# Translation Statement

Institute ofEcotoxicology Co., Ltd.

Sponsor:
Title: Bioconcentration Test of in Carp, Cyprinus carpio


# Test No.: C-186

I, the undersigned, hereby declare that this report provides a correct English translation ofthe Final Report (Study No. C-186, issued on March 1, 2011).

Date May 15,2012 Study Director

# k.ka~l Kenji Kaneko

# Final Report

Bioconcentration Test of in Carp, Cyprinus carpio

Report No. E4 -11006·C186·NS

March 1, 2011
Institute ofEcotoxicology Co., Ltd.

# Table ofContents

GLP Statement Quality Assurance Certificate

Preface 

# 1. 2. 

# 3. 4. Title Sponsor Test facility Objective ofthe test 5. Testmethods 

# 6. 7. 

# 8. 9. 10. 11. 

# 12. 13. 14. 15. GLP applied Name ofthe test substance Testperiods Testresults Evaluation, discussion ofthe results and conclusion Storage ofthe test source materials Environmental factors assumed to have an effect onreliability ofthe data Personnel related to the test and their assigned positions Name and address ofscientists having contributed reports to the final report Approval ofthe final report

Main Subject 1. 

# 1.1 1.2 

# 1.3 1.4 1.5 

# 2. 2.1

2.2 2.3 Test substance Name Structural formula Supplier and Lot No. Identification Physico-chemical properties Testmethods and analytical methods Acute toxicity test (96h LC50) 

# inZebrafish 

# (Danio rerio) for selecting the substance concentration Performing a bioconcentration test Analyses offish and test water Page
1
11



# 1 1 

# 1 1 1



# 2 2 2 2 

# 3 3 

# 4 

# 4 5 5

6 

# 6 6 6 6 

# 6 

# 7 

# 7 7

8 12

# 2.3.1 Analytical method for fish 12 2.3.2 Analytical method for test water 14 2.3.3 Measurement methods 16 2.4 Calibration curve by liquid chromatography mass spectrometry 16 2.4.1 Preparation ofstandard solutions 16 2.4.2 Preparation ofcalibration curve 17 2.5 Recovery test and blank test for 17 2.5.1 Recovery test from fish for 17 2.5.2 Recovery test from test water for 18 2.6 Quantifiable limit 19 2.6.1 Quantifiable limit for fish 19 2.6.2 Quantifiable limit for test water 20 2.7 Preliminary test 20 2.8 Schedule ofanalyses offish and test water 21 3. Testresults 22 3.1 Concentrations of intest water 22 3.2 Bioconcentration factors (BCFs) 23 3.3 Evaluation, discussion ofthe results and conclusion 24

Table ofcontents for references 25

This report includes 90 pages

# GLP Statement

Institute ofEcotoxicology Co., Ltd.

Sponsor:

Title ofthe test: Bioconcentration Test of in Carp, Cyprinus carpio



# Test No.: C-186

The aforementioned test was conducted in compliance with following GLP principles:

"Standards Concerning Test Facilities Performing Tests Concerning New Chemical Substances" on Japanese GLP (Yakushokuhatsu No. 1121003, Heisei 15 • 11 • 17 Seikyoku No.3 and Kanpokihatsu No. 031121004, Joint notification ofDirector General of Pharmaceutical and Food Safety Bureau in Ministry ofHealth, Labor and Welfare, Director General ofManufacturing Industries Bureau in Ministry ofEconomy, Trade and Industry and Director General ofEnvironmental Policy Bureau in Ministry ofthe Environment) (dated November 21, 2003)

"OECD Principles ofGood Laboratory Practice" (as revised in 1997).

March 1, 2011 Study Director: Signed in original Kenji Kaneko

# ReportNo. E4 -11006· C186 ·NS TestNo. C-186 

# Quality Assurance Certificate

Title: Bioconcentration Test of in Carp, Cyprinus carpio 

# Sponsor: Study Director: Kenji Kaneko Study Personnel: Kenji Kaneko Test Facility: Institute ofEcotoxicology Co., Ltd.
We certify that the present final report describes the test methods accurately and the contents were performed according to the test protocol and standard operation procedures, and the original data are accurately presented. The inspection and audit were performed

as follows.

Dates performed Status ofperformance Water/fish analysis on Day 7 from the start February 2, 2011 oftest inspected ofexposure Water/fish analysis on Day 14 from the February 9, 2011 start ofexposure Water/fish analysis on Day 21 from the February 16, 2011 start ofexposure Test protocol audited Protocol audited January 12, 2011 Protocol (revision) audited January 18,2011 Final report audited Report audited March 1, 2011



# Institute ofEcotoxicology Co., Ltd., Department ofQuality Assurance Unit Quality Assurance Director: Signed in original Mikiyasu Goto Signature date: March 1, 2011 (Inspections ofstudy conduct, protocol and final report) 

# Quality Assurance :**-~@ed** 

# in orig"--in_a_l

______ _

Quality Assurance : Mitsuko Takamatsu Signature date: March 1, 2011 (Inspections ofprotocol and final report) 

# Signed in original Miyuki Kurihara Signature date: March 1, 2011 (Inspection offinal report)

11 

## Dates reported to Test Facility Management

and Study Director February 2, 2011 February 9, 2011 February 16,2011 January 12, 2011 January 18, 2011 March 1, 2011

# 1. Title

2. Sponsor 

# Final Report-PrefaceBioconcentration Test of in Carp, Cyprinus carpio



# 3. Test facility Institute ofEcotoxicology Co., Ltd. 2-28-1 Yoshino-cho, Kita-ku, Saitarna City, 331-0811 Japan Representative Test Facility Management

4. Objective ofthe test Mikiyasu Goto Tadayoshi Kan

The objective ofthis test is to determine the degree ofbioconcentration ofthe test substance in fish in order to file for a Chemical Substance Control Law in Japan (No. 117 ofOctober 16, 1973). (abbreviated to 'CSCL' below)

5. Test methods The methods used in this test were based on "TestMethods Concerning New Chemical Substances< Bioconcentration Test ofChemical Substances in the Bodies ofFish and Shellfish>" (Yakushokuhatsu No.l121002, Heisei 15 • 11 • 13 Seikyoku No.2 and Kanpokihatsu No. 031121002, Joint notification ofDirector General ofPharrnaceutical and Food Safety Bureau at the Ministry ofHealth, Labor and Welfare, Director General ofManufacturing Industries Bureau in Ministry ofEconomy, Trade and Industry and Director General ofEnvironmental Policy Bureau in Ministry ofthe Environment, Japan) (dated November 21, 2003) and OECD Guidelines for the Testing ofChemicals, "No. 305 Bioconcentration: Flow-through Fish Test" (adopted on June 14, 1996).

# 6. GLP applied

7.



# 8. Japanese Chemical Substance GLP: "Standards Concerning Test Facilities Performing the Tests Concerning New Chemical Substances" (Yakushokuhatsu No.l121003, Heisei 15 • 11 • 17 Seikyoku No.3 and Kanpokihatsu No. 031121004, Joint notification ofDirector General ofPharmaceutical and Food Safety Bureau at the Ministry ofHealth, Labor and Welfare, Director General of Manufacturing Industries Bureau in Ministry ofEconomy, Trade and Industry and Director General of Environmental Policy Bureau in Ministry oftheEnvironment)(dated November 21, 2003)

OECD GLP: "the OECD Principles ofGood Laboratory Practice" (as revised in 1997).



# Name ofthe test substance Chemical name Abbreviation CAS No.



# Test periods (1) 

# Study initiation date January 12, 2011 (2) Duration ofthe test Date ofarrival offish August 23,2010 Acclimation completion date January 26,2011 Experiment starting date January 26,2011 Experiment completion date February 23, 2011 (3) Study completion date March 1, 2011


# 9. Test results The bioconcentration test ofthe test substance in carp, Cyprinus carpio 

# was conducted. The test substance is a salt consisting of

, and dissociates completely in water. In this test, the bioconcentration ofonly was confirmed. The bioconcentration factors of during the 28-day exposure period are summarized in the next page as a table.

2

# High exposure level Low exposure level Bioconcentration factor of (Test substance: 0.2 mgL- 1) 

# (Test substance: 0.02 mgL- 1) 

## ( 

# : 0.193 mgL- 1) 

# ( 

# : 0.0193 mgL- 1) 

# Bioconcentration factors under a detectable limit under a detectable limit (BCF) (less than 1-fold) (less than 11-fold)



# 10. Evaluation, discussion ofthe results and conclusion The test substance is a salt consisting of only was confirmed. Since In

# this test, the bioconcentration of listed as an existing chemical substance, is not high bioconcentration, the bioconcentration of is considered to be confirmed by analogy. The 96h LCso value ofthe test substance in Zebrafish 

# (Dania rerio) was greater than 101 mgL- 1

. 

# Since the solubility in water was>1.0 x 

# 105 

# mgL- 1

, 

# two concentrations ofthe test substance in water were set up to be 0.2 mgL- 1 ( 

# 0.193 mgL- 1) 

# for the high exposure level (Concentration Levell) and 0.02 mgL- 1

# ~ 

# 0.0193 mgL- 1) 

# for the low exposure level (Concentration Level2). According to the results ofthe bioconcentration test, the bioconcentration factors (BCFs) in both Concentration Levels 1and 2 were less than 50-fold. Thus, a steady state was assumed to be achieved within 28 days ofexposure. 

# Itwas confirmed that the bioconcentration was under a detectable limit and the bioconcentration factors were less than 1-fold in Concentration Level 1 and less than 11- fold in Concentration Level 2. Therefore, bioconcentration of anion ofthe test substance was confirmed to be low.



# 11. Storage ofthe test source materials The following records and samples concerning the tests will be stored for 10 years after receiving notification based on the provisions ofChemical Substances Control Law (CSCL) (No. 117, 1973): Article 4, Paragraphs 1 or 2, Article 4-2, Paragraphs 2, 3 or 8, Article 5-4, Paragraph 2, Article 24, Paragraph 2 orArticle 25-3, Paragraph 2, and the test substance, reference substance and other items will be stored for 1 0 years after receiving notification based on the provisions ofthe CSCL: Article 4, Paragraphs 1 or 2, Article 4-2, Paragraphs 2, 3 or 8, Article 5-4, Paragraph 2, Article 24, Paragraph 2 or



# 3

# Article 25-3, Paragraph 2 or the period wherein they canbe stored stably without causing any interference with quality, which is at least one oftheperiods that is shorter, at the Institute ofEcotoxicology Co., Ltd.. Regarding the storage after these periods, it will be determined separately after discussion. (1) 

# Protocols, records ofmodifications and changes (2) 

# Records concerning the test substance, etc. (3) 

# Experimental notes, raw data, charts (4) Test substance, reference substance, other items (stored at about 20°C in a dark place) (5) 

# Inspection and audit records recorded by the personnel in charge ofquality

assurance regarding the present test (6) 

# Final report (7) 

# Other items that are required

12. Environmental factors assumed to have an effect on reliability ofthe data 

# No particular abnormalities were found.

13. Personnel related to the test and their assigned positions 

# Study Director: Kenji Kaneko Study Personnel: Kenji Kaneko

• 96h LCso test
• Establishment ofanalytical methods for test water and fish
• Preparation ofcalibration curve for analysis oftest water and fish
• Recovery tests oftest water and fish
• Preliminary testto arrange the studyperiod and the sampling schedule
• Analyses oftest water and fish during the experimental period
• Preparation and maintenance oftest solution during the experimental period
• Measurement oflipid contents in fish body
• Measurement ofIR
• Observation ofthe conditions offish
• Measurement ofwater environment
Final report prepared by Kenji Kaneko

4

# 14. Name and address ofscientists having contributed reports to the final report Not applicable



# 15. Approval ofthe final report Study Director: Signed in original Kenji Kaneko Date signed: March 1, 2011



# 5

# Main Subject

1. Test substance 

# In the present report, the test substance has the following name and structural formula (based on the material provided by the sponsor).

1.1 Name 

# Chemical name: Abbreviation: CAS No.:

1.2 Structural formula



# The abbreviation words were used in this test as shown below.

Molecular formula:

1.3 Supplier and Lot 

# No. (1) 

# Supplier (2) 

# LotNo.

1.4 Identification 

# It was identified by the agreement oftheIR spectrum provided by the sponsor with the IR spectrum measured at our institution at the time ofthe start ofthe test. Further, the stability ofthe test substance during the test period was confirmed by the agreement of theIR spectrum at the end ofthe test with theIR spectrum measured at the start ofthe test (see, Ref. 12).

6

# 1.5 Physico-chemical properties (based on the data provided by the sponsor) Appearance*: Purity*: Solubility (25°C) measured at Institute ofEcotoxicology: water > 1.0 x 

# 105 

# mgL- 1 

# dimethyl sulfoxide > 1.0 x 

# 105 

# mgL- 1 

# acetone > 1.0 x 

# 105 

# mgL- 1 

# acetonitrile > 1.0 x 

# 105 

# mgL- 1 

# methanol > 1.0 x 

# 105 

# mgL- 1 

# dichloromethane 5.1 x 

# 101 

# mgL- 1 

# n-hexane 6.0 X 

# 1 

# o-2 

# mgL- 1


# 2. Test methods and analytical methods The methods used in this test were based on the Japanese Guideline, "Test Methods ConcerningNew Chemical Substances 

# < Bioconcentration Test ofChemical Substances in the Bodies ofFish and Shellfish>" (Yakushokuhatsu No. 1121002, Heisei 15 • 11 • 13 Seikyoku No. 2 and Kanpokihatsu No. 031121 002) (dated November 21, 2003) and the OECD Guidelines for Testing ofChemicals, "No. 305 Bioconcentration: Flowthrough Fish Test" (adopted on June 14, 1996)


# 2.1 Acute toxicity test (96h LC50) 

# in Zebrafish (Danio rerio) 

# for selecting the substance concentration (SOP 2.6) Based on the results ofa preliminary test, the concentrations ofthe test substance in test water ofa 96h LC50 

# test were selected. The 96h LC5

# ovalue was determined from the test result (the test was performed with reference to the method described in OECD Test Guidelines 203). 

# (1) Test fish Zebrafish 

# (Danio rerio) LotNo. 101202 Supplier Kamihata Fish Ind., Ltd.

Date received Mean total length Mean bodyweight 292-1 Chigusa-cho, Hanamigawa-ku, Chiba City, 262-0012 Japan December 2, 2010 3.2 

# ±0.1 

# em 0.24 

# ±0.03 g Acclimation period December2, 2010

- December 20, 201 0 Receiving and acclimatization procedures were based on SOP 2.1 and SOP 2.5.

7

# (2) Test solution (3) Test temperature (4) Test aquarium (5) Exposure method (6) Water for test

(7) Number offish Test water was prepared by dissolving the test substance in water for test. 24.9 

# ± 0.2°C Made ofglass, amount oftest water; 4 L Semi-static method (replacing test water after 48 hours) Dechlorinated city water ofSaitama Analysis ofwater quality: 

# itwas confirmed that water quality was adopted to 'the standard ofquality offisheries water' (see, Ref.10). 10 fish/4 L test aquarium 

# (8) Experimental period (9) Sensitivity test of December 20,2010- December 24,2010 A sensitivity test was conducted using CuS04 • 

# 5H2

# 0 as 

# Danio rerio



# (10) Test results a standard substance. The fish in the same lot which passed in the sensitivity test (96h LC50 

# value; 0.08 to 0.3 mgL- 1) 

# were used
as 96h LC5

# otest fish. Since the preliminary 96h LC50 

# value ofthe test substance in Zebrafish 

# (Danio rerio) was greater than 100 mgL- 1

, 

# the test was done by "a limited test" in which the concentration ofthe test substance was 100 mgL- 1• 

# As a result, it was confirmed that the 96h LC50 

# value was greater than 101 mgL- 1• 

# The concentration was an initial experimental value calculated from LC/MS analysis (see, the following table).

Test water Mortality 

# (%) Compound Concentration (mgL- 1) 

# Copper (II) sulfate pentahydrate 0.5 (sensitivitytest) 0.05 Test substance 101 Control (water for test)



# -

2.2 Performing a bioconcentration test (1) Test fish 24 48 72 96 hours hours hours hours 0 20 70 100 0 0 0 0 0 0 0 0 0 0 0 0

To confirm the bioconcentration in fish, the carp 

# (Cyprinus**ca~pio),** 

# representative fish species in Japan, was used (SOP 2.1.2) as the test fish for the bioconcentration test.
Common carp Supplier: 

# Cyprinus carpio (Lot No. 100823) Kitamura Carp Breeding Farm (12-388 Gunchiku, Yatsushiro-shi, Kumamoto Prefecture, Japan)



# 8

# Date purchased: August 23,2010 Feeding and acclimatization:

(2) Feeding 

# Kind:

Manufacturer: Feeding method: After receiving the fish at the Institute of Ecotoxicology Co., Ltd., those with a total length of approx. 8.0 

# ± 4.0 em were selected (SOP 2.4). After the fish were treated with Elevage® (4-[2-(5-nitro-2-furanyl)ethenyl]benzoic acid, sodium salt) and rock salt, the fish were raised. Fish were selected again on January 5, 2011 and 150 fish were transferred to an acclimation water-tank. After they were treated again with Elevage®, the process of acclimatization was started (SOP 2.3, SOP 2.5). Finally, the fish were acclimatized at water temperature 25 

# ± 2.0°C for 7 days and it was confirmed that the mortality was 5% or less before use in the test. Mean body weight Mean total length 3.36 ± 

# 0.21 g 6.9± 0.2 em Mmiality offish during the experiment High exposure level 0% Low exposure level 0% Control 0% (SOP 2.8) (SOP 2.8) (SOP 2.7)

Lipid contents (by chloroform/methanol extraction method) Before experiment 5.0% (mean value, n=2) (SOP 2.9) After experiment 4.8% (mean value, n=2) (SOP 2.9) (The conditions ofthe test fish were shown in Ref. 11)

Mixed feed for carp in pellet form (Swimmy) Crude proteins 37.0% Crude lipids 2.5% Nippon PetFood Company Fish were fed daily in an amount ofapproximately 1.5% ofthe bodyweight ofthe fish. However, no feed was given on the day before sampling ofthe test fish. (3) Performing the test 

# Water for test: Same as in Section 2.1 (6) Preparation ofa stock solution: A test substance was added to water for test to prepare 105 

# mgL" 1 

# stock solution. The stock solution was diluted with water for test to obtain test water. High exposure level:



# 9

# The stock solution was diluted with water for test to obtain a solution ofthe test substance (20 mgL- 1). 

# This was further diluted with water for test and continuously flowed through the test aquarium. Low exposure level:

Control: The stock solution was diluted with water for test to obtain a solution ofthe test substance (2 mgL- 1). 

# This was further diluted with water for test and continuously flowed through the test aquarium. Water for test was continuously flowed through the test aquarium.
Bioconcentration test equipment: see, Ref. I

Test aquarium: "Flow-through system" (made ofglass) Capacity 100 L (amount oftestwater; 50 L) Flow rate 300 mL min·' (432 L day" 1) 

# No. 9 aquarium for high exposure level, No. 8 aquarium for low exposure level and No. 7 aquarium for the control were used. (4) Test concentrations The 96h LCso of in Dania rerio 

# was greater than 101 mgL" 1 

# and the water solubility of was>1.0 x 

# 105 

# mgL- 1• 

# The setup concentrations for the bioconcentration test were determined to be less than the solubility in water. Based on the 96h LC50 

# value, 1/505 and 1/5050 were used as safety factors and the test concentrations were set as follows. High exposure Low exposure Control level level Concentration ofthe test 0.200 mgL- 1 

# 0.0200 mgL- 1 

# substance (concentration of (5) Test conditions Temperature oftestwater: pH oftest water: DO oftest water: (0.193 mgL- 1)



# 25.3 ± 0.2°C 7.5±0.1 7.4 ± 0.1 mgL- 1 

# (0.0193 mgL- 1)



# (Temperature, pH and DO oftest water were measured on each analysis day, see, Ref. 2-2)

10 -

-

# Measurement oftotal organic carbon concentrations in the test water (SOP 2.12):

Light type: Photoperiod: Experimental period: Since solvents or dispersant were not used in this test, the total organic carbon concentrations in the test water was not measured.
UV- 

# cut fluorescent lamp About 1 0 hours January 26, 2011 -February 23, 2011 The analysis schedule is presented in Section 2.8.
Number oftest fish: High exposure level Low exposure level Control 50 fish 50 fish 15 fish
Number offish analyzed and number repeated: 2 fish were paired for a single analysis (n=2). 2 fish were paired for analysis ofthe control group (n=l).



