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 · · · · ·, · : Figure 3-19 

· ·· · · · · · · ··· · 

: 

·

, 

, ·· ·.......... -.........----.. "' ... -.---... ---.....-..----~.--~--.--..--­

545 ·3 Additional dala 3­
2 

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 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 to Peak F by disulfide formation. 

Additionally, it was suggested that Peak A was stereoisomer of Peak B. 

Also, it was suggested that PEMB changed to Peak D by elimination of hydrogen 

sulfide, Peak C by both disulfide formation and elimination of hydrogen sulfide, and 

Peak E and F by disulfide formation caused by oxidation of thiol 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 

13.07 NL:

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 3­
94746 

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 of peak 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 


:sf200000 .. 


100000­

8.11 Mass chromatogram ofmlz =488 

Position of peak 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 of peak 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 of Peak 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 of peak 2 I 

2200000· 

2000000 

1800000 

1600000 


i 1400000 
 c: 

~1200000 

1000000 


800000 
 500.28 

600000 

400000. 323.10 


200000 l34 425.19
 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 of peak 2 

/ 

Mass chromatogram 

ofmlz =562 

Position of peak 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 of Peak 2 and Mass chromatogram (standard solution) 

549 -7 Additional data 3­
94746 

: 

2.33 

l.~i A\ 

Test solution 

RT: 0.00 - 30.00 

::=1' 
 7000000­

8500000­

60000001 

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 TIC chromatogram [5.0 mglL (at time that all test organisms died)] 

550 -8 Additional data 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 of peak 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 

7.40 NL:

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~ ~2000oo peak A 

o L...;,;,;.;.65;.::'_......:.:5.:;:::29:......",J 

150000 

i'100000 

~50000 

0- r""i"'1 I 1 

o 

7.40. 7,.61 

pOSit~ peak A 

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) 

Additional figure 3-5 Mass spectrum of Peak Aand Mass chromatogram [5.0 mgfL (at time that all test organisms died)] 

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 of peak B . i 

650000 


600000 


550000 

500000 


450000. 


400000 


1£ 

i 350000 

'E-300000 


250000 


200000 


150000 	 321.06 4.D7.24 

100000 	 I 441..,24

4!U).25 576.31 5000: ~17~'~1 , 	 rI ~4~'~r,2~~;47~r l~i~rT r, 4~3j~ ~, 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 of mlz =458 

" Position of peak 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 

of mlz =321 

Position of peak B 

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 of mlz =486 mIz= 

485.74­

486.74 F: 

us 

94746­

Position of peak B 090306­

H93hA 

!J.2 12.~7 14.97 1§.98 21.60 26.45 

Additional figure 3·6 Mass spectrum of Peak Band Mass chromatogram 15.0 mgIL (at time that all test organisms died)] 

552 -1 0 Additional data 3­
94746 

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 of peak 1 

488.27 

RT: 0.00 - 30.00 

600000 

:ir4oo000 I

c: -200000 

0-­

o 

200000 

I~= 60000 

o 

8.05 NL:

6.84E5 

Mass chromatogram of mlz =488 	 mIz= 

487.77­

488.nF: 

Position of peak 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 chromatogram of mlz =323 322.60­

459.75­

460.75F: 

Position of peak 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'*

2.9.5 6.1M ;Jj
 !.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 of Peak 1and Mass chromatogram [5.0 mgIL (allime thai alilesl organisms died)J 

553 -11 Additional data 3­
94746 

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 of peak 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 l .5.l?1.35 . 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..( ;'!' ~ Y11'+jIL.~.\li1l'i ~,\II ,1!~ .j J y.'1 

150 200 250 300 350 400 450 500 550 600 

mlz 

RY0,00 - 30.00 

10.42 NL: 

160000 

Position of peak 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 ofm/z =526 17.67 	 ~.~2E4 

10.42 

Position of peak 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 spectrum of Peak Cand Mass chromatogram [5.0 mgIL (at time tbat all test organisms died)] 

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

Ii 150000 I• 

.5 

100000 

50000 

556.29 

Mass spectrum of peak 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 of mlz =556 3.23E5 

rnIz= 

555.79­ Pilion of peak 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 of mlz =528 

7tion of peak 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) 

Additional figure 3-9 Mass spectrum of Peak Dand Mass cbromatogram [5.0 m!VL (at time that allIes! organisms died)] 

555 

-13 Additional data 3­

20000 
94746 

Test solution 

94746-090306-H93hA it733 RT: 12.59 AV: 1 S8: 39 14.27-14.92 NL: 4.35E5 

T: +C Q1 MS { 150.00-600.001 

588.23 

I Mass spectrum of peak 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 ,7ilion of peak E 94746­ ~250000 , c: 090306­

~2000OO H93hA

-'50000' 

100000 

50000 2.92 

o 2..'\Q ,54 6,89 9.43 	 k:o~.,.. .~"! !!'SII_...,"I,! 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 of peak E 	 MS 

94746­

090306­

H93hA 

I 

30 

Time (min) 

Additional figure 3,10 Mass speclrum of Peak Eand Mass chromatogram [5.0 mgIL (at time that all test organisms died)J 

556 

-14 Additional 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 of peak 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 ' / :'.76 F: 

~ 40.0000. I' 94746­ -	 090306­

2000.: L-2'~!__~'~lI. ____ ~~'~ ~S:-) \,,3.~1 _'-~:~___~2..:94 ~~:~~__ ~~LH93hA 

12.85 	 NL: 

'1' 12..98 	 nOES 

60.0000- Mass chromatogram I =72­

.~ f Iz 560' ',Position of peak F 560.72 F; 

S 	 ~ 40000.0."[' m = f / we MS

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 of peak F 3

M2

s

'59F: 

~ 94746­

50.000 . I 	 090306­ i 	

1805 ! ~ 1:-

/ 

.. .., 1908 H93hA 

0. 	 ""1"""I'i"·,'· ...3~_~~ ...r-.c i 1 I I I j , , , ~-""-L I , , I I ~i~1 "'I,~, ~~; 0. 5 10. 15 20 2S 30 

l1me(mIn) 

Additional figure 3-11 Mass spectrum of Peak Fand Mass chromatogram [5.0 mg/L (at time thai an test organisms 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 of peak 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: 

of mlz =590 

12..8§ 

Mass chromatogram 

of mlz =562 

4.59E5 

mJz=

Position of peak 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= 

Position of peak 2 561.75­

/' 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 of peak 2 MS 

~ 94746­ 100000-: 

090306­

50000. 29", H93hA 2.f!! •. 7.Q2L11 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 ofPeak 2and Mass chromatogram [5.0 mgIL (at time that all test organisms 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 of Peak 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 of Peak B 

559 -17 Additional data 3­
••• 

94746 


mlz Detected ion 

488 [M+NH4+HCOOH-HzOr 

460 [M+NH4r 

323 [M+H-C4HoOS-H20r 

Additional figure 3-15 Estimated structure of Peak 1 

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

. · · , 

.. · · .. · . · . · . · . · . · 

· 

: . .

· 

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

mlz Detected ion 

554 [M+NH4+HCOOH-H20]~ 

526 [M+NH4t 

Additional figure 3-16 Estimated structure of Peak C 

560 -18 Additional data )­
94746 


........ --.~----.-- ..----.---.---.------..-.. 


, 

,

, 

, 

,

· 


· 


· 


· 


· 

· 

, 

, 

,

, 

· 
 · 


m/z Detected ion 

556 [M+NH4+HCOOH-H20r 

528 [M+NH4r 

Additional figure 3-17 Estimated structure of Peak D 

~--.-- ...------.-------.. -... ----.--.--.-~ · . · · 

· .. . · . · , · , · · ., · . 

· 

· · 

· 

· 

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

mlz Detected ion 

586 [M+NH4+HCOOH.HzOr 

558 [M+NH4t 

Additional figure 3-18 Estimated structure of Peak E 

561 -19 Additional data 3­
94746 


m/z Detected ion 

588 [M+NH4+HCOOH-HzOr 

560 [M+NlLt 

321 [M+H-2C4lIt;OS-H20r 

Additional figure 3-19 Estimated structure of Peak F 

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 of Peak 2 

562 -20 Additional data 3­
Receipt number 662-08-E-4745 


Study number 94745 


FINAL REPORT 


A 48-hour Acute Immobilization Study of Karenz MT (Mixture of PEMB 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 of the Final Report (Study No. 94745, issued on April 10, 2009). 

