### 94746

..........

### -_.............. .......................... .................. '"'....."'. 

# · · : : 

# · · · · · Figure 3-8 : 

### 10.4 

# · C , 

### Figure**3~16** , : : 

# · · , 

# · · 

# · · · , 

# · · · 

# · · : 

# · · · · 

# · , 

### , 

# · · , 

# · , 

# · , , i 

# · I 

# · Figure**3~9** D 

### 10.7 

# · : t 

# · · Figure 3-17 I 

# · · · · : 

# · 

# · · , 

# · , 

# ·
, , , , 

# · · · ,

, 

# · · : 

### Figure 3-10 : 

### E 12.6 

### Figure 3-18 

# · 

# · · · · · · · · · · · · · : 

# · 

# · · 

# · · : 

# ·
· , 

# · · · · 

# · , , 

# · 

# · · · 

# · · · · · · Figure 3-11 

# · F 12.9 

# · · 

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### Figure 3-19 

# · · · · · · 

# · · · · · · · :



# · ,
, 

# · ·
·..........

### -.........

## ----.. "'...

## -.---...

## ---.....

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

## ..

# --­

545 

### ·3 Additional dala 3­2 94746

Figure 3·12 13.0 Figure 3·20

From these estimated structures, it was suggested that Peak A and B were the products converted from PE3MB by disulfide formation caused by oxidation ofthiol groups. In addition, there was possibility that Peak A and B were formed as a result ofdisulfide formation after PEMB changed to PE3MB by hydrolysis, and hydrolysis of a part of ester bindings after PEMB changed to Peak F by disulfide formation. Additionally, it was suggested that Peak A was stereoisomer ofPeak B. Also, itwas suggested that PEMB changed to Peak D by elimination ofhydrogen sulfide, Peak C by both disulfide formation and elimination ofhydrogen sulfide, and Peak E and F by disulfide formation caused by oxidation ofthiol groups. These converted products were not quantitative analyzed, because standard samples were not available.

546 -4 Additional data 3­

### 94746

Standard solution 50 mgIL

RT: 0.00

- 30.00 NL: 13.07 9000000 800000O 0·05 7000000 1

# ~
4000000 3000000 

### i 8.05

18.72 1 .47 20.64

. -­ 9.66E6 TIC F: MS 947e 09030&-501

..**_~.56** '><it 07**~~** 2000000j 1000000 0- I!" I ' I '--'--T-"--'-' I I I I I I ' I , I I , I 

### '---'1-,



### o 5 10 15 20 25 30 Time (min)



### Additional figure 3-1 TIC chromatogram (standard solution)
547 -5 Addilional data 3­94746

Standard solution 50 mgIL

94748-090306-501 #469 RT: 8.05 AV: 1 S8: 47 9.05-9.84 NL: 4.53E5 T:
... C 01 MS 1'150.00-600.00) 460.23 Mass**spec~rum** ofpeak 

### 1 400000 450000

350000
300000 2:'250000 'iii Ii .E 200000

150000 100000 488.28 

# I I 1

RT: 0.00 30.00 400000 350000 300000 :i250000 s::**~200000'** -150000'

100000 50000 

### o**PJ)7~~~::~** 400000 300000 

# :s 

# f200000
..

100000­ 8.11 Mass

### chromatogram of

### mlz =488

Positionofpeak 1 

# /

14.75 18.84 21.23 25.00 27.89

..".47 HI...: 4.38E5 mJz= 487.78-488.78 F: + C 01MS [ 15O.CJ0-600.00] MS 94746-0903()6.. S01

., •**·'1:.-'··~~NL:** 8.05 4.55E5 Mass chromatogram of

### mlz =460 mIz=: 459.73-460.73 F: MS 94746-090306­ Position ofpeak 1 501 

# /**O·~·P~~3~~~~\~~~~1~~.·~~~I~;6~:~T3~1~~~'J~ 3rl~I~23~1·3~~~I~~ir2~~~~~51**  

### o I) fO 15 20 25 30 Time (min) Additional figure 3-2 Mass spectrum ofPeak 1 and Mass chromatogram (standard solution)

548 ·6 Additiunal data 3­

### 94746

15 Standard solution 50 mgIL

94746-090306-S01 #761 RT: 13.07 AV; 1 SB: 49 14.46-15.28 NL: 2.59E6 T: +c 01MS (150.00-600.00) 562.25 2400000' Mass spectrum ofpeak 

### 2 

# I 2200000· 2000000 1800000 1600000 

## i 1 400000 c:**~1200000**

1000000 800000 500.28 600000 400000. 323.10 200000 425.19 

# l34 J 11



# ·**144~.20** 199.22 249.15 311.29 342.07 (516.42 58Q,1§. 

### o 1''1'' ",,1 I rr1 '" I ('f r ,-

# t'j"'''rrT I rT' "I " "f-'FJ"" I

150 200 250 300 350 400 450 500 55P 600 mlz RT: 0.00·3(1.(10 700000 600000 ;C; 500000**~** 400000­

- {! 300000 200000· 13.05 

### Mass chromatogram

,**1~.2I'f** mlz =590 Position ofpeak2 

# /

Mass chromatogram ofmlz =562 Position ofpeak 2 

# / NL: 1.68E5 II'It.t-I' 589.180690.18 F: +cQ1MS[ 150.00
..eOCtoo) MS**M7~** S01

mIz= 561.75-562.75 F: MS 94746-090306­ S01

Time (min) 

# ,f1500000

1000000 500000 ' 

### 0.

### o 10 .0.10

Additional figure 3-3 Mass spectrum ofPeak 2 and Mass chromatogram (standard solution)
549 -7 Additional data 3­

### 94746

: 2.33

## l.~i 

# A\ Test solution

RT: 0.00

- 30.00

7000000­ 

# ::=1'

8500000­ 6000000

# 1 5600000

# l SOOO()()(r" f4500000 

# i 4000000 -3500000"

30000007 2500000 • 1500000­ 1000000­ 500000 () -Y-T ,



### o NL: 8.22E6**2~3.03** TIC F: MS Genesis 94746­ 0903()6.. 

### F H93hA 

### B 

# !**12..~,** \ 8.05

'"T"" ,-- "1'"""' "I , : 1 '"1-'r I I I""'
.. T--'-,--T' I

5 10 15 20 25 30 Time (min)



### Additional figure 3-4 TICchromatogram [5.0mglL (at time that all test organisms died)]
550 -8 Additionaldata 3­

### 94746

Test solution 94746-690306-H93hA#431 RT: 7.40 AV: 1 S8: 51 3.73-4.58 NL: 7.83E5 T:
... CQ1 MS [ 150.00-600.00] 486.24 

### j

4!:1J.26**.4~j:I.22** 750000
. Mass spectrum ofpeak A 700000 850000 600000­ 458.21 550000 

# I 500000. 27450000 •**~400000** III !i350OOO

300000·. 250000-' 200000 150000 100000 : 50000 184.20 211.21 

### o I I ,'I I t'; I 1 I 'I 150 200 250 RT: 0.00

- 30.00 NL: 7.40 600000 '**~**

# pOSit~**~400000** peak A 

### £ 200000 

### o 1...- 1 _.

7

_ 3
__ 5 _,5,_1..... 7.59 Mass chromatogram of

### m/z =486 7.S5E5 mfz= 485.74­ 486.74 F: MS 94746­ 090306­ H93hA 8.72 12.!?1 14.97 1§.98 21.60 26.45 7.59 NL: 600000 7.40

### ~400000 c:

# POSi~ peakA

# ~2000oo o L...;,;,;.;.65;.::' _......:.:5.:;:::29:......",J 150000 i'100000

# ~50000 0- r""i"'1 I 1



### o 7.40. 7,.61

# pOSit~ peakA

3.31 6.QI 6.7SE5 Mass chromatogram of

### m/z =458 mlz= 457.71­ 458.71 F: MS 94746­ 090306­ H93hA**~·041P>25** __**18.,~** 20.74 24.95 26.42 --NL: 1.60E5 12.88 Mass chromatogram of

### m/z =321**~56.** 13.10 321.56 F:

" MS 94746· 090306­ 805 19.08 H93hA ".A \.,.1-3.60 r"'f I'" 'l" I 1 T-' 10 15 .J.I**~09 26,~28~7**  l' 1/Y1' "'-1'1", "'\ ;:;•

# .,..+, 20 25 30 Time (min) Additionalfigure 3-5 MassspectrumofPeak AandMasschromatogram [5.0 mgfL(attime thatall testorganismsdied)]
551 -9 Additional data 3­

### 94746

2$,i'\ 28 090306­ HDahA Test solution 94i46-090306-H93hA 11443 RT: 7.61 AV: 1 S8: 51 3.73-4.58 Nl: 6.91E5 T:
... C Q1MS [150.00-600.00] 486.24 458.18 Mass spectrum ofpeak B . i 650000 600000 550000 500000 450000. 400000 1£ 

# i 350000 'E -300000

250000 200000 150000 321.06 4.D7.24 100000 441..,24 

# I 4!U).25 576.31 

# 5000:

# ~17~'~1 rI

# ~4~'~r,2~~;47**~r l~i~rT** **4~3j~ ~,**  r , r, 

### Iq
..t1rl,-r .}" r 'l"f**~"i** 1..) 1.50 200 250 300 350 400 450 SOD 550 600 mtz RT: 0.00

- 30.00 7.40 NL:

600000 f4oooo0 c d) C -200000 7.{Q Mass chromatogram ofmlz =458 

# " Position ofpeak B
O· 1.66 20.74 24.95 28.42 150000 1 f100000 

# j 50000 3.31 6,87 

### o T'I I J 1'1 12.86 Mass chromatogram ofmlz =321 Position ofpeakB 1'1180 19.08 .)I!.**~.09** t I I 1 l' I I r 11 10 15 25 Time (min) 7.59 NL: 6.7BES mIz= 457.68­ 458.68 F: US 94746­ 090306­ 1-193hA NL: i.60E5 m/z= 320.56­ 321.56F: MS 94746­ 600000**~** I! 400000

### ~200000 1.73 5.51 0



### o 5 7.8SES 7.59 Mass chromatogram ofmlz =486 mIz= 485.74­ 486.74 F: us 94746­ Position ofpeak B 090306­ H93hA !J.2**12.~7** 14.97 1§.98 21.60 26.45

Additional figure 3·6 Mass spectrumofPeak Band Masschromatogram 

### 15.0mgIL(attime that all testorganismsdied)]
552 -10 Additional data 3­94746

Test solution 94746-090306-H93hA #469 -RT:8.05 AV: 1 S8: 51 3.13-4.58 NL: 8.99E5 T: + c Q1MS [ 150.()().600.00} 850000 800000 750000 700000 650000· 600000 550000· 

# t=**-~** 460.25 

### Mass spectrum ofpeak 1

488.27

RT: 0.00

- 30.00 600000 :ir

4oo000 

# I c: -200000 0-­



## o 200000

# I~=

60000 o NL: 8.05 6.84E5 Mass chromatogram of

### mlz =488 mIz= 487.77­ 488.nF: Position ofpeak 1 MS 947'* 090306­**7.~** H93hA**Q·!~L4.ro** 5.99**~~** ___**~V8 .~.~**  17.86 t9,97 24.21 28.18 8,04 NL: 9.35E5 Mass chromatogram of

### mlz =460 mIz=



### Mass chromatogramof

### mlz =323 322.60­ 459.75­ 460.75F: Position ofpeak 1 MS 947'* 090306­**7.~** H93hA**Q~ 5.~**  

### "'"f
......**,38_~13,.....1,...2_1..;.:~~** 11-65**1~.90** 23.97**~~** 13.05 NL: 

### PbSitionofpeak 1 2.39E5 miz= 05 ,,\8. 323.60F· MS 947'* 

# ;Jj
2.9.5 6.1M 

### !.f1'l, 11:13 16,18 18.99 24.44

# ~.2T ::::­**~!** 1 ,- l I '1 r I J' "'r I' 1, 

### o 5 10 15 20 25 30 Time (min) Additional figure 3·7 Mass spectrum ofPeak 1and Masschromatogram [5.0mgIL(allime thai alileslorganismsdied)J


### 553 -11 Additional data 3­94746

Test solution**94146-090306~H93hA'#60s** RT: 10.<41 AV: 1 SB: 30 8.9().9.39 NL: 1.60E5 T: +c Q1MS [ 150.0()..600.00] 554.24 Mass spectrum ofpeak C 150000 140000 130000 120000 110000. 100000**~9oooo** 

### '" c: 80000 

# i 10000 60000
. 5245023 50000.**~5.27** 40000· gie.25 30000· 20000 10000 171,21 20717 255.15 306.59 45118 .5.l?1.35 

# l . j l I'" 

# I I 369.36 I' 509,,21. 1 

## o .JI"

### ~Jn II

## r f 1 ,"\'t

### 1i1,~'<"'"1..f,,l) ",- r' l"'f,l..y..( ;'!'

### ~ Y

### 11'+jIL.~.\li1l'i**~,\II ,1!~**  .j

## Jy.'1 150 200 250 300 350 400 450 500 550 600 mlz RY0,00

- 30.00 10.42 NL: 160000 Position ofpeak C 140000 120000

# ~**~100000** co .**~** 80000 60000 40000 20000 G 60000 50()()(}: 

## .f 40000 230000 E**9.~ ~U7**  5.15 Mass chromatogram of

### m/z =554

### ~:E5 553,74­ 554.74 F: MS 9474&­ 090306­ H93hA
20.02**~~~_~~~~v~~~~~~~~~** Mass chromatogram of

### m/z =526 17.67

### ~.~2E4 10.42 Position ofpeak C .10.53 17.4t?**17~** .1.1
..96 24.63**~,55** .wrVV'
.......

# ""~ m/z= 525.73­ 526.73F: MS 94746­ 090306­ H93hA

25 30 Time (min) 

### Additional figure 3-8 Mass spectrumofPeakCandMasschromatogram [5.0 mgIL(attimetbatall testorganismsdied)]
554 -12 Additional data 3­

### 94746

Test solution 94746-090306-H93hAltl625 RT: 10.73 'A,,:'1 S8; 30 8.90-9.39 NL: i20E5 T: +c Q1MS [ 150.00-600.001 300000 280000'**~** 240000 220000 200000**~180000** 

## j160000 -140000

120000 100000

80000­ 60000'" 40000· 20Cl00-. 

### o 150 

### RT: 0.00

- 30.00

­ 300000 250000-­**~2oo000**

# I Ii 150000 • .5 100000 50000 556.29 

### Mass spectrum ofpeak D 

# I I
1

628.26

171.21

1185.17 272.48 3QI5.89 391.33
..

### ~.12 493.52 T'**·1r;~r"r·I'r+lt""l.tll'1"~P;'-I·r·rIJt-r+·ft** "·'i"r·tT"'l· hiT' \" 200 250 300 350 400 450 500 mIz 10.73 

### Mass chromatogram ofmlz =556 3.23E5 rnIz= 555.79­ 

# Pilion ofpeak

# D**~' 'l-0...;.fi2...;..",~4~_.;;.....;~~** **18.6~** ..19.11 25.86 .?T.39 O 10.1!'!a""·**~_"""f'._vJ_ -~NL:**  100000 80000 

## t 60000 t: S .5 40000



### o**~~~~~~~** 

### Mass chromatogram ofmlz =528 

### 7tionofpeak D**,~.27 .~26**  588.26 

# }
..,"I"i't1 550 500
NL:**~~~~~~~~~~~~~**1.08E5 m/z= 527.76­ 528.76F: MS 94746­**0903~** H93hA



## o 5 10 15 20 25 30 Time (min) Additionalfigure 3-9 Mass spectrum ofPeak Dand Masscbromatogram [5.0 m!VL(attime that allIes! organisms died)]
555 -13 Additional data 3­ 2000094746

Test solution 94746-090306-H93hA

### it733 RT: 12.59 AV: 1 S8: 39 14.27-14.92 NL: 4.35E5 T: +CQ1 MS { 150.00-600.001 588.23 I Mass spectrum ofpeak E ! 400000 t
350000 300000**~250000** 

## .., ii 

### sg 200000

150000 100000'
50000

# I
167.15 255.16 321.10 381.93 

### o**,~,** 

### .,"+''1"'",,:,'''r't"!"liJ**~** "1",\,,",' 'r" ,.,. r**i"'I"~r**J,.",:" \ 150 200 250 300 350 400 mJz RT: 0.00

- 30.00 12.59 Nl: 4.39ES 400000' Mass chromatogram of

### mlz =586 mlz:= 585.73­ 350000 586.73 F: 300000 MS 

### ,7ilionofpeakE 94746­**~250000** , 090306­ c: H93hA**~2000OO** -'50000'

100000 50000 2.92 2..'\Q ,54 6,89 9.43 

## !!'SII_...,"I,!

# k:o~.,..**.~"!** o**h.,.;;.~J""";;.......~t\,.",.** .. .... 12.59 Nl: 160000 140000
. 120000**~100000** ! 80000

### ~ 60000

40000 20000 5 10 15 20 10.13 1.61E5 Mass chromatogram of

### mlz =558 mIz= 557.70­ 558.70 F:**~ition** ofpeak E MS 94746­ 090306­ H93hA

I 30 Time (min)


### Additionalfigure 3,10 Mass speclrum ofPeakEand Masschromatogram [5.0mgIL(attime thatall testorganismsdied)J
556 -14Additional data 3­

### 94746

Test solution 94746-090306-H93hA #748 RT: 12.85 AV: 1 S8: 39 14.27-14.92 NL: 1.04E6 T: +c aiMS [ 150..00-600.001 588.26 1000000. 

# I Mass spectrum ofpeak F 900000; 80.0000 700000 ,6000.0.0. 

## i 

# I 500000 400000 300000
200® 560.22 I



## I 321,0.9 

# l

1000.00 ! ' 23 16 1,68,18 20.0..23 255,14 

## i

## 3~9,17 391.3:
. 441.18 558,,20. 0. i T J," II"} "'1'r"1',"1 I

## r'~' -t 1'"" ,.,"1' ,)., III T,,'" I I',-r'i'i', jill I 150. 200 250. 30.0. 350 400 450. 500 550. 600 mI% RT: 0..00

- 30..00 12.85 NL: 1000000 Mass chromatogram

## ~ p' . f k F

## ~E6 800000 OSItion 0 pea 587.76­ 

# f 600000 of

### m/z =588 ' / :'.76F:**~** 40.0000. I' 94746­ - 090306­ 2000.:

# L-2'~! __

# ~'~lI. ____

# ~~'~**~S:-) \,,3.~1** **_'-~:~** ___

# ~2..:94**~~:~~** __

# ~~L H93hA 12.85 NL: 

# '1' 12..98 nOES 60.0000- 

### Mass chromatogram I =72­**.~** 

### f 

### Iz 560' ',Positionof

### peakF 560.72F;

### ~ 40000.0."[' 

### m 

### = f / MS S 

## we E: , 090306:­ 200000, ',' 12..59) H93ilA 0. 1).31**3·~-** •

## ..!:Ef=·1!
.......:..-**!~~~_-J!44** ,23.18 "**28~6.4** Nt.: 150000.
. 7.61 12.86 

### Mass cmvmatogram ',,60E5 mfz=



# _ of

### m/z =321 320.59­ li'1o.oooo 

# I 13.1o.Position ofpeak F 3 M 2 s '59F: 94746­**~** 50.000
. I 090306­ 

# i 1805 

## !

# ~1:- 

# /

...., 1908 H93hA

...

## 3~_~~**~-""-L ~i~1** **"'I,~, ~~;**  0. ""1"""I'i"·,'·
...

# r-.c i 1 I j , , , I , , I I I I 0. 5 10. 15 20 2S 30 l1me(mIn) 

### Additionalfigure 3-11 MassspectrumofPeakFand Masschromatogram [5.0mg/L(attimethai

### an testorganisms died)}
557



### -15 Additional clata 3­

### 94746

Test solution 94746-()90306-H93hA#759 RT: 

### 13.03 AV: 1 S8: 

### 39 f4.27-14.92 NL: 1.61E6 T: + c Q1MS [150.00-800.001 1600000· 150000C). 14(10000" 

### 1300000**1~OO004F** 1100000 1000000 

# f=

- 700000 800000· 500000



### Fn: 0.00

- 30.00 562.25 

### Mass spectrum ofpeak 2

590.26



# I 400000. Ii' 300000 <I> 200000 100000' 0 

## 1 1.72 6118 I.j56 10.99 1500000'**~1000000'**

# ~ 500000 

### Mass chromatogram 13.02 NL: 

### ofmlz =590 12..8§



### Mass chromatogram ofmlz =562 4.59E5 mJz= 

### Position ofpeak 2 589.76­ 590.76F: 

# / MS 94746­ 0903()6. H93hA 1001.•56 18.01 23.63 25.69 28.64 ,- 13.03

-**--~....;."",.'",,,,,,.-.-** __ ••

_".
......-NL: 1.61E6 mJz= 561.75­ 

### Position ofpeak 2 

# /' 562.75F: MS 94746­ 090306­ H93hA 1.114 8.04 8. 11.13 13.43 19.87 24.28 27.25 

### o**I..-.;:'::;;;";'-~:;';';:"':"-;=~.** 

### 13,05'
. NI.: 

### Mass chromatogram 2.39E5 200000- mtz.= 8.05 ofmlz =323 322.57.**~** 150000 323.57F: <= ­ 

### Position ofpeak 2 MS 94746­**~** 100000-: 090306­ 50000. 

# 29", H93hA 2.f!! •. 7.Q2 

# L
11 

# J

# <7)1 18.....
... 

## o I**T~'1** I - 1" I I -I" I I I I "'1 "1 " V'I



### o 5 10 1520 25 3D TIme (min) 

### Additional figure 3-12 Mass spectrum ofPeak2and Masschromatogram [5.0 mgIL(attime thatalltestorganisms died)]
558 -16 Addilional data 3­

### 94746

.. " .. , ....... __ ..
_ ............ _,.,"',.... ::: 

### mlz Detected ion 

### 486**[M+N~+HCOOH-HzOr** 

### 458 [M+NH4r 

### 321**[M+H-C4~OS-H20r** Additional figure 3-13 Estimated structure ofPeak A

...........-._ ..

### -........ -- ...........

### -.. -..........

### -..

### ---_ .. "; 

# · ·**~~** 

# . . 

# · . · 

# . · : 

# . . · 

# . 

# · : 

# · . 

# · . 

# · . : 

# . · 

# . · . · :

",.,- .•........•' 

### mlz Detected ion 

### 486**[M+N~+HCOOH-H20r** 

### 458**[M+N~r** 321**fM+H-C4~OS-H20r** Additional figure 3-14 Estimated structure ofPeak B

559 -17 Additional data 3­••• 

### 94746

mlz Detected ion 

### 488 [M+NH4+HCOOH-H

### z Or 

### 460 [M+NH4r 323 [M+H-C4HoOS-H20r Additional figure 3-15 Estimated structure ofPeak 1

_c._c_.__________ ._.____ . ______ ...______• 

# . · · , 

# . . · · 

# . . · . · . · . 

# · . 

# · . 

# · · : 

# . 

# . ·

,"'.e.,
_ '"._,._.e' 

### mlz Detected ion 554**[M+NH4+HCOOH-H20]~** 526 [M+NH4t Additional figure 3-16 Estimated structure ofPeak C

560 -18 Additional data )­

### 94746



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

## .. ----.---.---.------

## ..

# -.. ,
, , ,

, 

# · · · · · · ,
, , , 

# · ·
m/z Detectedion 

### 556 [M+NH4+HCOOH-H20r 

### 528 [M+NH4r Additional figure 3-17 Estimated structure ofPeak D**~--.--** 

### ...------.-------

### ..

### -

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

# · . · · ·


# . . . · . 

# · , 

# · , 

# · · . , 

# · .

· · 

# · · · · h C! •••••••••• ,... • ••••• e.. '" 

# · mlz Detected ion 

### 586 [M+NH4+HCOOH.HzOr 

### 558 [M+NH4t Additional figure 3-18 Estimated structure ofPeakE

561 -19 Additional data 3­

### 94746

m/z Detected ion 

### 588 [M+NH4+HCOOH-HzOr 

### 560 [M+NlLt 321 [M+H-2C4lIt;OS-H20

# r Additional figure 3-19 Estimated structure ofPeakF**I·---················--~·--~·-·--·········-··~** 

# · . 

# · . 

# · . 

# · . · . 

# · . · . 

# · . 

# · : 

# . .

• " ••••••••••••r.' •• '":_e
__**._*~:.=c2.'.e'****reese** 

# · . . m/z Detected ion 

### 590 [M+NlL+HCOOH-HzOr 

### 562 [M+NlLt 323**[M+H-2C4~OS-H20t** Additional figure 3-20 Estimated structure ofPeak 2



### 562 

### -20 Additionaldata 3­

### Receipt number 662-08-E-4745 Study number 94745



# FINAL REPORT

A 48-hour Acute Immobilization Study of Karenz MT (Mixture ofPEMB and PE3ME) with 

### Daphnia magna

April 10, 2009
Kurume Laboratory Chemicals Evaluation and Research Institute, Japan



### 563

### 94745 

# STATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor Showa Denko K.K.


### Title A 48-hour Acute Immobilization Study of Karenz MT (Mixture of PEMB and PE3ME) with Daphnia magna 

### Study number 94745

I, the undersigned, hereby declare that this report provides a correct English translation ofthe Final Report (Study No. 94745, issued on April 10, 2009).



# JW7(z ($, 

### .;..009 Date 

# If a)u/u)'

# A /l'ttat--\ 

### Study Director Makiko Anai
564

### 94745 

# GLPSTATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor Showa Denko K.K.
Title A 48-hour Acute Immobilization Study of Karenz MT (Mixture of PEMB and PE3ME) with 

### Daphnia magna Study number 94745
The study described in this report was conducted in compliance with the following GLP principles: (1) "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 2003; No. 1121003, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 17, 2003, No.3, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry ofthe Environment) (2) "OECD Principles of Good Laboratory Practice (November 26, 1997, ENV/MC/CHEM (98)17)" This final report reflects the raw data accurately and it has been confirmed that the test data are valid.

Date April 10, 2009 Study Director Signed in original Makiko Anai
565

### 94745 

# QUALITY ASSURANCE STATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor Showa Denko K.K.
Title A**48~hour** 

### Acute Immobilization Study of Karenz MT (Mixture of PEMB and PE3ME) with 

### Daphnia magna Study number 94745

I assure that the final report accurately describes the test methods and procedures, and that the reported results accurately reflect the raw data of the study. The inspections and audit of this study were carried out and the results were reported to the Study Director and the Test Facility Management by Quality Assurance Unit as follows.
Item ofinspection 

### I audit Date of inspection 

### I audit Date of report to Study Director and Test Facility Management Study plan draft ,----._----_ 

### .. Study plan



# _ ..

### _--, Start of the exposure and after the exposure



## -----------­ - March 6, 2009 -----,---" _. 

### March 6, 2009 March 9, 2009

. - 

### March 11, 2009 -. 

### March 13, 2009



### - March 6, 2009 March 6, 2009 March 16, 2009 March 16, 2009 March 16, 2009 -,-­



## -­ --,

" 

### Raw data and final report draft April 10, 2009 April 10, 2009 Final report April 10, 2009 April 10, 2009

Date Head of Quality Assurance Unit April 10, 2009 Signed in original

Keiji Shiraishi

566

### 94745 

# CONTENTS Page Title
....................................................................················.. ···········..····1 

### Sponsor··
..······ .. ··.. ··········· ......····..·..·····..···....·······........·····..·....·.. ·..·....····1 

### Test facility
..··..······ ................................................................................ 1 

### Objective
............................................................................................... 1 

### Test method
........................................................................................... 1 

### Applied GLP
.......................................................................................... 1 Dates··
..··..··········....·······..·····..······· ..·· ..···.. ·........·····.. ··········· ..····· .. ····..

### ····2 Storage of test item, raw data, etc......................·........·......·..·.. ·..........

### ···2 Personnel····
.. ·........................................................................................3 Approval offinal report .........................................................................