# 11

# 2.3 Analyses offish and test water 2.3.1 Analytical method for fish (SOP 2.11) Pretreatment Each six fish in the Concentration Levell and Level 2 and two fish in the control group were sampled using a landing net on the analysis day. The fish were temporarily paralyzed by placing them in an ice water. The body surface was washed with tap water and wiped lightly by gauze. Then, the body weight was measured in a unit of 0.01 g using a balance (SOP/INS/4.2.2), and the body length from the mouth to the tail fin was measured in a unit of0.1 em. In the test group, two fish were used as one sample and analyzed in duplicate, and other sample was put into a plastic bag and stored in a freezer after the fish were wrapped with aluminum-foil and packed in a plastic bag (SOP 2.15). The fish were put into a homogenizer cup after slicing, and acetonitrile (60 mL) was added to the homogenizer cup. The mixture was homogenized at 9000 r.p.m. for 6 min. (SOP/INS/4.17.2). The lid and the interior ofthe homogenizer cup were rinsed with acetonitrile (4 mL) and the washings were transferred to a centrifugal tube. The interior ofthe said homogenizer cup was rinsed three times with 4 mL ofacetonitrile and the washings were also transferred to the said centrifugal tube. The weight ofthe centrifugal tube was adjusted by adding a small amount ofacetonitrile. After centrifugal separation for 15 min. (SOP/INS/4.16.3) (9000 r.p.m., at about l0°C), the supernatant was filtered into a 100 mL volumetric flask (a glass filter GC50 by Advantec MFS, Inc., Tokyo, Japan). The interior ofthe said centrifugal tube and the filter paper were rinsed three times with 4 mL ofacetonitrile and the washings were also filtered into the said volumetric flask. After waiting until the temperature ofthe solution reached room temperature, the volume was adjusted to 100 mL by adding acetonitrile. A portion ofthe constant volume solution was taken with a hole pipette (1 mL) and was transferred to a 10 mL volumetric flask. After adding 4 mL ofacetonitrile, the volume
was adjusted to 10 mL by adding ultra pure water. The constantvolume solution was filtered through a filter (EKICRODISK 

# 13 by Nihon Pall Ltd., Tokyo, Japan). The filtrate was analyzed by liquid chromatography mass spectrometer (LC/MS). The operations ofthe pretreatment are shown in the next page as a flow chart.

12

# I Residue Test fish (one sample consisting oftwo fish)

• Measurements ofthe total length and bodyweight
• Slicing
• Transferred to a homogenizer cup**~Acetonitrile** 

## 60 mL
• Homogenized (9000 r.p.m., 6 min.)**~Acetonitrile** 

## 4 mL

(washingthe lid and the interior ofthe homogenizer cup)
• Transferred to a centrifugal tube**~Acetonitrile** 

## 4mLx3 (washing the interior ofthe homogenizer cup)


## • The weightofthecentrifugaltube

## was adjustedbyadding asmall amountofacetonitrile.
• Centrifugal separation (9000 r.p.m., at about 10°C, 15 min.) 

# I Supernatant 

# I

• Filtration (glass filter GC50 by Advantec MFS, Inc.)

(filtered into a 100 mL volumetric flask)**~Acetonitrile** 

## 4 mLx3 (washing the interior ofthe centrifugal tube and the filter paper)
• Waiting until the temperature ofthe solutionreached room temperature
• Fixed volume 100 mL
Fixed volume solution (acetonitrile)**~Taken** 

## in 1mL (using a hole pipette)
• Transferred to a 10 mL volumetric flask**~Acetonitrile** 

## 4 mL**~Ultra** 

## pure water
• Fixed volume 10 mL
Fixed volume solution (acetonitrile/water(! :1))

• Filtration through a filter 

## (EKICRODISK 13 

## by Nihon Pall Ltd.) Sample for LC/MS analysis



# 13

# 2.3.2 Analytical method for test water (SOP 2.10) Pretreatment Test water was sampled (approx. 200 mL from the Concentration Levell and approx. 200 mL from the Concentration Level 2) from the central area ofeach aquarium with an Erlenmeyer flask. After leaving it standing, the supernatant (1 

# mL in the Concentration Level 1 and 2.5 mL in the Concentration Level 2) was taken using a hole pipette and transferred to a volumetric flask (20 mL in Concentration Level 1 and 5 mL in Concentration Level2). In Concentration Levell, ultrapure water (9 mL) was added to the volumetric flask. The volume was adjusted with acetonitrile to 20 mL in Concentration Level 1 and 5 mL in Concentration Level 2. The constant volume solution was filtered through a filter (EKICRODISK 13 by Nihon Pall Ltd., Tokyo, Japan). The filtrate was analyzed by liquid chromatography mass spectrometer (LC/MS). The operations ofthe pretreatment are shown below as a flow chart.

14

# I

Concentration Level 1 Test water 

# I

• Sampling water: Concentration Levell, 200 mL; Concentration Level2, 200 mL

(left standing for a while, waiting for suspended dirt to be deposited)
• Measurement: Concentration Level 1, 1mL; ConcentrationLevel 2, 2.5 mL

(measurement using a hole pipette)
Taken solution 

# _j

Concentration Level 2

• Transferred to a 20 mL volumetric flask**~Ultra** 

## pure water 9 mL • Transferred to a 5 mL volumetric flask**~Acetonitrile**

### ~Acetonitrile 

## • Fixed volume 20 mL
Fixed volume solution (acetonitrile/water(!:1))

• Filtration through a filter

(EKICRODISK 13 by Nihon Pall Ltd.) Sample for LC/MS analysis • Fixed volume 5 

## mL
Fixed volume solution (acetonitrile/water(1 :1))

• Filtration through a filter (EKICRODISK 13 byNihon Pall Ltd.) Sample for LC/MS analysis



# The operation ofwater analysis in the control group (when starting the test) was based
on the method described in Concentration Level 2.

15

# 2.3.3 Measurement methods <Analytical machine> Liquid chromatography mass spectrometer (SOP/INS/4.33) <Analytical apparatuses and conditions> < Liquid chromatography (LC)> Analytical apparatus High performance liquid chromatography LC-20AD (Shimadzu Co., Ltd.) Column Hypersil GOLD 2.1 mm i.d. x 

# 50 mm (5 J.lm) (Lot No. 9966) Thermo Scientific Inc. Mobile phase
Flowrate Temperature Injection volume A: 20 mM ammonium formate/formic acid (1000:1), B: acetonitrile, A/B(l:1) 0.2 mL/min 40°C 5 f.lL



# < Mass spectrometer (MS)> Analytical apparatus TSQ Quantum Access MAXtriple stage quadrupole mass spectrometer
Ionization method Vaporizer temperature Spray voltage Sheath gas AUX gas Ion sweep gas Capillary temperature Scan type (Thermo Fisher Scientific Inc.) Negative ESI 450°C 2500V 50 15 0 

# 220°C Selected ion monitoring (SIM) m/z 180 [M-Lif <Quantitative calculation> Instrument Xcalibur by Thermo Fisher Scientific Inc. Calculation method Two-point calibration method (absolute calibration method) The calibration using two standard solutions and the quantification ofsample were performed on the analysis day.


# 2.4 Calibration curve by liquid 

# chromatography mass spectrometry 2.4.1 Preparation ofstandard solutions The test substances (5.03 mg and 3.15 mg) were weighed using Mettler Toledo XS 1 05DU (SOP/INS/4.1.4) into 200 mL and 250 mL volumetric flasks and brought to the mark with acetonitrile/water(1:1), respectively. The concentrations ofthe solutions were 25.0242 mgL- 1 

# and 12.5370 mgL- 1 

# when considering the purity. The concentrations ofthe solutions were 24.0959 mgL- 1 

# and 12.0719 mgL- 1 

# when

16

# converting to the concentration of The solutions were diluted with acetonitrile/water(I:1) to prepare standard solutions for calibration curve.


# 2.4.2 Preparation ofcalibration curve The calibration curve was prepared using the standard solutions prepared 

# in Section 2.4.1 and the linearity was checked. Test substance Anion Solution concentration concentration Peak area

No. (mgL" 1) 

## [X] (mgL- 1) 

## [X] [Y]

1 0.0050 0.0048 1246726 2 0.0100 0.0096 2149557 3 0.0200 0.0193 3499884 4 0.0501 0.0483 7806108 5 0.1001 0.0964 15009834 Calibration curve Y = 

## 149267349.5345 X +616562.7686 (by the least square method) r = 

## 1.000 

# [Y]

c;j**~**

### ~ 8000000 

### f-------.rat'-+----......J
ll.) Q..



### 0 0. 06 0. 12 

## Concentration of (mgL" 1) 

## [X]

1111 

# The chromatograms used when preparing the calibration curve are shown in Ref. 6.


# 2.5 Recovery test and blank test for 2.5.1 Recovery test from fish for (SOP 2.11) Fish bodies (two fish paired as one sample) were crashed with a blender after mincing, added an acetonitrile solution (1 ml) 

# ofthe test substance , an analytical sample
was prepared according to Section "2.3.1 Analytical method for fish" and was analyzed by liquid chromatography mass spectrometer. The recovery rates of were found to be as follows. The amount ofan addition ofthe test substance (the amount converted to : 21.9 j..tg) corresponded to 3.40 j.lgg· 1

# relative to the fish 3.22 g (6.44 g/2 fish) and the bioconcentration factor in Concentration Level2 (the concentration converted to : 0.0193 mgL- 1) 

# was approximately 176-fold. Added group

Fixed Quantity ofaddition Recovery Recovery Sample Fish weight Dilution

No. (g) volume factor Measured Calculated measured rate (mL) concentrated areas (%) areas (mgL" 1) 

## 1 3.12 100 10 4025794 0.0219 3951983 98.2 3.21 2 3.14 100 10 4025794 0.0219 3953287 98.2 3.15 3 3.38 100 10 4025794 0.0219 3849419 95.6 3.22 

# Mean recovery rate: 97.3 

# ± 1.2%

17

# <Background ofthe fish> After measuring bodyweight and total length offish bodies (two fish were paired as one sample), the fish bodies were crashed with a blender after mincing. After adding 1mL of acetonitrile, an analytical sample was prepared according to Section "2.3.1 Analytical method for fish" and was analyzed by liquid chromatography mass spectrometer. No background peaks were detected atthe position ofthe peak of Control group Sample Fish Fixed Measured bodyweight volume Dilution factor No. 

## (g) (mL) areas



# 1 3.21 100 10 N.D. 

# 3.29 2 3.25 100 10 N.D. 

# 3.22 3 3.09 100 10 

## N.D. 

# 3.41


# • The chromatograms used when calculating the recovery rates of

bodies are shown in Ref. 7-1. from the fish



# 2.5.2 Recovery test from test water for (SOP 2.10) Water used for raising fish was sampled and a solution containing the test substance
was added. According to Section "2.3.2 Analytical method for test water", an analytical sample was prepared and analyzed by LC/MS. As shown in the following table, the mean recovery rates of were 96.2 ± 

# 1.1% in Concentration Level 1 and 95.5 ± 

# 1.2% in Concentration Level2. Recovery rates in Concentration Level 1 (Added group) Amount of Fixed Quantity ofaddition Recovery Recovery Sample water Calculated

No. measured volume Measured concentrated measured rate 

## (mL) (mL) areas (mgL-I) areas (%) 

## I 

# 1 20 2377235 0.0114 2270545 95.5 2 1 20 2377235 0.0114 2266589 95.3 3 1 20 2377235 0.0114 2321802 97.7 

# Mean recovery rate: 96.2 ± 

# 1.1%

18

# Recovery rates in Concentration Level 2 (Added group) Amount of Fixed Quantity ofaddition Recovery Recovery Sample water Calculated

No. measured volume Measured concentrated measured rate (mL) 

## (mL) areas (mgL-1) areas 

## (%)
1 2.5 5 2413659 0,0118 2334903 96.7 2 2.5 5 2413659 0.0118 2315855 95.9 3 2.5 5 2413659 0.0118 2264899 93.8 

# Mean recovery rate: 95.5 

# ± 1.2% <Background ofthe test water> Water used for raising fish was sampled and ultra pure water was added. According to Section "2.3.2 Analytical method for test water", an analytical sample was prepared and analyzed byLC/MS. No background peaks were detected at the position ofthe peak of either in Concentration Level 1orin Concentration Level 2. 

# contro group Concentration Level 1 Concentration Level 2 Amount of Fixed Amount of Fixed Sample water volume Measured Sample water volume Measured

No. measured (mL) areas No. measured (mL) areas (mL) (mL) 1 1 20 N.D. 1 2.5 5 N.D. 2 1 

## 20 N.D. 2 2.5 5 N.D. 3 1 20 N.D. 3 2.5 5 N.D.
111 

# The chromatograms used when calculating the recovery rates of

shown in Ref. 7-2. from water are

2.6 

# Quantifiable limit 2.6.1 Quantifiable limit for fish In the LC/MS chromatograms, there were no background peaks found due to fish body at the position of

. Ifthe peak area of800000 counts is set as a detectable limit, the quantifiable bioconcentration factor is shown in the following table.

Setup concentration in Detectable Quantifiable Bioconcentration factor-'' each concentration level concentration1 l limit2

## l Concentration Concentration (concentration of (mgL-1 ) (IJ.gg-1) Levell Level2 I 

## (0.193 mgL-1 ) (0.0193 mgL-1 ) Concentraion Level 1 0.193 mgL-1 0.0012 0.192 1-fold or greater 10-fold or greater Concentraion Level 2 0.0193 mgL-1 (Note I) The detectable concentrationwas calculated from the calibration curve in Section 2.4.2. (Note 2) The quantifiable limit in the fish bodies was calculated by the calculation formula shown in the nextpage.



# 19

## The quantifiable limit was calculated as the fish bodyweight; 6.44 g (3.22 g per fish, 2 fish
were paired as one sample), recovery rate; 97.3%, fixed volume; 100 ml and dilution factor; 10.
Quantifiable limit (f,!gg·I) = 

### Detectable concentration (mgL" 1) 

### x Fixed volume (mL) x Dilution factor x 100 Recovery rate(%) x Fish bodyweight (g) 

## (Note 3) Quantifiable limit (ugg· 1) 

### Bioconcentration factor (BCF) = 

### Setup concentration inthe test group (theoretical value) (mgL" 1)



# 2.6.2 Quantifiable limit for test water In the LC/MS chromatograms, there were no background peaks observed due to test water at the position ofthepeak of

. Ifthepeak area of800000 counts is set as a detectable limit, the quantifiable limit for test water is shown in the following table.
Setup concentration in each concentration level Detectable Quantifiable limit2

## l(mgL" 1) 

## (concentration of concentration1

## l Concentration Concentration (mgL"I) Concentration Concentration Levell Level2 Levell Level2 (0.193 mgL" 1) 

## (0.0193 mgL" 1) 

## 0.193 mgL" 1 

## 0.0193 mgL" 1 

## 0.0012 0.0249 0.00251 (Note 1) The detectable concentration was calculated from the calibration curve in Section 2.4.2. (Note 2) The quantifiable limit for test water was calculated by the following calculation formula. The quantifiable limit was calculated as the fixed volume; 20 mL in Levell and 5 mL in Level 2, recovery rate; 96.2% in Levell and 95.5% in Level 2 and amount ofwater measured; 1mL in Level 1 and 2.5 mL in Level 2.
Quantifiable limit (mgL" 1) 

### =



# 2.7 Preliminary test Detectable concentration (mgL" 1) 

### x Fixed volume (mL) 

### Recovery rate(%) x Amount ofwater measured (mL) X 

### 100



# Test fish (Cyprinus carpio) 

# were exposed to test water (the concentration converted to 0.0193 mgL" 1) 

# in Concentration Level2 (the flow-through system). On the ih 

# day ofexposure, the test fish (2 fish as a pair) and the test water were sampled and analyzed according to Sections "2.3.1 Analytical method for fish" and "2.3.2 Analytical method for test water". As a result, the BCF of was found to be under a detectable limit and less than 8-fold.

20

# 2.8 Schedule ofanalyses offish and test water Analyses oftest water and fish were conducted according to the following schedule:

Number of Sampling date days after Test group Control group exposure Analysis ofwater Exposure January 26,2011 

## DayO Analysis ofwater Analysis offish period Lipid contents (28 

## days) February 2, 2011 7th 

## day Analysis ofwater

- 

## Analysis offish February 9, 2011 14th 

## day Analysis ofwater

- 

## Analysis offish February 16, 2011 21 51 

## day Analysis ofwater



# - Analysis offish February 18, 2011 23rd 

## day Analysis ofwater



# - Analysis offish February 23,2011 28th 

## day Analysis ofwater Analysis offish Analysis offish Lipid contents


# On the day ofanalysis ofwater, pH, DO (adjusted to be approximately 7 mgL- 1) 

# and water temperature were measured for each aquarium and the data were recorded (pH meter SOP/INS/4.3.3, DO meter SOP/INS/4.4.4). In compliance with SOP 2.10 (measurement ofconcentrations ofthe test substance in the test water), analysis oftest water was conducted according to Section "2.3.2 Analytical method for test water". The changes in the concentration ofthe test substance in water were confirmed to be ±20% or less relative to the mean ofthe measured values during the exposure period. In compliance with SOP 2.11 (measurement ofconcentrations ofthe test substance in the fish), analysis offish was conducted according to Section "2.3.1 Analytical method for fish". In compliance with SOP 2.9 (measurement oflipid contents in the fish), lipid contents were measured according to the "chloroform/methanol extraction method". The changes in the lipid contents from the beginning ofthe experiment to the end ofthe experiment were confirmed to be 

# ±25% or less. Storage before analysis offish and treatment ofwaste during analysis offish were performed in compliance with SOP 2.15 (storage before analysis offish) and SOP 2.16 (discharging waste during analysis offish).

21

# 3. Test results 3.1 Concentrations of in test water The concentrations of in the test water (mgL- 1) 

# are shown in the following table with respect to the concentration levels.

e

# ~ DayO 7th day 14th day 21 51 

## day 23rd day 28th day p


## Concentration Level I 0.177 0.181 0.177 0.181 0.187 0.185
Concentration Level 2 0.0186 O.oi88 0.0184 0.0184 0.0193 0.0188 Umt. mgL·l



# • Analytical data during the analysis oftestwater are shown in Ref.5.
Based on the results shown above, the mean concentration during the test period and the standard deviation were as follows.
Concentration Level 

# 1: 0.181 ± 

# 0.00373 mgL-I 

# Concentration Level2: 0.0187 

# ± 0.000308 mgL" 1



# The following calculation formula was used.

Mean Standard deviation

11 

# L.>i 

# 2>**~=i::!____=-**



## n n

111 

# Changes over time in the concentrations ofthe test substance in water, water

temperature, pH and DO are shown in Ref. 2. The chromatograms at the time of analysis on Day 0, 7, 14, 21, 23 and 28 after exposure are shown in Ref. 9-1. Further, the chromatograms at the time ofanalysis ofwater in the control group are shown in Ref. 9-2.

22

# 3.2 Bioconcentration factors (BCFs) According to the results ofthe bioconcentration test, the bioconcentration factors (BCFs) of were under a detectable limit in both Concentration Levels (less than 1-fold in Concentration Level 1 and less than 11-fold in Concentration Level 2). Since it was confirmed that the steady-state was achieved within 28 days in both Concentration Levels, exposure was finished. The bioconcentration factors (BCFs) of are shown below with reference to the day ofanalysis.

Number of Concentration Level 1 Concentration Level 2 days after Sample No. Bioconcentration Sample No. Bioconcentration exposure (n==2) factor (BCF) (n==2) factor (BCF)

(Days)


## 7 No.1 <1 No.1 <10 No.2 <1 No.2 < 10 14 No.1 <1 No. I <11 No.2 <1 No.2 <10 21 No.1 <I No. I < 10 No.2 <1 No.2 < 10 23 No.1 <1 No. I < 10 No.2 <1 No.2 < IO 28 No.1 <1 No.1 < 10 No.2 <1 No.2 < 10 (Note 1) The sample numbers in Concentration Levels 1 and 2 are symbols given to the fish taken from each concentration group when the amount ofaccumulation was measured. (Note 2) The fish bodyweight during the analysis and other analytical data are shown in Ref. 4, and the chromatograms obtained at the time ofanalysis on the 7fu, 14th, 21 5\ 

## 23rdand 28th day are shown in Ref. 8-1. The chromatograms obtained at the time ofanalysis offish in the control group are shown in Ref. 8-2. (Note 3) The time-dependent changes in the bioconcentration factors in fish (BCF) are shown in Ref. 3. 

# (1) Mean ofbioconcentration factors (BCFs) and standard deviation The mean ofthe bioconcentration factors (BCFs) of during the experimentperiod and the standard deviation are shown below.
Concentration Level 1 BCF =The mean ofthe BCFs was not calculated because all the BCFs were less than the detectable limit. Concentration Level2 BCF =The mean ofthe BCFs was not calculated because all the BCFs were less than the detectable limit.

Bioconcentration factor (BCF) == Concentration of in the aquarium (mgL" 1) 

## Concentration of 

# Mean Standard deviation

n 

# 2>; 2>
;: == .!.::!.__ 

## == -- 

## n n n 

# n 

# 23

# (2) Confirmation ofa steady-state Since the bioconcentration factors (BCFs) of in both Concentration Levels were less than 50-fold during the 28-day exposure period, the steady state was assumed to be achieved on the 28th day ofexposure and exposure was completed on the 28th day.



# < Reference items> Test fish in the control group were analyzed before exposure Day 0 (n=1) and at the end ofexposure Day 28 (n=1). No background peaks due to fish were detected in the LC/MS chromatograms at the position ofthe peak of as in the control group in the added recovery test. These chromatograms are shown in Ref. 8-2.