JW7(z ($, .;..009 Date 

Ifa)u/u)' A/l'ttat--\ Study Director 

Makiko Anai 

564 
94745 

GLPSTATEMENT 


Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor Show a 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 of the 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 of inspection 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 of final 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 of test item 

Appendix 3 Calibration curve and chromatogram 

Additional data 1 Results of preliminary studies 

Additional data 2 Calculation results of concentrations of each component 

Additional data 3 Qualitative analysis of converted products 

567 

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Title 

Sponsor 

Test facility 

Objective 

Test method 

Applied GLP 

A 48-hour Acute Immobilization Study of Karenz MT (Mixture 


of PEMB 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 Principles 	of Good Laboratory Practice" (November 

26, 1997, ENV/MC/CHEM (98)17). 

568 -1­
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Dates 

Study 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 of Manufacture, 

etc. of Chemical Substances". Treatment of raw data and materials, etc. 

after the storage period will be discussed with sponsor. 

569 -2­
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Personnel 

Study Director 

Study personal 

Bio]ogy 

Ana]ytical chemistry 

Approval of final 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 of organisms Twenty daphnids/test level 

(five daphnids/test vessel) 

(6) Dilution water Dechlorinated tap water 

(7) Type of test Semi-static (renewal at 24 hours after the 

start of the exposure) 

(8) Preparation of test 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 of white fluorescent lamp, 

16-hour light/8-hour dark 

(13) Feeding No feeding 

(14) Aeration No aeration 

(15) Analysis of concentration of test item in test solution 

HPLC analysis 

(at the start of the exposure, before and after 

the renewal, and the end of the exposure) 

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Results 

(l)Solubility in dilution water (20:tl°C) 

10 mg/L (measured value in preliminary study) 

(2)Concentration of test item in test solution (percentage of the initial concentration) 

At the start of the exposure and after the renewal 

3.8 to 11 mg/L 

At the end of the 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% confidence interval: 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 on geometric mean ofmeasured concentration.] 

(6)Reference value: 48-hour ELso (Median Effective Loading); 


88 mg/L (loading rate) 


572 -5­
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1. Test item 

t···~·-·-·-~··~-----·····--·--······-·--·--·------···· ..----.......-.---.......---....----...........--.....-_._ ......_.....
 . . .

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2. Test sample 

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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 S 10 

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 
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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 of the test solution. 

(2) Test equipment 

Water bath Plastic tank 

Warming/cooling unit (Type HCA250, Sato craft) 

3.4 Test conditions 

(1) Conditions of exposure 

(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
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(f) 	 The number of organisms 


Twenty daphnids/test level (five daphnids/test vessel) 


(g) 	 Volume of test solution 


400 mL/test level (100 mLitest vessel) 


(2) Conditions of test 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 of white fluorescent lamp, 16-hour light/8-hour dark 

(e) 	 Feeding 


Test organisms were not fed during the exposure. 


3.5 Preparation of test 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 of test 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
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(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. 

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3.7 	Calculating method of ECso*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 of test 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 of tested population during exposure. 

*4 	 ELso (Median Effective Loading) is the loading at which causes 50% 

immobility of tested population during exposure. 

3.8 	 Validity of the 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 of water. 

(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) 

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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 

of the test (more than 3 mg/L at the end of exposure). 

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(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 of the 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
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Table 1 Immobility 

Measured Immobility (%)

Concentration"6 48 hour 

(mg/L) 

24 hour 

Replicate Test level 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 (-._.......__...__...
 5.3 "'--- .._..._-...._......................- o 
 40

C 0 20

1--,-" ......- ...----....... 


D 0 40 

A 0 o _._- .._...,........._.._._-...... _­

B 0 20 6.4 f-_...... f--...- ....... ............- ...-.. 
 o ...._.... _...-.... _.­ 10 

C 0 20 

t-'-......... ---"'_.__._--.._-­ t--------......­ D 0 o 

A 0 80 .,......_,_..__......_. B 0 .--..... I-..--... ..,..--.....-.... 

20 8.6 ............,..... t-.....-.........................-..............-.. 
 o 40

C 0 o .........om.... _-..........._,.""............ """' ........_ ...... 
 --_._..........,--­

D 0 60 

A 20 80 ....... ~ ._-..._..._._--....­ B 20 40 ....-.... _..........._..__.__.­ 9.5 20 t-'-...._-­ 60 

C 20 60

.-.......;. ..... ..._-_.. 
 D 20 60 

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

measured concentration) 

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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 

Before At the end 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 

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Table 3·2 pH of test solutions 

Measured 24 hours 

Concentra tion At the end 

(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 

Concentra tion 

(mg/L) 

At the start 

24 hours 

Before At the end 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 

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Table 4-1 ECso to Daphnia magna 

95% confidence interval Statistical procedure Exposure (mg/L) ECso (mg/L) used for determ ination duration (Slope of the

of ECso dose-response curve) 

- 24-hour >9.5 - ( - ) 

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

Not obtained 

Table 4-2 ELso'7 to Daphnia magna 

95% confidence interval Statistical procedure Exposure (mg/L) ELso (mg/L) used for determination duration (Slope of the

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
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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 -19­
94745 


Appendix 1 


Chemical characteristics of dilution water 

587 

94745 

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 - Organic phosphorous 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 of test 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. 

590 

94745 


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 of the test item concentrations in the test solutions. 

Recovery rate for pretreatment 


87%, 89% average 88% 


592 -3 Appeodix 2­
94745 

6. 	 Results of measurement 

The results of the measured concentrations of the test item in the test 

solution are shown below. 

Appendix table 2-1 Measured concentrations of test item in test solutions 

Nominal 

concentration 

(mg/L) 

Measured concentration (mglL) 

(Percentage of measured concentration versus that at each 

preparation %) 

At the 

start 

24 hours 

At the end Geometric 

Before mean 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 of test item for analysis by HPLC 

595 -1 Appendix 3­
94745 

Study No. 94745 

!!:.!h_50 miLL ___.. __.. -------.--­ PKHO 

1 

2 

Operating Date : Mar. 11.2009 

Time Area Helcllt Cone. 

(min) (j1 Y'6lIe) (/I V) (\) 

8.72 61716 5211 14.67 

15.28 35!lO54 39287 85.33 

Total 420770 100.00 

t 

--" 

. 

0.0 

A 

I ~. 11;'0 zO.o 30.0 Operator ; Hi royuki 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) (p V' Bec) (/I V) ('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!;:(07yk : T034\.1.14 

<I> 

Appendix figure 3-2-1 HPLC chromatograms at start of exposure 

596 -2 Appendill3­
94745 

Study No. 94745 

94145/0h/46 mg/.!. __ .~- --" ­ -- ---.. -----~-

PKNO 

1 

2 

Operating Dab: Mar. 11.2009 

TIme Area Height Cone. 

(min) (IIY·aec) (II V) ,,) 

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 : Hi royuk i TO(lIa 

94145LOhi59 mg!l --~-.-

PKNO 

1 

2 

Operat i nl Date ; Mlr. 11.2009 

Tilllll Area HeiJht COne. 

(min) (p V'sec) (Il Y) (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 : Hi rDYuk i T ogawa 

94745/0bLILlI]gLL - ..--~--~..~ 

PllNO 

1 

2 

Operat iiii Date : liar. I\. 2009 

Tima AreB Height COne. 

(min) (Il V'sec) (Il V) (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)'uk i Tala". 

Fig 

Date Mar. 12.2009 Name: Hi\,"o/""~{ T"'J/l1!?4

• 

Appendix figure 3-2-2 HPLC chromatograms at start of exposure 

597 -3 Appendix 3­
94745 


Study No. 94745 

24745LOhL1OO I1IiLL 	 Operating Date: IIlIr.1l.2009 

PKIIO 	 TIllie Area Height Cone. 

Ullin) (II V·;ee) (ll'/) ('4) 

1 8.70 381039 33507 94.84 

2 	 15.21 20735 2.2.41 5.16 

Total 401774 100.00 

Operator: HI rOl'ukl Togawa 

Fig 

Appendix figure 3-2-3 HPLC chromatogram at start of exposure 

598 -4 AppeDdix 3­
94745 

Study No. 94745 

Operatin, Date : Mar. 12. 2000 /ltd. 50 mgtl ."----~ 

PKNO Time Area Height Cane. 