### 3 SUMMARY
.............................................................................·· ..·····..···4 

### 1. Test item
..............................................................................................

### 6 2. Test sample
............................................................................................7 

### 3. Test materials and methods ·.. ·..··· ..·.. ··.. ·....··....···....···......··.. ·.. ·....·· .. ··..

### ·8 4. Results and discussion
.. ·........·....···......·· ....·· ..··........··....···......·· ..···..·.. ·13 5. 

### Factors that affected reliability of test results""""""'''''''''''''''''''''''''' 14 

### Tables and Figure of the test result Table 1 

### Immobility Table 

### 2 Observed abnormal response Table 3-1 Dissolved Oxygen Concentration of test solutions Table 3-2 

### pH of test solution Table 3-3 Temperature of test solution Table 4-1 

### EC50 

### to 

### Daphnia magna Table 4-2 

### EL50 to 

### Daphnia magna Figure 1 Concentration-immobility curve. Appendix 1 Chemical characteristic of dilution water Appendix 2 Analytical method and measured concentration oftest item Appendix 3 Calibration curve and chromatogram Additional data 1 Results ofpreliminary studies Additional data 2 Calculation results ofconcentrations ofeach component Additional data 3 Qualitative analysis of converted products



### 567

### 94745

Title
Sponsor

Test facility

Objective

Test method

Applied GLP A 48-hour Acute Immobilization Study of Karenz MT (Mixture ofPEMB and PE3ME) with Daphnia magna 

### Showa Denko K.K. 13-9, Shiba Daimon l-Chome, Minato-ku, Tokyo 105-8518, Japan Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 3-2-7, Miyanojin, Kurume-shi, Fukuoka 839·0801, Japan The objective of this study is to estimate the acute toxicity of Karenz MT (Mixture of PEMB and PE3ME) to Daphnia 

### sp. This study was performed according to the following test methods and guidance document. (1) Daphnia 

### sp., Acute Immobilization Test stipulated in the "Testing Methods for New Chemical Substances" (November 21, 2003; No. 1121002, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 13, 2003, No.2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, Environmental Policy Bureau, Ministry of the Environment) (2) OECD Guidelines for Testing of Chemicals, Section 2 : Effects on Biotic Systems, 202 "Daphnia sp., Acute Immobilisation Test (Guideline 202, April 13, 2004)" (3) OECD Guidance Document 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures" (September 2000) This study was conducted in compliance with the following GLP principles: (l) "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 2003; No. 1121003, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 17, 2003, No.3, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry of the Environment) (2) "OECD Principlesof Good Laboratory Practice" (November 26, 1997, ENV/MC/CHEM (98)17).


### 568 -1­

### 94745

DatesStudy initiation date March 6, 2009 Experimental starting date March 11, 2009 Experimental completion date March 13, 2009 Study completion date April 10, 2009 Storage of test item, raw data, etc.

(1) Test item The test sample"l will be sealed in a storage vessel and stored in archives in this laboratory for ten years after the receipt of notice specified under Clause 1 or Clause 2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 5-4 or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning Examination and Regulation of Manufacture, etc. of Chemical Substances". If it 

### is not stable for the storage period, it will be stored as long while it is kept stable and it will be disposed with approval of sponsor. Treatment of the test sample after the storage period will be discussed with sponsor. *1 It 

### will be stored as the common sample for storage of these studies (Study Nos. 94744,94745 and 94746). (2) Raw data and materials Raw data, the study plan, documents concerning the study presented by the sponsor, the final report and necessary materials will be stored in archives in this laboratory for ten years after the receipt of the notice specified under Clause 1 or Clause 2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 5-4 or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning Examination and Regulation ofManufacture, etc. of Chemical Substances". Treatment of raw data and materials, etc. after the storage period will be discussed with sponsor.

569 -2­

### 94745

Personnel Study Director Study personal Bio]ogy Ana]ytical chemistry

Approval offinal report Study Director Makiko Anai Section 4 (Eco-toxicity test area)
Makiko Anai, Mika Ono, Ald Nagano, Jun Sugimura Hiroyuki Togawa, Fumiko Kamiah, Tomoyo Kagamiyama, Mariko Tanaka, Hitomi Fukui, Hiromi Ando

Date April 10, 2009 Signature Signed in original Makiko Anai

570 -3­

### 94745 

# SUMMARY
Title A 48-hour Acute Immobilization Study of Karenz MT (Mixture of PEMB and PE3ME) with

### Daphnia magna
Test conditions (1) Test item Karenz MT (Mixture of PEMB and PE3ME) (2) Test organism 

### Daphnia magna (3) Exposure duration 48 hours (4) Test concentration Five exposure levels of 100, 77, 59, 46 and 35 mg/L (a geometric series with a factor of 1.3) as loading rate [respectively 9.5, 8.6, 6.4, 5.3 and 3.7 mg/L as measured concentration] and control (5) The number oforganisms Twenty daphnids/test level (five daphnids/test vessel) (6) Dilution water Dechlorinated tap water (7) Type oftest Semi-static (renewal at 24 hours after the start ofthe exposure) (8) Preparation oftest solution Test sample and dilution water were mixed and stirred to prepare each test concentration. After about 48 hours, test solution was prepared by filtrating them with a glass fiber filter. (9) Replicate Four replicates/test level (10) Volume of test solution 400 mLltest level (100 

### mLltest vessel) (11) Temperature of test solutions 20.0 to 20.2"C (12) Irradiation condition Artificial light ofwhite fluorescent lamp, 16-hour light/8-hour dark (13) Feeding No feeding (14) Aeration No aeration (15) Analysis of concentration oftest item in test solution HPLC analysis (at the start of the exposure, before and after the renewal, and the end ofthe exposure)

571 -4­94745

Results (l)Solubility in dilution water (20:tl°C) 10 mg/L (measured value in preliminary study) (2)Concentration oftest item in test solution (percentage ofthe initial concentration) At the start of the exposure and after the renewal 3.8 to 11 mg/L At the end ofthe exposure and before the renewal 3.0 to 9.5 mg/L (78 to 91%) (3)48-hour ECso (Median Effective Concentration) 9.0 mgIL (95% confidenceinterval: 8.2 to 11 mgIL) (4)Minimum concentration causing 100% immobility within 48 hours >9.5 mg/L (5)Maximum concentration causing no immobility within 48 hours <3.7 mg/L {The values of(3), (4) and (5) were based ongeometricmeanofmeasured concentration.] (6)Reference value: 48-hour ELso (Median Effective Loading); 88 mg/L (loading rate)

572 -5­

### 94745

1. 

### Test item**t···~·-·-·-~··~-----·····--·--······-·--·--·------····**..----.......-.---.......---....----...........--.....-_._......_ ..... 

# . 

# . 

# .

573 -6­

### 94745

2. Test sample

574 -7­

### 94745

2.5 Storage condition and confirmation of stability at storage condition Storage condition Dark storage place at room temperature and avoided contacting with oxidizing agent. Confirmation of stability The stability of the test item during the test period was confirmed by no alteration in the IR spectra of the test item before the experimental start and after the experimental completion. 3. Test materials and methods 3.1 Test organism (1) Species 

### Daphnia magna (Clone A) (2) Reason for selection of species Species recommended in the test guidelines (3) Source Young daphnids produced by parents which were cultured in the Kurume Laboratory were used. Daphnids 

### [Daphnia magna (Clone A)] originally came from the University of Sheffield (Address: Sheffield S10 2UQ, United Kingdom). The parents to obtain young daphnids were bred in the same quality of water (dechlorinated tap water), water temperature (20±1°C), photoperiod (16-hour Iight/8-hour dark) as used in the test. Parents used for the test were same lot and bred more than 14 days, and their age and survival rate were 21-days old and 100%, respectively. 

### Chiorella vulgaris of 0.1 to 0.2 mgC/day per 

### daphnia was fed to the parents once a day. A 48-hour acute immobilization test of K2Cr207 (Reagent grade, Wako Pure Chemical Industries, Ltd.) with the test organisms was conducted (on February 2

- February 4, 2009) to confirm the reproducibility of the test conditions. The 48-hour ECso of K2Crz07 was 0.28 mg/L. This value was within the normal range in this laboratory (mean ± 2S.D.: 0.13

- 0.36 mg/L) [mean ± S.D.: 0.24± 0.056 mglL (n=66)]. (4) Selection of young daphnids Less than 24-hour old daphnids were used for the test. (5) Allocation to the test groups Test organisms were placed at random to each test vessel.

-8­ 

### 575

### 94745

3.2 Dilution water Dechlorinated tap water, aerated sufficiently and temperature-controlled, was used. Some chemical characteristics of the dilution water measured regularly are listed in Appendix 1. 

### 3.3 Test apparatus and equipment (1) Test apparatus Test vessel: 100 mL glass beaker The test vessels were covered with lid in order to prevent dust, and volatilization ofthe test solution. (2) Test equipment Water bath Plastic tank Warming/cooling unit (Type HCA250, Sato craft) 3.4 Test conditions (1) Conditions ofexposure (a) Type of test The test organisms were exposed to the test solution containing the test item. The test solutions were renewed 24 hours after the exposure, as semi-static regime. (b) Exposure duration 48 hours (c) Test concentration Five exposure levels of 100, 77, 59, 46 and 35 mg/L (a geometric series with a factor of 1.3) as loading rate were used in the test. The exposure levels and the factor were decided based on the results of preliminary studies. The results of the preliminary studies are shown in Additional data. (d) Control The dilution water without the test item, which was treated in the same manner as the test solution except for one hour settlement and sampling form middle layer at 48 hours after mixing, was used as the control. (e) Replicates Four replicates/test level 

### 576 -9­

### 94745


### (f) The number oforganisms Twenty daphnids/test level (five daphnids/test vessel) (g) Volume oftest solution 

### 400 mL/test level (100 mLitest vessel) (2) Conditions oftest environment (a) Water temperature 20:!:1°C (b) Dissolved oxygen concentration The study was performed in the condition where dissolved oxygen concentration was more than 3 mg/L. No aeration was used for the test during the exposure. 

### (c) pH The test was performed without adjusting pH. (d) Irradiation condition Artificial light ofwhite fluorescent lamp, 16-hour light/8-hour dark (e) Feeding Test organisms were not fed during the exposure. 3.5 Preparation oftest solution The test sample and dilution water were mixed and stirred by magnetic stirrer for about 48 hours under about 20"C to prepare each test concentration (loading rate). The prepared solutions were settled for one hour after cease of stirring, and then the solution taken out from the middle layer of the settled solution was filtrated with a glass fiber filter (ADVANTEC, GB-140, 0.4 !1-m filter unit) by suction. After the filtration, the filtrate was stirred for about 30 minutes under about 20°C in order to recover decreased dissolved oxygen concentration by suction and then used for test solution. The prepared test solution was divided into each test vessel. 3.6 Observation and measurements (1) Observation oftest organisms Immobility and symptom were observed at 24 and 48 hours after the exposure. Daphnids were considered immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessel. 577 

### -10­

### 94745
(2) Appearance of test solution Appearance of the test solutions was observed at the start of the exposure and before renewal (after 24 hours of the exposure). (3) Condition of test solutions Dissolved oxygen concentration, pH and water temperature of the test solutions were measured at the start of the exposure, before and after the renewal, and at the end of the exposure (that is twice of measurement at the preparation and 24 hours after). At the preparation, another solution sampled from the container for preparation was used for the measurement. At the 24 hours after, the measurement was carried out for one test vessel in each level. The dissolved oxygen concentration measurements were carried out with an oxygen meter (YSI Model 58, YSI Incorporated.). The pH measurements were carried out with a pH meter (Model HM·21P, DKK-TOA). The water temperature measurements were carried out with a calibrated red alcohol thermometer of glass stick type. (4) Concentration of test item in test solution The concentration of the test item in the test solutions was measured at the start of the exposure, before and after the renewal, and at the end of the exposure (that is twice of measurement at the preparation and 24 hours after). At the preparation, another solution sampled from the container for preparation was used for analysis. At the 24 hours after, the test solutions taken out same volume from the middle layer of the test solution in test vessels in each test level were combined and used for analysis. The concentration of the test item was analyzed by gas chromatography (GC). The concentration of PMP was analyzed by high-performance liquid chromatography (HPLC). Analytical method and measured concentration of test item are shown in Appendix 2, and analytical calibration curve and chromatograms are shown in Appendix 3.



### 578 -11­

### 94745

3.7 Calculating method ofECso*3 

### The 48-hour ECso value was calculated by Moving average analysis, and also the 95% confidence limits were calculated. For 24 hours, the ECso was estimated as "> 

### the highest test concentration" since immobility over 50% was not observed. The ECso was estimated using Computer Program (running on Microsoft software "Excel") developed by our laboratory. The estimation of ECso was performed based on geometric mean of measured concentration oftest solution during the exposure. In addition, ELso·4 

### estimated using the nominal loading rate was also described as a reference. *3 

### ECso (Median Effective Concentration) is the concentration at which causes 50% 

### immobility oftested population during exposure. *4 ELso (Median Effective Loading) is 

### the loading at which causes 50% 

### immobility of tested population during exposure. 3.8 Validity ofthe test (1) The immobilization rate must not exceed 10% in control group during exposure. (2) More than 10% of the control daphnids should must not the signs of disease or stress, for example, discoloration or unusual behavior such as trapping at surface ofwater. (3) Dissolved oxygen concentration must be more than 3 mg/L at the end of the exposure. 3.9 Treatment of numerical values Values were rounded off in accordance with 1IS Z 8401 rule B, 1999. (118 ; 

### Japanese Industrial Standards)

579 

### -12­94745

4. Results and discussion The test concentrations in the following sentence is expressed using geometric mean of the measured concentrations which are 9.5, 8.6, 6.4, 5.3 and 3.7 mg/L, which are respectively 100, 77, 59, 46 and 35 mg/L as the loading rate. 4.1 Immobility Immobility at 24 and 48 hours arc shown in Table 1. The lowest concentration causing 100% immobility was expressed as >9.5 mg/L since 100% immobility was not obtained in the exposure levels. The highest concentration causing no immobility was expressed as <3.7 mg/L since no immobility was not obtained in the exposure levels. The concentration -response of immobility was not linear between 3.7 and 8.6 mg/L. In the control during the exposure, no symptom and abnormal behavior (such as discoloration and trapping at the surface of the water) was observed and immobility was 0%, which meets the criterion for the validity of the test (Le. not more than 10%). 4.2 Observed abnormal response The abnormal responses observed during exposure are shown in Table 2. There was no abnormal response in the control. The following results of observation were based on the comparison with the control organisms. The abnormal responses obtained in the test level during exposure were lethargic immobilization and reduced activity. 4.3 Observation and measurement of test solution (1) Appearance of test solution At the start of the exposure, the test solution was colorless and clear. The appearance was kept until the renewal. (2) Condition of test solutions Conditions of the test solutions are shown in Table 3-1,3-2 and 3-3. The measured values of dissolved oxygen concentration, pH, and temperature of the test solutions during the exposure ranged from 8.4 to 8.9 mg/L, 7.3 to 7.8, and 20.0 to 20.23°C, respectively. The measured values of dissolved oxygen concentration met the criterion for the validity ofthe test (more than 3 mg/L at the end of exposure).

580 -13­

### 94745
(3) Concentration of test item in test solution The results of the measured concentrations of the test item are shown in Appendix 2. The measured concentrations of the test item in the test solution were from 3.8 to 11 mg/L at the start of the exposure and after the renewal, and they were from 3.0 to 9.5 mglL (78 to 91% of the initial measuredconcentration) before the renewal and at the end ofthe exposure. 4.4 ECso and ELso The ECsos and ELsos at each observation time are shown in Table 4. The 24-hour and 48-hour ECso were respectively >9.5 mg/L and 9.0 mg/L (95% confidence interval: 8.2 to 11 mg/L). As reference value, the 24-hour and 48-hour ELso estimated based on the loading rate were> 100 mg/L and 88 mg/L (95% confidence interval: 77 

### to 110 mg/L). 4.5 Discussion This study was conducted as a test in order to estimate ECso of the test item for the test organisms below the solubility of the test item in dilution water. As a result, the 48-hour ECso was 9.0 mg/L. The concentration -response of immobility was not linear between 3.7 and 8.6 mg/L. The dissolved concentrations of PEMB*S, being poorly water-soluble and toxic, which were not proportional to the loading rate seems to be the reason for the nonlinear response since the test solutions were prepared at every exposure level (reference: Additional data 2). The concentration of the test item in the test solution was maintained during the exposure due to the renewal. The environmental conditions were within the suitable range, therefore, it is concluded that this study complied with the applied test guidelines. *5 U.S EPA OPPT(2004) ECOSAR Class Program Information, ECOWIN vO.99h
5. Factors that affected reliability of test results There were no factors which might have affected the reliability of the test.

581 

### -14­

# _ _ 94745

Table 1 Immobility Immobility (%) Measured Concentration"6 

### 48 hour (mg/L) 24 hour Test level Replicate Replicate Test level A 0 

# o f-._-
_ .............._._-_..­ 1--"-'-'-'-­

B 0 

## o

....

### "_
..... 

### _ .....

# _--_
..

# _­ Control 

# o 1--'''-.---• .-- 

## o C 0 

# o

--- t-'._
..._......__._-_ ..­ 1---""'''''''''''''-'''-'';­ D 0 

## o A 0 

### 40 ."....

# -_...

# _ .....

# _ ...

# -..

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

## -................

### _ ..

## ---_._.. B 0 

## o

_......._.t-'
......_ ..._-_.........__...­



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

### _ ...

# _--.......

# ­ 10 

### C 0 

### 3.7 

# o o

.....

# _._.......

# _._-­ D 0 

# o A 0 

### 60 B 0 

### 40 (-._.......

# __ ...

# __... 

# "'---

..

# _ ...

# _-...._ ......................

# - o 40 

### 5.3 20 

### C 0 1--,-"
......- ...----.......

D 0 

### 40 A 0 

# o 

# _._- ..

# _ ... ,.........

# _ ..

# _._-......

# _­ B 0 20

....

# _ ....

# _ ...

### -....

# _.­ 6.4 f-_
......f--
...- ....... ............- ...-.. 

# o 10 

### C 0 20 t-'-
......... ---"'_.

__._--.._-­ 

## t--------......­

D 0 

# o 80 

### A 0
.,......

## _,_..

# __......

# _. B 

### 0 .--..... I-
..--... ..,..--.....-.... 20

............,.....t-
.....-.........................-..............-.. 

### 40 

### 8.6 

# o C 0 

# o

......... om.... _-.... ......._,.""
............"""'
........_ ..... . 

# --_._.........

# .,--­

D 0 

### 60 A 20 

### 80

....... 

## ._-..._ ...

# _._--....

# ­**~** B 20 

### 40

....

# -

....

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

### _ ..

# __. 

# __.­ 20 t-'-
...._-­ 

### 9.5 60 C 20 

### 60 

## .-.......;. 

### ..... ...

# _-_..

D 20 

### 60 *6 Geometric mean of measured concentration (followings are described as measured concentration)

582

..15·

### 94745

Table 2 Observed abnormal response Measured Observed response (number of abnormal daphnia) Concentration (mg/L) 24 hours 48 hours Immobility Others Immobility Others Control 

### - 

## - 

# - 

# - 3.7 

# - - JM(2) RA(4) 5.3 

# - - IM(8) LETH(8) RA(1) 6.4 

# - - IM(2) RA(3) S.6 

## - RA(4) IM(S) LETH(S) RA(3) 9.5 IM(4) RA(4) IM(12) LETH(11) RA(3) -; No abnormal response Abbreviation of symptom 1M: Immobilization LETH: Lethargic RA: Reduced activity

Table 3-1 Dissolved oxygen concentration of test solutions Measured Concentration (mg/L) At the start 

### 24 hours At the end Before the

renewal After the renewal Control S.7 8.7 S.7 8.8 3.7 8.6 8.7 8.6 8.9 5.3 8.6 8.7 8.4 8.8 6.4 8.5 8.8 8.7 8.8 8.6 8.6 8.7 8.7 8.8 9.S 

### 8.6 8.7 8.6 8.9 Unit: mglL



### 583 -16­

### 94745

Table 

### 3·2 pH oftest solutions Measured 24 hours At the end Concentration (mg/L) At the start Before the renewal After the renewal Control 

### 7.7 7.6 7.7 7.6 3.7 7.7 7.7 7.7 7.6 5.3 7.7 7.7 7.7 7.7 6.4 7.8 7.7 7.8 7.7 8.6 7.8 7.8 7.8 7.7 9.5 7.8 7.8 7.8 7.3

Table 

### 3-3 Temperature of test solutions Measured Concentration (mg/L) At the start 24 hours At the end Before the

renewal After the renewal Control 20.0 20.0 20.0 20.2 

### 3.7 20.0 20.0 20.0 20.2 

### 5.3 20.0 20.0 20.0 20.2 

### 6.4 20.0 20.0 20.0 20.2 

### 8.6 20.0 20.0 20.0 20.2 

### 9.5 20.0 20.0 20.0 20.2 Unit: °C

584 -17­

### 94745

Table 4-1 ECso to Daphnia magna 

### 95% confidence interval Statistical procedure Exposure (mg/L) used for determination ECso(mg/L) (Slope of the duration ofECso dose-response curve)



# - 24-hour >9.5 

# - ( - 

## ) Moving average 8.2 to 11 48-hour 9.0 analysis 

## (

# -) Not obtained

Table 4-2 ELso'7 

### to Daphnia magna 

### 95% confidence interval Statistical procedure Exposure (mg/L) used for determination ELso (mg/L) (Slope ofthe duration of ECso dose-response curve)**~** 

### 24-hour > 

### 100 

## (

# -) 77 to 110 Moving average 48-hour 88 analysis 

## (

# -)
-: Not obtained *7 Reference value (Based on loading rate)

585 

### -18­

### 94745

100
-9-Z4-hour -6-48-hour 

### 80



# -**~** "-' 

# .c­ 

# .... 

# - ..... ,t;) 

# j 60



### 40

20



# o 1 

### Measured concentration (mgIL) 10

Figure 1 Concentration-immobility curve.

586 

### -19­94745



# Appendix 1

Chemical characteristics of dilution water

58794745

Chemical characteristics of dilution water (Sampling on January 14, 2009) Parameter Unit Resulls Lower limit of d Total hardness (as Dle03) mg/L 42 1 Suspended solid mg/L < 1 1 pH 

### - 7.9(22°C) 

### - Total organic**c~rbon** mg/L 0.4 0.1 Chemical oxygen demand mg/L 0.6 0.5 Residual chlorine mg/L < 0.02 0.02 Ammonium ion mg/L < 0.01 0.01 Tota1 cyan mg/L < 0.01 0.01 A1kalinity mg/L 29 1 Eleclric conductivity mS/m 17.1 

## - Organicphosphorous mg/L < 0.1 0.1 Alkylmercury mg/L < 0.0005 0.0005 Mercury mg/L < 0.0005 0.0005 Cadmium mg/L < 0.001 0.001 Chromium (VI) mg/L < 0.01 0.01 Lead mg/L < 0.005 0.005 Arsenic mg/L < 0.001 0.001 Boron mg/L 0.08 

### O.ot Fluorine mg/L < 0.1 0.1 Iron mg/L < 0.01 0.01 Copper mg/L < 0.005 0.005 Cobalt mg/L < 0.001 0.001 Manganese mg/L < 0.01 0.01 Zinc mg/L < 0.005 0.005 Aluminum mg/L 0.042 0.001 Nickel mg/L < 0.001 0.001 Silver mg/L < 0.0001 0.0001 Sulfate ion mg/L 20.1 0.1 Chloride ion mg/L 12 1 Sodium mg/L 14.9 0.01 Potassium mg/L 1.8 0.01 Calcium mg/L 11.5 0.01 Magnesium mg/L 3.3 0.01 1,2-dichloropropane mg/L < 0.0001 0.0001 ChloTolhalonil mg/L < 0.0001 0.0001 Propyzamide mg/L < 0.0001 0.0001 Chlornitrofen mg/L < 0.0001 0.0001 Simazine mg/L < 0.0001 0.0001 Thiobellcarb mg/L < 0.0001 0.0001 Diazinon mg/L < 0.0001 0.0001 Isoxathion mg/L < 0.0001 0.0001 Fenilrolhion mg/L < 0.0001 0.0001 EPN mg/L < 0.0001 0.0001 Dichlorvos mg/L < 0.0001 0.0001 lprobenfos mg/L < 0.0001 0.0001 PCB mg/L < 0.0005 0.0005

-1 Appendix 1­ 588

### 94745



# Appendix 2

Analytical method and measured concentration oftest item



### 589

### 94745

1. Pretreatment of test solution After no treatment or dilution with dechlorinated tap water, the test solutions sampled were pretreated according to the following flow scheme to prepare the sample for high-performance liquid chromatography (HPLC) analysis. Flow scheme 

# I Test solution 100 mL 

# I**~-Chloroform** 

### 30 mL (graduated cylinder) • Shaking (10 min) 

# I 

# IWater layer

# I !Chloroform layer!
• Transferred chloroform layer to the eggplant flask with

a beaker • Dryness (rotary evaporator, about 40°C, nitrogen stream) -Acetonitrile / water'! (1/1 v/v) about 1 mL (pasteur pipette) • Ultrasonic irradiation (ultrasonic cleaner, about 30 sec) • Filling up to 2 mL [acetonitrile / water*l (1/1 v/v),

volumetric flask] 

# I Sample for HPLC analysis 

# I *1 

### Tap water was treated with ultrapure water system.

59094745

2. Method of analysis The pretreated samples for HPLC analysis were quantitatively analyzed by HPLC under the following conditions to determine the concentration of the test item. The test item was detected as two peaks on the chromatogram. Therefore, these were analyzed as the test item. The concentration of the test item in each sample for HPLC analysis was determined on the basis of a comparison of the total peak area on the chromatogram of the sample solution with that of a standard solution. Some chromatograms obtained are shown in Appendix 

### 3. Analytical conditions Instrument High-performance liquid chromatograph SHTMADZU LC-201OA (Equipped with UV-VIS detector) Column L-column ODS (15 cm 

### x 2.1 mm I.D., Chemicals Evaluation and

Research Institute) Column temp. 40uC Eluent A: Acetonitrile / phosphoric acid (1000/1 v/v) B: Water'l / phosphoric acid (100011 v/v)

Gradient condition Time (min) A (%) B (%) 0.00 50 50 20.00 100 

## o 40.00 100 

# o Flow rate 0.2 mL/min Wave length 210 nm Injection volume 10,uL Detector output 2 V/AU

3. Preparation of standard solution The standard solution to determine the concentration of the test item in the sample for HPLC analysis was prepared as follows. Test sample of 100 mg was precisely weighed with an electronic balance and dissolved in acetonitrile to obtain 1000 mg/L solution of the test item. The solution was diluted with acetonitrile / water· 1 (111 v/v) to prepare 50 mg/L of standard solution.

591 -2 Appendix 2­

### 94745

4. Calibration curve The standard solutions of 5.0, 25, 50 and 100 mg/L were prepared for HPLC analysis by the same procedure as described in section 3. These solutions were analyzed according to the quantitative analytical conditions described in section 2. A calibration curve was drawn from the relationship between the concentrations of standard solution and the total peak area on the chromatogram, and the determination was confirmed. The calibration curve is shown in Appendix 3. The determination limit of the test item was the lowest concentration of the standard solution (5.0 mg/L) within the calibration range. Therefore, the

- determination limit of the test item in the test solution was 0.11 mg/L in consideration of pretreatment.
5. Recovery test and blank test 5.1 Method The recovery test was conducted by adding the test item (prepared with acetonitrile) to dechlorinated tap water according to pretreatment of test solution described in section 1. The blank test was also conducted using dechlorinated tap water (added acetonitrile) without the test item in the same way as the recovery test. The recovery test and the blank test were performed in duplicate. Amount of the test item added 100f..lg 5.2 Result As a result of analysis by the method of section 5.1, no peak of the test item appeared in the chromatogram of the blank test. Two individual recovery rates and their average on the analytical procedure are shown below. The average of recovery rate was used as correction factor for the determination ofthe test item concentrations in the test solutions. Recovery rate for pretreatment 87%, 89% average 88%

592 

### -3 Appeodix 2­

### 94745

6. Results ofmeasurement The 

### results of the measured concentrations of the test item in the test solution are shown below. Appendix table 

### 2-1 Measured concentrations oftest item in test solutions

Nominal concentration (mg/L) Measured concentration (mglL) (Percentage ofmeasured concentration versus that at each preparation %) 

### At the

start 

### 24 hours At the end Geometric mean Before the

renewal After the renewal Contro] n.d. n.d. n.d. n.d. 