# 3.3 Evaluation, discussion ofthe results and conclusion The test substance is a salt consisting of

. In this test, the bioconcentration of only was confirmed. Since lithium bromide [1-110], listed as an existing chemical substance, is not high bioconcentration, the bioconcentration ofLi is considered to be confirmed by analogy. The 96h LC5

# ovalue ofthe test substance in Zebrafish 

# (Dania rerio) was greater than 101 mgL- 1• 

# Since the solubility in waterwas >1.0 xl05 

# mgL- 1

, 

# two concentrations ofthe test substance in water were set up to be 0.2 mgL- 1 ( : 

# 0.193 mgL- 1) 

# for the high exposure level (Concentration Levell) and 0.02 mgL- 1 ( : 

# 0.0193 mgL- 1) 

# for the low exposure level (Concentration Level2). According to the results ofthe bioconcentration test, the bioconcentration factors (BCFs) in both Concentration Levels 1 and 2 were less than 50-fold. Thus, a steady state was assumed to be achieved within 28 days ofexposure. It was confirmed that the bioconcentration was under a detectable limit and the bioconcentration factors were less than 1-fold in Concentration Levell and less than 11- fold in Concentration Level 2. Therefore, bioconcentration of ofthe test substance was confirmed to be low.

24

# Table ofcontents for references

Page
Reference 1 Bioconcentration test equipment outline 26 Reference 2 Aquatic environment (concentrations, watertemperature, pH and DO) 27 2-1 Concentrations of in water 27 2-2 Measurement oftemperature, pH and DO in water 27 Reference 

# 3 Time-dependent changes in bioconcentration factors 28 Reference 4 Fish analysis data 29 Reference 

# 5 Water analysis data 32 Reference 6 Chromatograms for the preparation ofcalibration curve 35 Reference 7 Chromatograms of for recovery rate 38 7-1 Chromatograms of for recovery rate from fish 38 7-2 Chromatograms of for recovery rate from water 43 Reference 8 Chromatograms in fish 52 8-1 Test group 52 8-2 Control group 67 Reference 9 Chromatograms used when measuring the concentrations in water 70 9-1 Test group 70 9-2 Control group 82 Reference 10 Water quality test 84 Reference 11 Records ofobservations ofthe conditions ofthe test fish (carp) 86 Reference 12 IR spectrum for the test substance 88

25

# Reference 1 Bioconcentration test equipment outline

A B

A: Tap water B: Filter/activated charcoal tank (dechlorination equipment) C: Tank for water for test (dechlorinated water) 

## E



# G



# .............................................................................. ... ....................................................................... ... .. 

# ........................................................................ .... 

# ... ........................................................................ ... ... ........................................................................ ...


# ... ······································································· ... . ,.. 

# ...

# ~11**~11111~~1~j~1111111j1111111jjjjj~1~1l111111111~jjj~jjjjj1jjj~jj 1~j**  

# m**111~~~1111111~11jj~jjjj1j1mmmlml~H~11111mm1l** m

# 11U~!~~~~~~~~~~~~lmrrrmmmmmm11l11llll1Wl111111llll!~~~!~Ull

E: Tank for stock solution oftest substance (lOL) F: Mixing tank (2L) (mechanical stirrer) G: Test aquarium (lOOL) G-1: Aeration D: Regulator for controllingthe flow rate and mixing ratio G-2: Temperature regulator/heater
D-1 : Water for test D-2: Stock solution oftest substance



# 26

# Reference 2 Aquatic environment (concentrations, water temperature, pH and 

# DO) 2-1 Concentrations of in water


# + Concentration Level 1Group Ill 

## Concentration Level 2 Group


## ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

"""""""""""""""""""""""""""""""."""""""""""""""""""""""""""""""."""""""""""""""""""""""""""""""

..

"""""""$""""""""""""""""""""" 

### 0. 1 ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::



# ----------------------------------------------------------------------------------------------------------------------------------**11'"""""""""""""""""""""""""""""""1111""""""""""""""""""""""""""""""11111""""""""""""""""""""""""""""""1!1"""""""1111"""""""""""""""""""""1~** 

### 0. 01 1;::=::.=·::=::=::=::=:::=::=::=::=::·=.::=::=::=::=::=:::=::=::=::=:::=::=::=::=::=:::=::""::=::=:::=::=::=::=::=:::=::=::=::=:::=::=::=::=::=:::=::=::=::=::=:::=::=::=::=:::=::=::=::=::=:::=1::



# ----------------------------------------------------------------------------------------------------------------------------------
----------------------------------------------------------------------------------------------------------------------------------
----------------------------------------------------------------------------------------------------------------------------------

0.001**~------------~------------~--------------~------------~**



## Number of days after 0 

### 7 

### 14 

### 21 28 

## Time after exposure (days)



# 2-2 

# Measurement oftemperature, pH and DO in water The aquatic environment was measured at a fixed time (10 a.m.). For pH, water was sampled from the center ofthe aquarium and measured with a pH meter (SOP/INS/4.3.3). Further, DO in water was measured at the center ofthe aquarium using a DO meter (SOP/INS/4.4.4). The results are shown in the following table.
Concentration Level 1 ConcentrationLevel 2 Control group Water DO 

## Water DO 

## Water DO 

## exposure temperature pH (mgL" 1) 

## temperature pH (mgL" 1) 

## temperature pH (mgL" 1) 

# (OC) CCC) CCC) 0 7 

## 14 21 23 28 24.8 7.4 7.5 25.1 7.4 7.5 25.5 25.4 7.4 7.5 25.2 7.5 7.3 25.3 25.5 7.5 7.4 25.5 7.5 7.4 25.4 25.1 7.5 7.5 25.4 7.4 7.4 25.1 25.4 7.6 7.5 25.4 7.6 7.4 25.4 25.2 7.6 7.4 25.1 7.5 7.5 25.3 

# [DO in water]



# + Concentration Level 1 Ill 

## Concentration Level 2 .A 

## Control Group 

## 10

9

# ~ 8 

# 5 

# 8 7
6
5 

# a

0 7 

# Ill lfll lfll**~**



## 14 21 28 Time after exposure (days) 

# 27 7.4 7.3 7.5 7.3 7.5 7.5 7.4 7.4 7.6 7.4 7.6 7.5

# Reference 3 Time-dependent changes in bioconcentration factors
[Concentration Level 1]

..!- indicates less than a detectable limit. 

# 100 :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::



# ----------------------------------------................................................................................................................................................................... """" (.;.. 

# u 

# 6

.... 

# 10 ..9 u 

# ..::: :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::



# ---------------------------------................................................................................................................................................................................... !::: 0

## ·~ !::: 0 u !::: 0 u 0 

# co 0.1 0 7 14 

## 21 

# 28

Number ofdays after exposure (days)



# [Concentration Level 2]

..!- indicates less than a detectable limit. 

# 100 G:' 

## u 

# 6

.... 

# 10 ..9 u 

# ..::: !::: 

# .g 0::

## ~ 0 u ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::



## ............................................................................................................................................................................................................................................................... 

# ---------------------------------------------------------------------..................................................................................................... :::: 

# ------------------------------------------------------------------------------------------------------------------ 0 u 0 

# co 0.1 0 7 14 

## 21 

# 28
Number ofdays after exposure (days)



# 28

### N \0 

# Reference 4 Fish analysis data Fish analysis data (Concentration Level 1) Items 

# I A B 

# c D E 

# I F 

# I G 

# I H 

# I I 

# I J 

# I K 

## Fixed Concentration Amount volume Dilution calculated by two- deposited in Fish Recovery rate Concentration Concentration Bioconcentration 

## 1 

## D f ofthe Peak area point calibration weight in fish body in water tank ateo factor fish body (%) factor 

## Samples ""- !solution curve (J.tg) (g) (J.tgg-1) (mgL-1 ) (BCF) measurement (mL) (mgL- 1 ) Day 7No.1 

# I 100 10 N.D. <0.0012 <1.2 

# I**~.69** [3.24, 3.45] 

# I 97.3 

# I <0.184 

# I 0.181 

# I <1 

# I 2011.2.2



# ....**P..~~-.?.**

# ..**~.?.:.~---······** 

# ......**~_gg** 

# ______.................!.?.

.......................**~:P.:**

# ....................**::':?..:.~9.** 

# ..**~-~**

# .......................**:!.:.~**

# ..............**?.:.~?.**

# ..**P.:.~~:.**

# ..**~.:.?.?.J..** 

# .............?..?.:?

.......................**~?..:}**

# ..**~-~**

# ..................**~.:.!.~.!**

# .............................::':L

...................**~-~}--~**

# ..:
..**~.:**

# ....**~**

# ... Day 14 No.I 100 10 N.D. <0.0012 <1.2 6.87 [3.58, 3.29] 97.3 <0.180 0.177 <1 2011.2. 9



# ...**P.~~-J-~**

# ..**~~-:~**

# ..... ....}

..**~~------** 

# ..............J9.

.......................**~:P.:**

# ....................**::':?..:.~~--~-~** 

# ........................**~.!..:.~**

# .............. ?.:.?.?.

..

# E:.!}.:.

..**~.:.?.~J..** 

# ............**.?..?.:.~** 

# .......................

# ::':?..:.!.

..**~-~--------·** 

# .........**~.:.!.?..?.**

# ...........................:::L

..............**----~g-~**

# ..!.:

...**~.:**

# ....?.

... Day 21 No.I 100 10 N.D. <0.0012 <1.2 6.83 [3.41, 3.42] 97.3 <0.181 0.181 <1 2011. 2.16



# ..**P.~?**

# ..**~L-~.?..:~** 

# ...... ...JQg

......................**!..~**

# ......................**~.:.P..:**

# ....................**::':~.:-~~.!.-~**

# .......................**::':.!..:.~**

# .............**~.:-~~**

# ..**P.:~.?..~.-~.:-~**

# ..**~L**

# .............**?.?..:.~**

# .......................**:::~.:.!..~.!**

# ...................**~.:.!..~.!.**

# ............................**::':.~**

# .....................**~-~-~).:** 

# ...**~.:-~**

# ..

# ?.

... Day23No.1 100 10 N.D. <0.0012 <1.2 6.62[3.14,3.48] 97.3 <0.186 0.187 <1 2011.2.18



# ...**P.~t~.~---~~-:~**

# ..... .....**L~~-----·**

# .................**~-~** 

# .......................**~:P.:**

# ....................**::':?..:.~~--~-~** 

# ........................**~.!.:.~**

# .............. ?.:.?..?.

...**P.:.~?.-~**

# ..**~.:~~1.** 

# ............**.?..?.:.~**

# ......................

# ::':?..:

..**~**

# ..**~-~**

# ..................**~.:.!..~.?.**

# ...........................::':L

...................**~.~-~**

# ..!..:

...**~.:-~-~**

# ... Day 28 No.1 100 10 N.D. <0.0012 <1.2 6.91 (3.11, 3.80] 97.3 <0.178 0.185 <1 2011.2.23 Day 28 No.2 100 10 N.D. <0.0012 <1.2

Calculation methods: Amount deposited in fish Concentration in fish

Bioeoneentration factor (BCF) 6.81 (3.53, 3.28]

E =AxBxD 

# H= EX 100 

## FxG



## J = 

### I 

## 97.3 <0.181 0.185 <I 2011.2.23

Total value of2 fish was used for F.

(Note) Sample No. is a symbol given when two fish were paired when sampling fish and analyzed at n=2. Forthe samples showing less than the detectable limit (peak area 800000), N.D. is recorded in the column C and the calculation was made by replacing the detectable limit of0.0012 mgL- 1in the aforementioned D.

## w 0 

# Fish analysis data (Concentration Level 2) Items 

# I A B 

# c D E 

# I F 

# I G 

# I H 

# I I 

# I J 

# I K 

## Fixed Concentration Amount volume Dilution calculated by two- deposited in Fish Recovery rate Concentration Concentration Bioconcentration 

## 1 

## D

. f ofthe Peak area point calibration weight in fish body in water tank ateo factor 

## "'- !solution factor fish body (g) (%) 

## (flgg-1) (mgL"1 ) (BCF) measurement Samples curve (f.!g) 

## (mL) (mgL" 1 ) Day 7No.1 

# I 100 10 N.D. <0.0012 <1.2 6.76 [3.58, 3.18] 

# I 97.3 

# I <0.1 82 

# I 0.0188 

# I <10 

# I 2011.2. 2



# ...**P.~~-.?.** 

# ..**~.?.:.~**

# .........**···---~--~~---··· ················-~-~---··--·········**  

# .......**~.:P.:**

# ....................**::~.:-~~--~-~---·········**

# ...........**:.!.:.~**

# .......... ....**~:-~~** 

# ...**P.:.~.?..~.-~.:.?..~1** 

# .............**?.?:.~**

# ......................**::~.:**

# ..**~.-~g·········** .........

# ~.:-~} ..

# ~~---······ ..............

# :.!.~

# ..............**··---~-~-~--~-:** 

# ...**~.:**

# ....**~---** Day14No.l 100 10 N.D. <0.0012 <1.2 6.25[2.89,3.36] 97.3 <0.197 0.0184 <11 2011.2.9

# ·---~-~~-}-~

# ..**~-?..:.~**

# .........**J.~~------** 

# .................**.~** ................ .......

# ~.:P.:

# ....................**::~.:.?.~** 

# ..**~-~**



# ........................**:::~.:-~**

# .......... ....**~.:.?.** 

# ..**~**



# ....**P.:.~.?.:.**

# ..**~.:-~.?..1..** 

# .............**?..?.:.~**

# ......................**::.~:}**

# ..**~.?.**

# ..................**~.:-~.~--~~** 

# ........................**:::~-~**

# ..................**~.?.-~** 

# ..**~**

# ..: ..**~.:**

# ....?.

... Day 21 No.I 100 10 N.D. <0.0012 <1.2 6.89 [3.24, 3.65] 97.3 <0.179 0.0184 <10 2011. 2.16

# ----~-~x

# ..**~~---~-?..:~**

# .....

# L.

...**~**

# ..**~~--** 

# ....I.

.............

# J~ 

# ................L..

....**~.:P.:**

# ........I.

...........

# ::~.:.?.~ 

# ..**~-~------**

# .......I..

.........

# :::~.:~..........

# L..?..:.?..~

# ..

# E:.?..?.:.

..**~.:.?..~.LI...**

# ..........**?..?.:.~**

# .............I.

........

# ::.?.:}

..**~-~----**

# .....l.

........**~.:-~-~--~~** 

# ..........1..

............

# :::~.~--------------L-~-~-~---~

# ..: ..**~.:.~** 

# ..?.

... Day 23 No.I 100 10 N.D. <0.0012 <1.2

Day 28 No.2 100 10 N.D. <0.0012 <1.2

Calculation methods: Amount deposited in fish Concentration in fish

Bioconccntration factor (BCF) 6.49 (3.29, 3.20]

6.76 (3.42, 3.34]

E=AxBxD 

# H= EX 

## 100 FxG

J = 

# _lL I 

## 97.3 <0.190 0.0193 <10 2011. 2.18

97.3 <0.182 0.0188 <10 201 I. 

## 2.23

Total value of2 fish was used for F.

(Note) SampleNo. is a symbol given when two fish were paired when sampling fish and analyzed at n=2. Forthe samples showing less than the detectable 

## limit (peak area 800000), N.D. is recorded in the column C and the calculation was made by replacing the detectable limit of0.0012 mgL·' in the aforementioned D.

## w



## ....... 

# FishItems

Samples

DayO

--------------------- Day28 

# . d 

# (C ' 

## A Fixed volume ofthe solution

(mL)

100



# ------------------
100 

# )

/ 

## B 

# c D Concentration calculated by Dilution Peak two-point

factor area calibration curve (mgL- 1)



## 10 N.D.

-



# ------------------------- ------------- ----------------------------- 10 N.D.


## - E F G H I J 

## K 

## Amount deposited Fish Recovery Concentration Concentration Bioconcentration 

## Date of

in fish weight rate in fish body in watertank factor 

## body (g) 

## (%) (!lgg-') (mgL- 1) 

## (BCF) measurement (!lg)

- 

## 6.84 [3.53, 3.31]

- 

## - - - 

## 2011. 1.26

------------------- --------------------- ------------------- ------------------------- -------------------------- 

# ------------------------- ----------------

- 6.62 [3.66, 2.96]

- - - 

# - 2011. 2.23

## w N 

# Reference 5 Water analysis data Water analysis data (Concentration Level I) Items A B 

# c D E 

## Fixed Concentration Absolute amount Amount of volume calculated by in the fixed water ofthe Peak area two-point solution calibration curve volume solution measured Samples (mL) (mgL- 1) 

## (f!g) (mL)

DayO 20 1873661 0.0085 0.170 1 

## Day7 20 1921305 0.0087 0.174 1 Day 14 20 1895936 0.0085 0.170 1 Day21 20 1931160 0.0087 0.174 1 Day23 20 1967483 0.0090 0.180 1 Day28 20 1964054 0.0089 0.178 1
Calculation methods: Absolute amount in the fixed volume solution
Concentration in the aquarium 

## F

Recovery rate (%)

96.2 96.2 96.2 96.2 96.2 96.2

D 

# = AxC G 

# =__ D X 

### lQO_ ExF 

## G H



## Concentration Date of in water tank

(mgL- 1) 

## measurement

0.177 2011. 1.26 0.181 2011.2. 2 0.177 2011.2. 9 0.181 2011.2.16 0.187 2011. 2.18 0.185 2011. 2.23

### w 

## w 

# Wat tration L 

# · data (C 12) ' / 

## Items A B C D E F G H Fixed Concentration Ab 1 t 

## A f 

## so u e amount mount o . volume calculated by

. Concentration

fth P k t . t m the fixed water Recovery rate . k Date of o e ea area wo-pom . 0 

## m water tan

I . J"b

. volume solutiOn measured (Yo) ( 

## L. 1) 

## measurement so utJOn ca 1

### rat10~**curve (~g)**  

## (mL) mg Samples (mL) (mgL )

Day 0 5 1937105 0.0089 0.0445 2.5 95.5 0.0186 2011. 1.26 Day 7 5 1970474 0.0090 0.0450 2.5 95.5 0.0188 2011.2. 2 Day 14 5 1941077 0.0088 0.0440 2.5 95.5 0.0184 2011.2. 9 Day21 5 1943578 0.0088 0.0440 2.5 95.5 0.0184 2011.2.16 Day23 5 1988219 0.0092 0.0460 2.5 95.5 0.0193 2011.2.18

... Day**2~** 

## _5_**198577~L_** 

## 0.0090 0.0450 2.5 95.5 0.0188 2011.2.23
Calculation methods: Absolute amount in the fixed volume solution
Concentration in the aquarium D = 

### AxC G = 

## _I)__2_!QQ_ ExF

## w ..p. 

# w Items

Samples
DayO 

# · data (C ' 

## A Fixed volume ofthe solution

(mL)

5 

# )

/ 

## B 

# c Concentration calculated by Peak area two-point calibration curve (mgL-1)



## N.D.


## - D E 

## F G H


## Absolute amount Amount of in the fixed water Recovery rate Concentration Date of in watertank volume solution measured (%) (mgL- 1) 

## measurement (!!g) (mL)



# - 2.5

- - 

## 2011. 1.26

# Reference 6 Chromatograms for the preparation ofcalibration curve
Calibration curve

Standard solution



## 0.0048 mgL" 1



### Calibration curve

Standard solution



## 0.0096 mgL" 1 Dafafile: C186CALS1·2 Acquisition Date: Original Oala Path: C:\XCAliBUR\DATA\KANEKOIC Samplo!O: 186\CAL Sampla Name: Sid. 0.0046 mg/1.. Comments: Sample Type: Std Bracket Operator:
'" ANALYTICAL CONDITIONS (HPLC) "' : SHIMADZU LC-20AD HPLCTYPE COLUMN ELUENT : Hyparsil GOLD 2.1 x50 mm (5 um) : A; 20 mM Amrrumlum !orrnale aqJformlc acid (1000:1)

B; Acefonlttife FLOW RATE COLUMN TEMP INJECTVOL NB(1:1) : 0.2mllmln : 313K : 5uL
"' ANALYTICALCONDITIONS (MS) "' MS 1YPE : Thermo Fisher ScientificTSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILlARYTEMP : 493 K SCAN TYPE : SIM (mlz 160)

Data File: C186CALS2·2 Acqulslllon Oata:- Or!glnal Oala Path:**fJ)l(~IBUR\DATA\KANEl(O\C** Sample!D: Sampla Namo: Std. 0.0096 mgt1.. Comments: Samplo Typo: Sid Bn>cket Operator:



# ................................................................................................................................... "' ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE : SHIMADZU LC-20AD COLUMN : Hyporsll GOlD 2.1 x50 mm (5 um) ELUENT : A: 20 mM Ammonium formate aqJformfco<'.d (1000:1) B; Acetonllrlle AIB(1:1) FlOW RATE : 0.2 mllmln COLUMN TEMP : 313 K INJECTVOL : 5 ul
"' ANALYTICALCONDITIONS {MS)

- MS TYPE : Thermo FtsherSclenUrreTSQ Quantum Access MAX IONIZATION : Nagalive ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM {m/z 1 80)



# ...................................................................................................................................