Unin) (/I V·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'uk i Togua 

94745/24hLQontrQUlMslil (jperatiTlf Oate : Mar. 12. 2009 -...... """-'-'~-'" "­

PKMl 	 Time Area Hei,ht Cone. 

emi n) (/I V·ne) (f! V) (I) 

n.d. n.d. 

_..:A..__ ~ ___t____._~_

: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 : Hi royuki 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 

94745/24h/46 mg/LLML~___ Operati ng Dlte : liar. 13. 2009 .. ---_.._­ ~,,-"--

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 	 20.0 	 )0.0 Operator: Hiroyuki yo,aWl 

Operat i 1'1& 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 : Hi royuki Tocawl 

Operatinc Date: Mar. 13, 2009 947A?L?4!:l/'ZIl1!gL!JJl~WI .­

PKNO 	 Time Area Heiaht Conc. 

(min) (j.( V-sec) (II V) (') 

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 Appendix 3­
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 3­
94745 


Additional data 1 


Results of preliminary 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 of test 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 of measured concentration stirred for 24 hours and 

48 hours. 

603 -1 Additional data 1­
94745 


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) Result 

Nominal 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 of organisms: 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 of the initial 

Nominal measured-concentration, %)

concen tration 

(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 of each component 

606 

94745 


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 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 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 of PEMB (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 

Before mean 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 of converted 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 5 jiL 

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 of mass 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 Peak on TIC Estimated structure Additional molecular No. chromatogram and molecular fomula figure No. weight (min) ... __ ... "' ..... __ ......... _ ..... a. ......... .. ................................... 


· 

·· · · 

· · 

· · · 

· ·· Figure 3-5 A · 7.6 · · · · Figure 3-9 · · · · 

· · 


· 
 · 

· 


· 


· · 

· · · Figure 3-6 B 7.8 Figure 3-10 

· 

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


611 -2 Additional data 3­
94745 


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 

13.22 Nt:

4500000 4.56ES

TICF:+c 

Q1MS[ 

400000(}­ 150.00­

600.00) MS 

94745­

3500000-· 090312-801

1 

~ f25OCXro. 8.21 

.52000000 

11.25 

.18.82 23~ 25. __4499 .25..25 .. ,88 

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 -4 Additional data 3­
94745 

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 of peak 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= 

Mass 459.79-460.79 F: chromatogram +cQ1MS[ 

of m/z = 460 150.0().600.00] 

MS 

94745-{)90312­

Position of peak 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 of Peak 1 and Mass chromatogram (standard solution) 

614 -5 Additional data 3­
94745 

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

110000D I, 

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': 

t1I 800000. 

600000 

400000 

200000':: 

1127 

3.40 6.89 10.~ Il 

13.22 . NL: 

590.30 

NL: 

2.73E5 

Mass chromatogram mlz= 

589.80-590.80 F: 

of m z I = 590 +cQ1MS(

150.00-600.00] 

MS 

94745-090312­ Position of peak 2 S01 / 

1oJ3.89 1L31 21,,48 24.30 2§.82 

1.66E6 

mlzMass chromatogram 561.71-562.77 F: 

+cQ1MS[ of mlz =562 150.00-600.00] 

MS 

94745-090312­

Position of peak 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 11600000

-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 -7 Additional 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 

300000 /. Mass chromatogram of mlz =486 mIz=

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 of mlz =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 of mlz =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) 

Additional figure 3-5 Mass spectrum of Peak Aand Mass chromatogram 1100 mglL (at 24 hours before renewal)] 

617 ·8 Additional dala 3­
94745 


Test solution 

94745-090312-H24hA-oldtt453 RT: irs' A\i:1"SB: 34 6.47-7.D4'NL: 3.41E5 '._--' ..... 

T: + c Q1MS [ 150.00-600.00] 

486.29 340000· 

Mass 458.26 spectrum of peak B 

280000 


2I0000. 


240000 


220000: 


200000· 


.180000 1. 1eoooo

140000 

12000D 

1 

80000 321.15 4t1.28

!1U.28 

eoooo- 40000 	 J 
 441.,33

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 of peak B 

O.-,--~1.S....,~=_..;;;t..:...~_-= ~§.::;..;,~_2__ 1.;..;;3.;....1..;..O._--,-17..;;..4.:.;:5--.:..19... ""1.:.,;;80....-;;2;;.;,3,;.;;:.25;;;......_ 26.40

1.18 	 NL: 

7n 	 NL: 

7.S8E4 

Hi] Mass chromatogram of m/z =321 mIz'" 

320.65-321.65 

F: MS 

• • 	 94745-090312­ PositIOn of peak 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 of peak B H24hA-old 

.&•.33 14.65 20.01 23.27 01 

Additional figure 3-6 Mass spectrum of Peak 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 ,-', T r- ,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 of peak 1 

323.11 443...30 

341.17 . 

191.25 221.19 268.29 r-~2.23 425", 1 25 1

8.17 _M5 

7.5.£t 

~.31 

488.30 .30 

572.52 kT~ 'T r I I I -. f~r ,- '1

500 550 600 

NL: 

2.2SES 

Mass chromatogram of mlz =488 m~80

488.80 F: 

MS 

Position of peak 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 of mlz =460 ~~~80

480.80 F: 

MS 

94745­

Position of peak 1 	 090312­

H24hA-okl 

o h 1r·", 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: 1 and Mass chromatogram [100 mgIL (at 24 hours before renewal)] 

619 -10 Additional data 3­
94745 


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 of peak 2 


180000 


160000 


590.33 

12bOOO . 

I

i 100000 

80000 

60000 

40000 

RT: 0:00: 30.00­

13.22 NL: 

140000 


120000 


100000­

f 80000·" c 

g 60000 

40000-:­

20000 11.23

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 of peak 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 of Peak 2 and Mass chromatogram [100 mgIL (at 24 hours before renewal)] 

620 -11 Addilional data 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-C4H60S-H20r 

Additional figure 3-10 Estimated structure of Peak B 

621 -12 Additional dala 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-H20 t 

562 [M+NH4t 

323 [M+H-2C4H60S-HzOr 

Additional figure 3-12 Estimated structure of Peak 2 

622 -13 Additional data 3­
CERI 


FINAL REPORT 


Algae Growth Inhibition Study of Karenz MT (Mixture of PEMB and PE3ME) 

with Pseudokirchneriella subcapitata 

April 14,2009 

Kurume Laboratory 

Chemicals Evaluation and Research Institute, Japan 

623 
94744 


STATEMENT 


Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor 	 Showa Denko K.K. 

Title 	 Algae Growth Inhibition Study of Karenz MT (Mixture of 

PEMB and PE3ME) with Pseudokirchneriella subcapitata 

Study number 	 94744 

I, the undersigned, hereby declare that this report provides a correct English translation of 

the Final Report (Study No. 94744, issued on April 14. 2(09). 

Date J UtiV' 1'1 'leG ~ 

Study Director 

MikaOno 

624 
94744 


GLPSTATEMENT 


Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor 	 Showa Denko 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 of the 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 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 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor Showa Denko KK. 

Title Algae Growth Inhibition Study of Karenz MT (Mixture of 

PEMB and PE3ME) with Pseudoldrchneriella subcapitata 

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 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. 

Date of report to 


Item of inspection I audit 
 Date of inspection I audit Study Director and 

Test Facility Management 

Study plan draft March 24, 2009 March 24, 2009 

Study plan March 26, 2009 March 26, 2009 


Start of the 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 of Quality Assurance Unit 

Apri1IS, 2009 

Signed in original 

Keiji Shiraishi 

626 
94744 

CONTENTS 

Page 


Title .............................................................. · ..····..················ .. · ..··..·.. · .... ······ ..··1 


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


Test facility ...... ··· .... ··....·..· ................................................................................ ] 


Objective ........................................................................................................... 1 


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


Applied GLP ..................................................................................................... 1 


Dates ............................................................................ · ...... ··...... · ..···.. ······..···..·2 


Storage of test item, raw data, etc ..................................................................'" 2 


Personnel··..···..· .... ······ ...................................................................................... 3 


Approval of final 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 of test results · .... ···· .. ······ .. · ..··..···.. ··..····· .... · .... 15 

Tables and figures 

Table 1 pH of test solutions 

Table 2 Culture temperature and light intensity in incubator 

Table 3 Value of biomass at each time 

Table 4 Growth rate and growth inhibition 

Table 5 ErCso and NOEC 

Table 6 Result of statistical analysis on growth rate between control level and 

exposure levels 

Table 7 Variation of growth rates in control 

Figure 1 Concentration-response curve 

Figure 2 Growth curve 

Appendix 1 Composition of medium 

Appendix 2 Analytical method and measured concentration of test item 

Appendix 3 Calibration curve and chromatogram 

Additional data 1 Results of preliminary studies 

Additional data 2 Qualitative analysis of converted 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 of PEMB and PE3ME) on growth of algae. 