# /
35 3.8 3.0 (79) 4.2 3.7 (88) 3.7 46 5.9 4.9 (83) 5.5 5.0 (90) 5.3 59 6.8 5.3 (78) 7.0 6.4 (91) 6.4 77 8.8 7.6 (86) 

### 9.5 8.6 (90) 8.6 100 11 

### 9.5 (88) 9.5 8.4 (88) 9.5 n.d. : < 

### 0.11 mg/L

593 -4 Appendix 2­

### 94745



# Appendix 3

Calibration curve and chromatogram

594

## o 94745

900000



## o 50 

### 100 Concentration (mgIL)

y 

### =

### 8519x r

## =1.00 Concentration Peak area (mg/L) (.ltV
.sec)



### 5.0 41878 25 212165 50 429373 100 850423

Appendix figure 3-1 Calibration curve oftest item for analysis by HPLC
595 -1 Appendix 3­94745


### Study 

### No. 94745 !!:.!h_ 50 

### miLL

___..__.. 

### -------.--­ PKHO 1 2 Operating Date : Mar. 11.2009 Time Area Helcllt Cone. (min) (j1 Y'6lIe) (/IV) (\) 8.72 61716 5211 14.67 15.28 35!lO54 39287 85.33 Total 420770 100.00
t

--" 

# . 0.0 I

# ~. A 11;'0 zO.o 30.0 Operator ; Hiroyuki TOlaw.**~41.1QlOh/Contto**I

- ­ --_.--­ - .._-.---->­ ... I'I(NO Operatlnl Date: Mar. II.2009 TI_ Ar'l He isilt Cone. 6ain)**(eV·se~) ~_~** 



### n.d. n.d.



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

# ~10.0

# ~**~-** 

### ---'-

..

### _-_._-,

20.0 30.0 Operator : Hiroyuki Togawe**~4745/0nL35** mgfb
___**--~-** ..-.-----"'.""'

.. '--­ I'I(NO 1 2 Oporating Date : liar. 11. 2009 Time Area Heicllt Cone. (mi n) (pV' Bec) (/IV) ('4) B.10 121122 10835 85.88 15.21 19921 206S 14.12 Total 141043 100.00

I**~.-L.** 

### ___. 

# . 0.0 10'.0 !**,~-** .....

## -.--­ 20.0 30.0 Operator: Hiroyuki Togen Oate Mar. 12.2009

_

### ~ame :

## 1!;:(07

# yk: T0

# 3 4\.1.14 

# <I>

Appendix figure 3-2-1 HPLC chromatograms at start ofexposure
596 -2 Appendill3­94745


### Study No. 94745 94145/0h/46 mg/.!.
__**.~-** --"­ -- ---..**-----~-** PKNO 1 2 Operating Dab: Mar. 11.2009 TIme Area Height Cone. (min) (IIY·aec) (IIV) ,,) 8.70 180213 15994 81.110 15.28 39581 4229 18.01 Total 219795 100.00

I

### ~A 0:0 111.0 2 

# . , 20.0 

# . 3It0 Operator : Hiroyuki TO(lIa 94145LOhi59 mg!l**--~-.-** PKNO 1 2 Operatinl Date ; Mlr. 11.2009 Tilllll Area HeiJht COne. (min) (pV'sec) (IlY) (i) 8.70 232691 20658 92.30 16.28 19409 2043 1.70 Total 262100 100.00

1 .A.....-.•:..

__

# l_..--L-.

0:0**1~0** 20.0 30.0 Operator : HirDYuki T ogawa 94745/0bLILlI]gLL 

### - ..**--~--~** ..**~** PllNO 1 2 Operati iii Date : liar. I\. 2009 Tima AreB Height COne. (min) (Il V'sec) (IlV) (S) 8.70 296366 26511 91. 00 15.21 29318 3137**g.~** Total 325684 100.00

I

# ~~_.~. ___-L.**o.~** 1cl.0 , 20.0 3l'io Operator : Hi ro)'uki Tala". Fig Date Mar. 12.2009 Name:

## H**i\,"o/""~{** 

## T"'J/l1!?4 

# •

Appendix figure 3-2-2 HPLC chromatograms at start ofexposure 

### 597 -3 Appendix 3­94745

Study No. 94745 24745LOhL1OO I1IiLL Operating Date: IIlIr.1l.2009 PKIIO TIllie Area Height Cone. Ullin) (IIV·;ee) (ll'/) ('4) 1 8.70 381039 33507 94.84 2 15.21 20735 2.2.41 5.16

Total 401774 100.00

Operator: HIrOl'ukl Togawa Fig



### Appendix figure 3-2-3 HPLC chromatogram at start of exposure
598 -4 AppeDdix3­

### 94745


### Study No. 94745 Operatin, Date : Mar. 12. 2000 /ltd. 50 

### mgtl**."----~** PKNO Time Area Height Cane. Unin) (/IV·sec) (Ull) (I) 1 8.72 63B34 5482 14.78 2 15.28 367963 40065 85.22**TotiT~i791-'-----;OO:OO**
2

# L_._J_~**~** , I 

### , I 0,0 10.0 20.0 30, a Operator : Hi rol'uki Togua (jperatiTlf Oate : Mar. 12. 2009 94745/24hLQontrQUlMslil 

## -
...... "­**"""-'-'~-'"** PKMl Time Area Hei,ht Cone. emin) (/IV·ne) (f!V) (I)



### n.d. n.d.



# ___ t

____**._~_** _..:A..
__

# ~ :0 10.0 20. D 30,1 Operator : Hi royuki To,alla**~4745/24h/~5****mg/lL~.*-L** 

### ____..__. ____ Operating Date: l\ar.12. 2009 PKHO Time Area Height Cone. (min) (/lV'leo) (j/. V) (%) 1 8.12 99813 9035 81.36 2 15,28 14447 1376 12.64 Total 114260 100, 00

A 

## J 1


### , 

# . 

# . 0.0 so.c 2O,e :ao.o Operator : Hiroyuki Tog.II. Date Mar.13.2009Nam!

## :M:t~!Ak;**T~!.wq** 

# •

Appendix figure 3·3-1 HPLC chromatograms before renewal at 24 hours


### 599 -5 Appendix 3­

# --- 94745

Study No. 94745 Operating Dlte : liar. 13. 2009 94745/24h/46**mg/LLML~** ___ .. ---_.._­**~,,-"--** PKNO TiIH Area Hel,nt COIlO. (IIin) (j.(V·seo) (j.( V) (l) 1 8.72 153704 14018 81.78 2 15.30 34240 3355 18.22 Total 187944 100.00

1 2 ........j 

## . oj 

# . 

# . i 0:0 10'0 )0.0 20.0 Operator: Hiroyuki yo,aWl Operati1'1& Date : liar. 13.2009 94145(24ht59 

### !!!GLLLtiJIJ. 

## .­ PKNO Ti
.. Area Heleht Cone. (IIin) (p V'seo) (p V) (Il 1 8.72 188066 16934 93.10 2 15.28 13939 1371 6.90 Total**~** 100.00



# , r

### ~. -- 

## 1



# . 0;0 100 20.0 la 0 ()perator : Hiroyuki Tocawl Operatinc Date: Mar. 13, 2009 94**7A?L?4!:l/'ZIl1!gL!JJl~WI** .­ PKNO Time Area Heiaht Conc. (min) (j.(V-sec) (IIV) (') 1 8.72 261405 23784 90.81 2 15.28 26457 2649 9.19

Total 287862 100.00



# , !

_ l •..

_______...__.A..-­ .)'t.

### A

... 

### _____._ 

# . I 0.0**10~** 0 tlie 30.0 Operator: Hiroyukl Tog8wa Date Mar. 13.2009 Name:

## Hi I'o/uk;

### 1~lJ.w(\ 

# •

Appendix figure 3-3-2 HPLC chromatograms before renewal at 24 hours


### 600 -6 Appendix3­

### 94745


### Study No. 94745 Operatinc Date : Mar. 13. 2009 PKNO Time Area lleilht Cone. (!lin) (pV'8eo) (pV) (I) 1 8. 73 337115 30668 93.37 2 15.28 23995 2414 6.63 Total 361710 100.00

I

# _~--L 

# __**.~_!",-"-** ____---,,,­ 0.'0 llio :W.o Operator : Hi roYuk I Togawa Fig Date: Mar. 13, 2009 Name:

## HiYOJY\ki 

# I"-tltl.wg 

# •

Appendix figure 3-3-3 HPLC chromatogram before renewal at 24 hours

601 

### -7 Appendix 3­94745



# Additional data 1

Results ofpreliminary studies

602

### 94745

1. Solubility of test item in dilution water It 

### was expected that the solubility of the test 

### item in dilution water was below 100 mg/L, therefore, the measurement of the solubility of the test item in dilution water was conducted. 1.1 Measurement oftest item solubility (1) Method The test sample and the dilution water were mixed to produce 100 mg/L and they were stirred for 24 and 48 hours under the test condition (20±1°C). After the mixture was settled for about one hour, the middle layer was collected and filtered· by suction filtration with a glass fiber filter (ADVANTEC, GB-140, 0.4 p.m filter unit). Then, the concentration was analyzed with high-performance liquid chromatography (HPLC) by same procedure described in Appendix 2. Test solutions were filtered by suction filtration after the filter was 

# '" washed with about 100 mL of the test solution. And it 

### was confirmed that the test item did not adsorb to the filter. (2) Result Measured concentration (mg/L) Arithmetic Sample Sample-l Sample-2 Sample-3 Sample-4 Sample-5 Stirring for 24 hours

9.1 11 

### 9.6 

# -----­ -----­ Stirring for 48 hours 

# -----­ -----­ ------­ 11 11 mean (mg/L)

10 

### Sample-6 

# -------­ 11 1.2 Summary It 

### was decided that the solubiHty of the test item in dilution water was 10 mg/L, which was the average ofmeasured concentration stirred for 24 hours and 48 hours.



### 603 -1 Additional data 1­94745

2. Study for effect on test organism 2.1 Preliminary study (1) Method The test sample and dilution water were mixed (nominal concentration: 100 mg/L) and stirred for about 48 hours under about 20°C. After the mixture was settled for about one hour, the middle layer was collected and filtered by suction filtration with a gJass fiber filter (ADVANTEC, GB-140, 0.4 pm filter unit). And then, the filtrate was stirred for about 30 minutes under 20°C in order to recover decreased dissolved oxygen concentration by suction filtration, and the filtrate was used for the test solution. The test organisms were exposed to the test solution in order to confirm effect of test item. The concentration of test item in the test solutions was also measured. (2) ResultNominal 24 hours 48 hours concentration (mg/L) Immobility 

### J%) Others Immobility 

### (%) Others 5.0 0 

# - 0 

# - 30 0 

# - 0 

## - 60 

### 0 

# - 0 

# - 100 

### 0 

# - 30 RA Type of test: Semi-static regime (renewal after 24 hours) The number oforganisms: ten daphnids/test level (five daphnids/replicate)

- shows that no abnormal response was observed. Abbreviation of symptom RA: Reduced activity Measured concentration(mg/L) (Percentage ofthe initial Nominal measured-concentration, %) concentration (mg/L) At the start At the end (48 hours after) 5.0 0.56 n.d 

### 3.9 60 5.3 (74) n.d. : < 0.11 mg/L

604 -2 Additional data 1­

### 94745

2.2 Summary Since it 

### was expected that the measured concentrations of the test item in the test solutions were decreased at 48 hours, the definitive study was plan,ed to conduct under semi-static regime of renewal at 24 hours. 3. Operation of definitive study Based on the preliminary study, a definitive study was conducted with five exposure levels of 100, 77, 59, 46, and 35 mg/L (a geometric series with a factor of 1.3) as loading rate and a control, and semi-static regime (renewal at 24 hours after the start of the exposure). The preparation method of the test solution was the same as used in the preliminary studies. The measurement of the test item in the test solution was performed at the start, before and after the renewal, and at the end of the exposure.

605 

### -3 Additional dala 1­

### 94745



# Additional data 2

Calculation results of concentrations ofeach component

60694745

1. Calculation results of concentrations of each component The test item was detected as two peaks in chromatogram. Calling Peak 1 and 2 in order of elution, each peak corresponded to PE3MB and PEMB (Information supplied by the sponsor). Based on the results of HPLC analysis in definitive study in each exposure level, the concentrations of each component were calculated. Results of calculation are shown below. The concentrations of each component in the each sample for HPLC analysis were determined on the basis of a comparison of the each peak area on the chromatogram of the sample solution with that of a standard solution. In addition, concentrations of each component in standard solution was calculated based on content percentage (PE3MB: 14.1%, PEMB: 81.7%). Additional figure 2-1 Concentration of PE3MB (Peak 1) in test solutions

Nominal concentration (mg/L) Measured concentration 

### (mg/L) (Percentage ofmeasured concentration versus that at each preparation %) At the

start 24 hours At the end Geometric mean Before the

renewal After the renewal Control n.d. n.d. n.d. n.d. 

# /
35 3.5 

### 2.8 (80) 3.7 3.3 (90) 3.3 46 5.2 4.3 (82) 

### 5.1 4.5 (89) 4.8 59 6.7 5.2 (78) 6.6 5.9 (90) 6.1 77 8.5 7.3 (85) 8.7 7.9 (91) 8.1 100 

### 11 9.4 

### (86) 9.2 8.1 (88) 9.4 n.d. : <0.018 mglL

607 -1 Additional data 2­

### 94745

Additional figure 2-2 Concentration ofPEMB (Peak 2) in test solutions

Nominal concentration (mg/L) Measured concentration (mg/L) (Percentage of measured concentration versus that at each preparation 

### %) At the

start 24 hours At the end Geometric mean Before the

renewal After the renewal Control n.d. n.d. n.d, n.d. 

# /
35 0.51 0.36 (71) 0.76 0.59 (78) 0.54 46 1.0 0.85 (84) 0.74 0.69 (93) 0.81 59 0.49 0.35 (70) 0.89 0.77 (87) 0.58 77 0.74 0.66 (88) 1.3 

### 1.1 (81) 0.92 100 0.53 0.59 (113) 0.88 0.73 (83) 0.67 n.d. : <0.013 mg/L

608 -2 Additional data 2­

### 94745



# Additional data 3

Qualitative analysis ofconverted products

609

### 94745

1. Qualitative analysis of converted products The peaks which were likely converted products except the test item were detected on the HPLC chromatogram of test solution in the definitive study. Therefore, the sample was qualitatively analyzed by liquid chromatographmass spectrometry (LC-MS) and estimated structures. 1.1 Method of analysis Standard solution and the pretreated sample of 100 mg/L at 24 hours before the renewal for HPLC analysis described in appendix 2 were qualitatively analyzed by LC-MS under the following conditions. Analytical conditions Instrument Liquid chromatograph

- mass spectrometer HPLC system SHIMADZU LC-20A Series Mass spectrometer Thermo Electron TSQ Quantum Discovery Pump (Post-column) HITACHI L-6200 Conditions of liquid chromatograph Column L-column ODS (15 em 

### x 2.1 mm I.D., Chemicals Evaluation and

Research Institute) Column temp. 40°C Eluent A: Acetonitrile / formic acid (1000/1 v/v) B: 

### Water"1 / formic acid (1000/1 v/v)

Gradient condition Time (min) A (%) B 

### (%) 0.00 50 50 20.00 100 

## o 40.00 100 

# o Flow rate 0.2 mUmin Injection volume 5jiL Post-column Solution Acetonitrile / water") (111 v/v) '2 

### Flow rate 0.1 mL/min

*1 Tap water was treated with ultrapure water system. *2 Containing 25 mmoIlL ammonium acetate.

610 

### -1 Additional dala 3­

### 94745

Conditions ofmass spectrometer Ionization mode Electrospray ionization (ESI) Detection ion Positive Measure mode Scan Scan range (m/z) 

### 150-600 Capillary temp. 300°C 1.2 Results Converted products, Peak A and B, detected on total ion current (TIC) chromatogram (Additional figure 3-4) were qualitatively analyzed by LC-MS and estimated as following structures. Also, it was recognized that Peak 1 and 2 were test item. Retention time Estimated Additional Peak Estimated structure on TIC molecular No. figure No. chromatogram and molecular fomula weight (min)

...__ ... "' .....__ ........._ .....a. ......... ..................................... 

# · 

# · · · 

# · · · 

# · · 

# · · · 

# ·Figure 3-5 

# · A 7.6 

# · 

# · ·Figure 3-9 

# · 

# · · · 

# · 

# · · · · · · · · 

# · · 

# · Figure 3-6 B 

### 7.8 Figure 3-10 

# ·

. .. .._ ....... .- 

### ..

611 -2Additional data 3­94745

1 8.2

, , ,

, ,
,
, , Figure 3-7 Figure 3-11

2 13.2 Figure 3-8


# · Figure 3-12 

# · ,
, , ,
, , , , , , , , ,

, , 

# · , , , , 

# · ,



# · 

# · · : 

# · , , , ,

, 

# · ,



# · -----­ From these estimated structures, 

### it was suggested that Peak A and B were the products converted from PE3MB by disulfide formation caused by oxidation of thiol groups. 

### In addition, there was possibility that Peak A and B were formed as a result of disulfide formation after PEMB changed to PE3MB by hydrolysis, and hydrolysis of a part of ester bindings after PEMB changed by disulfide formation. Additionally, it was suggested that Peak A was stereoisomer of Peak B. These converted products were not quantitative analyzed, because standard samples were not available.

612 -3 Additional data 3­

### 94745

1500000­ 15.58 

### Standard solution 50 mg/L

RT;**([oo~** 36.00 Nt: 13.22 4.56ES 4500000 TICF:+c Q1MS[ 400000(}­ 150.00­ 600.00) MS 94745­ 090312-801 3500000-·


### 1

# ~ 

# f25OCXro. 8.21 .52000000 11.25 .18.82**23~** 25. .25..25 ,88

__ 4499
..

## Jv.~~

0­**'"T·-I~r·f!-'---'** I I-r' I·"'·, I I 1 I r"j--'T, t-T·" T-,.....,-·:



### o 5 10 15 20 25 30 TIme (min)



### Additional figure 3-1 TIC chromatogram (standard solution)

613 

### -4Additional data 3­94745

Standard solution 50 mg/L 94745-090312-501**i4i8~RT:** 8.21 AV: 188:66'8.47=1.59 NL: S:71ES-----

­ T: +c QiMS( 160.00.000.00] 480.29

160000
'00000 

### ! 

### Mass spectrum ofpeak 

### 1

488.31

:!il8.31

mlz 8.26 HL: 2.S1ES J..33 Mass chromatogram m/z= 487.81-488.81 F: of

### m/z 

# =488 +cQ1MSI 150.00«10.00] MS Position of peak 1 94745-090312­ S01

3.50 6.15**~72** a.fi7 16.71**19.~** 21.52 26.!4§ 28.42 8.23 filL: 3.75ES mlz= 459.79-460.79 F: Mass chromatogram +cQ1MS[ of

### m/z = 460 150.0().600.00] MS 94745-{)90312­ Position ofpeak 1 S01**50000~** 22.60 27.07 0,**~r-r-T""T-r-+-r-i** 'r-:,r-r..,-l""-r-F'T-I-r"T---r--o--r-.,....,....,..-r--,!"'-'r-..,...,

o 10 20 25 30 360000 300000­ 25ODOO {2oooo0 GO lh50000

100000:

Additional figure 3-2 Mass spectrum ofPeak 1 and Mass chromatogram (standard solution)
614 -5 Additional data 3­94745

Standard 

### solution 50 

### mg/L 9474S:090312-s01#nO**RT:-i3:22~AV:** 1 SB: 66 6.47-7.59 Nl: 1.66E6 T: +c QU.1S [ 150.00-600.00] 562.27 1600000­ 

### Mass spectrum of

### peak 2 1500000: 1400000' 1300000: 1200000­ 

# I 110000D , 1000000
. fOOOOOO

­ i 800000'**~**



### o·**!r;05~!-r15~;~~1~~'·~=rI-r-~·~i~=rT.~O,1~122~I·~~9~f,~r·1T~'1~2rI7~·~~~~1** 

### o 5 10 15 20 25 30 Time (min) Fit: 0.00 - 

### 30.00

200000­ 

## f 150000' c

### ~ 100000­ 50000 

### o· 1800000­ 1400000-­ 1200000': 

# t1 

# I 800000. 600000 400000 200000':: 1127

3.40 6.89

### 10.~

# Il 13.22
. NL: 590.30

NL: 2.73E5 

### Mass chromatogram mlz= 589.80-590.80F: 

### of 

### I 590 +cQ1MS( m z = 150.00-600.00] MS 94745-090312­ 

### Position of peak 2 S01 

# /

1o J3.89 1L31 21,,48 24.30 2§.82 1.66E6mlz

### Mass chromatogram 561.71-562.77 F: +cQ1MS[ 

### of

### mlz =562 150.00-600.00] MS 94745-090312­ 

### Position ofpeak 2 S01 

# /

Additional figure 3-3 Mass spectrum of

### Peak 2 and Mass chromatogram (standard solution)

615 -6 Additional dala 3­

### 94745

Test solution
RT: 0.00

- 30.0('---

­ 3200000 3OOOOQO: 2800000 2600 2400000 2200000 ' 2000000 1800000 1600000 

# 1 -1400000:

1200000 1000000 800000 600000 400000 200000 0 NL: 8.21 3.2SES 

# / T1CF: MS 94745­ 090312­ H24hA-01d 

# \

"

# 7.~ 7.&'l 

# !

13.21



## , , " "'1 J I ,-'- I t I I' I -1-''1' I I' I t 1'1 1 l' I 1 I " I 1.80

0 5 10 15 20 25 30 Time (min)



### Additional figure 3-4 TIC chromatogram [100 mg/L (at 24 hours before renewal)]

616 

### -7Additional data 3­

### 94745

Test solution 94745-09031i.:ti24fiA.:o1d

### 1M41R'T:7.5'7 Av:TSe: 34 

### 6.47.7:04- Nt..: 

### a::fe -' T: +c Q1MS [**150.~.OO ]**  

### sooooo '·1 

### Mass spectrum of

### peak A 280000­ 260000 • 240000'. 220000 200000·**~180000.** 

# j160000

1 . 120000 100000
. 80000­ 458.26

m/z RT: 0:00'- 30.00 NL: 7 7.76 3.53E5 

### /. Mass chromatogram ofmlz =486 mIz= 300000 485.79-486.79 F: MS

### ~200000 Position

# Ofl

94745-090312­ 

### peak A H24hA-oid

## ~ -100000 ()_**1'~_1.92** 862**_~~.10~._** 17.45 11!:78 23.25 28.40 7.78 ---NL: 300000 

# / .Position of peak A o· 1.79 4.45 7. 3.22E5 mlzi= 

### Mass chromatogram ofmlz =458 457.76-4S8,76 F: MS 94745-090312­ H24hA-old Jl
.... 33 14.65 2O.Q1 23.27 29.{J1 

### 7:78 NL: 7.68E4 mIz= 

# ;yo Mass chromatogram ofmlz 

### =321**O~~321.85** 

### Position of 94745-090312­ H24hA-old 

### peak A 13.67 Ol-r-.--I-
................... , ....__

### r~.J,-.,.....,.16...".7_4,....,.. I

### ~1,~1 

# "f8, ,ZP.t o 5 

### ro _ 20**~** ® T1me(mln) 

### Additionalfigure 3-5 Mass spectrum ofPeakAand Mass chromatogram 

### 1100mglL(at 24 hoursbeforerenewal)]
617 

### ·8Additional dala 3­

### 94745

Test solution

94745-090312-H24hA-oldtt453 RT: 

### irs'A\i:1"SB: 34 6.47-7.D4'NL: 3.41E5 '._--'
..... T: + cQ1MS [ 150.00-600.00] 486.29 340000· 458.26 

### Mass spectrum ofpeak 

### B
280000 2I0000. 240000 220000: 200000· 

# . 180000



## . 1eoooo 

# 1

140000 12000D 1 4t1.28 80000 321.15 !1U.28 40000 441.,33 

## eoooo- 

# J

2OCiOI)-' 182.23 236.21 280.22

# 'f~~.14 423...23j o .. ,. I", 

## I,h",'" ',.'rr1'" 1 .J,: r"r"'f-TT 

### r

### '1~1

150 200 250 300 350 400 450 mIz NL: 3.S3ES 300000 

### Mass chromatogram of

### m/z =486 ='79-486.79 F: MS 94745-090312­ H24hA-oId 

### Position ofpeak B**O.-,--~1.S....,~=_..;;;t..:...~_-= ~§.::;..;,~_2** __ ... 26.40 1.;..;;3.;....1..;..O._--,-17..;;..4.:.;:5--.:..19 ""1.:.,;;80....-;;2;;.;,3,;.;;:.25;;;......_ 1.18 NL:

7n NL: 7.S8E4 Hi] 

### Mass chromatogram ofm/z =321 mIz'" 320.65-321.65 F: MS • • 94745-090312­ 

### PositIOn ofpeak B H24hA-old 13.87 

### o**~~~~~¥~~~t~~~~~-r~~-r~~~~~~**



### o 5 10 15 20 30 TIme (rnin) 300000 7.Sl**~2000oo·** 1/1 • 

## !i .5 100000 1.79 4.45**7.~** 

### o 3.22ES 

### Mass chromatogram of

### m/z =458 rnIl:= 457.76-458.76 F: MS 94745-090312· 

### Position ofpeak B H24hA-old .&•.

33 14.65 20.01 23.27 01



### Additional figure 3-6 Mass spectrum ofPeak Band Mass chromatogram [100 mglL(at 24 hours before renewal)]
618 -9 Additional data 3·

### 94745

Test solution**94745-090312~H24hdA-ok:tj478** RT: 

### a-#21-·'AV: 1 SB: 34 

### 6.47-7.04 NL:'1.18ES-­ T: +c Q1MS [150.Q0-600.00] 460.30 1100006· 1000000­

800000 7 

# i 600000­ 

### E 500000 400000 300000 200000 : 100000 0--1 ,- I , r 

### r.-r-T I 1 , r ,-', Tr- ,1'-,1 I r I I I I·LI-11 150 200 250 300 350 400 450 mIz 'RT: 0.00

- 

### 30.00-"
200000 150000

### ~ co**~** 100000­ .E 

### Mass spectrum ofpeak 1

323.11 443
... 30 341.17
. 191.25 221.19 268.29**r-~2.23** 425", 25

# 1 

# 1

8.17 _M5

7.5.£t**~.31** 488.30 .30 572.52**T~** 'T r I I I -.**f~r** ,- '1



# k

500 550 600
NL: 2.2SES 

### Mass chromatogram ofmlz 

### =488**m~80** 488.80 F: MS 

### Position ofpeak 1 94745­ 090312­ H24hA-old

### .~-,71 6.20§.,88**o~** 2.<C6
__**~.** J124
. .1Q,68 17.74**~a54** 25.11 27.41

# ~**~23~~~----------~------~------NL:** 1000000 800000 

# f 600000--­ !Q • 

### E 400000­ 2()()(J()(> U8E6 

### Mass chromatogram ofmlz 

### =460**~~~80** 480.80 F: MS 94745­ 

### Position ofpeak 1 090312­ H24hA-okl



### oh 1 r·",

79

-r-r--F1""'-r'-'l "'r"",9''r-12.,.-;1.,.1,.,82F-r1'-'i5F·2'T-5r1F7-r" 7:...;;6i--T
......**-r=M~....,--r2:::;8r=.2T7..,** 

## o 5 10 15 20 25 30 Time (min) 

### Additional figure 3-7 Mass spectrum ofPeal: 1and Mass chromatogram [100 mgIL (at 24 hours before renewal)]
619 -10 Additional data 3­94745

Test solution

9414s.:0903i:i:H24hA-old#769
.RT: 13.21 

### AV: 1 S8: 76·**f4~oo.15.28** 

### ·'NC-i.07E5· T: +c Q1MS [ 150.00-600.001 562.30 200000 Mass spectrum ofpeak 2 180000 160000 590.33 12bOOO
. 

# I 

# i 1

### 00000 80000 60000 40000



### RT: 0:00: 30.00­ 13.22 NL: 140000 120000 100000­ 

## f 80000·" c 

# g 60000 40000-:­ 11.23 20000 1.79 4.96 7.35 O·
........**~--..** .........W'O'
... --­ 1.41E5 Mass chromatogram mIz= 589.83­ of

### m/z =590 590.83 F: MS 94745­ 090312­ Position ofpeak 2 H24hA-oId 

# /

15.27 1?,48**21~** 24 2$.72 13.21 NL: 2O(lOO()-­ 2.07E5 Mass chromatogram mlz= 561.80­ of

### mlz =562 562.80 F: MS 94745­ Position of peak 2 090312­ 

# / H24hA-old



### o 1." 3.87 6.47 8.43 11.23 21,QO 25J.1 26.76 , I**~I** I , 

## rOt #£ J , I 

### o 5 10 20 25 30 

### Additional figure 3-8 Mass spectrum ofPeak2and Mass chromatogram [100 mgIL (at 24 hours before renewal)]
620 -11 Addilionaldata 3­

### 94745

mlz Detected ion 

### 486 [M+NH4

### +HCOOH-H20r 

### 458 [M+NH4t 321 [M+H-C4H60S-H201+



### Additional figure 3-9 Estimated structure of Peak A**------------------------------------~** •••••••••••••••••••••••••••••••••••••• r_i

---------------------------­ 

### mlz Detected ion 

### 486 [M+NH4+HCOOH-H20]+ 

### 458 [M+NH4t 321 (M+H-C4

### H60S

-H20r



### Additional figure 3-10 Estimated structure of Peak B

621 -12 Additionaldala 3­

### 94745

, _.__•••____._•••••**~_._**••____A.
______ •_____• 

### mlz Detected ion 

### 488 [M+NH4+HCOOH-H20t 

### 460 [M+NH4t



### Additional figure 3-11 Estimated structure of Peak 1



## ----_....
_............ ------.........

### -_..... , ,



### mlz Detected ion 

### 590 [M+NH4+HCOOH-H20t 

### 562 [M+NH4t 

### 323 [M+H-2C4H60S-HzOr



### Additional figure 3-12 Estimated structure ofPeak 2


### 622 -13 Additional data 3­

# CERI

FINAL REPORT

Algae Growth Inhibition Study ofKarenz MT(Mixture ofPEMB and PE3ME) withPseudokirchneriella subcapitata

April 14,2009

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan



### 623

### 94744 

# STATEMENT

Kurume Laboratory ChemicalsEvaluation and Research Institute, Japan
Sponsor Showa Denko K.K.