# 35

## Calibration curve
Standard solution

0.0193 mgL- 1



## Calibration curve
Standard solution

0.0483 mgL- 1 Data Fife: CfB6CAlS3·2 Acquisition Date: Original Data Path: C:\XCAUBUR\DATA\KANEKO\C SampleiD: 186\CAL Sampto Name: Ski. 0.0193

### mgn. Commenfs: Sample Type: Sid Bracket Oporator:

"' ANALYTICALCONDITIONS (HPLC) "' H?LC TYPE : SHIMADZU Lc-20AD COLUMN : HyporsiiGOLD 2.1x50mm(5um) ELUENT : A; 20 mM Alnrn<><'Jum fomtale aq.llormlc acid (1000:1) B;Acetcnlftl!e NS(f:f) FLOW RATE : 0.2 mVmin COLUMN TEMP : 313 K INJECT VOL : 5 ul
'" ANALYTICAL CO!IDITIONS (MS) "' MS TYPE : Thermo Fisher Sc!an!lticTSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZER TEMP : 723 K SPAAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARY TEMP : 493 K SCANTYPE : SIM (mlz 180)**1\T;O.CG·~OO**



# - ,._, lto:«rl

## -::=
·-

Data Fifo: Cf86CAlS7·2 Acqulsnlon Oate: Original Data Path: C:IXCAUBUR\OATA\KANEKO\C SampleiO: 186\CAL Sample Namo: Std. 0.0483 mgll Comments: Sample Type: Sid Bracket Operator:
"' AIIAL YTICAL CONDITIONS (HPLC) "'

: SHIMADZU LC.20AD HPlCTYPE COLUMN ElUENT : Hypersll GOlD 2.1 xEO mm (5 um) A; 20mM Ammoniumfo111U!Ie •qJformlc add(1000:1) Bi Ac:e!onitrile FLOW RATE COLUMN TEMP INJECTVOL NS(1:1) : 0.2mUmln : 313K : 5ul
'" ANALYTICAL CONDITIONS {MS) '" MS TYPE : Thermo FisherSclenllficTSQ Quantum Acc.,s MAX IONIZATION : Negallve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : EO AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM{mlz180)



## '""" 

## "''""**Jt~~** 

## '''"' 

# ..,., .....,·
..,...

0· 0.0



# 36

### Calibration curve

Standard solution

0.0964 mgL· 1



### Calibration curve Chromatogram of solvent for adjustment Data File: C166CA!.S4·2 Acqulsllfon Dalo: Original Data Path: 0:\XCAUBURIOATA\KANEKO\C SampleiO: 186\CAL Sample Name: Std. 0.0964 mg/L Comments: Sampltt Type: Std Bracket Operator:

'" ANAl \'TICAl CONDITIONS (HPLC) "' HPLC TYPE : SHIMADZU LG-20AD COI.UMN Hypersl GOI.O 2.1x50mm (5 urn) ELUENT A; 20mMAmmonium lormale aq.lfotmle aeld (1000:1) B;AcetonlkHs A/8(1:1) FLOWRATE : 0.2mlhnln COLUMN TEMP : 313 K INJECTVOL : 5uL**·~** ANALYTICALCONDinONS(MS) •·• MS TYPE : Thermo FisherScientificTSQ Quanlum Access MAX IONIZATION : Negallve ESI VAPORIZERTEI.lP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

Data Fils: C186CALS0·1 Acquisition Date: Original Data Path: C:IXCALIBUR\OATA\KANEKO\C SamplaiD: 186\CAL Sample Name: Solvent Comments: Sampte Type: Blank Operator:

"'ANALYTICALCONDITIONS (HPLC) "' : SHIMAOZU LG-20AO HPLCTYPE COI.UMN ELUENT : Hypersll GOL!l 2.1x somm (5 urn) : A; 20 mM Ammonium lormato eqJiorm!c acid (1000:1)

B;Aeelonll!lle FLOWRATE COLUMN TEMP INJECTVOL A/8(1:1) : 0.2mllmln : 313K : SuL
"' ANAL\'TICALCOIIDITIOilS (MS) "' MS TYPE : Thermo Asher Scitnlifi-cTSQ Qua."'otum Access:**~tAX** IONIZATION : NegaliveESI VAPORIZERTEI.IP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

__



# 37

# Reference 7 Chromatograms of for recovery rate 7-1 Chromatograms of for recovery rate from fish
Recovery test (Fish analysis)
Standard solution

0.0048 mgL· 1



### Recovery test (Fish analysis)
Standard solution

0.0483 mgL· 1 DalaF!Ie: Original Data Path: Sample Name: SampleType: C1B8FRS1 Acquisition Dale: C:IXCALIBUR\OATA\kaneko\C18 SampleiD: 6\FR Sld.0.0048 mgll Comments: Sid Bracket Opor.ator:
'" ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHIMADZU LC-2!lAO COlUMN : Hypersll GOLO 2.1 x50 mm (5 um) ElUENT : A; 20 mMAmmonlum formate aqJf01mk: acid (1000:1) B; Acelonllli!e NB(I:1) FLOW RATE : 0.2mllmln COLUMN TEMP : 313 K INJECTVOL : 5 uL
'" ANALYTICAL CONDITIONS (MS) '" MSTYPE : Tharmo'Fisher ScientificTSQ Quantum Access MAX IONIZATION : Negall\'0 ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : SO AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

•
.. '1-·

Oafa FUe: C1e6FRS2·1 AcquisiUon Oate: Original OalaPath: C:IXCALIBURIDATA\kaneko\018 SampleiD: 6\FR Sample Name: Std. 0.0483 mgJL Comments: Sample rype: Std Bracket Operator.
"' ANALYTICAL CONOtnONS (HPLC) "' HPLC TYPE : SHIMAOZU LC-20AD COLUMN : Hypetsl GOLD 2.1 >50 mm (Sum) ELUENT : A; 20mMAmmon!um fonnalcaq./form!cacld (1000:1) B; Acelonltrllo AIB(1:1) FLOW RATE : 0.2 mUmln COLUMN TEMP : 313 K INJECT VOL : 5 uL
"' ANALYTICAL CONDITIONS (MS) "' MS TYPE : Tharmo Asher SeienlilicTSQ. Quantum AcceS3 MAX IONIZATION : NegallveESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : o CAPILLARYTEMP : 493 K SCAN TYPE : SIM (m/z 180)



## "''" """'**''**



# 38

### Recovery test (Fish analysis)
Added Standard solution

Recovery test (Fish analysis)
Added group No.1 Data File: C1S6FRAS Acquisition Dal&: Otlginal Data Path: C:\XCALIBUR\DATA\kanoko\C1S SamploiO: 6\FR Samplo Name: Added Std. Comments: Samplv Typo: Unknown Operafor:

"' ANALYTICALCONDITIONS(HPLC) '" HPLC7YPE : SHJMADZU LC.ZOAD COLUMN HypetsU GOLD 2.1 xSO mm (5 urn) ELUENT A; 20 mMAmmonium formate aq./form!cacid (1000:1) B;Aeetonlkilo FLOWRATE :

## ~(~8ru, COLUMN TEMP : 313 K INJECTVOL : 6uL
'" ANALYTICAL CONDITIONS (MS) '" MS TYPE ! Thermo FisherSclenllfioTSQ Quantum Access MAX IONIZATION : NegaUva ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2SOOV SHEATH GAS : 50 AUXGAS : 15 JON SWEEP GAS : 0 CAPiLLARYTEMP : 493 K SCAN 7YPE : SIM (mlz 180)

i

## ··-

Data FiltH C185FAA1·1 Acqulsltfon Date; Otlgtnat Data•Path: C:\XCALIBUR\DATA\I<aneko\C18 Sampte!D: 6\FR Sample Name: Added No.1 Commanls: Sample Type: Unknown Operator.
"' ANALYT!CALCONDITIONS (HPLC) "' HPLC 7YPE SHIMADZU LC·20AD COLUMN Hypersll GOLD 2.1 x 50 mm (5 um) ELUENT A; 20 mMAmmonium**~rmale** aq.lform!c acid (1000:1) B;Acaton!trllc FLOWRATE :

## ~(~Dmln COLUMN TEMP ! 313 K INJECTVO!. : 5ul
"' AIIALYTICALCONDITIONS (MS) "' MS 7YPE : Thenno FisherScientificTSQ Quantum Acress MAX IONIZATION : Nega!lva ESI VAPORIZER TEMP ! 723 K SPRAYVOLTAGE : 2SOOV SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN 7YPE : SIM (mtz 180)



# 39

## Recovery test (Fish analysis)
Added group 

## No.2

Recovery test (Fish analysis)
Added group 

## No.3 Data Fifo: C186FRA2 Acqulslflon Da!e: Original Data Path: C:\XCAUBUR\OATA\l<aneko\018 Sample ID: 6\FR Sample Name: Added No.2 Comments: Sample Type: Unkno\\'tl Operator:

"' ANALYTICAL CONDITIONS (HPLC) "' HPLC 1YPE : SHIMAOZU LC.20AO COLUMN : Hypersl GOLD 2.1 x50 1r.m (5 um) ELUENT : A: 20 mMAmlron!um formate aqJformlc acld (1000:1) B:AcetcnitrHa AIB(1:1) FlOW RATE : 0.2 mlJmln COLUMN TEMP : 313 K INJECT VOL : 5 ul

.., ANALYTICAL CONOmONS (MS) '" MS lYPS. : Thermo FisherScle.nUffcTSQ Quantum Access MAX IONIZATION : Negailve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPilLARYTEMP : 493 K SCAN TYPE : SIM (mlz 160)



# .. u

DataFllo! C186FRA3 Acquisition Date: Original Data Path: C:\XCALIBUR\DATA\kaneko\C18 Sample ID: 6\FR Sample Name: Added No.3 Comments: SampleType: Unknown Operator:
"' ANALYTICAL CONDITIONS (HPLC) ••• HPLCTYPE COLUMN ELUENT
FLOWRATE COlUMN TEMP INJECTVOL SHIMAOZU LC-20AO Hyporsi GOLD 2.1X 50 mm (5 um) A; 20 mMAmmonium formate aqJforrnlo acld (1000:1) B;Ao<!lonl'.r!le AIB(1:1) :**0.2mUm~** : 313K l Sul
''' ANALYTICALCONOmONS (MS) "' MS TYPE : Therrr.o Asher ScientificTSQ Quantum Access MAX IONIZATION l Negallw ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE l 2500V SHEATH GAS : 50 AUXGAS : i5 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (m/z 1 80)



# 40

## Recovery test (Fish analysis)


## Control group No.1

Recovery test (Fish analysis)


## Control group No.2 DataFite: C165FRCH Acquls!Uon Date: Original Data Path: C:IXCALIBUR\DATA\I<aneko\C16 SamploiD: 6\FR SampJa Nama: Cfrl No.f Comments: Sample Type: Unknovm Operator:

"' ANALYTICALCONDITIONS(IiPLC)**·~** : SHIMADZU LC·20AO HPLCTYPE COLUMi'l ELUENT : Hypersll GOLD 2.1 x50 mm (5 um) : A; 20 mM Ammonium formate aq./fonnloecld (1000:1)

B:Acelonlttilc FLOW RATE COLUMN iEMP INJECTVOL NB(1:1) : 0.2mUmln : 3t3K : 5uL
"' ANALYTICAL CONDITIONS (MS) "' MS TYPE :**ThermoflsherScterrtir~eTSQQuao!umAccess** MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 250GV SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP :**~93** K SCANTYPE : SIM (m/z160)

DatafUe: C166FRC2 Acqulstuon Dale: Original Data Path: C:IXCALtBURIDATAikaneko\C16 SampleiO: 6\FR Sample Name: Ctrf No.2 Comments: Samplo. Type: Unknown Operator:

"' ANALYTICAL CONDITIONS (HPLC) m SHIMA02U LC.20AD HPLCTYPE COLUMN ELUENT Hyperolf GOLD 2.1 x 50 mm (5 um) A; 20 mM Ammonium formate aqlform!o acid (1000:1) 

### a: Acetonffrlfa fl.OWRATE COLUMN TEMP INJECTVOL NB(1:1) : 0.2mUmin : 313K : 5 uL 

### H' ANALYTICAL CONDITIONS (MS) '" MSTYPE : Thermo Fisher Sci.!lnL'ficTSQ Quanlum Access MAX IONIZATION : Negative ESJ VAPORIZERTEMP: 723K SPRAY VOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYiEMP : 493K SCANTYPE : SIM (rr!z 180)



# 41

## Recovery test (Fish analysis)
Control group 

## No.3 Dataflre: CI86FRC3 Acquisfl!on Date: Original Data Path:**~CAUBURIDATA1l<aneko\C18** SampieJD: SampJe Name: C!rl No.3 Comments: Sample Typo: Unknown Operator:



# .........:

# ........................_

# ......................................................... '" ANALYTICAL CONDITIONS (HPLC) '" HPLC TYPE : SHIMAOZU LC-21lAD COlUMN : HypersiiGOLO 2.1xo0mm(5um) ELIIENT : A;20mMAmrnon!umformale"'lJformicaeld(1000:!) B;Aceton!trlla FLOW RATE :

## 'ri:~(~Bmln COLUMN TEMP : 313 K INJECTVOL : 5 uL
"' ANALYTICALCONOtnONS (MS)"' MS TYPE : Thermo Fisher Sclenfir!CTSQ Quanfiim Access MAX IONIZATION ! Nega!lve ESI VAPORIZERTEMP: 723K SPRAYVOLTAGE : 2500 V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP ! 493 K SCAN TYPE : SIM (mlz 180)



## '""'·**tl**



# 42

# 7-2 Chromatograms of for recovery rate from water Recovery test (Water analysis)
Standard solution

0.0048 mgL- 1



## Recovery test (Water analysis)
Standard solution

0.0483 mgL- 1 Oata fife: C166WRHS1·2 Acqu1sfUon Data: Original DalaPalh:**~jfca!lbu!ldala\kaneko\C1B6\W** SamploiD: Sample Name: Std. 0.0048 mgfL Commen!s: Sampfa Type: Sfd Brac!ret Operator.



# ........................................................................................... "' ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHIMADZU LC-20AD COLUMN : HypersU GOLD 2.1 X50 mm (5 um) ELUENT**~i~~~~~moolum** formate sqJiorm!c acld (1000:1) Nll(1:1) FlOWRATE : 0.2 mUm!n COLUMN TEMP : 313 K INJECTVOL : 5 uL
"' ANALYTICAL CONDITIONS (MS) '" MS TYPE : Thsrmo FisherSclentifJCTSQ Quantum Access MAX IONIZATION : NegaUve ESI VAPORIZER TEMP : 7231< SPRAYVOLTAGE : 2500V SHEATH GAS : EO AUXGAS : 15 ION SWEEP GAS : 0 CAPILlARYTEMP : 493 K SCAN TYPE : SIM (m'z 180)

''''''"''"''''''''''"''"'''''''"'"''''''''""''''''"''''"'''''"''"'''''"''''"Hooouooohoo"'"''"'''"'''"''''''''

OafaFl1e: C186WRHS2·2 Acqubltro11 Date: Original Da!a Path: C:\Xcafibur\data\kaneko\C16tAW Sample ID: RH Sample Name: Std. 0.0483 mgit Commenls: Sample Type: S1dBtacket Opcrnlor:

"'ANALYTICALCONDITIONS (HPLC) 'M HPLCTYPE COLUMN ELUENT : SHIMADZU LC-20AD : Hypersa GOLD 2.1 x50 mm (5 um) : A: 20 mM Ammonium formalo aq.lformlo acid (1000:1)

B: Aoelonllrlle FlOW RATE COLUMN TEMP INJECTVOL Nll(1:1) : 0.2mUm!n : S1SK : 5UL
"' ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thermo Asher ScientificTSQ Q'Jantum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM (m'z 180)

RT:.C.Oih:1b2 ut1$0



## """' 

# ,.,.,.



# 43

### Recovery test (Water analysis) High exposure level

Added Standard solution

Recovery test (Water analysis) High exposure level Added group No.1 Data Fila: Original Data Path: Sample Name: Sample Type: C186WRHAS·2 C:\Xcallburldalalkaneko\C186\W RH Added Sid. Unknown
"' ANALYTICALCONDITIONS (HPLC) "' Acqufsltlon Oafe: SampleiO: Comments: Operator:

HPLCTYPe COLUMN ELUENT SHIMAOZU LC.20AO Hyper>UGOLO 2.1 x60mm(5um) A; 20 mMAmmonium fo1111ale aqlfOflllllacld (1000:1) B:Acalon!We FLOW RATE COLUMN TEMP INJEOTVOL A/8(1:1) ; 0.2mUmln : 313K : 5ul
'" ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thermo FisherSclenUfio TSQ Quantum Access MAX IONIZATION : NegaUva ESI VAPORIZERTE\lP : 723 K SPRAYVOLTAGE : 250DV SHEATHGAS ; 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

u

Dala Fila: C186WRHA1·2 Aequts!Uon Date: Orlglnal Data Path: C:\Xl:allburldala\kaneko\C1BS\W SamploiD: RH Sample Name: Added No.1 Comments: Sample Typo: Unknown Operator:



# ............................................................................................ '" Af/AL YTICAL COf/DITIONS (HPLC) "' HPLC TYPE : SHIMAOZU LC.20AD COLUMN : h)persHGOLO 2.1x50mm(5um) ELUENT : A; 20 mMAmmonium fo1mate aqJformlc acld (1000:1) B: Acetonitrile NB(1:1) FLOWRATE : 0.2mllmln COLUMN TEMP : 313 K INJECTVOL : 5 uL
'"ANALYTICALCONDITIONS {MS) "' MS 1YPE : Thermo Fishe-rSdtmtll1cTSQ Ol.mnhlm Acce.ss 

### ,.,tAX IONIZATION : NegaUve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAFILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)



# ....................................................................................................................................

"""'



# 44

### Recovery test (Water analysis) High exposure level Added groupNo.2

Recovery test (Water analysis) High exposure level Added group No.3 Oafa Frte: Original OalaPath: C1SSWRHA2·2 Aequt.tUon Date<**~lfcaliburldala\l<aneko\C186\W** Sample 10: Sample Name: AddedNo.2 Comments: Sample Type: Unknown Opa.ralon

"' ANALYTICALC0,1DITJONS (HPLC) "' HPLC TYPE : SHIMADZU Lc-20AO COLUMN : J{yparsl GOLD 2.1 x50 mm (5 urn) ELUENT : A;20mMAmmenlumformaleaqJfotmloacld(100D:1) a;Acelonllrila !VIl(1:1) FLOW RATE : 0.2 mllmln COLUMN TSMP : 313 I< INJECTVOL : 5uL**·~ANALYTICAL**CONDITIONS (MS) '" MS TYPE : Thermo Fisher SclentiflcTSQ QuanlumAccess MAX IONIZATION : NegaiivoESI VAPORIZERTEMP : 7231( SPAAYVOLTAGE : 2500V SHEATHGAS : 5<l AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARY TEMP : 493 K SCAN TYPE : SIM (miz 1eo)

DataFife: OriginalDataPath! C186WRHA3·2 CX<cali1lu!lda!alkanel<o\C1Bfii'l/ RH SampleName: SamploTypo: Added No.3 Unknown
'"ANALYTICALCONDITIONS (HPLC) "' : SHIMADZU LC.20AO : J{ypmUGOLO 2.h50mml5um) Acquisition Oato: SamplalO: Comments: Operator:

HPLCTYPE COLUMN ELUENT : A; 20 mM Ammonium founate aqJfotmlo acid (1000:1) B:AcelonlkHa FLOWAATE COLUMN TEMP INJECTVOL !VIl(1:1) : 0.2mUmln : 313 K : 5uL
"' ANALYTICAL CONDITIONS (MS) "' MSTYPE : Thermo FisherSclenfiria TSQ QuanlumAct:es' MAX IONIZATION : NegallveESI VAPORIZER TEMP : 723 K SPAAYVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS : 15 IONSWEEPGAS : 0 CAPILI.ARYTEMP : 493 K SCAN TYPE : SIM (mh ISO)



# 45

### Recovery test (Water analysis) High exposure level Control group No.I

Recovery test (Water analysis) High exposure level Control group No.2 O.afaFito: C166WRHC1-2 Acquisition Date: Original Dala Palh: C:\XIl<lubui\dala\l<aneko\C166\W SamplaiD: RH Sampla Name: Clrl No.1 Commants: SampleType: Unknown Operator:**•~** ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHIMAOZU LC20AD COLUMN : Hyper.UGOLD 2.1 x50mm(5um) ELUENT : A; 20 mM Ammonium formate aq./fc!mtc acid (1000:1) B;Acelonllrlle N8(1:1) PLOW RATE : 0.2 m!Jmin COLUMN TEMP ; 313 K INJECTVOL : 5uL
'" ANALYTICAL CONDITIONS (MS) "' MS lYPE : Thfrmo FisherSctenUf!CTSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZER TEMP : 723 K SPRAYVDLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (11'Jz 150)**U,**

Data File: C1B6WRHC2·2 Acquisition Date: Orlgtnal Data Path: C:\Xcalibur\data\kaneko\C1B6\W Sample fO: RH Sample Name: Cfrl No.2 Comments: SampleType: Unknown Operator:

'"ANALYTICALCONDITIONS (HPLC) '" HPLCTYPE COLUMN ELUENT SHIMAOZU LC20AD : HypersiiGOLD 2.1x50mm(5um) : A;20 mM Ammonium formate aqJ!ormlc acid (1000:1)



### a;Aceton!!rile PLOW RATE COLUMN TEMP INJECTVOL AIB(1:1) : 0.2rnUmln : 313K :5uL "'ANALYTICALCONDITIONS (MS) "' MS TYPE : Thermo FisherSO.nUfioTSQ Quanlcm**A~ss** MAX IONIZATION : Negative ESI VAPORIZER TEMP ; 

### n3 K SPRAY VOLTAGE ; 2500 V SHEATHGAS ; BO AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 1 BO)

ttr:o.n.::r.w



# -
"'''" 

# ,.,.,.



# 46

## Recovery test (Water analysis) High exposure level Control groupNo.3

Recovery test (Water analysis)
Standard solution

0.0048 mgL" 1 Data File: C186WRHC3..2 AcquisfUon Date: Original Oala Path: C:\XcaibUI\dala\kaneko\C186\W Sampla!O: RH Sample Nama: Clrt No.3 Comments: Sample Type: Unknown Operator:

'" ANALYT!CAL CONDITIONS (HPLC) '" HPLC TYPE SH!MADZU LG-20AD COLUMN HypersU GOLD 2.1 X50mm (5um) ELUENT A; 20 mMAmmon!um lonnaleaqJfOill11c ecld (1000:1) SjAI;elonilrifa FLOW RATE COLUMN TEMP INJECTVOL NB{1:1) : 0.2mUmin : 313K : SoL
"' AIIALYT!CALCOND!T!ONS(MS) "' MS TYPE : ThermoAsher SclenfifrcTSQ QuanlumAccess t.iAX IONIZATION : Negative ES! VAPORIZERTEMP : 723 K SPRAY VOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPilLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)**l'tT;o;tt.:uo**

Data File: C186WRLS1·2 Acqufsltlon Date; Original Data Path: C:\Xca!lbur\dala\kanako\C186\W SamplelD: RL Sample Name! Std. 0.0048 mgll Comments: Sampla Type: Std Bracket Oporalor:

"' ANALYTICAlCOND!TlONS(HPLC) '" HPLCTYPE COLUMN ELUENT : SHIMADZU LC.20AD : HypersH GOLD 2.1 X 50 mm (5 um) : A; 20mM Ammonium formale aqlformf<: acid (1000:1)

B; AcetonJ!rflo FLOW RATE COLUMN TEMP INJECTVOL A/8(1:1) : 0.2mUmln : 313K : 5uL
'" ANALYTICAL COIIDlT!ONS (MS) 

### "* MS TYPE : Thermo FisherS"Clanl!ficTSQ Ouanlum Atcess MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 60 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SlM (mlz 180)



# ...................................................................................................................................