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 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, 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 of the Environment) 

(2) 	 "OECD Principles of Good Laboratory Practice (November 26, 1997, 

ENV/MC/CHEM (98)17)". 

628 -1­
94744 


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 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 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 -2­
94744 


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 of final 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 of PEMB 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 of test 	 Incubation with shaking (approximately 100 rpm) 

(6) Preparation of test 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 of test solution 600 mL / control level (100 mL / test vessel) 

(Two test vessels were additionally prepared for 

analytical chemistry) 

300 mL / exposure level (100 mL / 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 of cell growth Chlorophyll fluorescence 

(12) Analysis of concentration of test item in test solution 

HPLC analysis 

(at the start, after 24 hours, after 48 hours and end of 

the exposure) 

631 -4­
94744 


Results 

(1) Solubility in medium (23 ± 1°C) 

1.3 mglL (measured value in preliminary study) 

(2) Concentration of test item in test solution 


[Percent of measured concentration versus that at the start of the exposure J 


At the start of the exposure 


Below the determination limit « 0.11 mglL) to 1.9 mgIL 


At the after 24 hours 

Below the determination limit « 0.11 mgIL) to 0.49 mglL 

[25% (in the stock solution content 100% test level)] 

At the after 48 hours 

Below the determination limit « 0.11 mgIL) to 0.21 mgIL 

[11 % (in the stock solution content 100% test level)] 

At the end of the 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 -5­
94744 


1. Test item :._._ •• ___ ~ •• ___ •••••• _____ ~_ •••• ~ •• __ •• _______ ••••••••_. __ ••• _ •••••• __ • ___ ••••• _. _____ •• _ ••• __ .~ __ •••• ___ ••••••• a ____ ••••••••

o : 

633 -6­
94744 


2. Test sample 

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

634 -7­
94744 


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 lR 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 

Pseu.dokirchneriella su.bcapitata (ATCC 22662) 

(The old scientific name "Selenastrum capricornutum") 

(2) 	 Reason for selection of species 

Species recommended in 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) of potassium dichromate was 0.80 mglL. This value was within the normal 

range of the 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 Culture medium 

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 -8­
94744 


3.3 Test apparatus and equipment 

(1) Test apparatus 

Test vessel 	 : Sterilized 500 mL Erlenmeyer flask 

(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 of exposure 

(a) Type of test 

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 100 rpm) during the exposure. 

(b) 	 Exposure duration 

72 hours 

(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. The results of preliminary 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 of approximately about 104 cells/mL. 

(g) 	 Operation 


All operations were carried out under sterile conditions. 


(h) 	 Volume of test 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 of test 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 light with wavelength range of 400 to 700 nm. 

3.5 Preparation of test 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 of the exposure. 

(2) Appearance of test 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 of the test solution was measured at the start and end of the 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 of the 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 of the 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. 

-11­ 638 
94744 


3.7 Treatment of results 

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 of concentration-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 

comparing with control values on growth rate. 

Comparison of growth rates 

The specific growth rate for a specific period was calculated as the logarithmic 

increase in biomass according to the following formula: 

InN i-InN i 

,U i-j tr t i 

where 

fl.i-] = Specific growth rate from time itoj (normally ((1) 

Ni =Value of chlorophyll fluorescence (relative unit) at ri. Set value of chlorophyU 

fluorescence (relative unit) was used at the start of the exposure (to). 

Nj =Value of chlorophyll fluorescence (relative unit) at tj 

ti = Time of i

lh measurement after beginning of test (d) 

tj =Time ofl measurement after beginning of test (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 of validity of the 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 -12­
94744 


(2) 	 Estimation of ECso 

The ECso was estimated as "> the highest test concentration" since no less 

than 50% of inhibition rate was not obtained within the exposure levels. The EC50 

was denoted as ErCso based on growth rate. 

(3) 	 Estimation of No 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 of the 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 of variation 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 of numerical values 

Values were rounded off in accordance with 1IS Z 8401 rule B, 1999. 

(JIS ; Japanese Industrial Standards) 

640 -13­
94744 


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 of test solution and measurement of water quality variables 

(1) Appearance of test solution 

At the start of the exposure, test solutions of exposure 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 of test vessel around surface of test solution. 

The solutions of controls were colorless and clear at the start of the exposure, 

and those at the end of the exposure were green due to the algae growth. 

(2) 	 Water quality and environmental conditions 

The measured values of pH 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 of pH were 7.7 to 7.9. Culture temperature in incubator 

ranged from 22.2 to 22.8°C and light intensity was 92 to 98/1E/m2

/s. 

(3) 	 Concentration of the 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 of the 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 of the 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 of exposure in the stock solution content 100% exposure level, respectively. 

Values of biomass 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 shown in Figure 1. 

ErCso of the test item based on the growth rate was> OAO mglL (95% confidence 

intervals: not calculated). 

-14­ 641 
94744 


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/L exposure 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 of cells was the same as the control. In 

the control, the condition of cells 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 of the test 

(1) Growth of the 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 of at least 16 times at 72 hours 

after the start of the exposure. 

(2) The specific growth rates of section-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 of variation in the control must not exceed 35%. 

(3) The specific growth 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 order to estimate the effect on the test organisms under 

the solubility of the test item in medium. As the result, the ErCso and NOEC of the test 

item were> OAO mgIL and 0.021 mgIL (reference value), respectively. Though the 

concentrations of the test item in the test solutions were decreased during the exposure, 

the concentration of stock solution content 100% exposure level (1.9 mg/L) was similar 

to solubility (1.3 mg/L; stirred 48 hours, measured concentration in preliminary study) of 

test item in medium, therefore it was considered that the effect of test 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 of the 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 of test results 

There were no factors which might have affected the reliability of the test. 

642 -15­
94744 


Table 1 pH of test 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 of the measured concentrations 

(also expressed as measured concentration in the following tables) 

Table 2 Culture temperature and light intensity in incubator 

Time At the start 1-day 2-day At the end 

Culture temperature ceq 22.6 22.2 22.2 22.8 

Ught intensity (pE/m7./s) 92 98 96 92 

643 -16­
94744 


Table 3 Value of biomass 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 of pre-culture 

The minimum cell growth in control (biomass at the end of exposure 1biomass at 

the start of exposure) 

160/3.9 =41 

-17­ 644 
----

--

--

--

94744 

Table 4 Growth rate and growth inhibition 

Inhibition 

concentration 

Measured Growth

rate 

(mw'L) 

No. rate

(%) 

A 

(0-3d) 

1.3 - B 1.3 - C 1.3 - D 1.2 - Control 

E 1.2 -

F 1.3 - -------..._-­

Mean ------~..--1.3 , 

- S.D. 0.020 " 

A 1.3 -0.22

B 1.3 -3.7 0.0067 


(reference 
 1.3 -3.2 

value) 

C 

Mean 1.3 -2.4

1.9 

A 

S.D. 0.024

1.2 2.9

B 1.2 2.7 0.021 


(reference 
 2.3 

value) 

C 1.2

Mean 1.2 2.6 

S.D. 0.0033 0.26

A 1.2 4.8

B 1.2 4.1

0.067 1.2 2.6 --C -----f-------­

Mean 1.2 3.8

1.2 

A 

S.D. 0.015 

1.1 10 

B 1.2 8.7

0.18 C 1.1 11

,-­

Mean 1.1 10 

S.D. 0.015 1.2

A 1.1 16

B 1.1 15

17 

Mean 

0.40 C 1.1

1.1 16

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 of statistical analysis on growth rate between control level 

and exposure levels 

Measured 

concentration 

(mg/L) 

Statistical 

analysis Statistical procedure 

0.0067 

(reference value) n.s. 

Bartlett's test. 

One-way ANOVA. 