Title Algae Growth Inhibition Study of Karenz MT (Mixture of PEMB and PE3ME) withPseudokirchneriella subcapitata
Study number 

### 94744

I, the undersigned, hereby declare that this reportprovides a correct English translation of the Final Report (StudyNo. 

### 94744, issued on April 

### 14. 2(09).

Date 

## J UtiV' 

### 1'1 'leG

### ~ StudyDirector MikaOno
62494744 

# GLPSTATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor ShowaDenko K.K.

Title Algae Growth Inhibition Study of Karenz MT (Mixture of PEMB and PE3ME) with Pseudokirchneriella subcapitata Study number 94744

The study described in this report was conducted in compliance with the following GLP principles:

(1) "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 2003; No. 1121003, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 17, 2003, No.3, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry oftheEnvironment) (2) "OECD Principles of Good Laboratory Practice {November 26, 1997, 

### ENV/MC/CHEM (98)17)" This final report reflects the raw data accurately and it has been confrrmed that the test data are valid.

Date April 14, 2009 Study Director Signed in original Mika Ono



### 625

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

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

--- 

## ----

## -----

----



# -- 94744 

# QUALITY ASSURANCE STATEMENT

Kurume Laboratory ChemicalsEvaluation and Research Institute, Japan Sponsor Showa Denko KK.

Title Algae Growth Inhibition Study of Karenz MT (Mixture of PEMB and PE3ME) with Pseudoldrchneriella subca pitata Study number 94744

I assure that the final report accurately describes the test methods and procedures, and that the reported results accurately reflect the raw data ofthe study. The inspections and audit ofthis study were carried out and the results were reported to the Study Director and the Test Facility Managementby Quality Assurance Unit as follows. Date ofreport to Item ofinspection 

### I audit Date ofinspection 

### Iaudit Study Director and Test Facility Management Studyplan draft March 24, 2009 March 24, 2009 Studyplan March 26, 2009 March 26, 2009 Start ofthe exposure and March 30, 2009

_ April 3, 2009 after the exposure April 2, 2009 April 

### 3, 2009

Raw data and April 

### 10, 2009 April 10, 2009 final report draft Final report April 14, 2009 Apri1IS,2009

Date Head ofQuality Assurance Unit Apri1IS, 2009 Signedin original Keiji Shiraishi



### 62694744 

# CONTENTS Page Title
..............................................................·..····..················..·..··..·..·....······..··1 Sponsor
..............................................................................·......·..·..·..·..·..······..·1 Test facility
......···....··....·..·................................................................................ ] Objective
........................................................................................................... 1 Testmethod
...................................................................................................... 1 Applied GLP
..................................................................................................... 1 Dates
............................................................................·......··......·..···..······..···..·2 Storage oftest item, raw data, etc ..................................................................'" 2 Personnel··
..···..·....······...................................................................................... 3 Approval offinal report .................................................................................... 3 SUMMARY
.................................................................................·..·......·..·......4 1. Test item
...................................................................·................······..········....···6 2. Test sample
......................................................................................................·7 3. Test materials and methods
.............................................................................. 8 4. Results and discussion
.................................................................................... 14 5. Factors that affected reliability oftest results ·....····..······..·..··..···.. ··..·····....·.... 15 Tables and figures Table 1 pH oftest solutions Table 2 Culture temperature and light intensity in incubator Table 3 Value ofbiomass at each time Table 4 Growth rate and growthinhibition Table 5 ErCso and NOEC Table 6 Result of statistical analysis on growth rate between control level and exposure levels Table7 Variation ofgrowth rates in control Figure 1 Concentration-response curve Figure 2 Growth curve Appendix 1 Composition ofmedium Appendix 2 Analytical method and measured concentration oftest item Appendix 3 Calibration curve and chromatogram Additional data 1 Results ofpreliminary studies Additional data 2 Qualitative analysis ofconverted product

627

### 94744

Title
Sponsor

Test facility

Objective

Test method

Applied GLP Algae Growth Inhibition Study of Karenz MT (Mixture of PEMB and PE3ME) with Pseudokirchneriella subcapitata


### Showa Denko K.K**13~9,** 

### Shiba Daimon**l~Chome, Minato~ku,**  

### Tokyo**105~8518** 

### Japan
Kurume Laboratory Chemicals Evaluation and Research Institute, Japan**3~2-7,** 

### Miyanojin, Kurume-shi, Fukuoka 839-0801,Japan
The objective of this study is to determine the effect of Karenz 

### MT (Mixture ofPEMB and PE3ME) on growth ofalgae.
The study was performed according to the following test methods and guidance document. (1) Algal Growth Inhibition Test stipulated in the "Testing Methods for New Chemical Substances" (November 21, 2003; No. 1121002, Pharmaceutical and Food Safcty Bureau, Ministry ofHealth, Labour and Welfare; November 13, 2003, No.2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, Environmental Policy Bureau, Ministry of the Environment, Partial amendment November 20, 20(6) (2) OECD Guidelines for Testing of Chemicals, Section 2: Effects on Biotic Systems, "Freshwater Alga and Cynobacteria, Growth Inhibition Test (Guideline 201, March 23, 2006)" (3) OECD Guidance Document 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures" (Septembcr 2000)
This study was conducted 

### in compliance with the following GLP principles: (1) "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21,2003; No. 1121003, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 17, 2003, No.3, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry ofthe Environment) (2) "OECD Principles ofGood Laboratory Practice (November 26, 1997, ENV/MC/CHEM (98)17)".


### 628 -1­94744

Dates Study initiation date March 

### 26, 

### 2009 Experimental starting date March 

### 30, 2009 Experimental completion date April 2, 

### 2009 Study completion date April 14, 

### 2009
Storage oftest item, raw data, etc. (1)Test item The test sample'l will be sealed in a storage vessel and stored in archives in this laboratory for ten years after the receipt ofnotice specified under Clause 1 or Clause 2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 5-4 or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning Examination and Regulation of Manufacture, etc. of Chemical Substances", If it is not stable for the storage period, it will be stored as long while it is kept stable and it will be disposed with approval of sponsor. Treatment of the test sample after the storage period will be discussed 'With sponsor.

*1 Itwill be stored as the common sample for storage ofthese studies (Study Nos. 94744,94745 and 94746). (2)Raw data and materials Raw data, the study planl, documents concerning the study presented by the sponsor, the final report and necessary materials will be stored in archives in this laboratory for ten years after the receipt of the notice specified under Clause 1 or Clause 2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 5-4 or Clause 2 

### in Article 24 or Clause 2 in Article 25-3 of "Law Concerning Examination and Regulation of Manufacture, etc. of Chemical Substances". Treatment of raw data and materials, etc. after the storage period will be discussed with the sponsor.



### 629 -2­94744

Personnel Study Director MikaOno Section 4 (Eco-toxicity test area) Study personal Biology Ryuta Adachi, Miwako Shikada, Akane Nagata, Mika Ono Analytical Chemistry: Hiroyuki Togawa, Fumiko Kamiaka, Tomoyo Kagamiyama, Mariko Tanaka, Hitomi Fukui, Hiromi Ando Approval offinal report Study Director Date April 14, 2009 Signature Signed in original Mika Ono



### 630 -3­

### 94744 

# SUMMARY
Title Algae Growth Inhibition Study of Karenz MT (Mixture of PEMB and PE3ME) with Pseudokirchneriella subcapitata


### Test condition (1) Test item Karenz MT(Mixture ofPEMB and PE3ME) (2)Test organism Pseudokirchneriella subcapitata 

### (3) Exposure duration 72 hours (4) Test concentration Five exposure levels of 100, 32, 10, 3.2 and 1.0% (a geometric series with a factor of 

## .J1O) as stock solution content [0.40, 0.18, 0.067, 0.021 (reference value) and 0.0067 (reference value) mgIL as measured concentration] and a control (5) Type oftest Incubation with shaking (approximately 100rpm) (6) Preparation oftest solution The test sample and medium were mixed to prepare 100 mgIL (nominal concentration), and then they were stirred for about 48 hours. After the mixture was centrifuged 

### (l0000xg, 30 min), the middle layer was collected for the stock solution. Then the test solution was prepared by diluting the stock solution with medium accordingly. (7)Replicate Six replicates / control level (Two test vessels were additionally prepared for analytical chemistry) Three replicates / exposure level (Two test vessels were additionally prepared for analytical chemistry) (8) Volume oftest solution 600 mL/ control level (100 mL/ test vessel) (Two test vessels were additionally prepared for analytical chemistry) 300 mL/ exposure level (100mL/ test vessel) (Two test vessels were additionally prepared for analytical chemistry) (9) Temperature in incubator 22.2 to 22.8°C (10)Light condition 92 to 98/J-E/m2 

### /s (11)Measurement ofcell growth Chlorophyll fluorescence (12)Analysis ofconcentration oftest item in test solution HPLC analysis (at the start, after 24 hours, after48 hours and end of the exposure) 631

-4­94744

Results (1) Solubility in medium (23 ± 1°C) 1.3 mglL (measured value in preliminary study) (2) Concentrationoftest item in test solution [Percent ofmeasured concentration versus that at the start ofthe exposure

## J Atthe start ofthe exposure Below the determination limit 

## « 0.11 mglL) to 1.9 mgIL At the after 24 hours Belowthe determination limit 

## « 0.11 mgIL) to 0.49 mglL [25% (in the stock solution content 100% test level)] At the after48 hours Below the determination limit 

# « 0.11 mgIL) to 0.21 mgIL [11% (in the stock solution content 100% test level)] At the end ofthe exposure Below the determination limit

## « 0.11 mglL) to 0.22 mgIL [11 % (in the stock solution content 100% test level)] 

### (3) ECso (ErCso) > 0.40 mgIL (95% confidence limit; not calculation) (4)NOEC (Growth rate 0-3d) 0.021 mgIL (reference value) [The above-mentioned concentrations (3) and (4) are based on geometric mean of measured concentrations.]



### 632 -5­94744

1. Testitem

:._._ •• ___ •• ___ •••••• _____ •••• •• __ •• _______ •••••••• _. __ ••• _ •••••• __ • ___ ••••• _. _____ •• _ ••• __ __ •••• ___ •• ••••• a ____ ••••••••**~ ~_** **~ .~**  o :



### 633 -6­

### 94744

2. Test sample



# .**~**

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

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

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

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

634 -7­94744

2.5 Storage condition and confirmation ofstability at storage condition Storage condition Dark storage place at room temperature and avoided contactingwith oxidizing agent. Confirmation ofstability The stability ofthe test item during the test period was confirmed by no alteration in the 

### lR spectra ofthe test item before the experimental start and after the experimental completion.

3. Testmaterials and methods 3.1 Test organism (1) Species Pseu.dokirchneriella su.bcapitata (ATCC 22662) (The old scientific name "Selenastrum capricornutum") (2) Reason for selection ofspecies Speciesrecommendedin the test guideline (3) Source Pseudokirchneriella subcapitata which originally came from the American Type Culture Collection (12301 Parklawn Drive Rockville, Maryland 20852-1776 U.S.A.) on Jun. 30, 1995 and has been cultured in this laboratory was used. An algae growth inhibition test of potassium dichromate (Reagent grade, Wako Pure Chemical Industries, Ltd.) with the test organisms was conducted to confirm the reproducibility of the test system (on January 6 - January 9, 2009). The ErCso (0-3d) ofpotassium dichromate was 0.80mglL. This value was within the normal range ofthe reference substance in this laboratory (mean 

### ±: 2S.D.: 0.53 to 1.4 mgIL) [mean 

### ±: S.D.: 0.96

### ±:0.21 mg/L(n:::11)]. 3.2 Culturemedium The medium recommended in OECD test guideline (Guideline 201, March 23, 2006). The composition of medium is shown in Appendix 

### 1. Medium was used under sterile condition.

635 -8­94744

3.3 Test apparatus and equipment (1) Test apparatus Testvessel : Sterilized 

### 500 mLErlenmeyerflask (with gas-permeable silicon rubber

### plug~ (2) Test equipment Incubator : Incubator with temperature control, continuous shaking and continuous illumination, maintained the uniform light intensity (Incubator with rotary shaker and artificial illumination, USI CO., LTD) 3.4 Test conditions (1) Conditions ofexposure (a) Type oftest The test organisms were exposed to the test solution containing the test item added to medium. The test vessels that contain test solution and test organisms were shaken (approximately 100rpm) during the exposure. 

### (b) Exposure duration 72hours (c) Test concentration Five exposure levels of 100, 32, 

### 10, 3.2 and 1.0% (a geometric series with a factor of 

### .JlO) as stock solution content prepared by diluting the stock solution described in section 3.5 with medium were used in the test. The test concentrations and the factor were decided based on the results of preliminary studies. Theresults ofpreliminary studies are shown in Additional data 1. (d) Control The medium without the test item, which was treated in the same manner as the stock solution, was used as the control. (e) Replicates Six replicates/ control level (Two test vessels were additionally prepared for analytical chemistry) Three replicates / exposure level (Two test vessels were additionally prepared for analytical chemistry)

-9­ 

### 636

### 94744
(f) Initial cell concentration The pre-culture, incubated under the same conditions as the test for 

### 3 days and exponentially growing was used as inoculum to prepare the initial cell concentration ofapproximately about 104 

### cells/mL. (g) Operation All operations were carried out under sterile conditions. (h) Volume oftest solution 600 mL 

### I control level (100 mL 

### I test vessel; Two test vessels were additionally prepared for analytical chemistry) 300 

### mL 

### I exposure level (100 mL 

### I test vessel; Two test vessels were additionally prepared for analytical chemistry) (2) Conditions oftest environment (a) Temperature in the incubator 21 to 24°C, not varied more than 

### ±2°C (b) Light Continuous il1umination with irradiation strength of 60 to 120 

### #E/m2/s (fluctuation range: mean ± 15%) at the test solutions surface was provided, using a fluorescent lightwith wavelength range of400 to 700nm. 3.5 Preparation oftest solution The test sample and medium were mixed and stirred by magnetic stirrer for about 48 hours to prepare 100 mgIL (nominal concentration). After the mixture was centrifuged 

### (lOOOOxg, 30 min), the middle layer was collected for the stock solution. Then, required volume of the stock solution and medium"3 were mixed and stirred in the preparation vessel to prepare the test solution. The test solution was divided into each test vessel. *3 Medium which was treated in the same manner as control



### 637 -10­

### 94744

3.6 Observation and measurements (1) Cell growth, etc. Cell growth was shown as chlorophyll fluorescence. Chlorophyll fluorescence was measured with spectrophotofluorometer (Model FP-6500, JASCO Corporation) at 24, 48, and 72 hours after the start of the exposure. In 

### order to measure the blank value of the test solution in each test level, it was measured simultaneously about test vessel (without algae) separately prepared to blank value measurement, and used for compensation. Furthermore, the cell condition for one vessel in each test level was observed under microscope (Model BX41, Olympus Co., Ud.) at the end ofthe exposure. (2) Appearance oftest solution The appearance of the test solutions was observed at the start and end of the exposure. (3) Water quality and environmental conditions The pH ofthe test solution was measured at the start and end ofthe exposure. For the measurement of pH, another solution sampled from the vessel for preparation was used at the start of the exposure and one test vessel in each test level was used for the measurement at the end of exposure. The culture temperature and light intensity in the incubator were measured once a day during the exposure. pH measurements were carried out with a portable pH meter (Model HM-21P, DKK-TOA Co.). Temperature was measured with a calibrated thermometer of glass stick type. Ught intensity was measured with quantum scalar laboratory irradiance meter (Model LI-250A, LI-COR). (4) Concentration oftest item in the test solution It 

### was considered that the measurement was necessary with time from the result of the preliminary study. Therefore, the concentration of test item in the test solution was measured at the start ofthe exposure, 24 and 48 hours after the start of the exposure, and the end of the exposure. At the start, the solution sampled from the container for preparation in each test level was used for the measurement. At 24 and 48 hours after the start ofthe exposure, the test solutions were sampled from the test vessel for analytical chemistry. and then the solution after removal of algae by centrifugation (3000 rpm, 10 min) was used for the measurement. At the end of the exposure, equal volume of the test solution was taken out from the test vessels in each test level and mixed, and then the solution after removal of algae by centrifugation (3000 rpm, 10 min) was used for the measurement. Analytical method and measured concentration of test item are shown in Appendix 2, and analytical calibration curve and chromatograms are shown in Appendix 3. -11­ 

### 63894744

3.7 Treatment ofresults The results of the study were estimated by geometric mean of measured concentration. However, the concentrations of the stock solution content 3.2% test level and 1.0% test level were calculated from the content rate of stock solution and the concentration of the stock solution content 10% test level which was the lowest test level that was able to be analyzed, was substituted as reference value, because it had been below the determination limit through the exposure duration. (1) Calculation ofconcentration-inhibition rates The mean value of biomass for each test level was plotted against time to produce growth curves. Using this curve, inhibition rates were calculated comparingwith control values on growth rate. Comparison ofgrowth rates The specific growth rate for a specific period was calculated as the logarithmic increase in biomass accordingto the following formula: InNi-InNi ,U i-j trt i where

fl.i-] 

### = Specific growth rate from time 

### itoj (normally ((1) 

### Ni =Value ofchlorophyll fluorescence (relative unit) at 

### ri. Set value ofchlorophyU fluorescence (relative unit) was used at the start ofthe exposure 

### (to). N

j =Value ofchlorophyll fluorescence (relative unit) at tj ti = Time ofilh measurement afterbeginning oftest (d) tj =Time oflmeasurement after beginning oftest (d)

Specific growth rate over the exposure duration (0-72 h) was calculated for determination of EC50"4. In control, specific growth rates for section-by-section were calculated for check ofvalidity ofthe test. The percentage inhibition of the cell growth at each exposure level 

### (Ill) was calculated as the difference between the average specific growth rate at control level (uc) and that at exposure level (ul) as:

*4 EC50 (Median Effective Concentration) is the concentration of the test item at which growth is inhibited by 50% as compared with the control.



### 639 -12­94744
(2) Estimation ofECso The ECso was estimated as "> the highest test concentration" since no less than 50% ofinhibition rate was not obtained within the exposure levels. The EC50 was denoted as ErCso based on growth rate. (3) Estimation ofNo Observed Effect Concentration (NOEC'5 ) Regarding the growth rate, Bartlett's test was done to determine the homogeneity of variance for the data. Then one-way analysis of variance and Dunnett's multiple comparison test were used to determine the significant difference between the control level and exposure levels. The statistical analysis was conducted using computer program (running on Microsoft software "Excel") constructed by our laboratory. NOEC was determined by the results of statistical analysis and cell condition.



### *5 NOEC (No Observed Effect Concentration) is the highest concentration of the test item that does not cause any observed adverse effects on growth of the test organisms during the exposure. 3.8 Validity ofthe test (1) The cell growth in the control cultures should have increased by a factor of at least 16 within the 72-hour exposure period. (2) The mean coefficient ofvariation for section-by-section specific growth rates in the control cultures must not exceed 35%. (3) The coefficient of variation of average specific growth rates in replicate control cultures must not exceed 7%. 3.9 Treatment ofnumerical values Values were rounded offin accordance with 1IS Z 8401 rule B, 1999. (JIS ; Japanese Industrial Standards)

640 -13­94744

4. Results and discussion An indication of exposure level is shown as the geometric mean of measured concentration [0.40, 0.18, 0.067, 0.021 (reference value) and (W067 (reference value) mglL] in each nominal concentration (the stock solution content: 100, 32, 10, 3.2 and 1.0%). 4.1 Observation oftest solution and measurement ofwater quality variables (1) Appearance oftest solution At the start ofthe exposure, test solutions ofexposure levels were colorless and clear. At the end of the exposure, the appearance of test solution in the 0.40 and 0.18 mgIL exposure levels were slightly light green due to the algae growth, other exposure levels were green due to the algae growth. Also in the 0.40 and 0.18 mgIL exposure levels, the sticking of white substance was observed on the inner wall oftest vessel around surface oftest solution. The solutions ofcontrols were colorless and clear at the start ofthe exposure, and those at the end ofthe exposure were green due to the algae growth. (2) Water quality and environmental conditions The measured values ofpH of test solution are shown in Table 1, and culture temperature and light intensity in the incubator are shown in Table 2. The measured values ofpH were 7.7 to 7.9. Culture temperature in incubator ranged from 22.2 to 22.8°C and light intensitywas 92 to 

### 98/1E/m2

### /s. (3) Concentration ofthe test item in test solution The results of the measured concentrations of the test item are shown in Appendix 2. The measured concentrations of the test item in the test solutions were below [he determination limit 

## « 0.11 mgIL) to 1.9 mgIL at the start ofthe exposure. The measured concentrations of the dissolved test item in the test solutions at 24 hours after the exposure, 

### 48 hours after the exposure, and at the end ofthe exposure were below the determination limit 

## « 0.11 mgIL) to 0.49 mglL, below the determination limit 

## « 0.11 mglL) to 0.21 mglI.., below the determination limit 

## « 0.11 mglL) to 0.22 mglL, respectively. They were 25, 

### 11 and 11% of measured concentration at the start ofexposure in the stock solution content 100% exposure level, respectively.

Values ofbiomass at each time, growth rate and growth inhibition rates, and the ErCso are shown in Table 3, Table 4, and Table 5, respectively. Concentrationresponse curve is shownin Figure 1. ErCso ofthe test item based on the growth rate was> OAO mglL (95% confidence intervals: not calculated).
-14­ 64194744

4.3 Growth curves in each test level, cell observations and NOEC NOEC, the result of statistical analysis of significant difference, and growth curve are shown in Table 5, Table 6 and Figure 2, respectively. In the 0.40 to 0.067 mgfL exposure levels, the algae growth was logarithmic, although inhibition was observed. The algae growth in the 0.021 (reference value) and 0.0067 (reference value) mg/Lexposure levels were close to the control. The following results of cell observation were based on the comparison with the control. In the exposure levels, the condition ofcells was the same as the control. In the control, the condition ofcells was not ahnormal. In the growth rate, there were statistically significant differences in the 0.40 to 0.067 mgIL exposure levels. Based on the results in statistical analysis and cell observation shown above, NOEC based on growth rate was 0.021 mgIL (reference value). 4.4 Validity ofthe test (1) Growthofthe control The cell in the control grew exponentially during the exposure (ref. Figure 2). At the end of exposure, it increased to 41 or more times of the number of initial cells in the control (ref. Table 3). This meets the validity of the test: the cell growth in control should have increased by a factor ofat least 16 times at 72 hours after the start ofthe exposure. (2) The specific growth rates ofsection-by-section in the controls The mean coefficient of variation for section-by-section specific growth rate in the controls was 23% (ref. Table 7). It meets the validity of the test: the mean coefficient ofvariation in the control must not exceed 35%. (3) The specificgrowth rates in replicate controls The coefficient of variation of specific growth rate in replicate controls was 1.6% (ref. Table 7). It meets the validity of the test: the mean coefficient of variation in controls must not exceed 7%. 4.5 Discussion This study was conducted in orderto estimate the effect on the test organisms under the solubility ofthe test item in medium. As the result, the ErCsoandNOEC ofthe test item were> OAO mgIL and 0.021 mgIL (reference value), respectively. Though the concentrations ofthe test item in the test solutions were decreased during the exposure, the concentration ofstock solution content 100% exposure level (1.9 mg/L) was similar to solubility (1.3 mg/L; stirred 48hours, measured concentration in preliminary study) of test item in medium, therefore it was considered that the effect oftest item is able to be evaluated. It was considered that the reduction of test item concentration is due to hydrolysis of test item, production of soluble convert product by oxidation and insolubilization ofthe soluble convert product (ref. Additional data 2). The environmental conditions were within the suitable range. Therefore, it is concluded that this study complied with the applied test guidelines.

5. Factors that affected reliability oftest results There were no factors which might have affected the reliability ofthe test. 642 -15­94744

Table 1 pH oftest solutions Measured concentration*6 (mgIL) pH At the start At the end Control 7.8 7.9 0.0067 (reference value) 7.8 7.9 0.021 (reference value) 7.8 7.9 0.067 7.7 7.9 0.18 7.7 7.9 

### 0040 7.7 7.9
*6 Geometric mean ofthe measuredconcentrations (also expressed as measured concentration 

### in the following tables)

Table 2 Culture temperature and light intensity 

### inincubator

Time Atthe start 1-day 2-day Atthe end Culturetemperature

# ceq 22.6 22.2 22.2 22.8 Ught intensity (pE/m7./s) 92 98 96 92

643 -16­94744

Table 3 Value ofbiomass at each time Measured concentration No. CmgJL) Chlorophyll fluorescence (relative unit) 

## ohour·? 24 hours 48 hours 72 hours

Control A B C 

### 0 E 

### F ,>0-.""""'•.._-­ Mean S.D. 3.9 3.9 3.9 3.9 3.9 3.9 

## -.------.­ 3.9 0 15 16 14 15 16 17**-.---~-** 16 1.2 76 71 61 65 61 78**.~** ..

## --..

## -----.­ 69 7.4 180 190 170 160'8 160"S 170**--.~.-~.---.-.-,,-** 170 

### 10 0.0067 (reference

value) A B C**-~--.** Mean S.D. 3.9 3.9 3.9 16 15 16



## --­ 16 0.52 74 70 69 180 200 200 c---­ 3.9 0 71 2.5 190 

### 13 0.021 (reference

value) 

### A B C r--"'-' Mean S.D. 3.9 3.9 3.9



### -------­ 3.9 0 14 15 14 ,--._",,--...,.­ 14 0.74 58 66 60 

### -.----....**~~-.-** ..-.­ 61 4.4 160 160 160 

### ----,_...-.,....,....'''--­ 160 1.6

0.067 A B C 3.9 3.9 3.9 15 12 15 66 54 55 140 150 160 Mean S.D. 3.9 0 14 1.5 -, 58 6.4 150 6.6

0.18 A B C .-­ Mean S.D. 3.9 3.9 3.9 12 14 13 52 51 48 

# --_.. 51 2.1 120 120 110



## --­ 3.9 0**~-.** 

### 13 0.81 120 5.3 

# -

0.40 

### A B C 3.9 3.9 3.9 14 

### 13 13 45 47 45 94 100 92
IMean S.D. 3.9 0 13 0.49 46 1.4 95 4.0 *7 

## *8 The value based on the measured value ofpre-culture The minimum cell growth in control (biomass at the end ofexposure 

### 1biomass at the start ofexposure) 160/3.9 =41 -17­ 644

### ----



## --

--

-- 94744

Table 4 Growth rate and growth inhibition Inhibition concentration Growth

Measured rate



### (mw'L) rate No. 

### (%) A 

### (0-3d) 1.3 

# - B 

### 1.3 

# - 1.3 C 

# - D 

### 1.2 

# - Control 

### 1.2 E 

### - 1.3 F 

### -

-------...