# 47

### Recovery test (Water analysis)
Standard solution



## 0.0483 mgL" 1



### Recovery test (Water analysis) Low exposure level

Added Standard solution Dala File: C166WRLS2·2 Acquisition Date: Original Data Path: C:\Xcalibur'dala\kaneko\Ctll6\W Sample 10: RL Sampf.a Name: Std. 0.0483 mglt Comments: Sample Type: Sid Bracket Operator:

'" ANALYTICAL CONDITIONS (HPLC) "' : SHIMAOZU LC.20AO HPLCTYPE COLUMN ELUENf : Hypersll GOLD 2.1 xso mm (Sum) : A; 20 mM Ammonium formale aqJiormlc acid (1000:1)

B; Ace1oni!rlre FLOW RATE COLUMN TEMP INJECTVOL NB(1:1) ; 0.2m\Jmln : 313K : 5oL
'" ANALYTICAL CONDITIONS (MS) '" MS 1YPE : Thermo Flsher SclenUlicTSQ Quantum Acteu MAX IONIZATION : Nejjalive ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE :**Sf~** (miz 1BO)

DataFile: C166WRLA8-3 Acquls!Uon Dale: Original DataPath: C:\Xcallbur\da!alkaneko\C165\W Sample ID: RL Sample Name: Added Std. Comments: Sample Type: Unkrnwm Operator:
"'ANALYTICALCONDITIONS (HPLC) "' HPLCTYPE SHIMADZU LC.20AO COLUMN H)llersi GOLD 2.1 xSO mm (5um} ElUENT A: 20 mM Ammonium formale aq./fctmlo acid (1000:1) B; Acetonltrifs NB(1:1) FLOW RATE : 0.2 mUmln COLUMN TEMP : 313 K INJECTVOL : 5oL
"" ANALYT!CALCONOITIONS(MS) "" MS TYPE : Therrr.o FisherSclentmcTSQ Quantum Access MAX IONIZATION : Negalive ESI VAPORIZERTEMP: 723K SFRAYVOLTAGE : 2500V SHEATH GAS : SO AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM(rr/z1BO)



# 48

### Recovery test (Water analysis)
Lowexposure level Added groupNo.1

Recovery test (Water analysis)
Low exposure level Added group No.2 Data File: C166WRLA1-4 AcqulsiUon Date: Original Data Path: C:\Xcallbul\data\IQlneko\C1BS\W Sample!O: Rl Sample Namo: Added No.1 Comments: SamptaTypa: Un!mov.n Operator:

'" ANALYTICAL CONDITIONS (HPLC) bo HPLC lYPE : SHIMADZU LC·20AO COLUMN : Hypersll GOLD 2.1 x5ll mm (5 om) ELUENT : A; 20**mMAmmon~.1111**furmale aq./formlc ocld (1000:1) BiAcetonitrile AIB(1:1) FLOWRATE : 0.2mUmfn COLUMN TEMP : 313K INJECTVOL : 5Ul
"' ANALYTtCALCONOITIONS(MS) "' MS TYf'E : Thermo FlsherSctenlif1cTSQ QuantumAccessA-fAX IONIZATION ; Negalive ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE ; SIM (mk 160)



# -

...

Oata Flle:**C186WRLA2~2** Acquisition Date: Original Data Path: C:\Xcalibul\dalalkaneko\C1B6\W Sample 10: RL Sampfe Name: Added No.2 Comments: Sample Typo: Unknown Operator:

"' ANALYTlCAL CONDITIONS (HPLC) "' Hf'LC TYPE : SHIMADZU LC-2MD COLUMN : Hypersi GOLD 2.1 x50 mm (5 um) ELUENT : A; 20 mM Ammonium formate aqJformlcacid (1000:1) 

### e; AcelonllrUa AIB(1;1) FLOW RATE : 0.2mllmln COLUMN TEMP : 313 K tNJECTVOL : 5Ul
"' ANALYTICAL CONDITIONS (MS) '" MS TYPE : TlMlrmo AsherSclent!trcTSQ Quanlt.lm Access MAX IONIZATION : NegaUva ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (m/z 160)



# 49

### Recovery test (Water analysis)
Low exposure level Added groupNo.3

Recovery test (Water analysis)
Low exposure level Control group No.1 Data File: C186WRlA3·2 Acqulslllon Date: Original Data Path: C:\Xcaiburldala\kaneko\C186\W Samp!o ID: RL Samplo Uame: Added No.3 Comments: SampleType: Unknown Operator:

'" ANALYTICAL CONDITIONS (IIPLC) "' HPLCTYPE SHIMAOW lC-20AD COLUMN Hyp""'U GOlD 2.1 X 50 mm (5 urn) ELUENT A: 20 mMAmmonium formal<! aqlformlo acid (1000:1) BiAcetonitrile AIB(1:1) FLOW RATE : 0.2 mllmln COLUMN TEMP : 313 K INJECTVOL : 5 uL**•~*** ANALYTICAL CONOrrtONS {MS) •u MS TYPE : Thermo FisherSclentincTSQ QuantumAccess MAX IONIZATION : Negallve ESI VAPORIZER TEMP: 723K SPRAYVOLTAGE : 2SOOV SHEATH GAS : 50· AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (m/z 180)

Data File: C186WRLC1-3 Acquisition Date: Original Data Path: C:\Xca!!burldnla\l<aneko\C186\W Sample ID: Rl Sample Nama: Cfrl No.1 Commonfs: Sample Typo: Unknown operator:
"' ANALYTICAL CONDITIONS (HPLC) "' H?LCTYPE : SHIMAOZV lC·20AD COLUMN : Hyporsll GOlD 2.1 X50mm (5 um) ELUENT : A;20 mMAmmonlum formate aqJfotrnlc acid (1000:1) B;Aceton!tnle AIB(1:1) FLOWRATE : 0.2ml!mln COLUMN TEMP : 313K !NJECTVOL : 5uL
"' ANALYTICALCONDITIONS (MS) '" MSTYPE : Thermo FisherSclenllncTSQ Qua:nlum Access MAX IONIZATION : !legallvo ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPillARYTEMP : 493 K SCANTYPE : SIM(mlz180)**RT:O.~·:tco** &.t15G



## '"'""



# 50

### Recovery test (Water analysis)
Low exposure level Control groupNo.2

Recovery test (Water analysis)
Low exposure level Control groupNo.3 Data FUe: C1B6WRLC2·2 Acquisition Dale: Original Data Path: C:\Xcalibui\da!all<aneko\C186\W SamploiO: RL Sample Name: Ctrl No.2 Comments: Sample Type: Unknown Operator:

'" ANALYTICAL CONDITIONS (HPLC) '" HPLC TYPE : SHIMADZU LC·20AD COLUMN : Hyp<lrsU GOLD 2.1 X50 mm (5 um) ELUENT : A;20mMAmmonlumfonnaleaqlfOlmlcacld(1000:1) B:AcelonittUe AIB(1:1) FLO\VRATE : 0.2mtlmln COLUMN TEMP : 313 K INJECT VOL : 5ul
"' ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thermo FtsherSclentlficTSQ QuantumAccess MAX IONIZATION : Negative ESI VAFORIZERTEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAFILLAAYTEMP : 493 K SCANTYPE : SIM (mlz 180)

Data FUe: C186WRLC3--2 Acquisition Date: Orlgfnal Data Path:**~L\Xcaflbu!\dala\kaneko\C1S6\W** Sample 10; Sampla Namo: Ctr!No.3 Comments: Sampfo Typo: Unknown Operator:



# ............................................................................................. , "' ANALYTICAL CONDITIONS (HPLC) '" HPLCTYPE COLUMN ELUENT : SHIMAOZU LC.20AD : Hypersll GCLO 2.1 X50 mm (5 um) : A; 20 mMAmmcn!um fOrmate aq./fonnicecld (1000:1)

DiAtelon!lrilo FLOWRATE COLUMN TEMP INJECTVOL AIB(1:1) : 0.2m!Jmtn : 313K 

### :SuL**•~** ANALYTICAL CONDITIONS (MS) "* MS TYPE : Thermo AsherScleotifio TSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLMYTEMP ! 493 K SCAN TYPE :**S~.l** (mlz 180)

### •1



# 51

# Reference 8 8-1 Chromatograms in fish Test group 

# .---------------· Fish analysis Day7
Standard solution

0.0048 mgL" 1 Da!a Fifo: Original Da!a Path: Sampht Name: sample Typo: C186WFA7S1 Acqu!siUon Dato: C:\Xcallbur\Oa!o\kaneko\C185\Da Sample 10: y7 Sid. 0.0048 mgll Comment.: Std Brackal Operator:
'" ANALYTICALCONDITIONS(HPLC} '" HPLC TYPE : SHIMADZU LC-20AD COLUMN : Hypersll GOlD 2.1 X50 mm (5 urn} ElUENT : A; 20 mM Ammonlum formate aq.tronnlc acid (1000:1} B;Acelonftrife Alll(1:1) FLOWAATE : 0.2 mUrnln COLUMN TEMP : 313 K INJECTVOL : 5ul
'" ANAlVTICAl CONDITIONS (MS) "' MS TYPE : Thermo Fisher Sciat.tifioTSQ Quantum Aa;ess MAX IONIZATION : Nega!lw ESJ VAPORIZER TEMP : 723 K SPAAVVOLTAGE : 25COV SHEATH GAS : 50 AUXGAS ; 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (rnlz 180}



## Fish analysis Day7
Standard solution

0.0964 mgL· 1 Data Filo: C1B6WFA7S2. At"qutsltton Date: Orlgfnal Data Path: C:\XcaUbur\Data\kaneko\C186\0a Sample fO: y7 Sample Name: 

### ·std. 0.0964 mg/L Comments; SampleTypo! Std Bracket Operator:

"'ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE : SHIMAOZU lC-20AO COLUMN : Hypersll GOLD 2.1 x50 mm (5 um) ELUEIIT : A; 20 mM Ammonium formate aq.trorrnloacid (1000:1}**B;Acelonl~llo** NB(1:1} FLOW AATE : 0.2 mUmln COLUMN TEMP : 313 K INJECTVOl : 5ul
'" ANAlYTICAL CONDITIONS (MS) "' MS TYPE : Thermo FisherSclenlifit:TSQ Quanlum ACCe$S MAX IONIZATION : NsgaUvo ESI VAPORIZER TEMP : 723 K SPAAVVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPillARYTEMP : 493 K SCAN TYPE : SIM (rnlz 180}



# 52

### Fish analysis Day7 High exposure level 

## No.1

Fish analysis Day7 High exposure level 

## No.2 Dafaflle: C1B6FA7H1 Acquisition Date: Original Data Path: C;\Xtallbur\Data\l<aneko\CIB6\0a Sample ID: y7 Ash anelysls Hlv No.1 Commanfs: Sample Namo: Sample Type: Unknown**Op~rator:**

"' ANALYTICAL CONDITIONS (HPlC) '" : SHIMADZU lC-20AO HPlCTYPE COlUMN ElUENT : Hyporsll GOlD 2.1 x50 mm (5 urn) A; 20 mM Ammonium formale aqJformlo acid (1000:1) B;Acelonltraa FlOWRATE COlUMi'llEMP INJECTVOl Ai!l(1:1) : 0.2mlfmln : 313K : 5uL
"' ANAlYTICAL CONDITIONS (MS) '" MS TYPE : Thermo FisherSclenllficTSQ Quantum Access MAX IONIZATION : Negallva ESI VAPORIZERTEMP: 723K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

Data File: C18SFA7H2 Acquls!Uon Dale: Original OataPath: C:\Xca11bur\Data\kaneko\C166\0a Samplo.IO: y7 Sampto Name: Fish analysls·H lv·No.2 Comments: Sample Type: Unknown Oparator.

"' ANALYTICALCONDITIONS (HPLC) '" HPLCTYPE : SHIMADZU lC-20AD COlUMN : Hypersll GOLD 2.1 x SO mm (5 um) ELUENT : A; 20 mMAmmon!um fonnale aqJfoll!11cacid (1000:1) a:Acelonitn1e NS(1:1) FlOWRATE : 0.2mUmln COLUMN TEMP : 313 K INJECTVOL : 5 ul
"' ANALYTICAL CONDITIONS (MS)

- MS TYPE : Thermo FisherScientificTSQ QuanlumAccess MAX IONIZATION : NegaUve ESI VAPORIZERTEMP : 723 K SPRAYVOlTAGE : 2SOOV SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 1 SO)**RT:o.c¢·~v.t:1SG** lCCIOOOO 

# '"'''"



# 53

### Fish analysis Day?
Low exposure level 

## No. I

Fish analysis Day?
Low exposure level 

## No.2 Oala File: C1S6FA7L1 Acqulsl!lon Date: Orlglnal Data Path:**C:\XcaHbu~Oata\l<aneko\C1S6\Da** SamploiD: y7 Sample Name: FJsh analysis LlvNo.1**Comments~** Sampte Typo: Unknown Opuator:

"' ANALYTICALCONDITIONS {HPLC) "' HPLCTYPE COLUMN ELUENT : SHIMADZU LC-20AD : Hypersll GOLD 2.1 x 50 mm (5 urn)

A; 20 mMAmmonlum formate aqJformlo acld (1000:1) S;Acelonllille FLOW RATE COLUMN TEMP INJECTVOL AIB(1:1) : 0.2mUmin : 313K : 5UL
"' ANALYTICALCONDITIONS (MS) "' MS TYPE : Thermo AsherSclcntlriaTSQ Quantum Access MAX IONIZATION : NegaUvo ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2oOOV SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM(mlz1SO)**1\TIO.tc~).OO** SltUG



# """' '"'""'**ur**

Data File: C186FA7l2 Acquisition Dato: OrlgtnaJ Data Path: Sampla Nama: C:\Xcafl:bur\Oala\kaneko\C186\0a Sample fD: y7 Fish analysis l Lv No.2 Comments: Sample Type: Unknown Operator;
"' ANALYTJCALCONDJTIONS(HPLC) '" HPLC TYPE : SHIMADZU Lc-20AD COLUMN : HypernO GOLD 2.1 X 50 

### mm (5 um) ELUENT : A; 20 mMAmmonium formata aq.lformlcacid (1C00:1)
FLOW RATE COLUMN TEMP INJECTVOl B;AcetonllrHa NB(1:1) : 0.2mUmln : 313K ! 5UL
"' AIIALYTICALCONDITIONS (MS) "' MS TYPE : Thermo FisherSc{entificTSQ Quan!um Access MAX IONIZATION : Naga!lve ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)**------**



# 54

### Fish analysis Day 14
Standard solution

0.0048 rngL·'

Fish analysis

Day 14
Standard solution

0.0964 rngL·' Data FOe: C186WFA14S1 Acquisition Date: Original Data Path: C:\Xcallbut\Da!a\keneko\C186\Da Samp!eiO: y14 Sample Name: Std. 0.0048 mg/L Comments: Sample Typa: Sid Erackat Operalor:
'" ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECTVOL : SHIMADZU lC.20AO : Hyporsi GOlD 2.1 x 50 mm (5 urn) : A; 20 mM Ammonium formate aqJformlo add (1000:1)

B; Acetonlll!le Al!l(1:1) : 0.2mUmln : 313K : 5Ul
'" ANALYTICAL CONDil!ONS (MS) m MS TYPE : Thermo FlsherSc!entlllcTSQ QuantumAccess MAX IONIZATION : Negal!vaESt VAPORIZERTEMP : 723 K SPAAYVOLTAGE : 2SOOV SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM (m'z 180)

Rf:O.C¢-:).00 .SIA:U3**u~**

# ~c.o'

Data File: C1S6WFA14S2 Acquisition Date: Original Data Path: C:\Xcalibut\Data\kanelro\C186\Da SampleiD: y14 Sample Name: Std. O.OSt64 mg!L Comments: Sample Type: Std**Brac.~et** Operator.
'" ANALYTICALCONDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECTVOL SHIMADZU LC.20AD Hypersll GOLD 2.1 x 50 mm (5 um) A; 20 mMAmmonlum fonnale oqJfonnlc acid (1000:1) Bi Acetonlln1e

## :-:<~]min : 313K : 5ul
'" ANAL YTlCAL CONDITIONS (MS) 

### •ri fi,1S TYPE : Thermo Fisher ScfenlificTSQ Quantum Access MAX IONIZATION : Negallve ESI VAPCRIZERTEMP: 723K SPAAYVOLTAGE : 2500V SHEATH GAS : 60 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

1\1;**0.00~),00** $l.t t$!J



# """ """

# ~~

55

### Fish analysis Day 14 High exposure level 

## No. I

Fish analysis

Day 14 High exposure level 

## No.2 Data File: C186FA14H1 Acqulslllcn Date: Original Data Path: C:\Xcallbu!l!lata\kaneko\C186\0a SamploiO: y14 Sampta Name: Fish analysis HLv No.1 Comments: Sample Type: UnJr.nown Operator:

HO ANALYTICAL CONDITIONS (HPLC)

... HPLCTYPE COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECTVOL SHIMAOZU LC.20AO Hypersil GOLD 2.1 X50 mm (5 urn) A; 20 mMAmmonium formate aq.Jformlc acid (1000:1) B;AC1llonltrllo NB(1:1) : 0.2mUmln : 313K : 5ul**•~** ANALYTICALCONDITIONS(MS) '" MS TYPE : Thenno FisherSclenllilc TSQ Quantum Access MAX IONIZATION : NegaUvo ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM (mlz 180)

U

Oatafllo: C185FA14H2 Acqulsl!lonOato: Original Data Path: C:\Xcalibw\OatsUianeko\C186\Da Sample ID: y14 Sample Nam-e:: Fish analysts HlvN-o.2 Comments: Sample Type: Unknown Operator:
"' ANALYTICAL COflDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECTVOL : SHIMAOZIJ LC·20AD HypersU GOLD 2.1 x 50 mm (5 um) A; 20 mMAmmonlum fotmale eq.Ifotmlc ecld (1000:1) B;AcelonUrile NB(1:1) : 0.2mUmin : 3i3K : 5ul
"' ANALYTICALCONDITIONS (MS) '" MS 1YPE : Thermo FisherSclenllficTSQ Quantum Access MAX IONIZATION : NegoUve ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : EO AUXGAS : 15 ION SWEEP GA9 : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

o



# 56

### Fish analysis Day 14
Low exposure level 

## No. I

Fish analysis

Day 14
Low exposure level 

## No.2 Data File: Original Dafa Path: Sampla Nama: SampleType: C186FA14L1 Acqulsll!on Date: C:\Xcalibur\Dala\kaneko\C1S6\Da Sample ID: yH Fish analysts L lvNo.1 Commenfs: Unknown Operator.
'" ANALYTICALCOND!TIONS(HPLC) "' HPLCTYPE : SHIMAOZU LC·2DAO COLUMN : Hyparsil GOlD 2.1 x50mm (5 urn) ELUENT : A;20 mMAmmoniumformate aqJTormloacid (1000:1) 8;Acefonitrlia AIB(1:1) FLOW RATE : 0.2 mUmln COlUMN TEMP : 313 K INJECTVOL ; 5 uL
'" ANALYTICAL COilDITIONS (MS) "' MS TYPE : Thermo FisherScientificTSQ QuantumAccess MAX IONIZATION : Naga!lva ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATHGAS ; 60 AUXGAS : 15 ION SWEEP GAS : 0 CAPillARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

.............................u ................................................................................................... .