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 -19­
94744 

Table 7 Variation of growth rates in control 

< Variation for section~bv-section specific growth rates in the 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 of average specific growth rates in replicate controls> 

0-3day 

Mean 1.3 

Standard deviation 0.020 

Coefficient of variation (%) 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) 

0.01 0.1 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 of medium 

650 

94744 

Composition of OECD medium 

[Guideline 201 (March 23, 2006)] 

Nutrient salts Amount 

H3BO] 0.185 mg 

MnCh' 4H20 0.415 mg 

ZnCb 0.003 mg 

FeCh' 6H20 0.064 mg 

Na2EDTA' 2H20 0.1 mg 

CoCh'6H2O 0.0015 mg 

Na2Mo04' 2H20 0.007 mg 

CuCh' 2H20 0.00001 mg 

CaCh' 2H20 18 mg 

NH4Cl 15 mg 

KH2P04 1.6 mg 

NaHC03 50 mg 

MgCh'6H20 12 mg 

MgS04' 7H20 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 of test item 

652 

94744 


1. 	 Pretreatment of test 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 100 mL I 


"'-Sodium chloride 30 g (electronic even balance) 

...-Chloroform 30 mL (graduated cylinder) 

• Shaking (10 min) 1.--------1 

IWater 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 (pasteur pipette) 

• Ultrasonic irradiation (ultrasonic cleaner, about 30 sec) 

• Filling up to 2 mL [Acetonitrile I water"l (1/1 v/v), 

volumetric flask J 

I Sample for HPLC analysis .1 

*1 Tap water was treated with ultrapure water system. 

653 -1 Appendix 2­
94744 


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 

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 210 run 

Injection volume 10,uL 

Detector output 2V/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 mgIL solution of the test item. The solution 

was di1uted with acetonitrile 1water*l (111 v/v) to prepare 50 mgIL of standard 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 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 medium according to pretreatment of test 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 of the 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 of the test item concentrations 

in the test solutions. 

Recovery rate for pretreatment 


97%, 98% average 97% 


655 -3 Appendix 2­
94744 


6. 	 Results of measurement 

The results of the measured concentrations of the test item in the test solution arc 

shown below. 

Appendix table 2-1 Measured concentrations of test item in test solutions 

Stock solution 

(%) 

Measured concentration (mglL) 

(Percentage of measured concentration versus that at the start %) 

At the start 24 hours 48 hours At the end Geometric 

mean 

Control 

1.0 

n.d. 

n.d. n.d. 

n.d. 

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, half the determination limit of 

the test item (0.055 mglL) was adopted for the calculation of geometric mean, because 

the test item was not detected and the detection limit (0.15 mgIL) was higher than half 

the determination limit of the test item. 

*3 	 In accordance with OECD Guidance Document No.23, half the determination limit of 

the test item (0.055 mg/L) was adopted for the calculation of geometric 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 of at the start, conc], .. " concn 

=The concentration of at the end 

656 -4 Appendix 2­
94744 


Appendix 3 


Calibration curve and chromatogram 

657 

o 

94744 


5000000 

o 	 250 soo

Concentration (mgtL) 

y =8339x 


r = 1.00 


Concentration Peak area 

Appendix figure 3-1 Calibration curve of test item for analysis by HPLC 

658 -1 Appendix 3­
94744 


Study No. 94144 

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

lo,n 2i.o ';,0 

PKHO 

1 

2 

Operat i 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 i ng Date : Mar, 30. 2009 

Tillie Area tleirllt Cone. .!lIIin),_(SlV'seo) (SlV) (') 

Operator : Hi royuki 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 : Hi royuk i Togawa 

Appendix figure 3-2·1 HPLC chromatograms at start of exposure 

659 -2 Appendix 3­
94744 

Study No. 94744 

Operating Datil: liar. 30. 2001) 9·~ 714/v.t./L2\\'_ .' 

PKNO Time Area liei,ht Cone. 

CIIinl (/I V·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 : Hi royuk j Togan 

~Jltlj.1QL..... _ ..._.______....._ Operati nl 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) (/l V-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 of exposure 

660 -3 Appendix 3­
94744 

Study No. 94744 

Operat Inl 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; HI royuki Togalla 

Fig 

Appendix figure 3-2-3 HPLC chromatogram at start of exposure 

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 ; Hi roy uk 1 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 : Hi royuki Toga"a 

94744/72h/l.0% 

n.d. n.d. 

_. __ ,.:A____ 

~ fo...-~ __~ -_____._________ -_...... ­

0.-0-'-'''- I~. 0 200 ;au 

PKHO 

Operot inf 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 HPLC chromatograms at end of exposure 

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 -­ .... ..,~,--~-------"-.. ~,-

Operati n, Date : Apr. 02. 2009 

!'KIlO Time 

(min) 

Area 

(jl Y·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 

Operat i"& 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 

Tau I 31887 100.00 

~~ I ____....-...­

0:0 I 10.0 20.0 ';'0 Operator : HI royuki Togan 

Fig 

Appendix. figure 3-3-2 HPLC chromatograms at end of exposure 

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 : Hi royuki Togawa 

Fig 

Appendix figure 3-3-3 HPLC chromatogram at end of exposure 

664 -7 Appendix 3­
94744 


Additional data 1 


Results of preliminary studies 

665 

94744 


1. Time-course analysis of concentration of test item 

The test item was predicted to transform in medium, so time-course of the test item 

concentration was measured to confirm the fluctuation of the concentration. 

1.1 Analysis of concentration of test 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 

Stirring for 

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 1­
94744 


PEMB 

PE3MB ! 

! 111 

~ 1 .1


0:0 ~--~--------~~--~--------~~------------~oo Hi. 0 0 

HPLC chromatogram (standard solution 50 mgIL) 

HPLC chromatogram (Stirring for one hour, decuplc dilutcd sample) 

PEMB 

1 

.2 

10'.0 'l.ci.o 30. 0 

HPLC chromatogram (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 stirring for 48 hours after mixing the test sample and medium. 

667 -2 Additional data 1­
94744 


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 (10000xg, 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 of the test item in the test 

solution without algae was also measured in order to confmn whether the test item 

was absorbed to algae or not. 

(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 of test item in test solutions> 

Stock solution content 

(%) 

Measured concentration (mglL) 

(Percent of the measured concentration 

versus that at the start of the exposure %) 

At the start of the 

exposure 

At the end of the 

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 of white substance on the interior waH of test 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 of the 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 of definitive study 

Based on the results of the 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 of the exposure, 24 and 48 hours after the exposure, and the end of the 

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 of converted products 

670 

94744 


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 chromatograph-mass spectrometry 

(LC-MS) and estimated structures. 

1.1 Method of analysis 

Standard solution and the pretreated sample of 0.40 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 of liquid 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 

Flow rate 0.2mUmin 

Injection volume 5#L 

Post-column 

Solution Acetonitrile / water"t (111 v/v) ·2 

Flow rate 0.1 mUmin 

*1 Tap water was treated with ultrapure water system. 

*2 Containing 25 mmollL ammonium acetate. 

671 -1 Additional data 2­
94744 


Conditions of mass spectrometer 


Ionization mode Electrospray ionization (ES1) 


Detection ion Positive 


Measure mode Scan 


Scan range (mlz) 150-600 


Capillary temp. 300"C 


1.2 Results 

Converted 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 molecular fonnula molecular figure No. weight 

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

Figure 2-5 

Figure 2-13 

Figure 2-6 

Figure 2-14 

,

,

, 

I 

! Figure 2-7 IFigure 2-15 

I, 

.l 

A 

B 

C 

4.7 

5.1 

5.4 

672 -2 Additional data 2­
94744 


· 


· 


· 


.' " Figure 2~8

D 7.5 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 2­
94744 

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 

of disulfide 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 of Peak E. 

Also, it was suggested that PE3MB and PE3MB fomled by hydrolysis from 

PEMB changed to Peak B by dehydration, Peak C by elimination of hydrogen sulfide, 

and Peak A by both disulfide fOmlation and elimination of hydrogen sulfide. And it 

was suggested that PEMB changed Peak F by disulfide formation caused by oxidation 

of thiol 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 

13.17 NL:

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 TIC chromatogram (standard solution) 

675 -5 Additional data 2­
94744 

Standard solution 50 mgIL 

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 of peak 1 

6500 460.32 

6000 I 

5500 l 

5000 I

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! IJ, 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

'TT'~~"-rl'T""1 ~:;.61 57~38 500 550 600 

NL: 

8000 . 8.17 Mass chromatogram ofmlz =488 8.14E3 

mlz= 

Position of peak 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 

6000 8.09.