# _-­ Mean**------~..--** 

### 1.3 ,



# - 0.020 S.D. " 

### -0.22 A 

### 1.3 B 

### 1.3 -3.7 0.0067 (reference 

### 1.3 -3.2

value) C 

### -2.4 Mean 

### 1.3 1.9

A 

### 0.024 S.D. 

### 2.9 1.2 2.7 1.2 B 

### 0.021 (reference 

### 2.3

value) 

### 1.2 C Mean 

### 1.2 2.6 0.26 S.D. 

### 0.0033 1.2 4.8 A 

### 4.1 B 

### 1.2 1.2 2.6 0.067



# -- C



## -----f-------­ 

### 1.2 3.8 Mean 

### 1.2

A S.D. 

### 0.015 1.1 10 

### 8.7 1.2 B 

### 1.1 11 

### 0.18 C 

## ,-­ Mean 

### 1.1 10 1.2 S.D. 

### 0.015 16

A 

### 1.1 1.1 15 B 

### 17 Mean 

### 1.1 0.40 C 

### 16 1.1 S.D. 

### 0.014 1.1

645



### -18­

### 94744

Table 

### 5 ErCso and NOEC
ErC50 (mg/L) NOEC(mg/L) >0.40 0.021 (reference value)

-

Table 6 Result ofstatistical analysis on growth rate betweencontrol level and exposure levels Measured concentration (mg/L) Statistical analysis Statisticalprocedure 0.0067 (reference value) n.s.
Bartlett's test. One-way ANOV 

### A. Dunnett's multiple comparison test. 0.021 (reference value) 0.067 n.s. 

## ** 0.18 

### ** 0.40 

### ** 

## * * : significant difference (p <0.01) n.s. : no significant difference

646 -19­94744

Table 7 Variation ofgrowth rates in control

<Variation for**section~bv-section** specific growth rates inthe controls> J Control No. Mean Standard deviation Coefficient of variation (%) A 1.3 0.37 29 23 (Mean) 

### B 1.3 0.27 21 C 1.3 0.21 17 D 1.2 0.29 23 E 1.2 0.23 18 

### F 1.3 0.41 33

< Variation ofaverage specific growth rates in replicate controls> 0-3day Mean 1.3 Standard deviation 0.020 Coefficient ofvariation (%) 

### 1.6

647 -20­

### 100 

### 94744

90
80 

### 70 60



# -**~** '-' <!.'I 

# - 50 t<:J
.... Cl 0 

### ..... 

## . ,t::l 

# .-**~** 

### 40 .r::: Cl 

### ..... 30
20


### 10



# o
-10



### 0.001 

### -e-(0-3day)

1 0.01 0.1 Measured concentration (mg/L)

Figure 1 Concentration-response curve
648 -21­

### 94744

1000**~Control**

,-b- 0.0067 (mgIL) 

### -e-O.021 (mgIL) : -+- 0.067 (mgIL) 

# 1- 0.18 (mgIL)

### i~0.40 (mgIL)



### 1

# o 24 48 72 Exposure duration (hours)



### Figure 2 Growth curve 

### 649 -22­

### 94744



### Appendix 1

Composition ofmedium

650

### 94744
Composition ofOECD medium [Guideline 201 (March 23, 2006)] Nutrient salts Amount H3BO] 0.185 mg MnCh'4H2

### 0 0.415 mg ZnCb 0.003 mg FeCh'6H2 0 0.064 mg Na2EDTA'2H20 0.1 mg CoCh'6H2

### O 0.0015 mg Na2Mo04'2H20 0.007 mg CuCh'2H20 0.00001 mg CaCh'2H20 18 mg NH4Cl 15 mg KH2 P04 1.6 mg NaHC03 50 mg MgCh'6H20 

### 12 mg MgS04'7H2 0 15 mg The constituents mentioned above were fined up to 1L with purified water.

651 -1 Appendix 1­

### 94744



### Appendix 2

Analytical method and measured concentration oftest item

65294744

1. Pretreatment oftest solution The test solutions sampled were pretreated according to the following flow scheme to prepare the sample for high-performance liquid chromatography (HPLC) analysis. Flow scheme 

# ! Test solution 100mL 

## I "'-Sodium chloride 30g (electronic evenbalance) ...-Chloroform 30mL(graduated cylinder) • Shaking (10 min) 

# 1.--------1 

# I Water layer 

# II Chloroform layer 

# I

• Transferred chloroform layer to the eggplant flask with a beaker • Dryness (rotary evaporator, about 40°C, nitrogen stream) +-Acetonitrile/ water"} (111 v/v) about 1 mL(pasteurpipette) • Ultrasonic irradiation (ultrasoniccleaner, about 30 sec) • Filling up to 2 mL [Acetonitrile 

### Iwater"l (1/1 v/v),

volumetric flask

## J 

# I Samplefor HPLC analysis 

## .1

*1 Tapwaterwas treated with ultrapure water system.



### 653 -1 Appendix 2­94744

2. Method of analysis The pretreated samples for HPLC analysis were quantitatively analyzed by HPLC under the following conditions to determine the concentration of the test item. The test item was detected as two peaks on the chromatogram. Therefore, these were analyzed as the test item. The concentration ofthe test item in each sample for HPLC analysis was determined on the basis of a comparison of the total peak area on the chromatogram of the sample solution with that of a standard solution. Some chromatograms obtained are shown in Appendix 3. Analytical conditions Instrument High-performance liquid chromatograph SHIMADZU LC-201OA (Equipped with UV-VIS detector) Column L-column ODS (15 ern x 2.1 mm 1.0., Chemicals Evaluation and Research Institute) Column temp. 40°C Eluent A: Acetonitrile 1phosphoric acid (1000/1 v/v) B: Water*t 1phosphoric acid (1000/1 v/v) Gradient condition Time (min) A(%) B(%) 0.00 50 50 20.00 100 

## o 40.00 100 

## o Flow rate 0.2mUmin Wave length 210run Injection volume 10,uL Detectoroutput 

### 2V/AU

3. Preparation ofstandard solution The standard solution to determine the concentration of the test item in the sample for HPLC analysis was prepared as follows. Test sample of 100 mg was precisely weighed with an electronic balance and dissolved in acetonitrile to obtain 1000 mgIL solution of the test item. The solution was di1uted with acetonitrile 

### 1water*l (111 v/v) to prepare 50mgIL ofstandard solution.

654 -2 Appendix 2­

### 94744

4. Calibration curve The standard s01utions of 5.0, 25, 50, 100, 250 and 500 mg/L were prepared for HPLC analysis by the same procedure as described in section 3. These solutions were analyzed according to the quantitative analytical conditions described in section 2. A calibration curve was drawn from the relationship between the concentrations of standard solution and the total peak area on the chromatogram, and the determination was confirmed. The calibration curve is shown in Appendix 3. The determination limit ofthe test item was the lowest concentration of the standard solution (5.0 mg/L) within the calibration range. Therefore, the determination limit of the test item in the test solution was 0.11 mg/Lin consideration ofpretreatment.
5. Recovery test and blank test 5.1 Method The recovery test was conducted by adding the test item (prepared with acetonitrile) to medium according to pretreatment oftest solution described in section 1. 

### The blank test was also conducted using medium (added acetonitrile) without the test item in the same way as the recovery test The recovery test and the blank test were performed in duplicate. Amount ofthe test item added 100

### fJ,g 5.2 Result As a result of analysis by the method of section 5.1, no peak of the test item appeared in the chromatogram of the blank test Two individual recovery rates and their average on the analytical procedure are shown below. The average of recovery rate was used as correction factor for the determination ofthe test item concentrations in the test solutions. Recovery rate for pretreatment 97%, 98% average 97%



### 655 -3 Appendix 2­94744

6. Results ofmeasurement The results ofthe measured concentrations ofthe test item in the test solution arc shown below. Appendix table 2-1 Measured concentrations oftest item in test solutions

Stock solution 

### (%) Measured concentration (mglL) (Percentage ofmeasured concentration versus that at the start 

### %) At the start 24 hours 48 hours At the end Geometric mean Control n.d. n.d. n.d. n.d.

# ~ 1.0 n.d. n.d. n.d. n.d.

# ~ 3.2 n.d. n.d. n.d. n.d.

# ~ 10 0.18 n.d. *J n.d. *3 n.d. '2 0.067 32 0.58 0.24 

## 141) 0.14 (24) "3 n.d. 0.18 100 1.9 0.49 (25) 0.21 (11) 0.22 (11) 0.40 n.d. : < 0.11 mg/L *2 In accordance with OECD Guidance Document No.23, halfthe determination limit of the test item (0.055 mglL) was adopted for the calculation ofgeometric mean, because the test item was not detected and the detection limit (0.15 mgIL) was higher than half the determination limit ofthe test item. *3 In accordance with OECD Guidance Document No.23, halfthe determination limit of the test item (0.055 mg/L) was adopted for the calculation ofgeometric mean, because the test item was detected but not quantified. The geometric mean value is calculated by the following expressions. antilog ( 	 ( 1_ ) [(log(conc,) + log(conci + ,)).(tl • , - ti }]) 2 t. t,**,~l** where t]

## =At the start < t]<· •. tn

## =At the end COnC1

# = The concentration ofat the start, conc],
.. " conc n 

## =The concentration ofat the end



### 656 -4 Appendix 2­94744



## Appendix 3

Calibration curve and chromatogram

657

## o 94744

5000000



## soo 

## o 250 Concentration (mgtL)

y

## =8339x r 

### = 1.00 Concentration Peak area



### Appendix figure 3-1 Calibrationcurve oftest item for analysis by HPLC

658 -1 Appendix 3­

### 94744
Study No. 94144

I 11 },_. - -_.**;"~-** ----- ..--,+

lo,n 2i.o ';,0 PKHO 1 2 Operati n&: Date : liar. 30. 2009 Time Area Height Cone, (min) (j,tV'6ec) (pVJ 'II) S,12 64096 5222 15,08 15,25 361026 39641 84.92 Total-" 42512-2-- --100, 00

Operator: Hiroyuki Togawa

• t.O '-'

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



### n.d. n.d.

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

_.. .-..--.-.**~-----""--~** PKHO Operat ing Date : Mar, 30. 2009 Tillie Area tleirllt Cone
. .!lIIin),_(SlV'seo) (SlV) (')

Operator : Hiroyuki To(awa

0,0**",.J,~** 

### n.d. n.d.

# ~

# ~ 

# . , .. 10' 0 20.0 ';.0 PKIIO Operating Date : Mar, 30, 2009 Tille Area Height Cono, J!l..ln)**(p,V'a.o)~** __**~**

Operator : Hiroyuki Togawa

Appendix figure 3-2·1 HPLCchromatograms at start ofexposure
659 -2 Appendix3­

### 94744

Study 

### No. 94744 Operating Datil: liar. 30. 2001)**9·~**714/v.t./L2\\'_ .' PKNO Time Area liei,ht Cone. CIIinl (/IV·sec) '(/1 V) (li) 1 8.10 6905 324 35.38 2 15: 23 12615 1043 64.62

Total 19521 100.00 

### n.d. n.d.

# ~

# ~

,.__._. -,--**,,-~'-** ,- .**.-J~\J._** '.

- ..___L . ,- .....**-~-"-** . .-,-,­ , O:D**IO~** 0 20.0 3Ci.o Operator : Hiroyuk j Togan**~Jltlj.1QL** ..... _ ..._. ______....._ Operatinl Date : Mar. 30. 2009 PKNO Tillie Area Height Coli<), (lIl;n) (IlY·.ec) (j/.V) (Ii) 

# , 8.10 18168 1312 26.91 2 15.25 49339 4354 73.09

Total 61507 100.00



## ___**J.~._JL.t~~** ------"'--­ ;).'0 100**~I41jQ~!~.f~** - _.,., 2,[0

- 30.0 PKNO 1 2 Operator: Hlroyukl Totawa Operatinc Date: liar. 30, 2009 Tille Area Height Cone. (min) (/lV-sec) (Ill{)**~)** 8.72 67483 47111 26.72 16.22 157613 14939 73.28 Total 215156 100.00

# ~~_--L __..__---,,_._ 0:0 .00**20~** 0 30:0 Operator: Hiroyukl Togawa Date Mar. 31. 2009 N8IIIlI:

## H,ro,YMk. 

# IOO,I!A\

Appendix figure 3-2-2 

### HPLC chromatograms at start ofexposure
660 -3 Appendix 3­94744
Study No. 94744 OperatInl D.te : liar. 31.2009 PKNO Time Area Height Corn:. (Join) (pV'seo) (pV) ('O) B. U 182731 14696 25.36 15.22 637786 49685 74.64 Total 120517 100.00

Operator; HIroyuki Togalla Fig



### Appendix figure 3-2-3 HPLCchromatogram at start ofexposure
661 -4 Appendix 3­

### 94744
Study No 94744 PKNO

1 2 Operatin, Date: Apr. 02. 2009 Time Area Height Cone. (lIio) (JjY·sec) ().IV) (') B. 67 63658 6271 14. 92 15.23 363085 39864 85.08 Total**·-~·--1-00.00**

Operator ; Hiroyuk1 Togawa**94744/]~.bL9Q!l~t()L.**__.,.,___...._._.._ ..__ .0,



### n.d. n.d.

# ~**~ ~~···---··--,~o.-o-----z""Ii.-c**  -----30. 0 PKNO Operating Date: Apr.02. 2009 Time Area Height Cooo. (min) ().IY·ssc) (1lY) (I)

Qperator : Hiroyuki Toga"a 94744/72h/l.0%



### n.d. n.d.

# ~**~** _.__,.:A
____**fo...-~** __-

_____ ._________ -_......­ 0.-0-'-'''-**I~.** 0 200 ;au PKHO Operotinf Datil : Apr. 02. 2009 lime Area llelgllt Cone. \JIlin) (/lV·ell) (/IV) 

### ('>

Operator ; Hi royuki TOK8wa Date Apr. 3. 2009



### Appendix figure 3-3-1 HPLCchromatograms at end ofexposure


### 662 -5 Appendix 3­

### 94744

Study No. 94744 94744/72h/3. 2% Operatinc Date : Apr. 02, 2009**-~.--.-----.-.-----** . PKNO nlDe Araa l\ei,ht Cone. (min) (/!_I!.:6.!lc) {IJ V} (I)


### n.d. n.d.

# ~**--~--"--~- ."~------"** **o~o** 10.0 211. 0 ,0.0 Operator: Hiroyuki TOlaD 9474402hL10l



### -­ ....**..,~,--~-------"-** ..**~,-** Operatin, Date : Apr.02. 2009 !'KIlO Time (min) Area (jlY·sec) l\eilht {eV} Cone. (I) 

## n.d. n.d.

# ~**~ ~~~**  

# ,,;. to'..o 94744/7211/32%



### n.d. n.d. 20.0 -'-­**sO~.o** 

# "NO 1 2 Operator : Hi royukl TOll8'l!ll Operati"& Date : Apr. 02. 2009 Time Area Heleht Cone. (min) (/tV-seo) (J.lV) (l) 8.68 12416 704 39.09 15.18 19421 1661 60.91 TauI 31887 100.00

# ~~ I

____....-...­ 0:0 I 10.0 20.0 ';'0 Operator : HIroyuki Togan Fig

Appendix. figure 3-3-2 HPLCchromatograms at end ofexposure


### 663 -6 Appendix 3­

### 94744
Study No. 

### 94744 Operating Date: Apr. 02,2009 PKNO TiI'lHl Area Height Cone. (m1n) (j/Y·sec) (Jl Y) (\) 1 8.68 34832 2511 43.01 2 15

. 23 46149 3339 56.99 Total 809B!l 100.00

Operator : Hiroyuki Togawa Fig

Appendix figure 3-3-3 

### HPLC chromatogram at end ofexposure
664 -7 Appendix 3­

### 94744



### Additional data 1

Results ofpreliminary studies



### 665

### 94744

1. Time-course analysis ofconcentration oftest item The test item was predicted to transform in medium, so time-course ofthe test item concentration was measured to confirm the fluctuation ofthe concentration. 1.1 Analysis ofconcentration oftest item (I) Content The test sample and medium were mixed to prepare 100 mgIL and they were stirred for 1, 3, 24 and 48 hours under the test condition (23 ± 

### 1°C). After that the mixture was settled for about one hour, and then the solution was collected from the middle layer. The collected solution was centrifuged (10000xg, 30 min) to remove insoluble matter for analysis with high-performance liquid chromatography (HPLC) as described in Appendix 2. (2) Results Sample name Measured concentration (mg/L) PEMB

### (peak ])' PEMB 

### (peak 2)' Test item (pEMB+PEMB Stirring for 1 hour Sample-1 4.6 1.2 5.8 Sample-2 5.5 1.1 6.6 Stirringfor

3 hours Sample-3 8.8 

### 1.6 10 

### Sample-4 8.8 1.6 10 Stirring for

24 hours Sample-5 1.3 0.8 2.1 Sample-6 1.4 0.8 2.2 Stirring for

48 hours Sample-7 0.1 1.2 1.3 Sample-8 0.4 0.8 1.2 

### * Calling Peak 1 and 2 in order of elution, each peak corresponded to PE3MB and PEMB (Information supplied by the sponsor). Concentrations of each peak was ca1culated by using content percentage of each peak (PE3MB: 14.1%, PEMB: 81.7%).



### 666 -1 Additional data 1­94744

PEMB PE3MB 

# ! ! 111 

# .1**~** 1**~--~--------~~--~--------~~------------~oo** 0:0 Hi. 0 0 HPLCchromatogram (standard solution 50mgIL)

HPLCchromatogram (Stirring for one hour, decuplc dilutcd sample)

PEMB



# 1 .2

10'.0 'l.ci.o 30. 0 HPLCchromatogram (Stirring for 48 hours) 1.2 Summary The concentration of PE3MB (Peak 1) was highest after stirring for 3 hours, and then recognized to decrease. The concentration of PEMB (Peak 2) was nearly constant all through the stirring times. Increase of converted products was recognized after stirring for 24 hours on HPLC chromatogram. Therefore, 

### it was decided to assess the effects of the test item including converted products in definitive study, preparing test solution by stirringfor 48 hours after mixing the test sample and medium.

667 -2 Additional data 1­94744

2. Study for effect on test organism 2.1 Preliminary study (1) Method The test sample and medium were mixed and stirred by magnetic stirrer for about 48 hours to prepare 100 mg/L (nominal concentration). After the mixture was centrifuged (10000

### xg, 30 min), the middle layer was collected for the stock solution to prepare the test solution. The test organisms were exposed to the test solution to confirm the effect. The concentration of the test item in the test solutions was measured. 

### In addition, the concentration ofthe test item in the test solution without algae was also measured in order to confmn whether the test item was absorbed to algae ornot. (2) Result Stock solution content (%) Growth inhibition rate based on growth rate (0-3d) (%) 0.010 -2.6 0.10 0.83 1.0 -2.8 10 0.26 100 18 Replicates: two replicates/ test level Measurement method: Chlorophyll fluorescence

< Measured concentrations oftest item in test solutions>
Stock solution content 

### (%) Measured concentration (mglL) (Percent ofthe measured concentration versus that at the start ofthe exposure 

### %) At the start ofthe exposure At the end ofthe exposure 10 0.31 n.d.
100 0.68 (20) 100 (no algae) 3.4 0.68 (20) n.d. : < 0.10 mgIL



### 668 -3 Additional data 1­

### 94744

2.2 Summary Growth inhibition was 18% in the stock solution content 100% exposure level with the sticking ofwhite substance on the interiorwaH oftest vessel around surface of test solution. It was considered that no observed effective concentration was around the stock s01ution content 1.0 to 10% exposure levels. The concentrations ofthe test item in the test solutions were decreased during the exposure. In addition, the concentrations of the test item in the test solutions between with and without algae were almost same, therefore it 

### was considered that the test item was no absorbed to algae.

3. Operation ofdefinitive study Based on the results ofthe preliminary study, the definitive study was conducted with five concentrations of the stock solution content 100, 32, 10, 3.2 and 1.0 % (a geometric series with a factor of 

### .JW) and the control. The preparation method of the test solution was the same as the preliminary study and stirring time was 48 hours, because the test item has poor water solubility. The concentrations of the test item in the test solution were measured at the start ofthe exposure, 24 and 48 hours after the exposure, and the end ofthe exposure, because the significant decrease in the concentration of the test item in 

### the test solution during the exposure was expected.



### 669 -4 Additional data 1­

### 94744



## Additional data 2

Qualitative analysis ofconvertedproducts

670

### 94744

1. Qualitative analysis ofconverted products The peaks which were likelyconverted products except the test item were detected on the HPLC chromatogram of test solution in the definitive study. Therefore, the sample was qualitatively analyzed by liquid chromatograph-mass spectrometry (LC-MS) and estimated structures. 1.1 Method ofanalysis Standard solution and the pretreated sample of0.40 mglL (100% stock solution) at 24 hours after the start of the exposure for HPLC analysis described in appendix 2 were qualitatively analyzed by LC-MS under the following conditions. Analytical conditions Instrument Liquid chromatograph

- mass spectrometer HPLCsystem SHIMADZU LC-20A Series Mass spectrometer Thermo Electron TSQ Quantum Discovery Pump (Post-column) HITACHI L-6200 Conditions ofliquid chromatograph Column L-column ODS (15 cm x 2.1 mm 

### lD., Chemicals Evaluation and Research Institute) Column temp. 40°C Eluent A: Acetonitrile / formic acid (1000/1 v/v) B: 

### Water· 1 / 

### formic acid (1000/1 v/v)

Gradient condition Time (min) A(%) B(%) 0.00 50 50 20.00 100 

## o 40.00 100 

# o Flowrate 0.2mUmin Injection volume 5#L Post-column

Solution Acetonitrile/ water"t 

### (111 v/v) ·2 

### Flow rate 0.1 mUmin *1 Tapwaterwas treated with ultrapure water system. *2 Containing 25 mmollL ammonium acetate.

671 

### -1 Additional data 2­94744

Conditions ofmass spectrometer Ionization mode Electrospray ionization (ES1) Detection ion Positive Measure mode Scan Scan range 

### (mlz) 150-600 Capillary temp. 300"C 1.2 ResultsConverted products, Peak A, B, C, D, E and F, detected on total ion current 

### (TIq chromatogram (Additional figure 2-4) were qualitatively analyzed by LC-MS and estimated as following structures. Also, it was recognized that Peak 1 and 2 were the test item.

Peak No. Retention time on TIC chromatogram (min) Estimated Estimated structure Additional and molecularfonnula molecular figure No. weight



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

### ..-­
Figure 2-5 Figure 2-13

Figure 2-6 Figure 2-14



### , 

## , , I 

# ! Figure 2-7 

# I Figure 2-15 

# I

, 

# . l 

### A

B

C 4.7

5.1

5.4

672 

### -2Additionaldata 2­

### 94744



# · · · .' Figure** 2~8** " 

### 7.5 D 

### Figure**2~16**

E 

### 7.7 Figure**2~9** 

### Figure 2-17

1 8.1 Figure**2~1O** 

### Figure 2-18



# · 

# · · F 12.9

, 

# · · 

# · · 

# · · 

# · , 

# · · · · · · · · · · · · · · 

# · · 

# · 

# · 2 13.1 Figure 2-11 Figure 2-19

Figure 2-12 Figure 2-20



### ._._----..--------------_..._----------_...

### _-_. __.--- .. -­ 

### 673 -3 Additional data 2­94744

From these estimated structures, it was suggested that Peak D and E were the products converted from PE3MB by disulfide formation caused by oxidation of thiol groups. In addition, there was possibility that Peak D and E were formed as a result ofdisulfide formation after PEMB changed to PE3MB by hydrolysis, and hydrolysis of a part of ester bindings after PEMB changed to Peak F by disulfide formation. Additionally, it was suggested that Peak D was stereoisomer ofPeak E. Also, it was suggested that PE3MB and PE3MB fomled by hydrolysis from PEMB changed to Peak B by dehydration, Peak C by elimination ofhydrogen sulfide, and Peak A by both disulfide fOmlation and elimination ofhydrogen sulfide. And it was suggested that PEMB changed Peak F by disulfide formation caused by oxidation ofthiol groups. These converted products were not quantitative analyzed, because standard samples were not available.

674 -4 Additional data 2­

### 94744

Standard solution 50 mglL
RT:·O.OO - 30.00 NL: 13.17 220000 

# 2'\. 200000 180000 160000 140000 :i'12ooo0 :

# ~ 100000 

### 1 80000

# ~ 8.14 60000­ 40000 20000 

### o I I I I I . 1 I I I I I I



### o 5 10 2.28E5 TleF: +G Q1MS( 150.00­ 600.00) MS 94744­ 090331-801

I I I , ·7 I j r" I ! I I I , : I

15 20 25 ;ro Time (min)



### Additional figure 2-1 TICchromatogram (standard solution)

675 

### -5 Additional data 2­94744

Standard solution 50mgIL 94744-090331-501 #474 RT: 8.14 AV: 1 SB: 138 8.74-11.09 NL: 7.12E3 T:
... c Q1MS [ 150.00-000.oo} 488.37 7000 Mass spectrum ofpeak 1 6500 460.32 6000 

# I 5500 

# l I 5000 4500 

# f4ooo.**~3500** 17126

3000· 2500· 2OQO 1500 1000. 500­



### o·**,~,**

150 11$U1

# 1,J~·52 298.18 392.34**~~06** 

# II J 239.24

## 1~ t.j

## ~ 

## "I! 

# I J, t, , ," ,
..1"

. I I 34.2,J.: ,3r ,', • 3

# ll3,~3,**.~,** rl,
. -'-" 200 250 300 350 400 450 m/z RT: 0.00

- 30.00 8.11**~.B.12 ~1**  .18 

# J**~:;.61 57~38** **'TT'~~"-rl'T""1** 500 550 600
NL: 8000
. 8.17 Mass chromatogram ofmlz =488 8.14E3 mlz= Position ofpeak 1 487.87-488.87 F: + cQ1MS [ 

# 1 '1,8.28/"**~5~.oo..eoO.OOl** 

# f= 94744-090331­ 8.35 S01 -3000

### 8'40

2000· 1000 0.0.09 3 . .P4 5.74 \ 11.13

___**~~~_. ~~.84**  22
..**~5** 24;66**~:').37** .__

# ~ 8.23 NL: 8000 7000 8.09. 6000

1000 8.67E3 Mass chromatogram of

### mlz =460 mlz= 459.82-480.82 F:

... c Q1MS [ 150.00-600.00) MS Position ofpeak 1 94744-090331­ 

# / S01

10

## ~:.,.:.........,.:;13:;:;.O:;.;;9 .......1:::.;6 • .:r6,

## 9::..-.,.....,....:14T!.:.-.:03~24..,J...i34,...........,..:2:;:8=;.39;:...,· I • I t I j I r 1 I til 15 20 2S 30 Time (min)
Additional figure 2-2 Mass spectrum ofPeak 1 and Mass chromatogram (standard solution)
676 -6 Additional data 2­

### 94744

Standard solution 50 mg/L 94744-090331-S01 #767 RT: 13.17 AV; 1 S9: 138 8.74·11.09 NL: 9.65E4" T: + C a1MS [150.Q0..600.001 562.27 Mass spectrum ofpeak 2 90000

# j

. j 80000 70000 60000· f50000

# ~ 40000

20000 10000 160.99 221,33 

### o 

### 1 -'/:\', I , ',",'1 I f , 150 200 250 RT: 0.00

- 30.00 323.18 283.20 

# l34P 4 391.59 443.31 484.55

### 560.~ , ,',' I ' i : 

## !. I ' , , " I ' ,lie' ,I.I ' ;--1", I ,,',.-, i"'\ 300 350 400 450 500 550 m/z 13.19 NL: 9.69E4 mJz= 

# I Mass chromatogram of 9000(}' 561.77-562.77 F: 

### m/z 

### =562 +cQ1MS [ 150.00-600.00] 8000(}' MS 94744-090331­ 

# / Position ofpeak 2 501 70000 60000

## ~50000 .!l ..E 40000

30000 20000 10000

n. '1.49 4.65 \r j I • I

(} 5
Additional figure2-3 Mass spectrum ofPeak 2 and Mass chromatogram (standard solution)
677 

### -7 Additional data 2­94744

Test solution RT: 0.00· 30.00
600000 550000

450000­ 400000

25CKM>O 200000 1501lOO 100000 50000 0" NL: '**7.71~** 6.46E5 D E TIC F: +c



# '\ Q1MS[ 150.00­ 7.52 600.00) MS 94744­ 090331· H24hA



## i 1 

# f 

# ·il**1~52** 09 

# .:""1

# f lJ-r

# r! 2 '. 6.41

# ~.54 I· 12.93 

# J 

# 1/, -13.14**3.~3** 8 64 I

. .. 16.14 1&.10 21,28**~a.10 ~05** **v-.......J-..I.\..'\t-~......,......,.~~~** j I , 1 I • I I 'T" ! I I I ' I I '1 j I T "T-'r" " I 1· I 05 10 15 20 25 SO Time (min)

Additional figure 2-4 TIC chromatogram [0.40mg,IL (at 24hours after the start ofthe exposure)] 

### 678 -8 Additionaldata 2­94744

Test solution

94744-()90331-H24hA#273 RT: 4.69 AV: 1 S8: 59 3.00-4.00 NL: 3.70E4 T: +c 01MS [ 150.00-600.00) 488.28 Mass spectrum ofpeak A 3500D
30000
25000 440.25
i'2oooo 

# I c:

### ~ 15000

10000

1 5000 321.36 171.34 212.22

### _~6.35 299.491324.36 391 .43 1 

# I . I I o 

### ·\··r·, Tt-I l r , .., "', r' rJ I I r ," r I 1'1 1 rUt '"," 150 200 250 300 350 400 450 mfz RT: 0.00

- 30.00 4.67**~9.26**

.470.32 471.32 

# ." 509.16

h,--'r-'-;:'=r, r

500 550
NL: 35000 30000 25ODO 

# ,::

10000 5000**~'72** Mass chromatogram of

### m/z =468 3.86E4

# 1 mIz= 487.16-468.78 F: 83 +cQ1MS[ 4. '" 150,oo-aoo.OOJ ! Position ofpeak A MS 94744-090331­ H24hA
\ 5.24 0.,-......;..3'..;...14-, \... 6.96 1Q.46 16.04 19.,,34 21.69 26.57 3()()(){)-· 25000 ,,20000
. 