Data File: C186FA14l2 Acquisition Date: Orlglnal Dala Path: Sample Name: C:\XcaU!rur\Data\kanako\C186\Da SamplaiD: y14 Frsh analysis LLv No.2 Comments: Sample Typo: Unknown Operator:

"' ANALYTICAL CONDITIONS {HPLC) '" : SHII.'.ADZU LC.20AD HPLCTYPE COLUMN ElUENT ; Hypersll GOLD 2.1 

### x50 mm (5 um) : A; 20 mMAmmonium formato aqJformic ac!d (1000:1)

B;Acetonlfrlla FLOW RATE COLUMN TEMP INJECT VOl A/8(1:1) : 0.2mUmin : 313 K : 5uL
'" ANALYTICALCONDITIONS(MS) •u MS lYPE : Thermo FisherSci&ntificTSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP: 723K SPRAYVOI.TAGE : 2500V SHEATH OAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARY TEMP : 493 K SCAN TYPE : SIM (mh: 180)



# 57

### Fish analysis Day21
Standard solution



## 0.0048 mgL" 1



### Fish analysis

Day21
Standard solution



## 0.0964 mgL· 1 Data Fllo: C166WFA2tS1 Aequfsltlon Dale: Original Data Palh: C:\Xcalibur\Oalalkaneko\0166\Da Sample ID: y21 Sample Nama! SampleTypo: Std. 0.0048 mg/L Comments: Std Broc-ket Operator:

••• AMALYTICALCONOITIONS(HPLC) '" HPLCTYPE COLUMN ELUENT : SHIMADZU LC·20AD liyper;UGOLO 2.1 x50mm(5um) A; 20 mMAmmonlum formals aqJformlc acid (1000:1) B;Acelonlln1e FLOW RATE COLUMN TEMP INJECTVOL NB(1:1) : 0.2mUmlo : 313K :5Ul
'" ANALYTICAL CONDITIONS (MS) '" MS TYPE : Thermo Fisher**S<:!en~fio**TSQ Quantum Aceess MAX IONIZATION : N'!)allvo ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (m/z 180)

Data Fife: Original Dala Path: Sample Name: Sample Type: C186WFA21S2 AequlsiUon Dale: C:\XcallburlDala\kaneko\C18S\Da Srunp!o!D: y21 Sid. 0.0964 mg/L Comments: Sid Bracket Operator:

wo ANALYTICALCONDITIONS {HPLC) '" HPLC TYPE SHIMADZU LC·20AO COLUMN liypersH GOLD 2.1 x50 mm (5 um) ELUENT A: 20 mM Ammonium fonnale eqJformlo acid (1000:1) B;Acotonllrllo A/6(1:1) FLOW RATE : 0.2 mUmln COLUMN TEMP : 313 K INJECTVOL : 5ul

••• ANALYl1CAL CONDITIONS (11.5) •ri MS TYPE : Thenno FisherScientificTSQ QuantumAccess Mt-.X IONIZATION : NegaUva ESI VAPORIZER TEMP ; 723 K SPRAYVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPIULARYTEMP : 493 K SCAN TYPE : SIM (mlz ISO)**~.l**



# 58

### Fish analysis Day21 High exposure level 

## No.!

Fish analysis

Day21 High exposure level 

## No.2 Data File: C186FA21H1 Acqulsltfon Dale: Original OalaPath: C:\XcallbU!IDala\kaneko\C186\0a SampleiD: y21 Sampfo Name! Ffsh ana.fysfs HLvNo.1 Comments: Sampfo Typo: Unknown Oporafor:

'" ANALYTICAL CONDITIONS (HPLC) "' : SHIMADZU LC.20AO HPLC1YPE COLUMN ElUENT Hypersl GCLO 2.1 x 60 mm (5 um) A; 20 mM Ammonium formate aqJformlc acid (1000:1) BiAcelonifrlle FLOW RATE COLUMN TEMP INJECTVOL NB(1:1) : 0.2mUmin : 313K : 5ul 

### H. ANALYTICALCONDITIONS (MS) 

### •» MS lYPE : Thermo Fisher Scientific TSQ QUanlumAccess MAX IONlZATlON : Negallve ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mil 160)

nr:o.oo.uo



## '"""' 

# ,...,.

___

Data File: C185FA21H2 AcqutslUon Date: Original Data Path: C!\Xca4'but\Oata\kaneko\C186\0a Sampte 10: y21 Sample Na:me: Fishanalysis: HlvNo.2 Comments: Sample Typo: Uni'.nown Operator:

'" ANAL YT!CALCONDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT : SHIMADZU LC.20AD : Hype!Sll GOLD 2.1 x50 mm (5 um) : Ai 20 mMAmmonlum fc:mc:te aqJformic ae-!d (1000:1}

B;Acelonltrile FLO\'/ RATE COLUMN TEMP INJECTVOL Ml(1:1) : 0.2mUmln : 313K : 5UL

m AIIALYTICALCONDITIONS (MS) "'**~.iS**TYPE : Thermo FfsherSclentllio 1SQQuanlum Access MAX lONlZATION : NegaUve ESI VAPORIZER TEMP: 723K SPRAYVOLTAG;; : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARVTEMP : 493K SCAN TYPE : SIM (mlz 180)



# 59

### Fish analysis Day21
Low exposure level 

## No. I

Fish analysis

Day 21
Low exposure level 

## No.2 DalaFJJo: C166FA21 L 1 Acquisition Date: Orfglnal Data Palh: C:\Xcallbur\Data\kaneko\C166\Da Sample to: Y21 Sample Name: Fish analysis llvNo.1 Comments: Sample Typo: Uokna.vn Operator:

"' ANALYTICAL CONDITIONS (HPI.C) "' : SHIMADZU LC·20AO HPLCTYPE COLUMN ELUENT Hyparsll GOLD 2.1 x 50 mm (5 um) k. 20 mMAmmonlum f0!1!1ale aqHormlc**a~d** (1000:1) B;Aceloni!rila FLOW RATE COLUMN TEMP INJECTVOL N8(1:1) : 0.2mUmln : 313K :Sui.
"' ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thermo FisherSclenUfro TSQ Quanlum Access MAX. ION12ATION : Negallve ESI VAPORIZER TEMP : 72.l K SPRAY VOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

Dalaflle: Or(gfnal Data Palh: C1B6FA21L2 AcqulsiUon Date: C:\Xcafibtir\Data\kaneko\C186\0a SamplsiD: y21 Sampfe Name: Sample Type: Fish anaiJ.'Sis llvNo.2 Ccmmonls: · Unkr.own Operator:



# ........................................................................................... , "' ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHIMADZU LC.20AD COLUMN : Hyper.;U GOLD 2.1 x 50 mm (5 um) ElUENT : k. 20mMAmmoniL'fll formola aqJformlo acid (1000:1) 

### a;Ac-etonllrRo FLOWRATE :

## :<~Vmln COLUMN TEMP : 313 K INJECTVCL : 5 ul
'" ANALYTICAL CONDITIONS (MS) '" MS TYPE : Thermo F1sher Selent'!icTSQ Quantum Act:ess MAX IONI2ATION : NegaUve ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILI.ARYTEMP :**4~3** K SCAN TYPE : SIM (mlz 180)



# ...................................................................................................................................



# .

60

### Fish analysis Day23
Standard solution

0.0048 mgL' 1



### Fish analysis Day23
Standard solution

0.0964 mgL· 1 Data File: C186WFA23S1 Acqutslllon Date: Original Data Path: C:\Xcalibur\Daia\kaneko\C186\Da SompleiD: y23 Sampro Name: Std. 0,0048 mg/L Commonfs: Sample Type: Sid Bracket Operator:

"'ANALYTICALCONDITIONS (HPLC)**•~** HPLClYPE COLUMN ELUENT
FLOW RATE COLUMN TEW.P INJECTVOL : SHIMAOZU LC·20AD : HypersKGOLD 2.1x50mm(5um) : A;20 mM Ammonium formalo nqJ!o<mlc acid (1000:1)

a:Acelcnllillo A/8(1:1) : 0.2mUmfn : 3131< : 6uL
"' ANALYTICAL COtllliTIONS (MS) '" MS TYPE : Thermo FisherScfenllficTSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP! 723 K SPRAYVOLTAGE ! 2500V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM (mh: 180)



## ""':

DataFlle: C186WFA23S2 Acquisition Dale: Original DataPa!li: C:IXoalibiR\Data\kaneko\C186\Da Samplo 10: y23 Sample Nama: Std. 0.0954 mgn.. Comments: Sample 'fYpo: Sid Bracket Operotor:

OM ANALYTICAL CONDITIONS (HPLC)

... HPLOTYPE COLUMN aUENT
FLOW RATE COLUMN TEMP INJECTVOL : SHIMADZU LC·20AO : Hypersll GOLD 2.1 X50 mm (5 urn) : A;20 mMAmmonium formale aqJformlc ecld (1000:1)

B;Acelcnllrile AIB(1:1) : 0.2mUmln : 3131< : 5ul

w• ANALYTICAL CONDITIONS {1\'.S) "' MS TYPE : Thermo FisherScientificTSQ QuantumAccess MAX IONIZATION : Negalive ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : tro AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM {mlz 180)**:**



# 61

### Fish analysis Day23 High exposure level No.1

Fish analysis

Day 23 High exposure level No.2 Data File: C186FA23H1-1 Acquisition Date: Original Data Path: C:\XcallburlDala\kaneko\C186\0a Sample ID: y23 SampleName: Fish analysis H Lv No.1 Comments: SampleType: Unkrw....n Operator:

'" ANALYTICAL CONDITIONS (HPLC) '" SHIMAOZU LC-20AO : HyporsifGOLO 2.1x50mm(5um) HPLCTYPE COLUMN ELUENT : A; 20 mMAmmonium formala aqJfarmlc acid (1000:1) B: Acaloo!UIIa FLOW RATE COLUMN TEMP INJECTVOL NB(1:1) : 0.2rnllmln : 313K : 5uL "'ANALYTICALCONDITIONS (MS) '" MSTYPE : Thermo Flsbe:rScientific TSQ Quanlt!m Access MAX IONIZATION : Nogallvo ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM (mlz 160)

DataFile: C1B6FA23H2-1 Aequlslllon Data: Original Data Path: C:\Xcallbur\Oafa\kaneko\C186\Da Samplo 10: y 23 Sample Name: Fish analysis H LvNo.2 Comments: Sample Type: Unknown**Op~ralor:**

'" ANALYriCAlCONDITIONS (HPLC) "' H?LCTYPE SHII.'ADZU LC-20AD COLUMN : Hyporsll GOLD 2.1 x50 mm (5 urn) ELUENT : A: 20 mMAmmonlum formale aq./formlc acid (1000:1) B;AcafonUrl!a NB(1:1) FLOIVRATE : 0.2mUmln COLUMN TEMP : 313 K INJECTVOL : 5ul "'ANALYTICALCONDITIONS (MS) "' MS TYPE : Thermo Fisher SclentifJCTSQ QuantumAccess MAX IONIZATION : NogaUve ESI VAPORIZERTEM?: 723K SPRAYVOLTAGE : 2500V SHEATH GAB : 60 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 1 SO)



# 62 ..,

## Fish analysis Day23
Low exposure level 

## No. I

Fish analysis Day23
Low exposure level 

## No.2 OalaFIIe: Original Data Palh: Sample Name: SamploType: C186FA23L1-1 C:\Xcallbur1Dala\kaool<o\C186\Da

## t1~**analys~** L Lv No.1 Unknov.n
"' ANALYTICAL CONDITIONS (HPI.C) "' HPLC ll'Pg SHIMADZU LC·20AD Acquisition Dato: Samplo!D: Comments: Operator:

COLUMN Hypersll GOLD 2.1 x 50 mm (5 urn) ELUENT A: 20 mMAmmonlum formate aqlfonnlo acid (1000:1) B: Acetonltrllo NB(1:1) FLOW RATE : 0.2 mllmln COLUMN TEMP : 313 K INJECTVOL : 5u!.
"' ANALYTICAL CONDITIONS (MS) '" MSTYPE : Thermo Asher SclenU!icTSQ Quantum Access MAX IONIZATION : NegaUva ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mil 180)

DataFile: C186FA23l2-1 Acqulslllon Date: Original Data Path: C:\Xca!bur\Data\lcanel<o\C186\0a SamplaiD: y23 Sampla Name: Ffsh analysis Llv No.2 Comments: Sample Type: Unknown Operator:

"' ANALYTICAl CONDITIONS {HPLC) "' HPLC TYPE : SHIMADZU LC-20AD COLUMN HypersH GOLD 2.1 X60 mm (5 um) ELUENT A; 20 mMAmmonium formalo aq.lfonnlo ac!d (1000:1) B; Acetoru1rile NB(1:1) FLOW RATE : 0.2 mt.lmln COLUMN TEMP : 313 K INJECTVOL : 5 L-1.

••• AIIAI.YTICALCONOITIONS (MS) '" MS TYPE : Thermo FisherScientific TSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZER TEMP : 723 K SPRAY VOLTAGE : 2500 V SHEATH GAS : 60 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN ll'PE : SIM (mlz 160)**}Fi**



# 63

### Fish analysis Day28
Standard solution

0.0048 mgL" 1



### Fish analysis Day28
Standard solution

0.0964 mgL· 1 Data Fife: C186WFA28S1 Acqulslflon Date: Original Data Path: C:\Xcalibur\Oala\kaneko\C16S\Da Sampla 10: y28 Sampfe Name: Sfd. 0.0046 mglt.. Comments: Sampfa: Type: Sid Bracket Operator:



## H• ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHIMADZU Lc-20AO COLUMN : Hyperd GOLO 2.1 X50 mm (5 um) ELUENT : A; 20 mM Ammonium formato aq.lforrnlcac!d (1000:1) B; Ac.elonilri!a FtOWRATE :

## ~~(;:;/)min COLUMN TEMP : 313 K INJECTVOL : 5 uL
"' ANALYTICAL CONDITIONS (MS) "' MSTYPE : Thermo FisherSclenllfioTSQ QuanlumAccess MAX IONIZATION : Negative ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

Data Fila: C186WFA28S2-2 Acquisition Date: Original Data Path: C:lXcallilw\Oata\kaneko\C185\0a SampleiD: y28 SampJe Name: Std. 0.0964·mg!L Comments! Sampre Type! Std Braekef Operator:

'" ANALYTICAL CONDITIONS (HPLC)

... HPLCTYPE SHIMAOZU LC.20AO COLUMN Hypersil GOLD 2.1 x50 mm (5 umj ELUENT A;20 mM Ammonium formalo aqJformlo**a~'d** (1000:1) B: Acelonllri!e AIB(1:1) FLOW RATE : 0.2 mtlmln COLUMN TEMP : 313 K INJECTVOL : 5 uL

... ANALYTICALCONDITIONS (MS) 

### H• MS TYPE : Thermo FisherScleni!RcTSQ Quantum Access MAX ION12ATION : NegaUve ESI VAPORIZERTEMP: 723 K SPRAYVOLTAGE : 25COV SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 160)



# """

,. 

# ..

64

### Fish analysis Day28 High exposure level No.1

Fish analysis

Day28 High exposure level No.2 DalaFlle: CIB6FA26HI Acquisition Dale: Original Data Path: Samplo Nama; Sample Typo: C:\Xcallbut\Data\kaneko\0186\Da Sampla 10: y2B Fish analysJs H LvNo.1 Comments: Unknown Operator:
'" ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE : SHIMADZU LC.20AO

## ~~~~/: 

## f**~fo~Gk.~o~~~;:r:.~e<!:;l,m!cac!d(1000:1)** B;Acolonlfrua Al8(1:1} FLOW RATE : 0.2 mUmln COLUMN TEMP : 313 K INJECTVOL : 5 uL
'" ANALYTICAl CONDITIONS (MS) "' MS TYPE : Thermo FisherScfenUficTSQ Quan!um Access MAX IONIZATION : Negallva ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARY TEMP : 493 K SCANTYPE : SIM (m/z 160)

OalaF!Je: Orfglnal Data Path: CI86FA26H2 Acquls!Uon Date: C:\Xcalibur\Dallle<aneko',C196\Da SampleiD: y28 SampleName: Fish analysis HLvNo.2 Comments: Samplo Type! Unknown Operalor:

"' ANALYTICAL CONDiliONS (HPLCJ '" H?LCTYPE SHIMAOZIJ LC.20AD COLUMN Hype"D GOLD 2.1 x50mm (5 urn) ELUENT A;20 mMAmmonlum formate aqJformlc acid (1000:1} B;AcalllnllrUa Alil(l:l) FLOW RATE : 0.2 mUmln COLUMN TEMP : 313K INJECTVOL : 5 uL
"" ANALYTICAlCONDmONS(MS} "' t.m TYPE : 1hflrrno FisherScle:nlificTSQ QuantumAccess MAX IONIZATION : Negaliva ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATHGAS : 56 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : Sh\1(rnlz180)



# ................................................................................................................................... RnC.t<l•'-t<llt1.tlSC



## '""" 

# '"""

0· a



# 65

### Fish analysis Day28


## Low exposure level No. I

Fish analysis Day28


## Low exposure level No.2 Data File: C1B6FA2BL1 Acquisition Date: Original Data Path: Sample Name: C:\Xcallbur\Oala\kaneko\C186\Da SampleiD: y2B Fish analysis LlvNo.1 Commenls: Sample Type: Unknown Operator:

'" ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHIMADZU LC-20AO COLUMN : Hypersl GOLD 2.1 X50 mm (5 um) ELUENT : A;20 mMAmmonium fonnale aqJform!o acid (1000:1) B;Acefonltrlla A!B(1:1) FLOW RATE : 0.2 ml!mln COLUMN TEMP : 313 K INJECTVOL : 5 ut
"' ANALYTICAL CONDITIONS**(~lS)** '" MS TYPE : Thenno FisherSclentlllcTSQ QuantumAccess MAX IONIZATION : Nega!lve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATHGAS : 00 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 100)



## ''''"

Data File: C186FA28L2 Aoqulslllon Date: Original Data Path: Sample Name: Sample Type:**C:\Xclllibur\Dala\~aneko\C166\0a** SampleiD: y2B Flsh anatys!s llvNo.2 Commenfs: Unknown Ope-rator.
'" At/AL YTICALCONDITIONS (HPLC) "' HPLC TYPE : SHII.'.ADZU LC·ZOAD COLUMN : HypersQ GOLD 2.1 x50 rr.m (5 um) ELUENT : A; 20 mMAmmonlum formals aq.trom!le acid (1000:1) B;Aca!onitrllo AIB(1:1) FLOWRATE : 0.2mllmln COLUMN TEMP : 313 K INJECTVOL : 5uL
"'**A~IAL** YTICAL CONDITIONS (MS) m MS TYPE : Thermo Fl•herSdenUiicTSQ Quantum Access MAX IONIZATION : NegaUve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz180)



# ·-- =

u



# 66

# 8-2 Control group**.-------------------~** 

## Fish analysis DayO
Standard solution

0.0048 mgL" 1



## Fish analysis DayO
Standard solution

0.0964 mgL· 1 Da!aFife: C1BSWFAOS1 Acqulslt!on Dalo: Original Data Palh: C:\Xcallbur\Oa!a\kaneko\C166\0a Sample 10: yO Sampfo Name: Std. 0.0048 mgll. Commsnts: Sample Typo: Sid Bracket Operator:

'" ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECTVOL SHIMADZU LC·20AD Hyper<B GOLD 2.1 x 50 mm (5 urn) A: 20 mMAmmoniumformate aqlfonnfcacld (1000:1) B;Acetonlltile A/8(1:1) : 0.2mUmln : 313K : suL**•~** ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thermo Fisher Sclenlific TSQ QuanlumAccess MAX IONIZATION : Negalive ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (rnlz 1 80)



# ...................................................................................................................................

Dala File: C186WFAOS2·2 Acquisition Dale: Original Dala Palh: C:\Xcallliut\Dala\kaneko\C186\Da Sample 10: 

### yo Sampfe Name: Std. 0.0964mgJL**Comment~:** Sample Typa: StdBracket Operator:

"'ANALYTICALCONDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECTVOL : SHIMAOZU LG-20AD : Hypersll GOLD 2.1 x50 mm (5 um) : A; 20 mMAmmonium fonnale aqlfo;mlc acid (1000:1)

B; Acelon!trlle NB(1:1) : 0.2 mllmln : 313 K : 5uL "' ANALYTICAL CCNDITIOtiS (MS) ••• MS TYPE : Thermo FlsherSclenUficTSQ QuantUJ"' Access MAX IONIZATION : Nega!lvo ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 16 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)



# 67

### Fish analysis DayO (before starting exposure)
Control group

Fish analysis Day28
Standard solution

0.0048 mgL" 1 DafaFtla: C185FAOC Acqulslllon Data: Original OalaPalh: C:\Xcalibuf\Dala\kanel<o\C186\0a Sampl< 10: SamptG Name: SampleType:

### ~~h analysis Ctrl Comments: Unknown Oporalor:



# ..........................................................................................., "' ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE COLUMN aUENT
FLOW RATE COLUMN TEMP INJECTVOL : SHIMADZU LC.ZOAD : Hype!SII GOlD 2.1 x50 mm (5 um) : A; 20 mM Ammoniuml01111a!e eqJform!c acid (1000:1)

B; Acelonllrile AIS(1:1) : 0.2mUmln : 313K : 5ul '"ANALYTICALCONDITIONS (MS) "' MS TYPE : Thermo FisherSclantl!ioTSQ Quanlum Accass MAX iONIZATION : Negallve ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM(mlz180)



# ...................................................................................................................................