1000 

8.67E3 

Mass chromatogram of mlz =460 mlz= 

459.82-480.82 F: 

... c Q1MS [ 

150.00-600.00) 

MS 

Position of peak 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 of Peak 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 of peak 2 
 90000 
j . 

j 

80000 


70000 


60000· 


f50000 


~ 40000 


20000 


10000 


160.99 221,33 

o 1-'/:\', f I , ',",'1 I
 , 

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

m/z =562 	 +cQ1MS [ 

150.00-600.00] 8000(}' MS 

94744-090331­

/ 

Position of peak 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 of Peak 2 and Mass chromatogram (standard solution) 

677
 -7 Additional data 2­
94744 

Test solution 

RT: 0.00· 30.00 

600000 

550000 

450000­

400000 

25CKM>O 

200000 

1501lOO 

100000 

50000 

0" 

7.71~ NL: ' 

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-rr! 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 

[0.40 mg,IL (at 24 hours after the start of the exposure)] 

678 -8 Additional data 2­
94744 


Test solution 

94744-()90331-H24hA#273 RT: 4.69 AV: 1 S8: 59 3.00-4.00 NL: 3.70E4 

T: + c 01 MS [ 150.00-600.00) 

488.28 

Mass spectrum of peak 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 o ·\··r·, Tt-I . I I l rUt '"," r , ..,"', r' rJ I I r ," r I 1'1 1

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 of peak 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 of peak A 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 of Peak A and Mass chromatogram 

[0.40 mgIL (at 24 hours after the start of the exposure)] 

679 -9 Additional data 2­
94744 


Test solution 

94744-090331-H24hA #296 RT: 5.08 AV: 1 58: 59 3.00-4.00 Nl: 7.61 E3 

T: "" cQ1MS I 150.0Q.600.00} 

470.43 

7500 

Mass spectrum of peak 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 •. , Jr. I I~ 'I-r-; ~~~lj\L.. I I '/·'l-l\'",·,I­ l t

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 of peak 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 of mlz =442 mIz= 

441.75-442.75 F: 

+cQ1MS( 

150.00-600.00) Position of peak B MS 

94744-<l90331­

H24hA 

7.89 7,.81

j.,,91 10.97 

I .....-r-r--+t-......-..--r15,,-.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 of Peak B and Mass chromatogram 

[0.40 mgiL (at 24 hours after the start of the exposure)] 

680 -10 Additional 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 of peak 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 of m/z =454 	 mfz.: 

453.87-454.87 F: 

-t-cQ1MS ( 

150.00-600.00] Position of peak 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 of m/z =426 	 425.89-426.89 F: 

-t-cQ1MS [ 

150.00-600.00]

Position of peak C MS 

94 744-090331­

H24hA 

~.85 

1000 , 1 

o ~~~~~~~~~~~~~rT~~~~~~~~ 

o 	 5 15 25 .30 


Time (min) 


Additional figure 2-7 Mass spectrum of Peak C and Mass chromatogram 

[0.40 mgIL (at 24 hours after the start of the exposure)] 

681 -11 Additional data 2­
94744 


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 ofpeak D 

3.211:~ 2" .,,<32 ·;'l~i 576·20

! 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 

Mass mJz= 457.73458.73 chromatogram of F:+c01MS[ 

m/z 150.00-600.oo} MS =458 947~331

H24hA 

'..21 12.28 16.28 21.64 2.4.78 28.17 

7.71 Nl: 7.28E4 

60000 7.54, 'f 40000 Position of

~ 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 of Peak D and Mass chromatogram 

[0.40 mglL (at 24 hours after the start of the exposure)] 

682 -12 Additional data 2­
94744 


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 of peak E 

180000 


160000 


~140000 

~t20000 

321.14 

7 :: 5 6·19 

l 1B 441..23 486.32 20000 . I ,. f l' 

210.52 

t T II 

2~.-45 I' I 1 l"t''f _.21 .• "r"r ~.19 I I' I T 424,,31 

f rL! I 1 I l·!;t.-rrl" 

1~17.27 54j.16 

1 I 'If'1 l

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......... 'l§· 9.79


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 of peak 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 of peak 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 of Peak E and Mass chromatogram 

[0040 mg/L (at 24 hours after the start of the exposure)] 

683 -13 Additional aala 2­
94744 


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 of peak 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 of peak 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 

Mass 4.14E4 chromatogram of mJz= 

mlz =460 459.84-480.84 F:

+cQ1MS[ 

150.00-600.00] Position of peak 1 	 MS 

94744-090331­ ,/ 	 H24hA 

~,12 

8.23 

\..;9~·~T"~11r44~rT,16~.50~19~.~;· -~ -,-~. i ~~~.:;:~;-~~3=r;~~ J', t f i i J ., 

10 15 20 25 30 

Time (min) 

Additional figure 2-10 Mass spectrum of Peak 1 and Mass chromatogram 

[DAD mgIL (at 24 hours after the start of the exposure)] 

684 -14 Additional data 2­
94744 

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 of peak 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 of peak F 150.00-600.00] 

f1OO00 

B 8000 '" ~~44-090331 .5 \.. 13.33 H24hA

6000 

12.]9 4000 

2000 2.21 

14j22, 16'~j 10'[r:5 o·'I-r-+-r-T~' :,-,-,-,..,=r26J?9, 2~.~61 1

o 15 20 25 30 

TIme (min) 

Additional figure 2-11 Mass spectrum of Peak F and Mass chromatogram 

[0.40 mg/L (at 24 hours after the start of the exposure)] 

685 -15 Additional data 2­
94744 

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 of peak 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 of Peak 2 and Mass chromatogram 

[0.40 mgIL (at 24 hours after the start of the exposure)] 

686 -16 Additional data 2­

8000-· 

t 6000 

i 4000 

.E 

2000 

o 

15000 

o 

Position of peak 2 

"-.. 

12.97 

2.21 3.42 6.37 12:1$ 

13.09 Mass chromatogram of NL: 1.67E4 

mlzc 559.82-560.82 

12,97 "'--- m/z =560 F: ... C Q1 MS [ ____ 	 150.00-600.00] MS 

94 744-090331­ Position of peak 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 of Peak A 

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

· . · . · . · . · . · . · . · . · . · . · . · . · . · . ----~-:----" ·

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

mlz Detected ion 

470 [M+N14+HCOOH-H20r 

442 [M+NH4r 

Additional figure 2-14 Estimated structure of Peak B 

687 -17 Additional data 2­
________________________ __ 

94744 

mlz Detected ion 

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

426 [M+NH4t 

Additional figure 2-15 Estimated structure of Peak C 

~- .........-..---.----.-.-.....
¥----.-.-~~

· . · . · · .. · : . . · · · 

· · · · · · · · 

· · 


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

mlz Detected ion 

576 Unknown 

458 [M+NH;t 

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

Additional figure 2-16 Estimated structure of Peak D 

688 -18 Additional data 2­
94744 


Detected ion 

576 Unknown 

458 [M+NlLtt 

321 [M+H-C4H60S-H20]+ 

Additional figure 2-17 Estimated structure of Peak E 

· --.~ . -.. ..--........-......----............... · . · . · . · . · · ..

· 

· · · · · 

mlz Detected ion 

488 [M+N~+HCOOH-H20]+ 

460 [M+NH4t 

Additional figure 2-18 Estimated structure of Peak 1 

689 -19 Additional data 2­
94744 

mlz 	 Detected ion 

588 [M+N~+HCOOH-H20r 

560 [M+NH4t 

Additional figure 2-19 Estimated structure of Peak F 

. 	 ~-.------------------.----------------.. . 	 . 

· 

· 

· · · · 

------------------------------------~-.-.-- ....-...-......--...-......... -~-----------------------------

mk 	 Detected ion 

590 [M+NI4+HCOOH-H20 t 

562 [M+NHd+ 

560 Derived from peak F 

Additional figure 2-20 Estimated structure of Peak 2 

690 -20 AddiLionaJ dara 2­
94744 


2. Qualitative analysis of white substance 

In the test vessel of 0.40 and 0.18 mglL, sticking of white matter was observed on 

the interior wall of the test vessel around surface of the test solution at end of exposure. 

Therefore, the white matter was qualitatively analyzed with LC-MS. 

2.1 Method of analysis 

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 of liquid 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 of mass 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 by LC·MS and estimated as following structures. 

