102 8 13 1020069337 102 10 15 NIEA B901.14B Daphnia 48 lethal concentration 50%, LC 50 acute toxic unit, TU a Daphnia pulex Daphnia magna 25 2 2 L 1 20
30 ml polystyrene 4 mm ph 0.1 mg blender 1. 1 2. 3. 4. 10 M -cm D. pulex D. magna 20 L 2 20
1. 18 L 2. 2 L 18 L 8 14 1 10% v/v NIEA W103 NIEA W104 NIEA W109 500 ml 4 2 36 1. 2. 1 1 3 20
2 10% v/v 1 3 A. 15 2 B. 10% v/v 1 C. 2 D. 1 E. 3 F. 1 3. 1 2 25 2 3.0 mg/l 3.0 mg/l 4. 24 2 range - finding test 1. 2. 10 25 ml 1 3. 5 24 5 4. 25 ± 2 16 ± 1 4 20
5. 8 24 definitive test 1. 5 2 5 100% 80% 60% 40% 20% 100 ml 2. ph ph 3. 20 4 25 ml 5 24 2 4. 4 25 ml 100% 5 24 5. 25 ± 2 16 ± 1 6. 48 48 ph 48 LC 50 1. 48 = 20 4 = 20 100% 2. 4 48 LC 50 graphic method probit method Spearman - Karber method trimmed Spearman - Karber 5 20
method 48 TU a 1. TU a LC 50 TU a = 100% 48 LC 50 2. 5 50% 48 LC 50 > 100% TU a < 1.00 3. 5 50% 48 LC 50 < 20% TU a > 5.00 4. 2 48 LC 50 TU a 10% 1. 5 2 g/l 5 50% 50% 1 2. 5 LC 50 coefficient of variation, CV CV 50% 3. 4. LC 50 15 SD 2 SD 3 SD 15 5 6 20
15 15 15 5. 3 SD 20 2 2 SD 3 D. magna 48 LC 50 4.11 g/l 12.6% n = 5 U.S. EPA, Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, EPA-821-R-02-012, 2002. American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 22 nd Edition, Part 8000, 2012. OECD, Guidelines for the Testing of Chemicals, Test No. 202: Daphnia sp. Acute Immobilisation Test, 2004. 1 2 3 Ephippium 7 20
mg/l NaHCO 3 MgSO 4 7H 2 O KCl CaSO 4 2H 2 O mg CaCO 3 /L 96.0 123.0 4.0 60.0 80-100 192.0 246.0 8.0 120.0 160-180 0.5 mm Daphnia magna 8 20
0.5 mm Daphnia magna 9 20
5 2 1 48 LC 50 10 20
24 1. 3 3 L 4 L 2 2. 25 ± 2 16 ± 1 3. 2 4. 3 L 25 200 5. 5 mg/l A B C 1 ml/l 1. A 6.3 g 2.6 g 0.5 g 500 ml 5 1 300 ml 7 2. B 15 g 5 g 5 g 1 L 7 3. C 7 1 5 g 1 L 1 2 L 11 20
7 7 1 300 ml 2 6 5 g 1 L 5 300 ml 3 7 1.5 g 300 ml 4. NIEA B906 1.0 g/ 1L 7 10 3 10 7 /ml 1.5 ml/l 24 24 25 ± 2 16 ± 1 24 24 12 20
LC 50 1. 5 50% 50% 2. 5 2 2 0 100% 1. probit.exe 2. probit.exe 1 2 Chi - square test for heterogeneity LC 50 Chi - square for heterogeneity (calculated) tabular value at 0.05 level LC 50 3. Overflow 1. 1 1 40% 60% 2. probit.exe 1 2 1 5.151 7.815 2 LC 50 56% TU a 1.79 13 20
1. 5 50% 2. 5 1 3. 0% 100% 1. tsk.exe 2. tsk.exe 3 4 SPEARMAN - KARBER TRIM LC 50 1 SPEARMAN - KARBER TRIM 0% 2 SPEARMAN - KARBER TRIM 0% 1. 1 2 40% 100% 2. tsk.exe 3 4 1 SPEARMAN - KARBER TRIM 20.51% 2 LC 50 92% TU a 1.09 5 50% 50% 14 20
1. 1 p 0 5 p 1 p 2 p 5 p 0 p 5 2 p 1 p 2 p 3 p 0 p 4 p 5 p 0 p 1 p 2 p 3 p 0 p 3 p 0 p 3 p s 0 p s 1 p s s 2 p 3 p 0 p 1 p 2 p 3 / 4 2. 0 p s 0 a s p i p i p s 0 / 1 p s 0 p a i i p s i i p s 0 3. 50 % 50 % LC 50 1. 1 3 5% 0% 0% 0% 100% 100% 2. p 0 p 3 p 0 p 5 1.25% 1.25% 1.25% 1.25% 100% 100% 3. p 0 s 0 0% 0% 0% 0% 100% 100% 4. 15 20 p 0 s
5 60 % 80 % 50 % 69.28 % LC 50 69% TU a 1.45 1 20 1 2 3 0 1 1 20% 0 0 0 40% 3 2 0 60% 9 0 0 80% 20 0 20 100.0% 20 16 20 16 20