""" """"

OataFne: C186WFA2BS1 AcqulsiUon Date: Original Data Path: Sampfe Name: Sampfo Type: C:\Xcalibur\Dala\kancl<o\C186\0a Sampla!O: y2a Std. 0.0048 mgJL Comments: Std Bratket Operator:
"' ANALYTICAl COilCITIONS (HPLC) '" HPLC TYPE : SHIM

.. OZU lC.20AO COlUMN : HypmUGOlD 2.1 x50mm(5um) ElUENT : A; 20mMAmmcnlum f01111ala aqJformlc acid (1000:1) B;Acetonlllllo N8(1:1) FLOW RATE : 0.2 mtlmin COLUMN TEMP : 313 K INJECTVOL : 5 uL
'" ANALYTICAl CONDITIONS (MS) "' MS TYPE : Thermo FisherSc!en!ilicTSQ QuanttJJ11 Access MAX IONIZATION : Nega!lve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS ! 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 160)



# 68

## Fish analysis Day28
Standard solution

0.0964 mgL- 1



## Fish analysis

Day28 (at the termination ofexposure) Control group Data File: C186WFA28S2·2 Acquisition Dale: Original Data Path:**C:1Xcal~u~Oalalkaneko\C1B6\Da** Sampla!D: y2B Sampro Nama: sample Type: Std. 0.0964 mgll.. Comments: Sid Br.u:kel Operator:
'" ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE : SHlMADZU LC-20AO COLUMN : Hypersll GOLD 2.1 X50 mm (5 um) ELUENT : A: 20 mMAmmonium rormalo aqJformlcacid (1000:1) B;Acetoni!tito AIB(1:1) FLOWRATE : 0.2m!lmln COLUMN TEMP : 313 K INJECTVat. : 5 uL**~•** ANAlYTICALCONDITIONS (MS) '" MS TYPE : Thermo AsherSdenlifioTSQ Quanlum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOI.TAGE : 2500V SHEATHGAS : SO AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (m/z 180)

Data Flto: C1B6FA28C Acquisition Date: Original Dala Palh:**C:\Xcalibu~aio\kaneko\C185\Da** Sample ID: y2B Sample NamG: Flsh analysis Ctrf Comments: Sample Typo: Unkno\vn Operator:
'" ANAlYTICAl CONDITIONS (HPLC) '" HPlCTYPE COLUMN ELUENT SHIMADZU tC-20AO : Hypersil GOLD 2.1 x50 mm (5 um) : A: 20 mMAmmonium forrnalo aq./formlo aold (1000:1)

B;Acetonlltllo FLOW RATE COLUMN TEMP INJECT VOL AIB(1:1) : 0.2mlJmln : 313K : 5uL
"' ANALYTICAL CONDITIONS (MS) "' MS lYPE : Thermo Fisher Sc!ontif'JOTSQ OuanlumAccess MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)



# 69

# Reference 9 9-1 Chromatograms when measuring the concentrations in water Test group**r-------------------~** 

### Water analysis DayO
Standard solution



## 0.0048 mgL" 1



### Water analysis DayO
Standard solution



## 0.0964 mgL" 1 Data File: Orlglnnl Data Path: Sample Name: Samplo Typo: C166WFAOS1 Acqulslllon Dale: C:\Xcallbw\Dalo\kaneko\C16o\Da SampteiD: 

### yo Sid, 0.0048 rr.gll Comments: Sid Braeket Operator:
"' ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHtMAOZU LC.20AO COlUMN : Hyparsi!GOLD 2.1x50mm(5um) ELUENT : A: 20 mM Ammonium formalo aq./formlo acid (1000:1) B; Aoetonlltila IVB(1:1) FLOW RATE : 0.2 mUmln COlUMN TEMP : 313 K INJECTVOL : 5 ul
'" AIIALYTICAL CONDITIONS (MS) '" MS TYPE : Thermo FisherSclonlifr.TSO QuanlumAccess MAX IONIZATION : NegatiVe ESI VAPDRIZERTEMP: 723K SPRAYVOlTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE ; SIM (mlz 180)**RT:O.WJ~3.00** UI:1SG



## "'''"

OalaFife: Original Data Path: Sampla Name: Sample Type: C186WFAOS2·2 Acqulsl!lon Date: C:\XcaHbur\Dalalkaneka\C186\Ila SampleiD: 

### yo Std. 0.0904 mgJL Commenls! Std Bracket Operator:

••• AIIALYTlCALCONDITIONS (HPLC) "' HPLCTYPE : SHIMAOZU LC.20AO COLUMN Hypersl! GOLD 2.1 

### x50 mm (5 urn) ELUENT A; 20 mMAmmonium formate aq.lformlo acid (1000:1) B;Acetonilrlle Alll(1:1) FLOW RATE : 0.2 mUmln COLUMN TEMP : 313 K INJECTVOL : 5 uL
'" ANALYTICAL CONDITIONS (MS) ••• MS TYPE : Thermo FisherScientificTSQ QuantumAccess MAX IONIZATION : llegallve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2SOOV SHEATH GAS : 50 AUXGAS ; 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAli TYPE : SIM (mlz 180)



# 70

### Water analysis DayO High exposure level

Water analysis DayO
Low exposure level Data File: Original Data Path: Sample Name: Sampio Type: C186WAOH3 Acquls!Uon Date: C:\Xca!ibur\Dafa\kaneko\0186\0a SampleiD: yO Wale:ranalysTs HLv Comments: Unknown Operator:
"' ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHIMADZU LC·20AD COLUMN : HypersllGDLD 2.1x50rrm(5um) ELUENT : A;20 mMAmmonium formate aqJformlo acid (1000:1) B; Acetonitrile A/8(1:1) FLOWRATE : 0.2mUmln COLUMN TEMP : 313 K INJECTVOL : 5 uL
'" ANALYTICAL CONDmONS {MS) "' MS TYPE : Thermo**F~her**Scienlif!CTSQ Quanfum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS ! 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM (mlz 180)

Data File: C186WAOL Acquisition Dato: OrlglnaJ Data Path! C:\Xcnllbur\Oala\kaneko\C1 B6\Da Sample 10: yo Sample Name: Waferanalysis Ltv Comments: Sample Type: Unknown oP-arator.

"' ANAL YnCAl CONOmONS {HPLC) 

### ri• HPLC TYPE : SHIMADZU LC.2DAD COLUMN Hypl!tsD GOLD 2.1 x50 mm {5 um) aUENT A; 20 mMAmmoniumroll!lale aq./Tormlo ocld {1000:1) Bi Aeelonltdte A/8(1:1) FLOWRATE : 0.2ml!mln COLUMN TEMP : 313 K INJECTVOL : 5ul
'" ANALYTICAL CONDITIONS {MS) '" MS TYPE : Thermo FisherSclenllficTSQ Quantum Access MAX IONIZATION : Negallve ESI VAPORIZERTEMP : 723 K SPRAY VOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARY TEMP : 493 K SCANTYPE : SIM (m/z 1BO)



# 71

### Water analysis Day7
Standard solution

0.0048 mgL' 1



### Water analysis Day7
Standard solution

0.0964 mgL' 1 Oala Fila: C166WFA7S1 Acqulslllon Dalo: Original Oala Palh: C:\Xcaf:lxu\Oala\l<llnekDIC166\Da SampleiD: y7 Sample Namo: Std. 0.0048 mg/L Comments: Sampfe Typo: Std Bracl:et Operator.

"' ANALYTICALCONDITIONS (HPLC) ''' HPLCTYPE : SHIMADZU LC·20AD COLUMN : HypersU GCLD 2.1 X50 mm (5 urn) ELUENT : A: 20 mMAmmonium formate aq.ffonnle add (100o:1) B;Acelonllrife N8(1:1) FLOW RATE : 0.2 mllmln CCLUMN TEMP : 313 K INJECTVOL : 5uL
"' ANALYTJCALCONDITIONS(MS) "' MS TYPE : Thermo Fisher ScientificTSQ QuantumAccess MAX IONIZATION : Negallve ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 !ONSWEEPGAS : 0 CAPILlARYTEMP : 493 K SCAN TYPE : SIM (mh: 180)

DataFite: Orlglnar Data Path: Sample Name: SampleType: C186WFA7S2 Acqulslllon Dale:**C:\Xcau~ur\Dala\kanel<o\C1B8\Da** SamploiD: y7 Sfd. 0.0964 mg/L Comments: Std Bracket Operator:

PO ANALYTICAL CONDITIONS (HPLC)

- HPLC TYPE : SHIMADZU LC-2DAD CCWMN : Hypersll GOLD 2.1x50 mm (5 um) ELUENr : A; 20 mMAmmonlom fomrate aqJform!c acld (1000:1) B;AcalonlliUe N8(1:1) FLOW RATE : 0.2 mUm!n COLUMN TEMP : 313 K INJECTVOL : 5 uL

•u ANALYTJCALCONOITIONS (MS) '" MS n'PE : lhenno FfsherScientificTSQ Quantum Access MAX IONIZATION : Negatlve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPillARYTEMP : 493 K SCANTYPE : SIM(m/z!BO)

Jtr:II.LII.:f.M nt1!G



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# 72

### Water analysis Day7 High exposure level

Water analysis Day7
Low exposure level DalaFIIe: CiSSWA7H Acqulslllcn Oate: Original Oafa Path: C:\Xcall00r\Data\l:aneko\C186\Da Sample!D: y7 Sampla Nama: Wateranalysis H Lv Comments: Sample Type: Unknown Operator:

"' ANALYTICAL CONDITIONS (HPLC) «> HPlCTYPE : SHIMADZU LC·20AO COLUMN : HypersH GOLO 2.1 X50 mm (5 um) ElUENr : A: 20 mMAmmentum rormefe aqJrormk:acld (1000:1) B;Acelonltrll-a AJB(1:1) FLOWRATE : 0.21nl/mln COLUMN TEMP : 313 K INJECTVOL : 5ul
'" ANALYTICAL CONDITIONS (MS) '" MS TYPE : Tharmo Fisher SclenOITeTSQ Quantum Access MAX IONIZATION : Negative ESt VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

DafaFife: Origfnal Data Path: C186WA7L C:\Xcalilror\Oafa\l:aneko\C186\Da AcqulsJUon Data: SampleiO: SampJo Name: Sample Type:

## ~!er**ana~tsls**llv Unknovm Comments: Operator:



# ............................................................................................. "' ANALYTICAL CONDITIONS (HPLC) 

## k• HPLOTYPE : SHIMADZU LC-20AD COLUMN : Hypersff GOLD 2.1 x50 mm (5 um) ELUENT : A; 20 mMAmmonium ronmale aqJiormlc acid (1000:1) B;AcsfOfl«rita AJB(1:1l FLOW RATE : 0.2 mllm!n COLUMN TEMP : 313 K INJECT VOL : 5 UL
"' AIIALYTICALCONOITIONS(MS)'" MS TYPE : Thanmo Flsi>erSclentiffoTSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAY VOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM(mll:150)



# ................................................................................................................................... RT:OJIO•),Ill Si.t-UQ



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# 73

### Water analysis Day 14
Standard solution



## 0.0048 mgL" 1



### Water analysis Day 14
Standard solution



## 0.0964 mgL" 1 Data File: C186WFA14S1 Aequlsfllon Date: Original DataPath: C:\Xcallbw\Oala\kaneko\C186\0a SampleiO: y 14 Sample Namo: Std. 0.0048 rng!L Comments: Sampfe Typo: Std Bracket Opera!or.

'"ANALYTICAL CONDITIONS (HPLC) '" HPLC TYPE : SHIMADZU LC-20AD COLUMN : Hypers!GOLD 2.1x50mm(5um) ELUENT : A;20mMAmmorJumrormaloaq.lformicaeid(1000:1) 8;**Acelon~rlte** AIB(1:1) FLOW RATE : 0.2 

### rntimln COLUMN TEMP : 313 K INJECTVOL : 5 uL
'" ANALYTICALCONDITIONS (MS) "' MS TYPE : Thenno Fisher Scfentlfic TSQ Quan!um Access t.-i.AX IONIZATION ! Negallve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPilLARYTEMP : 493 K SCANTYPE : SIM(mlz1BO)

__

Data File: C1B6WFA14S2 AcqulsiUon Dale: Original Data Path: C:\Xcalibur\Oala\kaneko\C18S\Oa SamploiD: y14 Sample Nama: Std. 0.0964 mgll Comments: Sampre Typo: Std Bracket Operator.

"' ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT : SHIMAOZU LG-20AD : Hypersl GOlD 2.1 x50 mm (5 urn) : A; 20 mMAmmonium formate aq.lformlc acid {1000:1)

B;AC<>!onl!n1e FLOW RATE COWMN TEMP InJECT VOL N8(1:1) : 0.2 m!Jmln : 313K : 5 uL
"' ANALYTICAL CONDITIONS (MS}

- MS TYPE : Thermo fisher ScfenUficTSQ Quenlum Aecass MAX IONIZATION : Nogatve ESI VAPORIZER TEMP : 723 K SPRAY VOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPilLARYTEMP : 493 K SCANTYPE : SIM(mlz18G)**U~Q**

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# 74

### Water analysis Day 14 High exposure level

Water analysis Day 14
Low exposure level Data Ftfe! C1B6WA14H Acquisition Dale: Original Dala Path: C:\Xcallbw\Dala\kaneko\0186\Da SampleJO: yt4 Sampfo Namo: Wateranalysis Hlv Comments: Sample TYpo: Unknowl\ Operator:
"' ANALYTICAL CONDITIONS (HPLC) "' HPLC TYPE : SHIMAOZU LC-20AD COLUMN Hyporsl!GOLD 2.1x50mm(Sum) ElUENT A; 20 mMAmmonium formale aq./formlc acld (1000:1) SjAce!onllrile AIB(l:l) FLOWRATE : 0.2 mUmln COLUMN TEMP : 313 K INJECTVOL : 5ul
"' ANALYTJCALCONDinONS (MS) '" MS 1YPE : Thermo Flsher SclentlflcTSQ Quantum Access MAX IONIZATION : Negallve ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 1BO)

--

Oala File: C1B6WA14L Aequlslllon Dale: Original Data Path: C:\Xcalibu!IOala\kanekoiC18610a Sample to: y14 Sample Namo: Water analysts LLv Comments: Sample Type: Unknown Operator:

'" ANALYTICAL CONDITIONS (HPLC) '" HPLCTYPE : SHIMADZU lC-20AD COLUMN : HypersU GOlD 2.1 XSO mm (5 urn) ELUENT : A; 20 mMAmmonium formate eqJIO!l11k: acld (1000:1) Bi AcelonllrUa AIB(1:1) FLOWRATE : 0.2mVmln COLUMN TEMP : 313 K INJECTVOL : 5 ul
"' ANALYTICAL CONDITIONS (MS) "' MS TYPE : ThermoFisherSclenlllioTSQ QoanlomAccess MAX IONIZATION ; Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : SO AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (mlz 180)

# """'

75

## Water analysis Day21
Standard solution

0.0048 mgL" 1



## Water analysis Day 21
Standard solution

0.0964 mgL· 1 Data File: C186WFA21S1 Acquisition Date: Original Data Path: C:IXcafibur\Dala\kanel<o1C1SS\Da Sampto to: Y21 Sampto Name: Std. 0.0048 mg/L Comments: Sample Typo: Sld Bracket Operator:

"' ANALYTtCALCONDITIONS (HPLC) "' HPLC TYPE : SHIMAOZU LC-20AD COLUMN : Hypersll GOLD 2.1 x50 mm (5 urn) ELUENT : A: 20 mMAmmonium fcrmele aqJroon!c acid (1000:1} BiAcelanilrllo NS(1:1) FLOW RATE : 0.2 mUmln COLUMN TEMP : 313 K INJECTVOL : 5Ul
"' ANALYTJCAL CONDITIONS (MS) "' MS TYPE : Thermo FisherScler.flticTSQ Quantum Acass MAX IONIZATION : Negalive ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS • : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM(mlz100)

Datafila: C156WFA21S2 Acquisition Date: OrlglnaJ Dala Path: C:\Xcalibur\Oaia\keneko1C1S6\0a Samplo!D: y21 Sample Name: Std. 0,0964-mgll. Comments: Sample Typa: Std Bracl<et Operator:

"*ANALYTICALCONDITIONS (HPLC) '" HPLCTYPE COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECTVOL : SHIMAOZU LC-20AO : Hyparsn GOLD 2.1 x 50 mm (5 um)

A: 20 mMAmmonhnn formale oqJiormlc acid (1000:1) a;Acetonilrne NS(1:1) : 0.2mllmin : 313K : SuL

.,, ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thermo FisherScientificTSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAY VOLTAGE : 2500 V SHEATH GAS : 60 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANTYPE : SIM (m!z 180)



# 76

### Water analysis Day21 High exposure level

Water analysis Day21
Low exposure level Data File: C186WA21H Acqulslllon Date: Original Data Path: C:\XcallbU1\0ala\kaneko\C188\Da SamplaiD: y21 Sample Name: Waleranalysls H lv Comments: Sampro 1'ype: Unknown Operator:
"' ANALYTICAL CONDITIONS (HPLC) "' HPLClYPe COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECTVOL : SHIMADZU LC·20AD : HypersU GOLD 2.1 X 50 mm (5 um) : A; 20 mM Ammonium formate aq./formlc acid (1000:1)

B; AcetonUnla A/8(1:1) : 0.2mUmln : 313K : 5 ul
"' ANALY11CAL CONDITIONS (MS) "' MS TYPE ! Thermo FisherSclentincTSQ Quan!um Access MAX IONIZATION : Negative ESI VAPORIZER TEMP : 723 K SPRAY VOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 

### o CAPILLARYTEMP : 493 K SCAN TYPE : SIM (m/z 180)

DataFile: Orlgtnal O;ata Path: Sample Name: SampleTypo: C186WA21L Acquisition Dale:**C:IXcaL'bu~Oala\kaneko\C186\Da** Sample 10: y21 Wateranalysis L Lv Ccmmcnfs! Unl<nown Operator:
"' ANALYTICALCONDITIONS (HPLC) "' HPLCTYPE : SHIMADZU LC·20AD COLUMN : HypersU GOLD 2.1 x50 mm (5 urn) ELUENT : A; 20 mM Ammonium formate aqJformlc acid (1000:1) B;Acetonitrile Nl3(1:1) FLOW RATE : 0.2 mllmln COLUMN TEM? : 313 K INJECTVOL : 5uL
'" ANALYTICAL CONDITIONS (MS) '" MS TYPE; : Thanno FisherSclenllffaTSQ Quanlum Ac<:ess MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN lYPE : SIM (m/z 180)

RT:ilctlo:l.£10 $J.t:14G



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# 77

### Water analysis Day23
Standard solution

0.0048 mgL" 1



### Water analysis Day23
Standard solution

0.0964 mgL· 1 Data Ale: C186WFA23S1 Acquisition Dale: Original Data**Pat~:** C:\Xcallbu!\Dala\l<aneko\C18G\Oa SampleiO: y23 Sample Name: Std. 0.0046 mgfl Comments: SamptoType: Std Bracket Operator.

'" ANALYTJCALCONDITIONS(HPLC)**•~** HPLC lYPE : SHIMAOZU LC-20AO COLUMN : Hyporsn GOLD 2.1 XS<l mm (5 um) ELUENT : A;20 mMAmmonlum formals aqJfennlc ncld{1000:1) B: Acetonlln1e NS(1:1) FLOWRATE : o.2m!.lmln COLUMri TEMP : 313 K INJECTVOL : 5uL
"' ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thermo FisherSclentiflo TSQ Quantum Access MAX ICNIZATKlN : Negative ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : :!SOOV SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN lYPE : SIM (mlz 180)

U:!lOOO•

•,

___

DataFila: C186WFA23S2 Acqulailfon Dale: Ortgtnal Data Path: Sample Namo: C:\XcafibUI\Oala\l<aneko\C186\Da SampleiD: y23 Sid. 0.0954 mgll. Comments: Sample Typo: S!dBmcket Operator:

"' ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE SHIMAOZU LC-20AD COLUMN Hypersa GOLD 2.1 xSO mm (5 um) ELUENT A; 20 mM Ammonium fonnalo aqlfO!f111c acld (1000:1) B;**Acekm!~Ha** A/8(1:1) FLOW RATE : 0.2mUrn!n COLUMN TEMP : 313 K INJECTVOL : 5ul

... ANALYTICAL CONDITIONS (MS)**~** MS TYPE : Thermo FisherScientificTSQ QuantumAccess MAX IONIZATION : NegaUveESI VAPORIZERTEMP: 723K SPRAYVOLTAGE : 2SOOV SHEATH GAS : 50 AUXGAS : 15 IONSWEEPGAS : 0 CAPILLARYTEMP : 493K SCAN lYPE : SIM (m/z 160)



# """**o;+**

### --------



# 78

### Water analysis Day23 High exposure level

Water analysis Day23
Low exposure level Dataflfe: Original Data Path: Sample Name: Sample Typo: Cf85WA23H C:\XcelbUJ\Oe!a\kaneko\C186\Da

## W~tr analyslsH Lv Unknown Aequisltlon Date: Sample 10: Comments: Operator:



# ............................................................................................ "' ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT
FLOW RATE COlUMN TEMP INJECTVOL : SHIMAOZU lC·20AD : HypersD GOlO 2.1 x50 mm (5 um) : A;20 mMAmmon!um formate aqJformlc acld{1000:1)

B;Acelonlltile Ml{1:1) : 0.2mUmin : 313K : 5ul

ou ANALYTICALCONDITIONS (MS)

- MS TYPE : Thermo Asher SclentmoTSQ Quan!um Access t..tAX IONIZATION : Noga!Ne ESI VAPORIZERTEMP ! 723 K SPRAYVOlTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE ! SIM (mlz 160)



# ...................................................................................................................................