Retention time Estimated 

Peak on TIC Estimated structure Additional molecular 

No. chromatogram and molecular formula figure No. weight (min) 

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


· · ·· · I ·

· · 

· · · 

· 

· · Figure 2-22 

G 13.7 ·

· Figure 2-26 · 

· · · 

· 

· · 

· · · · · 

· · 

· · · 

· · · 

· · · Figure 2-23 H 13.9 · · ·· Figure 2·27 ·· ·· · 

· 

· 

· · 

· 

· · · 

Figure 2-24 I 17.0 Figure 2-28 

-

692 -22 Additional 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 of the test vessel was the 

dimmer of Peak D and E caused by intermolecular disulfide fonnation. Also, it was 

suggested that PEMB bound to PE3MB by action of disulfide 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 

of the substance. And it was suggested that Peak G was stereoisomer of Peak H. 

693 -23 Additional datil 2­
94744 


Insoluble converted products 

RT: 0.00 - 30.00 

NL: 

5.69E5 

550000 
 nCF:+c 

Q1MS[ 

500000 
 150.00-1200.00]

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 TIC chromatogram 

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 of peak 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] 50000 Position of peak G 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: +cQ1MS I 

15O.0(}'1200.00]

13.91 	 MS Position of peak G 94144-090402­

H72hA-Ring..2 

l-'+'-1-i'-T'"r-r-rB..,..2....,3,......,..1'-T1.;:..;.4r:-7J14.9'., !?-'?....:4..... , ...............=,25:;:.1:....;.~:::.,..::,29:;::,.:!j·'"""1 


5 10 15 20 25 so 

Time (min) 

Additional figure2-22 Mass spectrum of Peak G and Mass chromatogram 

695 -25 Additional data 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 of peak H 

1 

l 

100000 i

I 

90000 

80000 

70000­

~ 

c:'" 80000 

~ 50000 

40000 

30000· 

20000 

10000 


0 


13.81 

m/z 

Mass chromatogram ::1 ofm/z =924 Position Of:a:H ~40000 

j 	30000L 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 of Peak H and Mass chromatogram 

696 -26 Additional data 2­
94744 


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 of peak I 

1*.38 

mtz 

17.03 

Position of peak I 

17.10 

17.20 


, 17.64 


25000 17.03 

, 

mlz= 16.~7 17.13 1001.67-1002.67 F: 

20000 Mass + cQ1MSl chromatogram 150.00-1200.00] MS 

ofmlz =1002 94744-090402­

~15ooo· Position of peak I H72hA-Ring-2

.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 of Peak 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 of peak 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 

:ei 6000 

4000· 

2000 

a 

25000 

20000 

i'15000' 

c: 

S 

..5 ,0000 

5000 

Mass 17.08 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 of peak J94744-090402­

H72hA-Ring-2 

p.71 

18.27 II' 21.69 26.34

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 of peak J 94744-090402­

H72hA-Ring-2 

Additional figure 2-25 Mass spectrum of Peak J and Mass chromatogram 

698 -28 Additional data 2­
94744 


-...--...--..---.. -....-- ..-----..--....---..---......---_·----_·-t 

· 
 · 
 · 


· 


· 


· 
 · 


· 
 · 


mlz Detected ion 

924 [M+N1-l4+HCOOH-H20r 

896 [M+N1-l4r 

Additional figure 2-26 Estimated structure of Peak G 

••••••• ____ •••••••••• _. _____ •••• _ •••• __ ••••• _ ••• __ ••• k •••• __ •••• __ •• , 

· 

· · · 

· · · · · · 

mlz Detected ion 

924 [M+N1-l4+HCOOH-HzOr 

896 [M+Na.r 

Additional figure 2-27 Estimated structure of Peak H 

699 -29 Additional dala 2­
94744 


m/z Detected ion 

1030 [M+NR!+HCOOH-H20r 

1002 [M+NR!t 

Additional figure 2-28 Estimated structure of Peak I 

m/z Detected ion 

1028 fM+NH4+HCOOH-HzOt 

1000 [M+NR!t 

Additional figure 2-29 Estimated structure of Peak J 

700 -30 Additional data Z­
PAGE 1 OF 21 PAGES harlanrM 

.... 

Mixture of PEMB and PE3MB: 


ASSESSMENT OF THE INHIBITORY EFFECT ON 


THE RESPIRATION OF ACTIVATED SEWAGE 


SLUDGE 


PROJECT NUMBER: 0145/0201 


STUDY SPONSOR: 

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 

701 

PROJECT 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 of the data generated and of the 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 of this 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 of QA Signature Final Report Audit 

§ Evaluation specific to this study 

02 FEB 2009 ....t\....~~.................. DATE: 


For the Quality Assurance Unit* 

*Authorised QA Signatures: 

Deputy Head of Department: JM Crowther MIScT MRQA 

Senior Audit Staff: G Wren ONC MRQA 

702 

PROJECT 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~ f4 30 JAN 2009 ...........1.\1... .... ......................................... Date: ..................................................... . 


N Youngs B c 

Study Director 

This report may be presented in final fonn as a digital (pdf) documenl Such documents are prepared by scanning the paper original. 

and are considered of equivalent Integrity and authenticity to varsions pftlduced by optical photocopy. However. In aN cases the 

hand-signed paper original. hald in secure archives. Is the definitive document. 

703 
PROJECT 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 Test Water 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 ofthe Test Preparations at the Start and End of the 

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 of PEMB and PE3MB ­

3 Hours Contact Time 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 for Testing of Chemicals (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 of the study are of value in the assessment of the toxicity of the 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 of the Sponsor. 

706 

PROJECT 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 Test Water 

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 of the following composition, was added to each test vessel to act as 

a respiratory substrate: 

16 9 Peptone 

11 9 Meat extract 

3g Urea 

0.7 9 NaCI 

0.4 9 CaC12·2H20 

0.2 9 MgS04.7H20 

2.8 9 K2HP04 

dissolved in 1 litre of water with the aid of ultrasonication. 

707 

PROJECT 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 of the 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 of inoculum 

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 

708 
PROJECT 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 of test system 

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 

709 
PROJECT 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 of the two control respiration rates. 

% inhibition =[1- 2RS C ] x 100 

RC1 +R 2 

where 

oxygen consumption rate for test or reference sample 

oxygen consumption rates for controls 1 and 2 

The percentage inhibition values were plotted against concentration, a line fitted using 

the Xlfit software package (lOBS) and the EC15 and 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 or disposal. 

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 of the test preparations at the 

start and end of the 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 - - -

It was not possible to obtain ECoo or ECso values for the 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 of controls 1 and 2 after 3 hours contact time 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 O2/1 and 2.5 mg 02/1 respectively). 

This was considered to have had no adverse effect on the results of the study given that 

711 

PROJECT 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 of test 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. 

712 
PROJECT 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 0 2/1) 

Measurement 

Period 

(minutes) 

Final O2 

Reading 

(mg O2/1) 

O2 

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 

713 
PROJECT 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 of the Test Preparations at the Start and End of the 

Exposure Period 

Nominal pH
 Concentration 


(mgll) 
 oHours 3 Hours 

Control R1 7.7 7.9 

R2 7.7 7.8

Test Material 10 7.8 7.8 

32 7.8 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 1 to 2 

714 
PROJECT NUMBER: 014510201 PAGE 15 

Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT 


ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE 


Table 3 Observations on the Test Preparations Throughout the Test Period 

Nominal Observations on Test Preparations 

Concentration oHours 30 Minutes 3 Hours I 

(mg/l) Contact Time Contact Time 

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 of test globules of test globules of test 

material dispersed material dispersed material dispersed 

throughout· throughout throughout 

Slightly cloudy Dark brown Dark brown 

dispersion, small dispersion, small dispersion, small 

32 globules of test globules of test 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 of test globules of test globules of test 

material dispersed material dispersed material dispersed 

throughout· throughout throughout 

Cloudy dispersion, Dark brown Dark brown 

-oily layer of test dispersion, oily layer dispersion, oily layer 

320 material on surface of test material on of test 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 layer of test dispersion, oily layer dispersion, oily layer 

1000 material on surface of test material on of test 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 1 to 2 


• Observations prior to addition of activated sewage sludge and synthetic sewage 

715 

PROJECT NUMBER: 0145/0201 PAGE 16 

Mixture of PEMB and PE3MB: ASSESSMENT OF THE INHIBITORY EFFECT 


ON THE RESPIRATION OF ACTIVATED SEWAGE SLUDGE 


Figure 1 

'! 


sa7ei­, 

6ci5<f' , 4Q-­ I 

Concentration-Response Curve: 3,5-dichlorophenol- 3 Hours 

Contact Time 

-c______c_ -T-; __;_c_ _c -- 'I 

j 

I · ", 

-:,' . . .' ...... ~.. .: .: ...:. I ' ' , , .. , " .