# ! UiOO():

10000·· 4.72 Nl: 3.09E4

1ii.3& 

### Mass chromatogram of

### m/z =440 mtz= 439.75-440.75 F: +cQ1MS( 150.00-eOO.OO} Position ofpeakA 

### MS 94744.090331­ H24hA

10.37 14.60 11.88 2Q,23 24.51 29.75 r-r J r'i J r t ' i r**~"....::r'1**.:;;.=,-,-Y-:::;,n!:""I'""T!**""'il~"** "1t 10 15 20 25 30 Time (min) Additional figure 2-5 Mass spectrum ofPeak A and Mass chromatogram [0.40 mgIL(at 24hours after the start ofthe exposure)] 679 -9Additional data 2­94744

Test solution
94744-090331-H24hA#296 RT: 5.08 AV: 1 58: 59 3.00-4.00 Nl: 7.61E3 T: "" cQ1MS 

### I 150.0Q.600.00} 470.43 7500 Mass spectrum ofpeak B 7000 6500 442.25 6000 5500 5000" 4500 1'1000 .53500

3000 2500 2000· 1500. 172.19 222.06 440.Jll3
. 440.08 1000· 500 183,07 223.03 299.02 300.40 382.40



### o •. , 

## J

# r. I 

## I

# ~ 'I-r-;

# ~~~lj\L.. I I 

## l t '/·'l-l\'",·,I­ 150 200 250 300 350 400 450 mit. 5.13 8000· 7000
. 6000­**~5000-'** III c**~4ooo** -3000

2000 1000· 

### o**~6000** ID C CD 54000

2000

· O 4.55

# .~ Mass chromatogram ofmlz =470 8.78E3 

# " ml%= 469.93-470.93 F: +cQ1MS[ Position ofpeak B 150.00-600.00) MS 941#-090331­ H24hA 5.32

# L....;;8:.:...;.1.::..2_1;.;:0~.~.___1_5;g

# 1ft~ 21.38

# ~ 28.11 471.33

47.2.33**4~t4.26 ~16.58**  582.75

,.. ""j-',,--rr I' r-l'r," I

500 550 600 NL;

5.12 NL:

4."" (U.

_ 

### r**~,..** 1 i i t 1 I r I J 1 i t J r , -I 9.62E3 Mass chromatogram ofmlz =442 mIz= 441.75-442.75 F: +cQ1MS( 150.00-600.00) Position ofpeakB MS 94744-<l90331­ H24hA 7.89 7,.81 

# j.,,91 10.97 I
.....-r-r--+t-
......-..--r 15

,,-.1.,.O
..............1...;9.27 22.48 27.17

# i, .0 5 10 15 20 25 30 Time (min) Additional figure 2-6 Mass spectrum ofPeak B and Mass chromatogram [0.40mgiL(at 24 hours afterthe start ofthe exposure)] 680 -10Additional data 2­

### 94744

Test solution
947M:090331.H24hA#31"5-RT: 5.41 AV: 1 5B: 59 3.00-4.00 NL: 9.16E3 T: -t- C Q1MS [ 150.0O-Q00.00) 454.37 9000 Mass spectrum ofpeak C

7000 6000

426.39

3000

m/z 

### Ri: 0.00 30.00 5.43 NL: 10000 8000 

## ·f 6000 c: 

# ! ::

# L:'~7 o Q.67
_ 1.06E4 Mass chromatogram ofm/z =454 mfz.: 453.87-454.87 F: -t-cQ1MS ( 150.00-600.00] Position ofpeak C MS 94744-090331· H24hA 

### e.13 6.94 7.18 11.28 14.07 17Jl9**19i.~** _20,37 26;59 29.7§ 5.39**-...I>-...--==:::!::'::'-::=:L...:=~NL:** 4.69E3 mIzs 4000 Mass chromatogram ofm/z =426 425.89-426.89 F: -t-cQ1MS [ 150.00-600.00] Position ofpeak C MS 94744-090331­ H24hA**~.85** 1000 , 1 

### o**~~~~~~~~~~~~~rT~~~~~~~~**



### o 5 15 25 .30 Time (min) Additional figure 2-7 Mass spectrum ofPeak C and Mass chromatogram [0.40 mgIL(at 24hours after the start ofthe exposure)] 681 -11 Additional data 2­94744

Test solution 94744-090331-H24hA#438'RT: 7.52 AV: 1 SB: 118 9.00-11.01 NL: 2.10E5 T: + c 01 MS [ 150.00-600.001 200000

160000 140000**~120000** 'f/) c 100000 11OQ00



### o 458.23 

### Mass spectrum of

### peak D

# 3.211:~ 2" .,,<32

# ·;'l~i ·20 576 

# ! 

# l**~.,.. ~7.33**  167.40 21?17 296.95 1;MO.20 424.20 "**~.22** 'r'i rf .) j" f r-r"T" r i'Tj T 

### T+-r· j I r-r' I r'r-r ! T j'TO""rr" I ,'1 'I 150 200 250 

### aoo 350 400 450 500 550 600 

# 'I mJz RT: 0.0'0

- 30.00 NL:6.56E4 mlz= 575.70-576.70 '7.61 7.66 12.38 60000 

# I F: +c01MS [ 

## f 40000

# ~ 20000 

### o 

# 250000j 200000 

### t1500oo·, Position of c**~** 100000

### peakD~, 50000 1.73 5.43 

### o Mass chromatogram of 150.00-600.00] MS 94744-090331­

## Posjtio~f 

# l 12.;:/z =576 H24hA peakD
.... . 3.35 

### s:m

# I 12.88 16.61

## 1~,99 27,86 26.69._
. 7.71 NL: 2.57E5 mJz= 457.73458.73 Mass chromatogram of F:+c01MS[ 150.00-600.oo} MS m/z 

### =458**947~331** H24hA
'..21 12.28 16.28 21.64 2.4.78 28.17 7.71 Nl: 7.28E4 60000 7.54, Position of 

# 'f 40000

### ~**peakD~** 20000'**4.6~. ~.79**  m/z= 320:65-321.65 Mass chromatogram of F:+c01MS[ 150.00-600.(0) MS 

### mlz =321 94744-090331­ H24hA 11.87 13.50 19..12 

### 29;'t P.80 29.$+ 

### o 1-r-.-+-tJ·'r-.,....,rt+
.........r-r.L\-
..... rT-rf
...**~..·** ,........-r-T'i



### o 5 10 15 20 25 30 Time (min) Additional figure 2-8 Mass spectrum ofPeakD and Mass chromatogram [0.40mglL(at 24 hours after the start ofthe exposure)] 

### 682 -12 Additional data 2­94744

Test solution 947#-090331-H24hA#449 RT: 7.71 AV: 

### 1'58: 

### 11e 9.00-11.01 NL: 2.57E5 T: +cQ1MS (150.0()..600.00) 458.26 240000 Mass spectrum ofpeak E

180000 160000**~140000**

## ~t20000
321.14 7 :: 5 6·19 

# l 1B 441..23 486.32 20000 210.52

## 2~.-45 _.21

## ~.19 424,,31 

### I

## 1~17.27 54j.16

# l

. I ,. f l' t T II I' I 1 l"t''f .• 

## "r"r I I' I T f 

### rL! 1 I l·!;t.-rrl" 1 I 'If'1 150 200 250 300 350 400 450 500 550 600 mlz RT: 0.00 - 30.00 7.t11 7.66 12;38 Nl: S.56E4 60000 f40000 Iii 

### :E 20000 Position of peakE '--..


### o '--'-'1.;.;;.67'--_6.;;.;i
......... 9.79 'l§·

7.71 250000 200000

### ~150000 

# j 100000-., 50000 

### o 60000·**~** I!! 40000 

### .s .E 20000**7.~** mlz= 575.69-576.69 Mass chromatogram of F: +cQ1MS ( 150.00-S0(100] MS 

### m/z =576 94744-090331­ H24hA 12.88 16.61 19,,99**~,8$** 26.60
__ Nl:2.57E5 m/z= 457.76-458.76 Mass chromatogram of F:+cQ1MS[ 150.00-600.00] MS 

### m/z =458 94744-090331­ H24hA Position ofpeak E 0.94**~48** :-fS"""
.2;;;,.:1.......:..;12::;.28=-.._--=:.17:.;:..59::;.=...·......::2:.;:,1;,:=.64..:.......::2::.:4;:.;,.78.::;;.·**---=:29~.t§~** 7.71 NL;.7.2654. mlz= 320.64-321.64 Mass chromatogram of F: +cQ1MS [ 150.00-600.00] MS 

### m/z =321 94744-090331­ H24hA Position ofpeak E 4.t!9**.~J9 tr·I-r.....-1'-r..n.-r-~** **M-r-r"r-rT""'T-r--r-1r-t-"'-i=f=-r-r-r'~~~** 

### o 5 10 15 20
. 25 30 Time (min) Additional figure 2-9 Mass spectrum ofPeakE and Mass chromatogram [0040 mg/L (at 24hours after the start ofthe exposure)] 683 -13 Additional aala 2­94744

Test solution 94744-090331-H24hAiI47f"RT:S:09 AV: 1 S8: 1189.00-11.01 NL:1.01E4 T: +c Q1MS [150,00-600.00] 10000 9000­ 8000 7000 6000 .?1 ! 5000

### ~ 4000

2000 1000 

### o r

150 RT: 0.00

- 30.00 30000-. 25000·**~20000** 15000. 10000 5000 

### o 40000 35000 30000.**~25000** /I) i20000 E -15000

10Cl00-' 5000 460,34 Mass spectrum ofpeak 1

488.25 171.27

321.28

# 288.46133~ 48:': 577.06

# !.l·f~·~

# [I

# ~2~::~r" ..

# :-fI.,-I,-~**~5;!~ ,~~l,j**  250 300 350 400 450 500 m/z 12.54 Mass chromatogram of 

### mlz =488 Position ofpeak

# 11

# 7~

# J. I i/lS.31 

### I

0·
......**~r,6·P~,)**



### o 5 

# [

# ~~~:~ill I**3.~** 5,01..1**t~ \1-_....:1.:;:6.;;;;23;;;......;1~7!.::.8;:,.8_2::.;SJ:;..,4:.::2:.....=.24.i.;;.**  ..4.:.;1:.......;2=.;8:.:..1:.::5_ 7.69 NL:**1.~.** 550 600
NL: 3.37E4 mlz= 487.75-488.75 F: +cQ1MS ( 150.00-600.00] MS 947#-090331­ H24hA

4.14E4 Mass chromatogram of mJz= 459.84-480.84 F: 

### mlz =460 +cQ1MS[ 150.00-600.00] Position ofpeak 1 MS 94744-090331­ 

# ,/ H24hA**~,12** 8.23**\..;9~·~T"~11r44~rT,16~.50~19~.~;· ~~~.:;:~;-~~3=r;~~**  i J', t f i i J**-~ -,-~.**  ., 10 15 20 25 30 Time (min) Additional figure 2-10 Mass spectrum ofPeak 1 and Mass chromatogram 

### [DAD mgIL (at 24hours after the start ofthe exposure)] 684 -14 Additional data 2­94744

Test solution 94744-090331-H24hA#753 RT: 12.93 AV: 1 58: 1189.00-11.01 NL: 1.54E4 T: +c Q1 MS [ 150.00-600.001 588.23 15000­ Mass spectrum ofpeak F 14000 13000­ 12000· 560.29 11000 10000 9000 

# I f '8000': 

# '

_ 7000. 6000 5000 4000 3000

# ~Ooo-: 171 :.21
..6'" 319.1. 556,§



## . II t'" 270·00 L 400.10 <165.f 1 504.85 I 0, 

### J 1'1 t '1l r 1'1' I I f' I I I ,.l' I 1 1 1 rr I I 1 I ' I" /'f"'fi""J-r 1'; I

# ~ 1 150 200 250 300 350 400 450 50Q 550 600 m/z RT: 0.00 - 30.00
14000­ 12000 1;**fOOOO~** 

### I! 8000.

### ~ 6000 4000· 2000-::
n. 12.91 NL: 

### Position of**p~**
12,66 10.85 1.96 4.67

### ~ 12.97 

# ::"E4 Mass chromatogram of

### m/z =588587

.73-588.73 F: +cQ1MS[ 150.00-600.00] MS 13.21 94744-090331­ H24hA

13.34 19.18 20.73 26.24 u-**'--.;.;.:;.;'--~~- 13.0!-::9;:----=:..:...:::-=·-·~--~-Nl:**  16000,: 1.67E4 mlz= 14000 12..97 Mass chromatogram of

### m/z**=560!S:Jr-~·79** F: 12000· Position ofpeak F 150.00-600.00] f1OO00 '"

# ~~44-090331 B 8000 \.. 13.33 H24hA .5 6000 12.]9 4000 2000 2.21 14j22,**16'~j** 

# 1 0

### '[r: 5 :,-,-,-,..,=r2 6J?9,**2~.~61 o·'I-r-+-r-T~'**  1 o 15 20 25 30 TIme (min) Additional figure 2-11 Mass spectrum ofPeak F and Mass chromatogram [0.40 mg/L (at 24 hours after the start ofthe exposure)] 685 -15 Additional data 2­94744

Test solution 94744-090331-H24hA#765 RT: 13.14 AV: 1 S8: 1189.00-11.01 NL: 1.28E4 T: + c Q1MS [ 150.00-600.00) 560.32 12000
. Mass spectrum of

### peak 2 11000**~j_51** 10000 9000 8000**~** 1000 

# I

_ 590.21 l!! 

### 6000.**5000~** 4000­ 3000 2000

-

. 322.98 1000 173.18 220.21 • J , .1 258.98 301.87 134()·84 356.15 426.97 450.69 538.33 

### o "I t

## l I'; f T I ,. I') I , I ; , j I I I ' I I" , r ,", r' I '

### ,.,-r', I I rT'j I

150 200 250 300 350 400 450 500 550 mJz 

### 'RT: 0.00

- 30.00

--­ 13.09 Mass chromatogram of NL: 6.46E3 6000 mlz= 589.71-590.71 

### m/z =590 F: + CQ1MS ( 12.90 '- 150.00-600.00] MS II 4000 

# j**~** 94744-090331­ H24hA Position ofpeak 2 2000 13.34 2.85 17.45 22.07 26.55 

### o**'----'----> 1L---'-1~3.17** **'-'-'~-'-'--'--NL.: 8.~** 



### o 5 10 15 20 25 30 Time (min) Additional figure 2-12 Mass spectrum ofPeak 2 and Mass chromatogram [0.40 mgIL (at 24 hours after the start ofthe exposure)] 

### 686 -16 Additional data 2­ 8000-· 

# t 6000 

### i 4000 

### .E 2000 

### o 15000



### o Position ofpeak 2 

## "-.. 12.97 2.21 3.42 6.37 12:1$ 13.09 Mass chromatogram ofNL: 1.67E4 mlzc559.82-560.82 12,97 "'--- m/z =560 F:
... C Q1 MS [

____ 150.00-600.00] MS 94744-090331­ Position ofpeak 2 H24hA 13.33 12.79 2.21 ?,.75 7.78 11.01 2 

### r-r-r-r J**1~,.-W~~.74 ,** **~,2~··crsl** Mass chromatogram of mlz:561.86-562.86 F: +cQ1MS [ m/z=562 150.00-600.00) MS 94744-090331­ H24hA 14.67 17..38 1$.68 25. 27.53

### 94744

mlz Detected ion 

### 468**[M+~+HCOOH-H20t** 

### 440 [M+NH4t Additional figure 2-13 Estimated structure ofPeakA**~-------** 

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

# · . · . · 

# . · . · . · 

# . · . · . · . · 

# . · . · . · . · . ·**----~-:----"**



# · &·.·e··.·.····.·.·.·······,···'.-=-=r.'.' 

# · mlz Detected ion 

### 470 [M+N14+HCOOH-H20r 442 [M+NH4r Additional figure 2-14 Estimated structure ofPeak B



### 687 -17 Additional data 2­________________________ __

### 94744

mlz Detected ion 

### 454 [M+NH;+HCOOH-H20]+ 

### 426 [M+NH4t 

### Additional figure 2-15 Estimated structure ofPeak C**~- ¥----.-.-~~**  

### .........-..---.----.-.-..... 

# · 

# . · . 

# · · 

# . . · : 

# . 

# . · · · · · · · 

# · · · 

# · · · 

# ·

____________________________________**~I** • ___ • _____ • _________ ••••• __ ._ •• __ ._. ____**~** 

# · · mlz Detected ion 

### 576 Unknown 458 [M+NH;t 

### 321**[M+H-C~60S-H20t** 

### Additional figure 2-16 Estimated structure ofPeak D



### 688 -18 Additional data 2­

### 94744

Detected ion 

### 576 Unknown 

### 458 [M+NlLtt 321 [M+H-C4

### H60S-H20]+ Additional figure 2-17 Estimated structure ofPeak E



# ·**--.~** 

# . -

## .. ..

### --........

## -......---- .............. . 

# · . 

# · . · 

# . · . 

# · 

# . · 

# . · · · · · ·

mlz Detectedion 

### 488**[M+N~+HCOOH-H20]+** 

### 460 [M+NH4t Additional figure 2-18 Estimated structure ofPeak 1



### 689 -19 Additional data 2­

### 94744



### mlz Detected ion 

### 588**[M+N~+HCOOH-H20r** 

### 560 [M+NH4t Additional figure 2-19 Estimated structure ofPeak F**~-.------------------.----------------.** 

# . . 

# . . · 

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

### ....

## -...

## -......

## --...

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

### mk Detected ion 

### 590 [M+NI4+HCOOH-H2

### 0

## t 562 [M+NHd+ 

### 560 Derived from peak 

### F Additional figure 2-20 Estimated structure ofPeak 2

690 

### -20 AddiLionaJ dara 2­

### 94744

2. Qualitative analysis ofwhite substance In the test vessel of0.40 and 0.18 mglL, sticking ofwhite matter was observed on the interior wall ofthe test vessel around surface ofthe test solution at end of exposure. Therefore, the white matter was qualitatively analyzed with LC-MS. 2.1 Method ofanalysis The white matter was dissolved with small amount of acetonitrile after the test solution of 0.40 mgIL (at end of exposure) was removed from the vessel. The acetonitrile solution was diluted to produce acetnitorile/water*l (1/1 v/v) for LC-MS analysis. The prepared sample was qualitatively analyzed with LC-MS under the following conditions. Analytical conditions Instrument Liquid chromatograph

- mass spectrometer HPLC system SHIMADZU LC-20A Series Mass spectrometer Thermo Electron TSQ Quantum Discovery Pump (Post-column) HITACHI L-6200 Conditions ofliquid chromatograph Column L-column ODS (15 cm x 2.1 mm J.D., Chemicals Evaluation and Research Institute) Column temp. 40°C Eluent A: Acetonitrile/ formic acid (1000/1 v/v) B: Water' l / formic acid (1000/1 v/v)

Gradient condition Time (min) A(%) 

### B(%) 0.00 50 50 20.00 100 

## o 40.00 100 

# o Flow rate 0.2 mUmin Injection volume 5,uL Post-column

Solution Acetonitrile /water*l (1/1 v/v)'2 Flow rate 0.1 mUmin



### *1 Tap water was treated with ultrapure water system. *2 Containing 25 mmoIlL ammonium acetate.

691 -21 Additional data 2­

### 94744

Conditions ofmass spectrometer Ionization mode Electrospray ionization (ESI) Detection ion Positive Measure mode Scan Scan range (mlz) 150·1200 Capillary temp. 300°C 2.2 Results Peak G, H, I and J detected on total ion current (TIC) chromatogram (Additional figure 2·21) were analyzed byLC·MS and estimated as following structures.
Retention time Estimated Additional Estimated structure Peak onTIC molecular figure No. and molecular formula chromatogram No. weight (min) 

## ------"

... "

### ..'.

## -_._------_.._ .. _"'

### ................. -...........

### "-_

.....

# · · 

# · · · I 

# · 

# · · 

# · · · · · Figure 2-22 

# · · G 13.7 

# · Figure 2-26 

# · · · · · · · 

# · · · 

# · · · 

# · · · · 

# · · · 

# · · Figure 2-23 

# · · 13.9 

### H 

# · · 

# · Figure 2·27 

# · · · · · · · · · 

# · · · ·
Figure 2-24 17.0 I Figure 2-28



### - 692

-22Additional data 2­

### 94744

,-----r------:- - --- ---- - ---- --- - --- -.. ----- -------- - ...... - - ... - -...... ....-------..,



### Figure 2-25 

### J 17.4 

### Figure 2-29

'--_.......!..

_____......._________ . __ . _.__•_________•••.••___•______._.__________"--

___---.J 

### From these estimated structures, it 

### was suggested that the major components of the white substance, Peak G and H, adhered to water surface ofthe test vessel was the dimmer ofPeak D and E caused byintermolecular disulfide fonnation. Also, it was suggested that PEMB bound to PE3MB by action ofdisulfide formation and became Peak I. 

### Additionally, the substance which had a molecular weight of 982 as Peak J 

### was detected, however three structures mentioned above are possible as the structure ofthe substance. And it was suggested that Peak G was stereoisomer ofPeak H.



### 693

-23 Additional datil 2­94744

Insoluble converted products RT: 0.00

- 30.00 NL: 5.69E5 550000 nCF:+c Q1MS[ 150.00-1200.00] 500000 MS 94744-090402­ H72hA-Ring-2 

# I 350000 2.13
..

## 2J~9 

# I i300000 i 

# I :5 250000

# '~1 

# I 

# 1

200000 150000 100000

# ~"·"W 50000 

### o I I ' ,.,.' T'I • I I I I I I : 'I ! i , i I \ J • Itt r", t



### o 5 10 15 20 25 30 Time (min)

Additional figure 2-21 

### TICchromatogram

694 -24 Additional dala 2­

### 94744

Insoluble converted products



### 94744-090402-H72hA
..Ring-2#783 RT: 13.69 AV: 1 S8: 58 15.00-15.99

### "NL: ""1.76E5 T: + cQ1MS [150.00-1200.00] 896.30 Mass spectrum ofpeak G 170000
.. 160000 150000 140000 130000: 120000: 

## I 

# I . 110000**~100000:** 

# I 9OODO**1~.27** 

### ..e 80000 70000­ 

## aoooo 5OODO'

RT: 0.00 - 30.00 13.81 NL: 6.56E4 m1z=923.88-924.88 60000 F: +cQ1MS [ 150.00-1200.00] Position ofpeak G 50000 MS 94744-090402­ 2:' 40000

# ~ H72hA-Ring-2 "!! 

### .s :!OOOO' 13.65 .£

# :::.1 I

13.<4Jt \ 14 19

## o-~6.01 1MB \. 13.89 NL: 1.76E5 mlz= 895.8o.B96.80 

# / F: +cQ1MSI 15O.0(}'1200.00] 13.91 MS Position ofpeak G 94144-090402­ H72hA-Ring..2

l-'+'-1-i'-T'"r-r-rB..,..2....,3,......,..1'-T1.;:..;.4r:-7

# J 14.9'., !?-'?....:4
..... , ...............=,25:;:.1

# :....;.~:::.,..::, 29:;::,.:!j·'"""1 5 10 15 20 25 

### so Time (min) Additional figure2-22 Mass spectrum ofPeak G and Mass chromatogram 695

-25 Additionaldata 2­ 4C)()()(r

m/z Mass chromatogram ofmlz =924

11.25 24:72 26.33 Mass chromatogram ofmlz =896

### 94744

Insoluble converted products

94744-090402-H72hA-Ring-2 #795 RT: 13.90 AV: 1 56: 58 1500-15.99 NL: i.l0ES T: + cQ.iMS{ 150.00-1200.oo} 896.33 120000

# 1 110000- Mass spectrum ofpeak H 1 l 100000 i

# I 90000 80000 70000­**~** c: 

### '" 80000

### ~ 50000 40000 30000· 20000 10000 0

13.81 m/z
Mass chromatogram ofm/z =924 

# ::1 Position

# Of:a:H**~40000** 30000

# L j i3.6p 

# I .5 20000 

### 1\ 10000 1348

# II**~~4.37**

13;17.42**.~30.55** 1183.70

....**J~r-M"'1"f"'t"'Y"tr~** 1000 1200

NL:6.56E4 mlz= 923.87-924.87 F:+cQ1MS[ 150.00-1200.001 MS 94744-090402­ H72hA-Ring-2

0-

## :U1~"**8.01_J~_~~** l'4.'9 17,25

## 24,7.~ 26.33**13.6':c9--:":":'::;"'---;;;;';';;:~~** -NL: 1.7eE5 mIz= 895.83-896.83 180000
. Mass F: +c QiMS [ 150.00-1200.00] 140000 13.91

### ~matogram MS 120000 94744..{)90402­ H72hA-Ring-2 f100000­ Position of peak**~** 80000 60000
. 

### 40000 20000 

### o 054 3.M 8.23

# '1.~J 1,0.9, '9.7< 25.45 29.lI!! r-r-r**I~....,.....,-rrr""'-T·I·** r "'---r-'! o 5 10 15 20 25 30 Time (min) Additional figure 2-23 Mass spectrum ofPeak H and Mass chromatogram


### 696

-26 Additional data 2­94744

Insoluble converted products

94744'..090402-H72hA-Ring-2 #975 

### R'r: 17.04 AV: 1 S8: 58 15.00-15.99 NL: 

### 2.44E4 T: +- cQ1MS (150.00-1200.00]**~**

ell 

### S

Mass chromatogram

15000

### .1. ofmlz =1030

### ~10000 c:

### ~ 5000 o 1.57 4.09 6.50 9.Q1 13·13 14.81 24000 22000 20000 18000 16000 14000 

### i 12000·

10000 8000 

### Mass spectrum ofpeak I

1*.38

mtz 17.03
Position ofpeak I 17.10 17.20

, 17.64

25000 17.03

# , mlz=**16.~7** 17.13 1001.67-1002.67 F: + cQ1MSl 20000 Mass chromatogram 150.00-1200.00] MS 94744-090402­ ofmlz =1002 Position ofpeak I H72hA-Ring-2**~15ooo·** 

