67 % 92 9.8 119 Trout058 67 % 104 13.2 69 Trout059 67 % 87 7.6 96 Trout060 67 % 100 11.3 62 KS I ISO 238932:2009 11 A.2 (SD) (SD) EROD (SD) EROD (95 % , CI95) mm g pmol/(minmg) proteins pmol/(minmg) proteins 10 936 10.11.9 92 8(6, 11) 10 1007 12.62.6 52713 488(347, 672) 2 % 10 927 9.62.

20、0 103 9(7, 13) 10 % 10 968 10.62.1 3516 32(24, 43) 33 % 10 926 9.01.8 6719 64(51, 79) 67 % 10 957 10.62.4 7722 74(60, 91) X % Y ERODpmol/(minmg) proteins a b A.1 A.2 EROD , (variance) (homogeneous) (Levene test)( A.1 ). EROD () ( 488/861). (effluent) 2 % EROD ( student t-test, p0.43). EROD ( student t-test, p 0.01). , (trout) 10 % ( ) 96 EROD KS I ISO 238932:2009 12 . A.2 Seine (bay) (dab, Limanda l