';ill' , . - - ."".. , .. -..-. ,", . : . ; : I , .. , 

· ;: : : , ." , · ,..... , . · , 

" , . , , , I , , . , 

. , 

-1- ___J .------ -- -------. __ lou :L__c_.____"___. ___ L oj]__ .J 

10 100 

Concentration (mgjl) 

716 

PROJECT 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 Contact Time 

90 

80 

70 

c:: 

:s

.2 50 

:.c 

.5 

0 ~ 

20 

0 

,.. . , , . , , , , 	 , 

~ ---.---- ¥-r~ --- --T~-- -1--~- r¥ -r-T- -r~ 

-10 

10 	 100 1000 

Concentration (mgll) 

717 

PROJECT 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 IIgn a <0.002 <0.003 <0.006 0.1 0 0.00 

Chlordane A ~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 

Endcsullan A 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 ar value for mandalOly parema1ers and specified limit or value for oon·rnandatory parameters (indicators and 


disinfectantll). 


"1 No numelical limit applies. 


"2 Guldanee levels only. Monitored for the calculation of Total 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. 


718 

PROJECT 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 OR VALUE 

(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 3 at 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 or value for mandatory parameters and specified limit or value for non-mandatory parameters (indicators and 


disinfectanlll). 


°1 No numeric::allimit applies. 


"2 Guidance levels only. Monitored for the calCulation of Total Indicative Dose, which Is the regulatoly parameter. No Hmples exceeded the TID 


In 2007. 


'3 The formula for the celculated NitrlIteINitrite levella [NitrateY5O + [Nitritey3. Thia number na. no units. 


719 

PROJECT 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 ~PLE PCV­ UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 

Trlclopyr <0,003 <0,003 <0.003 0,1 0,00 

Trletazine 

1.19" 8 0

8 <0,001 <0,001 <0,001 0,1 0,00 

Tfillul'8lin 

IJ9II 0

8 <0,002 <0,002 <0.002 0,1 0,00 

Turbidity 

Ilg/! 0

NTU 52 <0,050 <0,088 0.190 0,00 

Free Chlorine 

a .", 4 

180 0,030 0.231 0.550 '"9" -, · Total ChlOrine mgll 180 0.080 0.297 0.610 · 

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 NO.ll00 ml 26 0,000 0.000 0,000 a 0 0.00 

Coliform Bacteria No, 1100 ml 180 0.000 0,000 0.000 0 a 0.00 

Colony Counts after 48 -,

hours at 370C No, 1100 ml 52 0.000 3.096 83,000 - · Colony Count after 72 .. No.1 100 ml 52 0.000 1,654 142,000 hours at 22"C · · Conductivity 251.000 <421.385 542.000 0,00 

Gross Alpha Activity 

IlSicm at 20"C 28 2500 a 

BqII 8 0.063 0,087 0.125 0.1'2 · · (2 Gross Beta ActIvity Bq" 8 0.012 0,042 0.071 · · Sulphate mgll 8 52,100 69.783 ! 85.800 250 0 0.00 

Total Organic Carbon moll 8 0.810 U141 1,380 ." I · 

"PCV Prescribed concentration or value lor mandatory parameters and specified lmit or value lor non-mandatory parameters (lIldic:ators and 


dilllnlectanis). 


"1 No numericallimil applies. 


*2 Guidance levels only, Monitored for the calculation of Total tndicativa Dose, which it the 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 number has no units. 


720 

PROJECT NUMBER: 0145/0201 PAGE 21 

Appendix 2 Statement of GLP Compliance in Accordance with Directive 

2004/9/EC 

,oH) Department 

\.:.: of Health 

THE DEPARTMENT OF HEALTH OFTBE GOVERNMENT 


OF THE UNITED KINGDOM 


GOOD LABORATORY PRACTICE 

STATEMENT OF COMPUANCE 


IN ACCORDANCE WITH DIRECTIVE lOO4I9/EC 


TEST fACIUTY 

SefePhenn Laboratori .. Ltd. Analytical Chemistry 

Shardlow BUIIln ... Park Environmental Fe.. 

Sherdlow Environmental Toxicity 

Derbyshire Mutagenicity 

DE722GD ~hem ...ting 

Toxicology 

DATE OF INSPEC110N 

21M August 2007 

A general inspection for compliance with the Principles of Good Laboratory Practice 

was carried out at the above test facility as part of the UK GLP Compliance Programme. 

At the lime of inspection no deviations were found of sufficient magnitude to affect 

the validity of non-clinical studies perfonned at these facilities. 

Dr.Andrew J. Gra)' 

Head. UK GLP Monitoring Authority 

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. 


721 

PAGE 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 

Shard low Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

Telephone: +44 (0) 1332792896 

Facsimile: +44 (0) 1332799018 

722 

PROJECT 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 of the data generated and of the 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 of this 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 

723 

PROJECT 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: .......................................... . 

N Youngs 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 

724 
PROJECT 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 of the 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 

725 

PROJECT 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 of the 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 for validation 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 of test 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 of the 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 of test 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 of test 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 of test 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 of test 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 of 3 litres. Large 

globules of test 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 of test material (50 mg) was 

weighed onto a glass slide and added to 3 litres of deionised reverse osmosis 

purified water. Globules of test material were visible on the surface. 

When using GF/A filter paper, an amount of test material (50 mg) was weighed 

onto a filter paper and added to 3 litres of deionised reverse osmosis purified 

water. Globules of test 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 prior to dispersion in culture medium to aid dispersion of the 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 101 K3700) prior to dispersal in 

729 

PROJECT 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 lit res 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 of the 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 of the 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. 

730 
PROJECT 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.1 01 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 of the 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 

731 
PROJECT 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 of test system 

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 of culture medium. 

The culture vessels were sealed and CO2-free air bubbled through the solution at a rate 

of approximately 40 mllminute and stirred continuously by magnetic stirrer. 

732 

PROJECT 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 CO2immediately. 

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 absorber vessels 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 Dissolved organic carbon (DOC) analysis 

Samples (20 ml) were removed from the test material and toxicity control vessels on 

Day 0 prior to the addition of the 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. 

733 

PROJECT NUMBER: 014510215 PAGE 13 

DOC analysis of the test material dispersions after dosing was not possible due to the 

insoluble nature of the 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 wrw pHIOxi 3401 pH and dissolved oxygen meter. 

3.7 Evaluation of Data 

3.7.1 Calculation of carbon 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 of each component in the test material. 

NootC atoms xmol w: ofC x100% 

mol wt of test material 

734 

PROJECT NUMBER: 0145/0215 PAGE 14 

Component Molecular Weight 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 of test 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 C atoms x mol wt of C x 100% 

mol wt of sod ium benzoate 

7 x12.011 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 present for 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 egra a 10n = x {a 

2 mg TOC as test 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 prior to calculation of percentage degradation. 

735 

PROJECT NUMBER: 0145/0215 PAGE 15 

Percentage degradation =[1 _ rng DOC in test flask on Day 28] x 100% 


mg DOC in test flask on Day 0 


The total C02 evolution in the control vessels at the end of the 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 O2 test volume 

. 100 1 =mg IC In control x --x ­

27.29 3 

3.7.3 Validation criteria 

The results of the 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 of the 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 of the test material suspension in the mineral medium at the beginning of 

the test should be < 5% of the TC. 

736 

PROJECT 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, after which 

instructions will be sought as to further retention or disposal. 

737 

PROJECT 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 of the 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 of the Dissolved OrganiC Carbon analyses performed on Days 0 and 28 are given 

in Table 4. The pH values of the 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 of the 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. 

138 

PROJECT 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 of OECD Guideline No 301 B. 

The toxicity control attained 55% degradation after 14 days thereby confirming that the 

test material was not toxic 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 of the 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 of the 

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 

739 

PROJECT NUMBER: 0145/0215 PAGE 19 

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. 

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. 

740 

PROJECT 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 1 and 2 


Abs = CO2 absorber vessels 


741 

PROJECT 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 of CO2 detected in Absorber 2 

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PROJECT 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 

Test vessel Total Carbon* 

(mg/l) 

Inorganic Carbon· 

(mg/l) IC Content (% of TC) 

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 of the test material and sodium benzoate where applicable 

743 
PROJECT 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 

Test Vessel 

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