EPA PROBIT ANALYSIS PROGRAM USED FOR CALCULATING LC/EC VALUES Version 1.5 Do you wish abbreviated (A) or full (F) input/output? a Output to printer (P) or disk file (D)? d File name for output? Test1 Title? Test1 Number responding in the control group =? 0 Number of exposure concentrations, exclusive of controls? 5 Input data starting with the lowest exposure concentration Concentration =? 20 Number responding =? 0 Number exposed =? 20 Concentration =? 40 Number responding =? 3 Number exposed =? 20 Concentration =? 60 Number responding =? 9 Number exposed =? 20 Concentration =? 80 Number responding =? 20 Number exposed =? 20 Concentration =? 100 Number responding =? 20 Number exposed =? 20 Number Conc. Number Resp. Number Exposed 1 20.0000 0 20 2 40.0000 3 20 3 60.0000 9 20 4 80.0000 20 20 5 100.0000 20 20 Do you wish to modify your data? n The control response = 0 Do you wish to modify it? n Output stored in Test1 1 probit 17 20
EPA PROBIT ANALYSIS PROGRAM USED FOR CALCULATING LC/EC VALUES Version 1.5 Test1 Proportion Observed Responding Number Number Proportion Adjusted for Conc. Exposed Resp. Responding Controls 20.0000 20 0 0.0000 0.0000 40.0000 20 3 0.1500 0.1500 60.0000 20 9 0.4500 0.4500 80.0000 20 20 1.0000 1.0000 100.0000 20 20 1.0000 1.0000 Chi - Square for Heterogeneity (calculated) = 5.151 Chi - Square for Heterogeneity (tabular value at 0.05 level) = 7.815 Test1 Estimated LC/EC Values and Confidence Limits Exposure 95% Confidence Limits Point Conc. Lower Upper LC/EC 1.00 31.620 22.002 37.962 LC/EC 50.00 55.550 49.626 61.121 2 probit 18 20
TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5 ENTER DATE OF TEST: ENTER TEST NUMBER: WHAT IS TO BE ESTIMATED? (ENTER L FOR LC50 AND E FOR EC50) L ENTER TEST SPECIES NAME: ENTER TOXICANT NAME: ENTER UNITS FOR EXPOSURE CONCENTRATION OF TOXICANT: % ENTER THE NUMBER OF INDIVIDUALS IN THE CONTROL: 20 ENTER THE NUMBER OF MORTALITIES IN THE CONTROL 1 ENTER THE NUMBER OF CONCENTRATIONS (NOT INCLUDING THE CONTROL; MAX = 10) 5 ENTER THE 5 EXPOSURE CONCENTRATIONS (IN INCREASING ORDER): 20 40 60 80 100 ARE THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION EQUAL(Y/N)? Y ENTER THE NUMBER OF INDIVIDUALS AT EACH EXPOSURE CONCENTRATION: 20 ENTER UNITS FOR DURATION OF EXPERIMENT (ENTER H FOR HOURS, D FOR DAYS, ETC.): ENTER DURATION OF TEST: 48 ENTER THE NUMBER OF MORTALITIES AT EACH EXPOSURE CONCENTRATION: 0 2 0 0 16 WOULD YOU LIKE THE AUTOMATIC TRIM CALCULATION(Y/N)? Y 3 tsk 19 20
DATE: TEST NUMBER: DURATION: 48 TOXICANT : SPECIES: RAW DATA: Concentration Number Mortalities --- ---- (%) Exposed.00 20 1 20.00 20 0 40.00 20 2 60.00 20 0 80.00 20 0 100.00 20 16 SPEARMAN-KARBER TRIM: 20.51% SPEARMAN-KARBER ESTIMATES: LC50: 91.97 95% LOWER CONFIDENCE: 89.05 95% UPPER CONFIDENCE: 94.99 NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING. ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION. ------------------------------------------------------------------------------ WOULD YOU LIKE TO HAVE A COPY SENT TO THE PRINTER(Y/N)? 4 tsk (%) 5 20