### .E 10000 5000 1002.17

NL: 1.82E4 mlz= 1029.88-1030.88 F: +cQ1MS[ 150.00-1200.ooJ MS 94744-090402­ H72hA-Ring-2**2.0.~** 22.74 24.88 Nl: 2.50E4

Additional figure 2-24 Mass spectrum ofPeak I and Mass chromatogram
697

-27 Additional data 2­ 

# i

### 94744

Insoluble convclted products

94744.090402-H72hA-Ring-2#995 RT: 17.39 AV: 1 SB: 58 15.0()'15.99 NL: 1.99E4 T; +c Q1MS [150.0()'1200.00] 1000.31 

# ! Mass spectrum ofpeak

### J 18000 16000 14000 1028.31

# r12000 J;**~** 10000

### ~ 8000

m/z RT: 0.00

- 30.00 17.24 NL: 1.36E4 12000 1()()O(r J; 8000 

# :e i 6000 4000· 2000 

### a 25000 20000 i'15000' c: S ..5 ,0000 5000 17.08 Mass chromatogram ofmlz =1028 16·117



## o..so 2
.. 66 7.60 11.12 14.68 m1z= 1027.81-1028.81 F: +cQ1MS [**~** 150.00-1200.00] MS 17.59 Position ofpeak J94744-090402­ H72hA-Ring-2 p.71 18.2721.69 26.34 

# II' 17.13" NL:2.69E4



### o 1--r=r'1--1F""T'''-r-'''PT--rnr...-,.,-.','1-'­



### o 5 10 16 TIme (min) Mass chromatogram ofmlz =1000
16.99

2.20 .0\**15~** 1].46 m1z= 

# " 999.81-1000.81 F: + cQ1MS [ 150.0()'12oo.oo] MS Position ofpeak

### J 94744-090402­ H72hA-Ring-2

Additional figure 2-25 Mass spectrum ofPeak

### J and Mass chromatogram


### 698 -28 Additional data 2­94744

-... -- ... -- ..--- ..-.... -- ..-----..-- ....--- .. --- ...... ---_·----_·-t 

# · · · · · · · · ·

mlz Detected ion 924 

### [M+N1-l4+HCOOH-H20r 

### 896 [M+N1-l4r Additional figure 2-26 Estimated structure ofPeak G

••••••• ____ •••••••••• _. _____ •••• _ •••• __ ••••• _ ••• __ ••• k •••• __ •••• __ •• ,

# · 

# · · · · · · · · 

# ·

mlz Detectedion 924 

### [M+N1-l4+HCOOH-HzOr 

### 896 [M+Na.r Additional figure 2-27 Estimated structure ofPeak H 699 -29Additionaldala 2­

### 94744



### m/z Detected ion 1030 [M+NR!+HCOOH-H20r 

### 1002 [M+NR!t 

### Additional figure 2-28 Estimated structure ofPeak I



### m/z Detected ion 1028 fM+NH4+HCOOH-H

### z Ot 1000 [M+NR!t 

### Additional figure 2-29 Estimated structure ofPeak J
700 

### -30 Additional data Z­PAGE 1 OF 21 PAGES 

# harlan rM


# ....

Mixture ofPEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON

THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE PROJECT NUMBER: 0145/0201

STUDYSPONSOR: Showa Denko K.K. 13-9, Shiba Daimon 1-Chome Minato-ku TOKYO 105-8518 JAPAN

0145-0201.doclNY AUTHOR: N Youngs

TEST FACILITY: Harlan Laboratories Ltd Shardlow Business Park Shardlow Derbyshire DE722GD UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332799018

701PROJECT NUMBER: 014510201 PAGE 2 

### QUALITY ASSURANCE REPORT This study type is classed as short-term. The standard test method for this study type ("General Study Plan" in OECD terminology) was reviewed for compliance once only on initial production. Inspection of 

### the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases at or about the time this study was in progress. In addition, inspection of general facilities not specifically related to this study are done monthly or annually in accordance with QA 

### Standard Procedure. This report has been audited by the Quality Assurance Unit. and is considered to be an accurate account ofthe data generated and ofthe procedures followed. In each case, the outcome of QA 

### evaluation is reported to the Study Director and Management on the d'ay of evaluation. Audits of study documentation, and process inspections appropriate to the type and schedule ofthis study were as follows:



### § § 20 November 2008 Standard Test Method Compliance Audit 09 December 2008 Test Material Preparation 09 December 2008 Test System Preparation 09 December 2008 Exposure 09 December 2008 Assessment of Response 26 January 2009 Draft Report Audit Date ofQA 

### Signature Final ReportAudit 

### § Evaluation specific to this study 

## 02 FEB 

## 2009 

# ....t\
....**~~** 

# .................. DATE: Forthe Quality Assurance Unit*



## *Authorised QA Signatures: Deputy Head of Department: JM Crowther MIScT MRQA SeniorAudit Staff: GWren ONC MRQA

702PROJECT NUMBER: 014510201 PAGE 3 

### GLP COMPLIANCE STATEMENT The work described was performed in compliance with UK GlP standards (Schedule 1, Good laboratory Practice Regulations 1999 (SI 

### 1999/3106 as amended by SI 

### 2004/0994». These Regulations are in accordance with GlP standards published as OECD Principles on**G~od** 

### Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17): and are in accordance with, and implement, the requirements of Directives 

### 2004/9/EC and 

### 2004/10/EC. This report fully and accurately reflects the procedures used and data generated.



## A

# ~ 30 JAN 

## 2009 

# f4

...........1.\1...
.... ......................................... Date:
...................................................... NYoungs B c Study Director

This report maybe presented in final fonn as a digital (pdf) documenl Such documents are prepared byscanning the paper original.

and are considered ofequivalent Integrity and authenticity to varsions pftlduced by optical photocopy. However. In aN cases the hand-signed paperoriginal. hald in secure archives. Is thedefinitive document.

703PROJECT NUMBER: 014510201 PAGE 4 

### CONTENTS QUALITY ASSURANCE REPORT 2 GLP COMPLIANCE STATEMENT 3 CONTENTS 4 SUMMARY 5 1. INTRODUCTION 6 2. TEST MATERIAL 6 2.1 Description, Identification and Storage Conditions 6 3. METHODS 7 3.1 Test Species 7 3.2 TestWater 7 3.3 Synthetic Sewage 7 3.4 Procedure 8 4. ARCHIVES 10 5. RESULTS 11 5.1 Definitive Test 11 6. CONCLUSION 12 7. REFERENCES 12 Table 1 Oxygen Consumption Rates and Percentage Inhibition Values in the Definitive Test after 3 Hours Contact Time 13 Table 2 pH Values oftheTest Preparations atthe Start and End ofthe Exposure Period. 14 Table 3 Observations on the Test Preparations Throughout the Test Period 15 Figure 1 Concentration-Response Curve: 3,5-dichlorophenol - 3 Hours Contact Time 16 Figure 2 Concentration-Response Curve: Mixture ofPEMB and PE3MB ­ 3 Hours ContactTime 17 Appendix 1 Typical Water Quality Characteristics 18 Appendix 2 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 21



### 704·PROJECT NUMBER: 014510201 PAGE 5
Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE

RESPIRATION OF ACTIVATED SEWAGE SLUDGE SUMMARY 

### Introduction. A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines forTesting ofChemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Tesf', Method C.11 ,of Council Regulation (EC) No. 

### 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800. 

### Methods. Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10,32, 100,320 and 1000 

### mgll for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol. 

### Results. The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of greater than 1000 mgll. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 230 mgll. It was considered unnecessary and unrealistic to test at concentrations in excess of 

### 1000 mg/l. The reference material gave a 3-Hour EC50 value of 9.2 mgll, 95% confidence limits 7.2 - 12 mgll.

705

### 2.1 PROJECT NUMBER: 014510201 PAGES 

### Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE

RESPIRATION OF ACTIVATED SEWAGE SLUDGe 1. INTRODUCTION This report contains a description of the methods used and results obtained during a study to investigate the effect of the test material on the respiration of activated sewage sludge. The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Council Regulation (EC) No. 44012008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800. The results ofthe study are ofvalue in the assessment ofthe toxicity ofthe test material to sewage sludge micro-organisms. The study was conducted between 21 January 2009 and 22 January 2009. 2. TEST MATERIAL 

### Description, Identification and Storage Conditions Sponsor's identification Mixture of PEMB and PE3MB

.. 

### .......

## ---....................

## - .............

### -.-.... 

# ·**~** 

### Description 

# · 

# . · , 

### Batch number

........_a__....___ ...____ ..__....___'**~** 

### Date received 15 December 2008 Storage conditions :' room temperature in the dark The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility ofthe Sponsor.



### 706PROJECT NUMBER: 014510201 PAGE 7 

### 3. METHODS 3.1 Test Species A mixed population of activated sewage sludge micro-organisms was obtained on 

### 22 January 

### 2009 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage. 

### 3.2 TestWater The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 

### 500) and partly softened (Elga Nimbus 

### 1248D Duplex water softener) giving water with a total hardness of approximately 

### 140 mg/l as CaC03. 

### After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening. are given in Appendix 

### 1. 3.3 Synthetic Sewage A synthetic sewage ofthe following composition, was added to each test vessel to act as a respiratory substrate: 

### 169 Peptone 

### 11 9 Meat extract



### 3g Urea 

### 0.7 9 NaCI 

### 0.4 9 CaC12·2H20 

### 0.2 9 MgS04

### .7H20 

### 2.8 9 K2HP

### 04 dissolved in 

### 1 litre ofwaterwith the aid of ultrasonication.



### 707PROJECT NUMBER: 014510201 PAGE 8


### 3.4 Procedure 3.4.1 Test material preparation For the purpose of the definitive test, the test material was dispersed directly in water. The following test concentrations were assigned to the test: 10, 32, 100, 320, and 1000 

### mg/l. Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes followed by prolonged mixing for 24 hours. All test vessels were shielded from the light during mixing.· Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentrations of 10,32, 100,320 and 1000 

### mg/'. The control group was maintained under identical conditions but not exposed to the test material. Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline. 

### 3.4.2 Reference material preparation For the purpose of the test a reference material, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 15402DH-367) was used. Two stock solutions of 50 and 160 mgll were prepared by dissolving the reference material directly in water with the aid of ultrasonication for approximately 20 minutes. Aliquots (10 and 100 ml) of the 160 mg/l stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 

### mg/l. Similarly, a 100 ml aliquot ofthe 50 

### mg/l stock solution was used to prepare the 10 mg/l concentration. The volumetric flasks containing the reference material were inverted several times to ensure homogeneity of the solutions. 

### 3.4.3 Preparation ofinoculum The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and was used on the day of collection. The pH of the sample was 7.7 measured using a 

## wrw pH/Oxi 3401 pH and dissolved



### 708PROJECT NUMBER: 014510201 PAGE 9 

### oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.8 g/l prior to use. 

### 3.4.4 Preparation oftestsystem At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 500 ml conical flask (first contrOl). The mixture was aerated with clean. oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared as described in Section 3.4.1. Finally a second control was prepared. As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between apprOXimately 6.5 mg O2

### /1 and 2.5 mg 

### 02m. In the case of a rapid oxygen consumption. measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C. Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to the addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a 

# wrw pH/Oxi 3401 pH

709PROJECT NUMBER: 014510201 PAGE 10



### RS 

# = and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. 

### 3.4.5 Evaluation of Data In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage ofthe two control respiration rates.
% inhibition

## =[1- 2RSC ] x 100 RC 1 

### +R 2 

### where oxygen consumption rate for test or reference sample oxygen consumption rates for controls 1and 2 The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (lOBS) and the EC15and EC20 values for the test material and the EC15, EC20, 

### EC50 and EC80 

### values for the reference material determined from the equation for the fitted line. The EC50 and ECao values for the test material were determined by inspection of the inhibition of respiration rate data. 95% confidence limits were calculated for the reference material EC50 values only, using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949). The No Observed Effect Concentration (NOEC) was taken as being the EC15 value. The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 

### mg/l. 

### 4. 

### ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which instructions will be sought as to further retention ordisposal.

710

### 5.1 PROJECT**NUMBER~** 0145/0201 PAGE 11 

### 5. RESULTS 

### Definitive Test Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1. The pH values ofthe test preparations at the start and end ofthe exposure period are given in Table 

### 2, and observations made on the test preparations throughout the study are given in Table 

### 3. Percentage inhibition is plotted against concentration for the test and reference materials (Figures 

### 1 and 

### 2)
.. The following results were derived: Mixture of PEMB and PE3MB 3.5-dichlorophenol ECx(3 Hours) (mg/l) 95% Confidence limits (mgll) ECx(3 Hours) (mgll) 95% Confidence Limits (mg/l) EC20 690 

## - 3.1 

## - ECso >1000 

## - 9.2 7.2-12 EC80 >1000 

## - 27 

## - NOEC 230 

### - - 

# - Itwas not possible to obtain 

### ECoo or 

### ECso values forthe test material as no concentration tested resulted in greater than 50% inhibition. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mgt!. Variation in respiration rates ofcontrols 1 and 2 after 3 hours contacttime was 

### ±4%. The validation criteria for the control respiration rates and reference material 

### ECso values were therefore satisfied. In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O

### 2 /1 and 2.5 mg 

### 02/1 respectively). This was considered to have had no adverse effect on the results ofthe study given that



### 711PROJECT NUMBER: 014510201 PAGE 12 

### in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. Observations made at 0 hours (see Table 3) prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10, 32 and 100 mgtl contained slightly cloudy dispersion with small globules of test material dispersed throughout. The test concentrations of 320 and 1000 mgll contained cloudy dispersions with an oily layer oftest material on the surface and small globules dispersed throughout. Observations made after 30 minutes and 3 hours contact time showed that the test concentrations of 10, 32 and 100 mgtl contained dark brown dispersions with small globules of test material dispersed throughout. The test concentrations of 320 and 1000 mgll contained dark brown dispersions with an oily layer of test material on the surface and small globules dispersed throughout. 

### 6. CONCLUSION The effect of the test material on the respiration of activated sewage sludge microorganisms gave a 3-Hour ECso 

### of greater than 1000 mgll. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 230 mgll. 

### 7. REFERENCES Litchfield, 

### J T and Wilcoxon, F (1949) A Simplified Method of Evaluating Dose-Effect Experiments. J Pharmaco/Exp Ther96, 99-113. 

### Xlfit. ID Business Solutions Ltd.

712PROJECT NUMBER: 0145/0201 PAGE 13 Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE 

### Table 1 Oxygen Consumption Rates and Percentage Inhibition Values in the Definitive Test after 3 Hours Contact Time Nominal Concentration (mg/l) Initial O2 Reading (mg 02 /1) Measurement Period (minutes) Final O2 Reading (mg O2 /1) 

### O 2 Consumption Rates (mg 02/Vmin) % Inhibition Control R1 R2 6.3 7 2.8 0.50 

### - 3.9 5 1.6 0.46 

## - Test Material 10 32 100 320 1000 6.4 7 2.9 0.50 [4] 4.7 5 2.4 0.46 4 3.8 5 1.7 0.42 13 4.1 5 2.0 0.42 13 5.7 9 2.3 0.37 23 3,5-dichlorophenol 3.2 10 32 7.1 10 3.2 0.39 19 7.6 10 5.5 0.21 56 8.2 10 7.4 0.08 83

[Increase in respiration rate as compared to controls] Rl - R2

# = Replicates 1 to 2

713PROJECT NUMBER: 0145/0201 PAGE 14 Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE 

### Table 2 pH Values ofthe Test Preparations atthe Startand End ofthe Exposure Period Nominal pH Concentration (mgll) 

## oHours 3 Hours Control R1 

### 7.7 7.9 7.7 7.8 R2 Test Material 

### 10 7.8 7.8 7.8 32 7.9 100 7.8 7.9 320 7.8 7.8 1000 

### 7.7 7.8 3,5­ 3.2 7.7 7.9 dichlorophenol 

### 10 7.8 8.0 32 7.8 8.1

R1

- R2

# =Replicates 1to 2

714PROJECT NUMBER: 014510201 PAGE 15 Mixture ofPEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE 

### Table 3 Observations on the Test Preparations Throughoutthe Test Period Nominal Observations on Test Preparations Concentration 

### oHours 30 Minutes 3 Hours I (mg/l) Contact Time ContactTime Control 

### R1 Dark brown Dark brown Dark brown dispersion dispersion dispersion R2 Dark brown Dark brown Dark brown dispersion**di~ersion** dispersion Slightly cloudy Dark brown Dark brown dispersion, small dispersion, small dispersion, small Test Material 10 globules oftest globules oftest globules oftest material dispersed material dispersed material dispersed throughout· throughout throughout Slightly cloudy Dark brown Dark brown dispersion, small dispersion, small dispersion, small 32 globules oftest globules oftest globules of test material dispersed material dispersed material dispersed throughout· throughout throughout Slightly cloudy Dark brown Dark brown dispersion, small dispersion, small dispersion, smalt 100 globules oftest globules oftest globules oftest material dispersed material dispersed material dispersed throughout· throughout throughout Cloudy dispersion, Dark brown Dark brown

-oily layer oftest dispersion, oily layer dispersion, oily layer 320 material on surface oftest material on oftest material on and small globules surface and small surface and small dispersed globules dispersed globules dispersed throughout* throughout**thro~hout** Cloudy dispersion, Dark brown Dark brown

oily layeroftest dispersion, oily layer dispersion, oily layer 1000 material on surface oftest material on oftest material on and small globules surface and small surface and small dispersed globules dispersed globules dispersed throughout· throughout throughout Dark brown Dark brown Dark brown dispersion, no dispersion, no dispersion, no 3,5-dichlorophenol 3.2 undissolved undissolved undissolved reference material reference material reference material visible visible visible Dark brown Dark brown Dark brown dispersion, no dispersion, no dispersion, no 10 undissolved undissolved undissolved reference material reference material reference material visible visible visible Dark brown Dark brown Dark brown dispersion, no dispersion, no dispersion, no 32 undissolved undissolved undissolved reference material reference material reference material visible visible visible

R,

- R2 

## =Replicates 1to 2 • Observations prior to addition of activated sewage sludge and synthetic sewage
715PROJECT NUMBER: 0145/0201 PAGE 16 Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE Figure 1 

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716PROJECT NUMBER: 014510201 PAGE 17 Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLlIDGE 

### Figure 2 Concentration-Response Curve: Mixture of PEMB and PE3MB ­ 3 Hours ContactTime

90 80 70

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717PROJECT NUMBER: 014510201 PAGE 18 Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE 

### Appendix 1 Typical Water Quality Characteristics WATER SUPPLY ZONE ZD813 ALVASTON REPORTING PERIOD: 0110112007 - 3111212007 PARAMETER ANALYSIS UNITS NO. SAMPlE TAKEN CONCENTRATION OR VALUE ALL SAMPLES) PCV' SAMPLES CONTRAVENING PCV MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 1 ,2 Dichloroethane**~g/I** 8 <0.080 <0.080 <0.080 3 0 0.00 2,3,6-TBA**~g/I** 8 <0.002 <0.002 <0.002 0.1 0 0.00 2,4-08 IlQII 8 <0.002 <0.002 <0.002 0.1 0 0.00 2,4, 5, • TCPA IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 2.4-0 1Ig/1 8 <0.002 <0.002 <0.002 0.1 0 0.00 Aklrin IJg/I 8 <0.001 <0.001 <0.001 0.03 0 0.00 Alpha-HCH IJg/I 8 <0.001 <0.001 <0.001 0.1 0 0.00 Aluminium IJg/I 52 <11.000 <12.892 29.000 200 0 0.00 Ametryn 1Ig/1 8 <0.002 <0.002 <0.002 01 0 0.00 Antimony IJgll 8 <0.120 <0.529 1.200 5 0 0.00 ABenic 1Ig/1 8 <0.370 <0.370 <0.370 10 0 0.00 Attazine IJg/I 8 <0.001 <0.001 0.001 0.1 0 0.00 Benazolin 1Ig/1 8 <0.002 <0.002 <0.002 0.1 0 0.00 8entazone 1Ig/1 8 <0.002 <0.002 <0.002 0.1 a 0.00 Benzene 1Ig/1 8 <0.060 <0.080 <0.060 1 0 0.00 8enzo 3.4 Pyrene 1Ig/1 8 <0.001 <0.001 <0.001 0.01 0 0.00 Beta- HCH 1Ig/1 8 <0.001 <0.001 <0.001 0.1 0 0.00 Baton mgII 8 0.021 0.03<4 0.052 1 a 0.00 Bromacil 11911 8 <0.012 <0.012 <0.012 0.1 0 0.00 Bromate I19n 8 <0.600 <0.600 <0.800 10 a 0.00 8romoxynil**~gll** 8 <0.002 <0.002 <0.002 0.1 0 0.00 Cadmlum 1Ig/1 a <0.080 <0.111 0.150 5 a 0.00 Caplan 1Ig/1 a <0.007 <0.007 <0.007 0.1 0 0.00 CarbenaZim 1Ig/1 a <0.002 <0.002 <0.002 0.1 0 0.00 Carbelamide IIg

### n a <0.002 <0.003 <0.006 0.1 0 0.00 ChlordaneA**~g/I** a <0.002 <0.002 <0.002 0.1 0 0.00**Chlorolhalcn~** 11911 8 <0.003 <0.003 <0.003 0.1 0 0.00 ChIoroIoIuron IIgn 8 <0.002 <0.002 <0.004 0.1 0 0.00 ChrOmium IJg/I 8 <0.700 <0.700 <0.700 50 0 0.00 Cis- Permethrin IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 Clopyralid 1Ig/1 8 <0.001 <0.001 <0.001 0.1 0 0.00 Colour mgII PIICo 28 <0.400 <0.408 0.800 20 0 0.00 Copper mgIl 8 <0.003 <0.008 0.027 2 0 0.00 Cyanazine IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 Cyanide IJg/I 8 <0.400 <0.825 0.700 50 0 0.00 Cylluthrin 11911 8 <0.003 <0.003 <0.003 0.1 0 0.00 Cypermethrin Ilgll 8 <0.004 <0.004 <0.004 0.1 0 0.00 O.o.E - Ortho Para 11911 8 <0.001 <0.001 <0.001 0.1 0 0.00 O.O.E - Para Pare**Ilg~** a <0.001 <0.001 <0.001 0.1 0 0.00 D.D.T- Ortho Pare Ilgl1 8 <0.003 <0.003 <0.003 0.1 0 0.00 O.D.T - Pare Para jJg/I 8 <0.001 <0.001 <0.001 0.1 0 0.00 DlIlta- HCH 11911 8 <0.002 <0.002 <0.002 0.1 0 0.00 OaltametMn Ilg11 8 <0.005 <0.005 <0.005 0.1 0 0.00 Oicamba Ilgl1 8 <0.002 <0.002 <0.002 0.1 0 0.00 01ch1obenl1**~g/I** 8 <0.001 <0.001 <0.001 0.1 0 0.00 Dlchloprop IlIJil 8 <0.002 <0.002 <0.002 0.1 0 0.00 Dieldrin 11911 8 <0.002 <0.002 <0.002 0.03 0 0.00 Oillufenlcan IIg11 8 <0.004 <0.004 <0.004 0.1 0 0.00 Dluron IJg/I 8 <0.003 <0.003 <0.003 0.1 0 0.00 E. coil No./100ml 180 0.000 0.000 0.000 0 0 0.00 EndcsullanA 1Ig11 8 <0.002 <0.002 <0.002 0.1 0 0.00 Endoaulfan B Ilg/l 8 <0.002 <0.002 <0.002 0.1 0 0.00 Endrin jJgII 8 <0.002 <0.002 <0.002 0.1 0 0.00 Enteroc:occl Na./100ml 8 0.000 0.000 0.000 0 a 0.00 EPTC 1Ig/1 8 <0.001 <0.001 <0.001 0.1 0 0.00 Ethofumeaate IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 Feooprop Fenpropldin

## ~ 8 8 <0.002 <0.003 

## I <0.002 <0.003 <0.002 <0.003 0.1 0.1 0 0 0.00 0.00 "PCV Preecrlbed concentration arvalue for mandalOly parema1ers and specified limit or value for oon·rnandatory parameters (indicators and disinfectantll). "1 No numelical limitapplies. "2 Guldanee levels only. Monitored for the calculation ofTotal Indicative Dose, which Is Ihe ragulatOJy parameter. No samples exc.eded the TID in 2007. "3 The formula for the calculated NltraleiNllrite level i, [Nltrate)l50 + [Nitlltejl3. This number has no unlls.

718PROJECT NUMBER: 014510201 PAGE 19 Mixture of PEMB and**~E3MB:** ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE 

### Appendix 1 (continued) Typical Water Quality Characteristics WATER SUPPLY ZONE ZDB13 ALVASTON REPORTING PERIOD: 0110112007 - 31/1212007 PARAMETER ANALYSIS UNITS NO.

### 0~lE TAKEN CONCENTRATION ORVALUE (All SAMPLES) PCV"' SAMPLES CONTRAVENING PCV MINIMUM AVERAGE MAXIMUM NUMBER PERCENT F enpropimorph 1Ig/1 8 <0.006 <0.006 <0.006 0.1 0 0.00 Fenvalefate IJg/I 8 <0.003 <0.003 <0.003 0.1 0 0.00 Fluoride mgII 8 0.310 0.351 0.415 1.5 0 0.00 Fluroxypyr**~g/I** 8 <0.002 <0.002 <0.002 0.1 0 0.00 Flutrialol IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 Gamma-HCH 1Ig/1 8 <0.001 <0.001 <0.001 0.1 0 0.00 Heptachlor IJg/I 8 <0.001 <0.001 <0.001 0.03 0 0.00 Heptachlor epoxide J.IIln 8 <0.002 <0.002 <0.002 0.03 0 0.00 Hexachlorobenzene IJg/I 8 <0.001 <0.001 <0.001 0.1 0 0.00 Hexachlorobutadiene IJg/I 8 <0.001 <0.001 <0.001 0.1 0 0.00 loxyrlil**~g/I** 8 <0.001 <0.001 <0.001 0.1 0 0.00 Iron IJg/I 52 <7.000 <13.481 82.000 200 0 0.00 lsodrln IlOn 8 <0.001 <0.001 <0.001 0.1 0 0.00 lsoproruron J.IIln 8 <0.002 <0.004 <0.004 0.1 0 0.00 lead J.IIlII 8 <0.500 <1.138 5.100 25 0 0.00 Linurcn IlOA e <0.003 <0.003 <0.005 0.1 0 0.00 Manganese Ilg/l 52 <1.500 <1.546 2.600 50 0 0.00 MCPA IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 MCPB**~gJ1** 8 <0.002 <0.002 <0.002 0.1 0 0.00 Mecoprop IJg/I B <0.002 <0.002 <0.002 0.1 0 0.00 Mercury IJg/I e <0.012 <0.012 <0.012 1 0 0.00 Metamilron J.IIlII 8 <0.002 <0.002 <0.003 0.1 0 0.00 Metazachlor IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 Methabenzlhiazuron**~gll** 8 <0.002 <0.002 <0.003 0.1 0 0.00 Methoxychlor IJg/I 8 <0.001 <0.001 <0.001 0.1 0 0.00 Monolinuron IJg/I 8 <0.002 <0.002 <0.004 0.1 0 0.00 Nickel**~gJ1** 8 <0.900 <1.788 2.500 20 0 0.00 Nittale rngJI 8 10.200 14.n5 17.900 50 0 0.00 Nittite rngJI 8 <0.002 <0.003 0.004 0.5 0 0.00 NitrltelNitIate Calculated _"3 8 0.200 0.295 0.360 <fJ 0 0.00 Odour Dilution Number 26 0.000 0.000 0.000 3 at 25°C 0 0.00 PAH 11011 8 0.000 0.000 0.001 0.1 a 0.00 PCB - Arochlor 1254 IlOII 8 <0.002 <0.002 <0.002 0.1 0 0.00 Pendlmethalin Ilgil 8 <0.003 <0.003 <0.003 0.1 0 0.00 pH pH value 52 7.260 7.719 8.240 Max 9.S, Min 8.S 0 0.00 Pirimlcartl IIgJ1 8 <0.001 <0.001 <0.001 0.1 0 0.00 Prometryn Ilgil 8 <0.002 <0.002 <0.002 0.1 0 0.00 Propachlor IlOII 8 <0.002 <0.002 <0.002 0.1 0 0.00 propazine liOn 8 <0.001 <0.001 <0.001 0.1 0 0.00 Propiconazoie IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 Propyzamitle IJg/k 8 <0.002 <0.002 <0.002 0.1 0 0.00 Selenium IJg/I 8 0.420 0.670 1.200 10 0 0.00 Simazine IJg/I 8 <0.001 <0.001 <0.001 0.1 0 0.00 Sodium mgJI 8 21.000 27.250 35.000 200 0 0.00 T.O.E - Ortho Para IJg/I 8 <0.001 <0.001 <0.001 0.1 0 0.00 T.D.E - Para Para IJg/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 Taate Dilution Number 28 0.000 0.000 0.000 3at 25°C 0 0.00 Tecnazene IJg/I 8 <0.001 <0.001 <0.001 0.1 0 0.00 Tertlutryn J.IIlI1 8 <0.002 <0.002 <0.002 0.1 0 0.00 Tetrac::hloroelhene and Trichloroelhene Ilgil 8 0.000 0.000 0.000 10 0 0.00 Tatrachloromethane Ilgil 8 <0.020 <0.030 <0.040 3 0 0.00 Total Pesticidea Ilgil 8 0.000 0.000 0.001 0.5 0 0.00 Total Trihalornelhanes J.IIlII 8 22.200 34.138 50.600 100 0 0.00 Trans - Permelhrln**~gJ1** 8 <0.002 <0.002 <0.002 0.1 0 0.00 Triadimefon Ilgil 8 <0.002 <0.002 <0.002 0.1 0 0.00 TriaiJate 8 <0.001 <0.001 <0.001 0.1 0 0.00 Trichloroben:tene

## ~~ 8 <0.001 <0.001 <0.001 0.1 0 0.00 °PCV Prescribed concentration orvalue for mandatory parameters and specified limit orvalue for non-mandatory parameters (indicators and disinfectanlll). °1 No numeric::allimit applies. "2 Guidance levels only. Monitored for the calCulation ofTotal Indicative Dose, which Is the regulatoly parameter. No Hmplesexceeded theTID In 2007. '3 The formula forthe celculated NitrlIteINitrite levella [NitrateY5O + [Nitritey3. Thia number na. no units.