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Data File: C166WA23L Aequlslllon Pate: Orlgtnal Dnla Path: C:\Xca!ibur\Oala\kaneko\C166\0a Sample 10: y23 Sample Name: Waleranalysis Llv Comments: SamptaTypt: Unknown Operator:

"' ANALYTICAL CONDITIONS (HPLC) '" HPLC TYPE SHIMAOZU lC-20AO COlUMN HypeiSR GOLD 2.1x 50 rnm (5 urn) ELUENT A; 20 mMAmmonium fonnalo aqJformlc sold (1000:1) B;AcetonltrUa FlOWRATE :

## ~{~Bmln COlUMN TEMP : 313 K INJECTVOL : 5uL**•~** ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thenno Fisher SclannncTSQ QuantumAccess MAX IONIZATION : NegaUva ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 25COV SHEATHGAS : 5I) AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : S!M (mlz 180)**flf;O.W·~$1.1:1~0**



# .

79

### Water analysis Day28
Standard solution



## 0.0048 mgL" 1



### Water analysis Day28
Standard solution



## 0.0964 mgL" 1 Data File: Origrnal DataPath: C1BGWFA2BS1 Acquisition Date: C:IXcallbUIIOala\kanako\C1B6\Da Sample ID: y29 Sample Name: Std. 0.0048 mg/L Comments: SamplaT)'pe: Sid llmket Operator.



# ........................................................................................... "' ANAL YTfCAL COI>IOJTIONS (HPLC) "'**HPLCTYP~** COLUMN ELUENT
FLOWRATE COLUMN TEMP INJECTVOL SHIMADZU LC.2ilAO Hypersl GOLD 2.1 x50 mm (5 urn) A; 20 mMAmmoolum formaloaqJformlc acid (1000:1) B;AcelonlltHe Al!l(1:1) : 0.2mUmln : 313K : 5ul
"' ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thermo FisherScfenlificTSQ QuantumAccess MAX IONIZATION : Nega!We ESI VAPORIZER TEMP: 723K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 IONSWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (rrk180)



# ...................................................................................................................................

"""

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# ..

___

Data File: C1BoWFA28S2·2 AcqulsfUon Date: Original Data Path: C:\XcaUbur\Oata\kaneko\C165\0a Sample JO: y2B Sample Name: Std. 0.{)964 mgJ1. Comments: Sampf& Type: Sid Bracket Operator.

"' ANALYTICAL CONDITIONS (HPLC) "' HPLCTYPE COLUMN ELUENT
FLOW RATE COLUMN TEMP INJECT VOL**•~** ANALYTICAL CONDITIONS (MS) "' MS TYPE : Thenno FisherSd&ntfficTSQ Quantum Access MAX IONIZATION : Nesallve ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARY TEMP : 493 K SCAN TYPE : SIM (ml< 180)



# ..



# 80

### Water analysis Day28 High exposure level

Water analysis Day28
Low exposure level Data File: C185WA26H Acquisition Date: Original Dala Path: C:\Xcallbu11Data\kaneko\C165\Da SamploiD: y28 Sample Name: Wateranalysis H Lv Comments: Sample Type: Unknown Oporator:

'" ANALYTICALCONDITIOilS(HPLC) "' HPLC TYPE : SHIMADZU LC-20AD COLUMil : Hypersll GOLD 2.1 X 50 mm (5 um) ELUENT : A; 20 mM Ammonium formal• aqJfotmlc acid (1000:1) B;Ac:etonltn1a FLOW RATE :

## ~(~Umln COLUMN TEMP : 313 K INJECTVOL : 5ut

•u ANALYTICAL CONDITIONS (MS) •u MS lYPE : Thermo FisherScienllficTSQ Quantum Access MAX IONIZATION : Nogallvo ESI VAPORIZERTEMP : 723 K SPRAY VOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (11'1Z 180)

Data Fllo: C166WA28t Acquisition Date: OrfglnafOataPath: C:\XcaffburiDala\lcane!:olC1S6\0a Sampte JD: y26 Sample Name: Water anafysls l Lv Comments: Sample Type: Unknown Operator:

u• ANALYilCA!.. CONDITIONS (HPLC) u• HPLCTYPE : SHIMADZU LC-20AD COLUMN H1Jl'!I>U GOLD 2.1 x50 mm (5 um) ELUENT A; 20 mMAmmonltJm formate aqJformla ackl (1000:1) B;Acelonl!rlle A/!!(1:1) FLOWRATE : 0.2mUmln COLUMN TEMP : 313 K INJECTVOL : 5ul

HO ANALYT!CALCONDITIONS (MS) '" MS lYPE : Thermo FisherSclenfirlcTSQ Quantum Access MAX IONIZATION : Negative ESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATHGAS : 50 AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCAN TYPE : SIM (m/z 160)

Rr:;.oo.:t..OO tJ.t1SG



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# 81

# 9-2 Control group .---------------, Water analysis DayO
Standard solution



## 0.0048 mgL" 1



### Water analysis DayO
Standard solution



## 0.0964 mgL" 1 Data Ale: C1S6WFAOS1 Acqulsl!lcn Dala: Original Dala Path: C:\Xcalibur\Dala\kanekc\C1B6\0a SamplaiD: 

### yo Samplo Name: Std. 0.0048 mg/L Comments: SamploTypo: Sid Bracket Operator:

"'ANALYTICALCONDITIONS (HPLC)**~·** HPLOIYPE COLUMN ELUENT
FLDIVRATE COLUMN TEMi' INJECTVOL : SHIMADZU l0-20AD : Hypar.dl GOlD 2.1 X50 mm (5 um) : A: 20 mMAmmoniumfcrmala aqJfcrmlc acid (1000:1)

B; Ace!onilllla AIB(1:1) : 0.2mllmlll : 313 K : Sci.
"' ANALYTICAL CONDITIONS (MS) ••• MS TYPE : Thermo FisherSdenti!icTSQ Quantum Access MAX IONIZATION : NegativeESI VAPORIZERTEMP : 723 K SPRAYVOLTAGE : 2500V SHEATH GAS : SO AUXGAS : 15 ION SWEEP GAS : 0 CAPILLARYTEMP : 493 K SCANIYPE : SIM (mlz 160)



# '"""' 

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Data Fila: C1e6WFAOS2·2 Acquisition Da1o: Original Data Path:**C:\Xcallbur\Dal2\kane~o\C186\0a** SamploiD: yO Sample Name: Sid.0.0964 mg1L**Comments~** Sample Type; Std Bracket Operator.

"' ANALYTICAL CONDITlONS (HPLC) "' HPLCIYPE COLUMN ELUENT
FLOWRATE COLUMN TEMP INJECTVOL : SHIMADZU LC·20AD : Hypetsll GOLD 2.1 x50 mm(5 um)

A: 20 mMAmmonium fCimolo aq.fformlc acid (1000:1) B;Ace!onltriJe IVB(1:1) : 0.2mtlmln : 313K : 5ul
'" ANALYTICAL CONDITIONS (MS) "' MS TYPE : Th<nr.c FbherSclenUfc TSQ Ouanlum Access MAX IONIZATION : Nega!lvo ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : 50 AUXGAS : 15 ION SWEE? GAS : 0 CAPILLARY TEMP : 493 K SCANIYPE : SJM (mlz 180)



# 82

## Water analysis DayO
Control group Data Fife: C186WAOC1 AcqulsiUon Dalll: OtJginaJ DafaPath: C:\Xcatihur\Oala\kaneko\C1BS\Da Sample 10: 

### yo Sample Nama: Water analysts Ctrt Comments: Samplo Typo: Unknown Operator:



# H. ANALYTICAL CONDITIONS (HPLCJ

... HPLCTYPE SHIMAOZU LC-20AD COLUMN Hypers» GOLD 2.1 x 50mm (5 urn) ElUENT A; 20 mMAmmonlum formal• aq.Jrormla acid (1000:1) B;Acelonilrife A/8(1:1) FLOW RATE : 0.2 mUmin COLUMN TEMP : 313 K INJECTVOL : 5 uL
"' ANALYTICALCONDITIONS (MS) "' MS TYPE : Thermo Fisher ScfenllfioTSQ Quanlum Access MAX IONIZATION : Negal!ve ESI VAPORIZER TEMP : 723 K SPRAYVOLTAGE : 2500 V SHEATH GAS : SO AUXGAS : 15 ION SWEEP GAS : 0 CAPiLLARYTEMP : 493 K SCAN TYPE : SIM (m'z 150)

<>3



# 83

# Reference 10 Water quality test Analyzed at Institute of Ecotoxicology Co., Ltd. <Quantitative analysis ofthe residual chlorine> The quantification ofthe residual chlorine in water used in this study was performed according to JIS K0102 33.1 (a-Tolidine colorimetric method). The concentrations ofthe residual chlorine during the experimental period were determined to be 0.01 mgL- 1 

# or less.
Concentration ofresidual Date ofquantitative chlorine (mgL- 1) 

# analysis

<0.01 1.26.2011 <0.01 2. 2.2011 <0.01 2. 9.2011 <0.01 2.16.2011 <0.01 2.23.2011

<Quantitative analysis ofcopper> The quantification ofcopper in water used in this study was perfonned according to

# ns KO 101 51.1 (Absorptiometry). The concentration ofcopper was confinned to be 0.005 mgL- 1

# or less. Copper (mgL- 1) 

# Date ofquantitative analysis < 0.005 6.20.2010

<Quantitative analysis ofdissolved organic carbon concentrations> The quantitative analysis was performed using a total organic carbon meter TOC

- 5000A (Shimadzu Corporation) (SOP/INS/4.9). The dissolved organic carbon concentration was confirmed to be 2 mgL- 1 

# or less.
Dissolved organic carbon

concentration (mgL

-1) 

# <2 Date ofquantitative analysis
2.14.2011

84

## No. 106020 April16,2010 

# Report ofthe test results

To the Institute ofEcotoxicology Inc. 3-15-1 Komazawa, Setagaya-ku, Tokyo Shinnihon Environmental Research Corporation (Registration ofMeasurement Certification Business, Tokyo, No. 882) Akira Kishi, Executive Director
The results ofthe test conducted on the sample as requested on April1, 2010 were as follows. Subject: Analysis ofwater used for raising carp Organization which Institute ofEcotoxicology, Inc. collected the sample: Type ofsample: Date when sample was collected: Water quality April 1, 2010

# ~ Water Quantifiable used for lower limit raising d carp Nitric nitrogen and (mg!L) 2.2 0.02 nitrous nitrogen Potassium (mg!L) 1.5 0.5 permanganate consumed Cyan (mg!L) Not 0.001 detected* Total mercury (mg!L) <0.0005 0.0005 Organic phosphorous (mg!L) Not 0.05 detected* Iron (mg!L) <0.001 0.001 Manganese (mg!L) <0.001 0.001 Zinc (mg!L) 0.014 0.001 Lead (mg!L) <0.001 0.001 Hexavalent (mg!L) <0.005 0.005 chromium Cadmium (mg!L) <0.001 0.001 Arsenic (mg!L) <0.001 0.001 Fluorine (mg!L) 0.10 0.05 Total hardness (mg!L) 63 0.02 Phenols (mg!L) <0.005 0.005 Anionic surfactant (mg!L) <0.02 0.02 pH( ) indicating water 7.4 0.1 temperature atthetime of (21.1) analysis Test methods

Service watertest method (2001) VI-2 13.2
Service watertest method (2001) VI-1 17.2

Service watertest method (200 1) VI-2 22.5
Service watertest method (200 1) VI-3 25.2 1974 Environmental Agency Notification No. 64 Appendix 1 Service watertest method (2001) VI-3 13.4 Service watertest method (200 1) VI-3 12.4 Service watertest method (2001) VI-3 16.4 Service watertest method (2001) VI-3 27.2 Service watertest method (200 1) VI-3 11. Reference 2 Service watertest method (2001) VI-3 21.4 Service watertest method (2001) VI-3 17.2.2 Service watertest method (200 1) VI-2 3.2 Service watertest method (200 1) VI-I 15.2.3 Service watertest method (200 1) VI-4 10.5 Service watertest method (200 1) VI-4 11.2 Service watertest method (200 1) VI-1 9.2

* "Not detected" means when measured by the analytical method hsted above, the result was less than the quantifiable limit.



# 8500 0\ 

# Reference 11 Records ofobservations ofthe conditions ofthe test fish (carp)

Records ofobservations ofthe conditions ofthe test fish No.1

Date Feeding Concentration Level I group period (Water tank No. 9) (days) Swim Food Health Water Mortality 

### 2011 status intake flow 

### 1.26 0 O.K.



## - O.K. O.K. 0 1.27 I O.K. O.K. O.K. O.K. 0 1.28 2 O.K. O.K. O.K. O.K. 0 1.29 3 O.K. O.K. O.K. O.K. 0 1.30 4 O.K. O.K. O.K. O.K. 0 1.31 5 O.K. O.K. O.K. O.K. 0 2.1 6 O.K.



## - O.K. O.K. 0 2.2 7 O.K. O.K. O.K. O.K. 0 2.3 

### 8 O.K. O.K. O.K. O.K. 0 2.4 9 O.K. O.K. O.K. O.K. 0 2.5 10 O.K. O.K. O.K. O.K. 0 2.6 II O.K. O.K. O.K. O.K. 0 2.7 12 O.K. O.K. O.K. O.K. 0 2.8 13 O.K.


### - O.K. O.K. 0 2.9 14 O.K. O.K. O.K. O.K. 0 

## Test substance

Concentration Level 2 group (Water tank No. 8) Swim Food Health Water Mortality status intake flow 

### O.K. 

## - O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K.



## - O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K.



## - O.K. O.K. 0 O.K. O.K. O.K. O.K. 0**~** 

### Flow rate: Level I: 300 ml/min, Lcvel2: 300 ml/min, Control: 300 ml/min to be confirmed. Swim status 

### O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K.**~** 

### Food intake: 0.050gper fish**~**

### Fcedprovided: Swimmy_(Japan Pel Food Col]l_., Lot No. 20 13.09.E3)**~Cumulative** 

### mortality at the end ofthe test: Level I: 0%, Levcl2: 0%, Control: 0%**~Remarks** 

### (Situation at the time ofabnormality and treatments taken): Not applicable Control group (Water tank No. 7) Food Health Water intake flow



## - O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K.



## - O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K.



## - O.K. O.K. O.K. O.K. O.K. 

## Test No. 

## C-186

Temperature/°C Observed by Mortality 

### Level Level Control I 2 0 24.8 25.1 25.5 Kaneko 0 25.1 25.5 25.6 Kaneko 0 

### 252 25.3 25.5 Kaneko 0 25.3 25.3 25.5 Kaneko 0 25.5 25.3 25.5 Kaneko 0 25.4 25.1 25.5 Kaneko 0 25.5 25.3 25.5 Kaneko 0 25.4 25.2 25.3 Kaneko 0 25.5 25.5 25.3 Kaneko 0 25.5 25.4 25.3 Kaneko 0 25.5 25.4 25.5 Kaneko 0 25.5 25.4 25.5 Kaneko 0 25.3 25.5 25.5 Kaneko 0 25.5 25.5 25.5 Kaneko 0 25.5 25.5 25.4 Kaneko00 .....:) 

## Records ofobservations ofthe conditions ofthe test fish No.2

Date Feeding Concentration Level 1 group period (Water tank No. 9) (days) Swim Food Health Water Mortality 

### 2011 status intake flow 

### 2.10 15 O.K. O.K. O.K. O.K. 0 2.11 16 O.K. O.K. O.K. O.K. 0 2.12 17 O.K. O.K. O.K. O.K. 0 2.13 18 O.K. O.K. O.K. O.K. 0 2.14 19 O.K. O.K. O.K. O.K. 0 2.15 20 O.K.



## - O.K. O.K. 0 2.16 21 O.K. O.K. O.K. O.K. 0 2.17 22 O.K.



## - O.K. O.K. 0 2.18 23 O.K. O.K. O.K. O.K. 0 2.19 24 O.K. O.K. O.K. O.K. 0 2.20 25 O.K. O.K. O.K. O.K. 0 2.21 26 O.K. O.K. O.K. O.K. 0 2.22 27 O.K.



## - O.K. O.K. 0 2.23 28 O.K. O.K. O.K. O.K. 0 

## Test substance

Concentration Level2 group _(Water tank No.8) Swim Food Health Water Mortality status intake flow 

### O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K.



## - O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. 

# - O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. 

## - O.K. O.K. 0 O.K. O.K. O.K. O.K. 0**~** 

### Flow rate: Level I: 300 ml/min, Level 2: 300 mllmin, Control: 300 mllmin to be confirmed. Swim status 

### O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K.**~** 

### Food intake: 0.050 g per fish**~Feed** 

### provided: Swimmy (Japan Pet Food Corp., Lot No. 2013.09.E3)**~** 

### Cumulative mortality at the end ofthe test: Level 1: 0%, Level 2: 0%, Control: 0%**~Remarks** 

### (Situation at the time ofabnormality and**treatrnenl~** 

### taken): Not applicable 

## Test No. C-186

Control group Tempcraturef'C Observed (Water tank No.7) by Food Health Water Mortality 

### Level Level Control intake flow 

### 1 2 O.K. O.K. O.K. 0 25.3 25.6 25.5 Kaneko O.K. O.K. O.K. 0 25.5 25.6 25.5 Kaneko O.K. O.K. O.K. 0 25.5 25.5 25.5 Kaneko O.K. O.K. O.K. 0 25.5 25.4 25.5 Kaneko O.K. O.K. O.K. 0 25.5 25.5 25.4 Kaneko



## - O.K. O.K. 0 25.5 25.6 25.4 Kaneko O.K. O.K. O.K. 0 25.1 25.4 25.1 Kaneko



## - O.K. O.K. 

### 0 25.1 25.4 25.5 Kaneko O.K. O.K. O.K. 0 25.4 25.4 25.4 Kaneko O.K. O.K. O.K. 0 25.4 25.6 25.3 Kaneko O.K. O.K. O.K. 0 25.3 25.5 25.3 Kaneko O.K. O.K. O.K. 

### 0 25.5 25.5 25.3 Kaneko



## - O.K. O.K. 0 25.3 25.5 25.2 Kaneko O.K. O.K. O.K. 0 25.2 25.1 25.3 Kaneko00 00

### lt~rian**t-rr,~-:.~~znuT'l'"'fl''ln~**



## l'l c 

### 2 .id 

### E**~** 

### !!! 1-**~**



## )_ Namcofthctestsubstance:
2. LotNo.: 

## 3. Date ofmeasurement: 4. Operator: Yasunori Okumura

# ~**~ ~** 

## ~ 

# = t':) (!) 

# ,....**~ II.>~**  

### "'0

### ~ (!) II.> 

# a"''

.., (!) 

# = t':) 

# !3 ::;- "'0 

# = 6 !3 <**~ Q:""~**  (!) """ 

### Q..=" (!)

# ~~ """II.> 

# =-""" (!) II.> II.> 

# = "'00"' 0 

### ;!4. 

# =**~** II.> 

# = 0 t':) ..:::;.,(!)

# IR spectrum (at the beginning ofthe study) measured at our location

52.5

45


### 37.5



### 30

22.5

15


### 7.5



### -7.5 4ooo 3aoo 3ioo 2s'oo 24oo 2000 1e'oo 16oo

[C-186]


### File Name:c186s5 Sample: [C-186] Date/Time: 2010/12/03 14:43:06 Type: IRPrestige-21 Units: 1/cm Min: 399.26 No. ofDatapoints: 1868 Gain: auto

2 3 4 5 

### 6 7 8 

### No. ofScans: 20 Ordinate: 

### %T Max: 4000.36 

### Data Interval: 1.93 

### Aperture: auto



# 89 I
.... I
.... l. 

# I'.,
.... I' I I I I 

### 1400 1200 1000 800 600 400



### Operator: Method: KBr 

### Detector: standard Apodization: Happ-Genzel Resolution: 4 

### [1Iem] SpeedStd: 2.8 1/cm

# IRspectrum (at the end ofthe study) measured at our location

30



### 22.5

•• I I
.. 

### I. I' I I I I' I I I I I I I I I' I I I I j It I I I J I I I' I I I l' I I,-, I 

## l-,,**~ ~** **~ ~** **~ ~**  1B 1B

### 1~**1~ 1~**  D B

### ~ [0-186] 1/cm
File Name:c186e1 

### Sample: [C-186; Date/Time: 2011/02/25 9:39:17 Type: IRPrestige-21 Units: 1/cm Min: 399.27 No. 

### of

### Datapoints: 3735 

### Gain: auto



### 1 2 3 

### 4 

### 5 6 

### 7 8 

### No. 

### ofScans: 20 

### Ordinate: %T Max: 

### 400o.43 

### Data Interval: 0.96 

### Aperture: auto



# 90 Operator: Method: 

### Detector: standard Apodization: 

### Happ-Genzel Resolution: 2 [11om] SpeedStd: 2.8