719PROJECT NUMBER: 0145/0201 PAGE 20 Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE 

### Appendix 1 (continued) Typical Water Quality Characteristics WATER SUPPLY ZONE Z0813 ALVASTON REPORTING PERIOD: 0110112007 - 3111212007 CONCENTRATION OR VALUE SAMPLES NO, ANALYSIS ALL SAMPLES) CONTRAVENING PCV PARAMETER PCV­**~PLE** UNITS TAKEN MINIMUM AVERAGE MA XIMUM NUMBER PERCENT Trlclopyr <0,003 <0,003 <0.003 0,1 0,00 Trletazine 8 0 1.19" 0,1 <0,001 <0,001 <0,001 8 0,00 Tfillul'8lin 0 IJ9II <0,002 <0,002 0,1 8 <0.002 0,00 Turbidity 0 Ilg/! <0,050 <0,088 NTU 52 0.190 0,00 Free Chlorine a 

### .", 4 180 0,030 0.550 0.231 '"9" 

### -, · mgll 180 0.297 0.610 Total ChlOrine 0.080 

### · 0,00 Chloride Ammonium mg/! 52 <0.009 <0.012 0.021 0.5 a mgll 8 17.600 24.788 32.500 250 a 0.00 Closlrldlum perfrlngens 0,000 NO.ll00 ml 26 0.000 0,000 a 0 0.00 Coliform Bacteria 0,000 No, 1100 ml 180 0.000 0 0.000 a 0.00 Colony Counts after 48 

### -, No, 1100 ml 83,000 52 0.000 3.096 

### - · hours at 370C Colony Count after 72 

# .. 1,654 No.1 100 ml 52 0.000 142,000 · 

### · hours at 22"C Conductivity 251.000 <421.385 542.000 0,00 Gross Alpha Activity IlSicm at 20"C 28 2500 a 0.1'2 BqII 0,087 0.125 8 0.063 · · (2 8 0.012 0,042 0.071 Gross Beta ActIvity Bq" · 

### · 52,100 Sulphate mgll 8 69.783 ! 85.800 250 0 0.00 Total Organic Carbon U141 1,380 8 0.810 moll 

### ." I 

### · "PCV Prescribed concentration orvalue lor mandatory parameters and specified lmit or value lor non-mandatory parameters (lIldic:ators and dilllnlectanis). "1 No numericallimilapplies. *2 Guidance levels only, Monitored for the calculation ofTotal tndicativa Dose, which itthe regulatory pal'8mater. No samples exceeded the TID in 2007, "3 The formula for the calculated Nitrate/Nilrlte level is [Nilrate]/5O + [Nilrite]l3, This numberhas no units.

720PROJECT NUMBER: 0145/0201 PAGE 21 

### Appendix 2 StatementofGLP Compliance in Accordance with Directive 2004/9/EC 

# ,oH) Department \.:.: ofHealth

THEDEPARTMENTOFHEALTH OFTBEGOVERNMENT OFTHEUNITED KINGDOM 

### GOODLABORATORYPRACTICE STATEMENTOFCOMPUANCE INACCORDANCEWITH DIRECTIVElOO4I9/EC
TEST fACIUTY SefePhenn Laboratori
.. Ltd. Analytical Chemistry Shardlow BUIIln
... Park Environmental Fe
.. Sherdlow Environmental Toxicity Derbyshire Mutagenicity DE722GD**~hem** ...ting Toxicology

DATEOFINSPEC110N 21M August2007 A general inspection for compliance with the Principles ofGood Laboratory Practice was carriedoutattheabove test facility as part ofthe UK GLPCompliance Programme. At the lime of inspection no deviations were found of sufficient magnitude to affect the validityofnon-clinical studiesperfonned at these facilities.

Dr.AndrewJ.Gra)' Head.UK GLPMonitoringAuthority

On 9'" November 2008. Safepharm Laboratories Ud changed its name to Harlan Laboratories Ltd. 111erefOl8, until a new GLP Certificate is issued from the UK GLP monitOring authority the certll'lcate in this report still statea the name as Safepharm Laboratories Ltd.

721PAGE 1 OF 28 PAGES 

# harlanT
" 

# .,

Mixture of PEMB and PE3MB:

ASSESSMENT OF READY BIODEGRADABILITY; CO2 

### EVOLUTION TEST PROJECT NUMBER: 

### 0145/0215

STUDY SPONSOR: Showa Denko 

### K.K. 13-9, Shiba Daimon 1-Chome Minato-ku TOKYO 105-8518 JAPAN

0145-0215.docln 

### AUTHOR: N Youngs

TEST FACILITY: Harlan Laboratories Ltd Shardlow Business Park Shardlow Derbyshire DE722GD 

## UK Telephone: +44 (0) 1332792896 Facsimile: +44 (0) 1332799018

722PROJECT NUMBER: 014510215 PAGE 2 

### QUALITY ASSURANCE REPORT This study type is classed as short-term. The standard test method for this study type ("General Study Plan" in OECD terminology) was reviewed for compliance once only on initial production. Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases at or about the time this study was in progress. In addition. inspection of general facilities not specifically related to this study are done monthly or annually in accordance with QA Standard Procedure. This report has been audited by the Quality Assurance Unit. and is considered to be an accurate account ofthe data generated and ofthe procedures followed. In each case. the outcome of QA evaluation is reported to the Study Director and Management on the day of evaluation. Audits of study documentation. and process inspections appropriate to the type and schedule ofthis study were as follows: 02 February 2009 Standard Test Method Compliance Audit 02 April 2009 Test Material Preparation 01. 02 April 2009 Test System Preparation 02 April 2009 Exposure 21 April 2009 Assessment of Response 

### § 22 May 2009 Draft Report Audit 

### § Date of QA Signature Final Report Audit 

### § Evaluation specific to this study

\ 

## 02J !H'''long**u~'lI** 1.., iI 

### For the Quality Assurance Unit'"

'Authorised QA Signatures: Manager. Quality Assurance: J G Riley eSe (Hons) MRQA Deputy Head of Department: JM Crowther MISeT MRQA Senior Audit Staff: G Wren ONC MRQA


### 723PROJECT NUMBER: 014510215 PAGE 3
GLP COMPLIANCE STATEMENT 

### The work described was performed in compliance with UK GLP standards (Schedule 1, 

### Good Laboratory Practice Regulations 1999 (SI 1999/3106 

### as amended by SI 2004/0994». 

### These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC 

### and 2004/10/EC. 

### This report fully and accurately reflects the procedures used and data generated.



### - 1 

## JUN 2009



# ..........

# ~.~ .......................................... Date:
........................................... NYoungs BSc Study Director

This report may be presented in final form as a digital (pdf) document. Such documents are prepared by scanning the paper original, and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the hand-signed paper original, held in secure archives, is the definitive document

724PROJECT NUMBER: 014510215 PAGE 4 

### CONTENTS QUALITY ASSURANCE REPORT 2 GLP COMPLIANCE STATEMENT 3 CONTENTS 4 SUMMARY 5 1. INTRODUCTION 6 2. TEST MATERIAL 6 2.1 Description, Identification and Storage Conditions 6 3. METHODS 7 3.1 Pre-Study Solubility Work 7 3.2 Experimental Preparation 8 3.3 Standard Material 9 3.4 Toxicity Control 9 3.5 Test Species 10 3.6 Procedure 10 3.7 Evaluation of Data 13 4. ARCHIVES 16 5. RESULTS 17 6. CONCLUSION 18 7. REFERENCES 19 Table 1 Inorganic Carbon Values on Each Analysis Occasion 20 Table 2 Percentage Biodegradation Values 21 Table 3 Total and Inorganic Carbon Values in the Culture Vessels on Day 0 22 Table 4 Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 28 23 Table 5 pH Values ofthe Test Preparations on Day 28 24 Table 6 Observations on the Test Preparations Throughout the Test Period 25 Figure 1 Biodegradation Curves 26 Appendix 1 Culture Medium 27 Appendix 2 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 28



### 725PROJECT NUMBER: 014510215 PAGES


### Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILITY; CO2 

### EVOLUTION TEST SUMMARY 

### Introduction. A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; C02 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 

### 44012008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)). 

### Methods. The test material, at a concentration of 1 0 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days. Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley 

### et aI, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test material in the test medium and to increase the surface area ofthe test material exposed to the test organisms. The degradation 

### of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used forvalidation purposes. 

### Results. The test material attained 70% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.

726

### 2.1 PROJECT NUMBER: 014510215 PAGES


### Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILITY; C02 EVOLUTION TEST 1. INTRODUCTION This report contains a description of the methods used and results obtained during a study to investigate the ready biodegradability of the test material when exposed to sewage sludge micro-organisms under aerobic conditions. The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (1992) No 301 B "Ready Biodegradability; C02 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 

### 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m». The results of the study are of value in the assessment of the persistence of the test material in the aquatic environment. In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (ISO 1995) and the published literature (Handley et aI, 

### 2002) is to adsorb the test material onto an inert support prior to dispersion in the test vessels. Using this method the test material is evenly distributed throughout the test medium and the surface area oftest material exposed to the test organisms is increased thereby increasing the potential for biodegradation. The study was conducted between 31 March 2009 and 29 April 2009. 

### 2. TEST MATERIAL Description, Identification and Storage Conditions Sponsor's identification Description Mixture of PEMB and PE3MB



## ..--.---.-.-

### ....

# ­ ........**---~~** 

### : Batch number : Date received 15 December 2008 Storage conditions room temperature in the dark

727

### 3.1 PROJECT NUMBER: 0145/0215 PAGE 7 

### The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility ofthe Sponsor. 

### 3. METHODS Pre-Study Solubility Work 

### Data supplied by the Sponsor indicated that the test material was insoluble in water. Therefore the following pre-study solubility/dispersibility work was conducted to confirm this: (i) Ultrasonication: An amount oftest material (50 mg) was dispersed in 1 litre of deionised reverse osmosis purified water with the aid of shaking by hand for approximately 20 seconds prior to ultrasonication for 30 minutes. This formed a clear 

### colourless water column with an oily layer of test material on the surface and small globules dispersed throughout. (ii) High shear mixing: An amount oftest material (50 mg) was dispersed in 1 litre of deionised reverse osmosis purified water and subjected to high shear mixing at approximately 7500 rpm for 30 minutes. This formed a cloudy dispersion with small globules oftest material on the surface. This work confirmed that the test material was poorly soluble in water. Therefore the following additional solubility work was conducted to ascertain the method that would give the best testable dispersion: (iii) 

### Ultrasonication: An amount oftest material (50 mg) was dispersed in 400 ml of deionised reverse osmosis purified water with the aid of ultrasonication for 15 minutes. The volume was then adjusted to a final volume of3 litres. Large globules oftest material were visible on the surface. (iv) High shear mixing: An amount of test material (50 mg) was dispersed in 400 ml of deionised reverse osmosis purified water with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. The volume was then adjusted to a final volume of 3 litres and small globules of test material were visible on the surface.

728

### 3.2 PROJECT NUMBER: 014510215 PAGE 8 

### (v) High shear mixing using an inert carrier, silica gel (an approved inert carrier, ISO 1995 and in the published literature (Handley at aI, 

### 2002»: An amount of test material (50 mg) was adsorbed onto silica gel (100 mg) and dispersed in 400 ml of deionised reverse osmosis purified water with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. The volume was then adjusted to a final volume of 3 litres and very small globules of test material were visible on the surface. (vi) Addition onto solid supports: The use of a glass microscope slide and glass fibre filter papers was attempted. When using a glass microscope slide, an amount oftest material (50 mg) was weighed onto a glass slide and added to 3 litres ofdeionised reverse osmosis purified water. Globules of test material were visible on the surface. When using GF/A filter paper, an amount oftest material (50 mg) was weighed onto a filter paper and added to 3 litres of deionised reverse osmosis purified water. Globules oftest material were visible on the surface. A volatility check was performed on the test material by passing a steady stream of compressed air over a known weight of test material for 3 hours as high shear mixing cannot be performed on volatile test materials. This resulted in no loss of test material over this time so the test material was considered not to be volatile. As such it was concluded appropriate to adsorb the test material onto silica gel and disperse with the aid of high shear mixing as this method appeared to increase the dispersibility of the test material over the other methods. 

### Experimental Preparation Following the recommendations of the International Standards Organisation (ISO 1995) and in the published literature (Handley at

### a'. 2002) the test material was adsorbed onto silica gel priorto dispersion in culture medium to aid dispersion ofthe test material in the test medium and to increase the surface area of the test material exposed to the test organisms. An amount of test material (66.6 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No 101K3700) prior to dispersal in

729PROJECT NUMBER: 014510215 PAGE 9 

### approximately 400 ml of culture medium with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. The test material/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 22.2 mg/l, equivalent to 10 mg carbon/I. A test concentration of 10 mg carbonll was employed in the test following the recommendations ofthe Test Guideline. Control vessels were prepared containing 100 mg silica gel per 3 litres of inoculated culture medium in order to maintain consistency between the control and test material vessels. Data from the control vessels was shared with similar concurrent studies. Analysis of the concentration, homogeneity and stability of the test material in the test preparations were not appropriate to the Test Guideline. 

### 3.3 Standard Material For the purposes of the test, a standard material, sodium benzoate (CaHsCOONa) (Sigma Aldrich lot No 096K1231), was used. An initial stock solution of 1000 mg/I was prepared by dissolving the standard material directly in culture medium with the aid of ultrasonication for approximately 2 minutes. An aliquot (51.4 ml) of this stock solution was added to the test vessel and the volume adjusted to 3 Htres to give a final test concentration of 17.1 mgll, equivalent to 10 mg carbonll. The volumetric flask containing the standard material was inverted several times to ensure homogeneity ofthe solution. Silica gel (100 mg) was also added to the standard material vessels in order to maintain consistency between the test and standard material vessels. Data from the standard material vessels was shared with similar concurrent studies. 

### 3.4 Toxicity Control For the purposes of the test, a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.

730PROJECT NUMBER: 0145/0215 PAGE 10 

### An amount of test material (66.6 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No.101 K3700) prior to dispersal in approximately 400 ml of culture medium with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. The test material/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and an aliquot (51.4 ml) of the sodium benzoate stock solution added. The volume was adjusted to 31itres to give a final concentration of 22.2 mg test materialll plus 17.1 mg sodium benzoatell, equivalent to a total of 20 mg carbonll. 3.5 Test Species A mixed population of activated sewage sludge micro-organisms was obtained on 30 March 2009 from the aeration stage ofthe Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK. which treats predominantly domestic sewage. 3.6 Procedure 3.6.1 Preparation of inoculum The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.3 gil prior to use.

• Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven

731PROJECT NUMBER: 0145/0215 PAGE 11 

### 3.6.2 Culture medium 

### The culture medium used in this study (see Appendix 1) was that recommended in the 

### DECO Guidelines. 

### 3.6.3 Preparation oftestsystem 

### The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution: a) A control, in duplicate, consisting of inoculated culture medium plus 100 mg silica gel. b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbonll. 

### c) The test material, in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final concentration of 10 mg carbonll. d) The test material plus the standard material in inoculated culture medium plus 100 mg silica gel to give a final concentration of 20 mg carbonll to act as a toxicity control (one vessel only). Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids 

### (ss)/1. The test was carried out in a temperature controlled room at approximately 21

### oC, in darkness. Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 39.1 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition ofculture medium. The culture vessels were sealed and CO2

### -free air bubbled through the solution at a rate ofapproximately 40 mllminute and stirred continuously by magnetic stirrer.



### 732PROJECT NUMBER: 0145/0215 PAGE 12 The C02-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules. The C02 produced by degradation was collected in two 

### 500 ml Dreschel bottles containing 

### 350 ml of 

### 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water. 

### 3.6.4 Sampling and analysis 

### 3.6.4.1 C02 analysis Samples (2 ml) were taken from the control, standard and test material first CO2 absorber vessels on Days 

### 0, 6, 

### 8, 10, 14, 21, 28 and 

### 29 and from the toxicity control first C02 absorber vessel on Days 

### 0, 2, 6, 8, 

### 10 and 

### 14. The second absorber vessel was sampled on Days 

### 0 and 

### 29 for the control, standard and test material and on Day 

### 0 for the toxicity control. All samples taken were analysed for CO2 immediately. On Day 

### 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganiC carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorbervessels on Day 

### 29. The samples were analysed for C02 using a Tekmar-Dohrmann Apollo 

### 9000 TOC analyser and a Shimadzu TOC-VcsH TOC analyser. Samples 

### (300 or 

### 50 IJI) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2C03). Each analysis was carried out in triplicate. 

### 3.6.4.2 Dissolvedorganic carbon (DOC) analysis Samples 

### (20 ml) were removed from the test material and toxicity control vessels on Day 

### 0 prior to the addition ofthe test material in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through Gelman 

### 0.45 IJm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.



### 733PROJECT NUMBER: 014510215 PAGE 13 

### DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature ofthe test material in water. On Days 0 and 28 samples (20 ml) were removed from the control and standard material vessels and filtered through Gelman 0.45 IJm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis. The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser and a Shimadzu TOC-VcsH TOC analyser. Samples (27 or 13**~I** 

### or 50 IJI) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (CaHsK04) and sodium carbonate (Na2C03) in deionised water. Each analysis was carried out in triplicate. 

### 3.6.4.3 pH 

### measurements The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a

## wrwpHIOxi 3401 pH and dissolved oxygen meter. 

### 3.7 Evaluation of Data 3.7.1 Calculation ofcarbon content The test material consists of two components which contribute to the total carbon content. The amount of carbon present in each component was calculated using the following equation and corrected for the amount ofeach component in the test material. NootCatoms xmol

# w: ofC x100% mol 

### wt oftest material

734PROJECT NUMBER: 0145/0215 PAGE 14 Component MolecularWeight Amount Present 

### (%) % Carbon C21H360aS4 

### 544.77 83 38.43 C17H3007S3 

### 442.61 14.3 

### 6.6 Total 

### 45.03 Therefore for a concentration of 

### 10 mg 

### C/I (a total of 

### 66.6 mg oftest material in 

### 3 litres) the total organic carbon present was 

### 30 mg C. The theoretical amount of carbon present in the standard material, sodium benzoate (CsH5COONa) was calculated as follows: No of Catoms x mol wt of C x 

### 100% mol wt of sodium benzoate 

### 7 

## x 12.011 

## x100

## =58.34% 144.11 Thus for a 

### 10 mg 

### C/I test concentration (a total of

### 51.4 mg of sodium benzoate in 

### 3 litres) the total organic carbon presentfor sodium benzoate was 

### 30 mg C. 

### 3.7.2 Percentage degradation The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThC02 ) produced is calculated by substituting the inorganic carbon values, given in Table 1, in the following equation: The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation.

Ol ThCO ( Ol ddt' ) mg IC in test flask - mg IC in control 1000l 10 

## = {a eg ra a 10n 

## = x {a 2 mg TOC astest material The percentage degradation from the results of the DOC analysis, see Table 4, is calculated from the equation below. Replicate values are corrected for the mean control value priorto calculation of percentage degradation.



### 735PROJECT NUMBER: 0145/0215 PAGE 15 Percentage degradation 

## =[1
_ rng DOC in testflask on Day

### 28]x 

### 100% mg DOC in testflask on Day

### 0 The total C02 evolution in the control vessels atthe end ofthe test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 

### 28 are meaned before substitution into the equation. 

### 10 Total C02 evolution 

# =mg IC in control x 0 C fOC x

__ 

### 1

__ 

### Yo 0 O 2 test volume



### . 100 1 

# =mg IC In control x -- x ­ 

### 27.29 3 3.7.3 Validation criteria The results ofthe degradation test are considered valid if in the same test the standard material yields**~** 

### 60% degradation by Day 

### 14. The test material may be considered to be readily biodegradable if**~** 

### 60% degradation is attained within 

### 28 days. This level of degradation must be reached within 

### 10 days of biodegradation exceeding 

### 10%. The toxicity control (test material and sodium benzoate) should attain**~** 25% degradation by Day 

### 14 for the test material to be considered as non-inhibitory. The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end ofthe test is less than 20%. The total CO2 evolution in the control vessels at the end of the test should not normally exceed 40 mgll medium. The IC content ofthe test material suspension in the mineral medium at the beginning of the test should be < 5% ofthe TC.

736PROJECT NUMBER: 014510215 PAGE 16 

### 4. ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, afterwhich instructions 

### will be sought as to further retention or disposal.

737PROJECT NUMBER: 014510215 PAGE 17 

### 5. RESULTS Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley 

### et aI, 2002). the test material was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test material in the test medium and to increase the surface area ofthe test material exposed to the test organisms. Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 

### 2 and the biodegradation curves are presented in Fjgure 1. Total and Inorganic Carbon values in the culture vessels on Day 

### 0 are given in Table 3, and the results ofthe Dissolved OrganiC Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values ofthe test preparations on Day 28 are given in Table 5. The total C02 evolution in the control vessels on Day 28 was 25.80 mgll and therefore satisfied the validation criterion given in the OECD Test Guidelines. The IC content of the test material suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines. The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines. Acidification ofthe test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved C02 present in the test vessels. Therefore any additional C02 detected in the Day 29 samples originated from dissolved CO2that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.



### 138PROJECT NUMBER: 0145/0215 PAGE 18 

### The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels with the exception of standard material Replicates R1 and R2. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 

### into the second absorber vessels occurred. The test material attained 70% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions ofOECD Guideline No 301 B. The toxicity control attained 55% degradation after 14 days thereby confirming that the test material was nottoxic to the sewage treatment micro-organisms used in the test. Sodium benzoate attained 84% degradation after 14 days and 86% degradation after 28 days thereby confirming the suitability ofthe inoculum and test conditions. Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 100% for Replicates R1 and 

### R2. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 

### evolution occurring. Observations made throughout the test period (see Table 6) showed the contents ofthe control vessels to be light brown slightly cloudy dispersions and the contents of the standard material vessels were light brown slightly cloudy dispersions with no undissolved standard material visible. The test material vessels were observed to contain cloudy dispersions with very small globules of test material on the surface. The toxicity control vessel contained a cloudy dispersion with very small globules of test material on the surface and no undissolved standard material visible. 

### 6. CONCLUSION 

### The test material attained 70% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of



### 739PROJECT NUMBER: 0145/0215 PAGE 19 

### the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable underthe strict terms and conditions ofOECD Guideline No 301 B. 

### 7. REFERENCES 

### Handley 

### J W, Mead C, Rausina G A, Waid L 

### J, Gee 

### J C and Herron S 

### J (2002) - The Use of Inert Carriers in Regulatory Biodegradation Tests of Low Density Poorly Watersoluble Substances, Chemosphere 

### 48 

### 529-34. International Standards Organisation (ISO). ISO 10634 (1995) Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium.



### 740PROJECT NUMBER: 0145/0215 PAGE 20 Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILITY; CO2 EVOLUTION TEST Table 1 Inorganic Carbon Values on Each Analysis Occasion

Control (mg IC) Day

8 10.66 10 11.40 14 14.62 16.22 28 21.06 21.17



### ... 29 20.37 1.85 20.15 1.63 Sodium Benzoate (mg IC)**~**

2 Abs 1 Abs2 1.40 1.05 30.74 34.14 31.14 36.23 33.94 35.57 34.77 38.S7 38.53 43.49 39.55 45.S1 44.13 47.09 1.63 44.98 1.63 Test Material (mg IC)

Rl R2 Abs 1 Abs2 Abs1 Abs2

0.93 1.28 1.05 1.75 8.35 6.26 10.27 10.61 15.14 14.33 19.72 15.39 25.61 20.29 36.73 34.14 41.10 41.66 41.08 1.74 Test Material plus Sodium

Benzoate Toxicity Control m IC

R1 Abs1 Abs2

1.40 1.05 20.77 36.44 43.00 44.01 46.81

R, - R2 

### = Replicates 1and 2 Abs =CO2 absorbervessels



### 741PROJECT NUMBER: 014510215 PAGE 21 Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILITY; CO2 EVOLUTION TEST 

### Table 2 Percentage Biodegradation Values Day % Degradation Sodium Benzoate % Degradation Test Material % Degradation Test Material plus Sodium Benzoate

Toxicity Control 

### 0 0 0 0 2 88 9 27 

### 6 83 9 48 

### 8 85 18 56 10 82 23 

### 56 14 84 31 

### 55 21 91 71 

# - 28 80 68 

# - 29' 

### 86 70 

## -

... Day 29 values corrected to include any carry-over ofCO2 detected in Absorber 2

742PROJECT NUMBER: 014510215 PAGE 22 Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILlTYj CO2 EVOLUTION TEST 

### Table 3 Total and Inorganic Carbon Values in the Culture Vessels on Day 0
Testvessel Total Carbon* (mg/l) Inorganic Carbon· (mg/l) IC Content (% ofTC) Sodium Benzoate 10 mg C/I R1 8.26 -1.14 0
Sodium Benzoate 10 mg C/I R2 10.34 0.24 2
Test Material 10 mg C/I R, 9.69** -1.16 0
Test Material 10 mg CII R2 9.68** -1.15 0 Test Material plus Sodium Benzoate Toxicity Control 20 mg CII 20.58­ 0.53 3

R1 - R2 = Replicates 1 and 2 * Corrected for control values. Negative values are due to measured concentrations being less than control values **Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution ofthe test material and sodium benzoate where applicable

743PROJECT NUMBER: 0145/0215 PAGE 23 Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILITY; CO2 EVOLUTION TEST 

### Table 4 Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 28

TestVessel mg CII DOC· Concentration Day 0 Day 28 % of Nominal % of Initial Carbon mg CII Carbon Content Concentration % Degradation
Sodium Benzoate 10 mg CII R1 9.39 94 <Control 0 100
Sodium Benzoate 10 mg CII R2 10.09 101 <Control 0 100

R1 - R2 

# =Replicates 1 and 2 * Corrected for control values

744