P R O T O C O L

A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW ( Pimephales promelas )

OECD Guidelines, Section 203
U.S. EPA OPPTS Number 850.1075 Annex to Commission Directive 92/69/EEC, Method C.1.- 2 -
A 96- HOUR STATI C

- R ENEWAL ACUTE TOXI CI TY TEST WI TH THE FATHEAD MI NNOW ( Pi me phal e s pr ome l as )

SPONSOR :

SPONSOR’S REPRESENTATI VE :
TESTI NG FACI L I TY :

STUDY DI RECTOR :

L ABORATORY MANAGEMENT :

FOR LABORATORY USE ONLY P r op osed D a tes: Expe r i m e nt a l St ar t Da t e :

________________________ Expe r i m e nt a l Te r m i n a t i o n Da t e :

_________________ Pr o j e c t No .:

________________________ Te s t Co nc e n tr a t i o ns :

_________________________________________________________ Te s t Subs t a nc e No .: ___________ Re f e r e nc e Subs t a nc e No . ( i f appl i c ab l e ) :

____________
PROTOCOL APPROVAL

____________________________________ ___________________________ STUDY DI RECTOR DATE

____________________________________ ___________________________ L A BORATORY MANAGEMENT DATE

____________________________________ ___________________________ SPONSOR’S REPRESENTATI VE DATE- 3 -
I NTRODUCTI O N

w ill c o nduc t a s t a tic

- r e n e w a l a c u te to xic ity te s t w ith the fa the a d

m i nno w , Pi me phal e s pr ome l as , for th e S p on sor at th e aq u a tic tox i cology facility in E a ston , Mary lan d

. T h e stu d y will b e p e rform ed b a sed on p r oced u r es in O E C D G u id elin e for Te s t i n g o f Che m i c a l s 203: F i sh , A c u t e T o xicity T e st ( 1 ) ; U.S. EPA Se r i e s 850

- Ec o l o g i c a l Ef f e c t s Te s t Gui d e l i n e s OPPTS Num b e r 850.1075: Fi s h Ac ut e Tox i c i t y Te s t , Fre s hwat e r and Mar i ne (2 ); U

. S

. E n viron m en tal P r otection A g en cy , S t an d ard E v alu a tion P r oced u r e, A c u t e T o xicity T e st fo r F resh w a t er Fi s h (3 ); ASTM St andar d E729- 96 St andar d Gui d e f o r

C

onduc t i ng Ac ut e Tox i c i t yTe s t s on Te s t Mat e r i als wi t h Fi s h e s , Mac r oi nv e r t e br at e s and Amphi bi ans (4 ) an d O fficial J o u r n a l of th e E u rop ean C o m mu n ities No . L 383 C.1. A c u t e T o xicity fo r F i sh (5 ). R aw d ata for all work p erform ed at

an d th e origin al fin a l rep o rt will b e filed b y p r oj ect n u m b e r in arch ives located on th e site or at an altern ative location to b e sp ecified in th e fin a l rep o rt.

OBJECTI V E T h e ob j ective of th is stu d y is to d e term in e th e acu te effects of a test su b s tan ce on th e fath ead

m i nno w , Pi me phal e s pr ome l as , unde r s t a tic

- r e n e w a l te s t c o nditio ns
.

EX PERI MENTAL DESI GN F a th ead min n o ws will b e ex p o sed to a geometric series of at least five water accommod a ted fraction s (WA F ) of th e test su b s tan ce an d a n e gative (d ilu tion water) con trol for 9 6 h o u r s. T w o rep licate test ch amb e rs will b e main tain ed in each treatmen t an d con trol grou p , with 1 0 min n o ws in each ch amb e r f o r

a

t o t a l o f 2 0 m i n n o w s p e r t e s t c o n c e n tr a t i o n
. W A F s o l u t i o n s an d con t rol water will b e ren e wed on D ay 2 of th e test. A t each ren e wal p e riod , fresh WA F solu tion s will b e p r e p a r e d and t h e f i s h w ill b etran sferred from th e old solu tion s to th e n e w solu tion s
.

WA F load in g rates will b e selected in con s u ltation with th e S p on sor, an d will b e b a sed u p o n in form ation su ch as th e resu lts of ex p l oratory ran g e- fin d i n g tox i city d a ta, kn own tox i city d a ta, p h y sical/ch e m i cal p r op erties of th e test su b s tan ce or oth e r relevan t in form ation

. F o r th is test, th e term load in g rate m ean s th e total am ou n t of test su b s tan ce ad d e d to t h e di l u ti o n w a t e r v o l um e ( m g / L ) t o a c h i e v e th e resp ective WA F solu tion

. G e n e rally , each test su b s tan ce load in g rate in th e d e fin itive test will b e at least 6 0 % of th e n e x t h i gh er load in g rate u n l ess in formation con cern i n g th e con cen tration

- effect cu rve- 4 -
in d i cates th at a d i fferen t d ilu tion factor wou l d b e m o re ap p r op riate. Water sam p les will b e collected at sp ecified in tervals for an aly s is of T o tal O r gan i c C arb on (T O C ).

T o con trol b i as, fath ead m i n n o ws will b e in d i scrim i n a tely assign ed to ex p o su re ch am b e rs at test in itiation

. N o oth e r p o ten tial sou r ces of b i as are ex p ected to affect th e resu lts of th e stu d y
. O b servation s of m o rtality an d oth e r clin ical sign s will b e m a d e th rou g h o u t th e 9 6

- h o u r test p e riod

. C u mu lative p e rcen t

m o rtality ob served in th e treatm e n t grou p s will b e u s ed to calcu late, wh en po s s i bl e , L L 50 v a l u e s a t 24, 48, 7 2 , an d 9 6 h o u r s. T h e n o
- m ortality level an d th e n o
- o b s erved

- effect level (N O E L) will b e d e term in ed b y visu ally in terp retin g th e m o rtality an d clin ical ob servation d a ta.

MATERI ALS AND METHODS Te s t Subs t a nc e I n formation on th e ch aracterization of test, con trol or referen ce su b s tan ces is req u i red b y G ood Lab o ratory P r actice (G LP ) S t an d ard s an d P r in cip l es. T h e S p on sor is resp on sib l e for p r ovid i n g

v e r i f i c a t i o n t h a t t h e t e s t s u b s t a n c e h a s b e e n c h ar a c t e r ized accord in g to G L P s p r ior to its

u s e in th e stu d y
. I f verification of G L P test su b s tan ce ch aracterization is n o t p r ovid e d to it will b e n o ted in th e com p lian ce statem en t of th e fin a l rep o rt.

T h e S p on sor is resp on sib l e for all in formation related to th e test su b s tan ce an d agrees to accep t

a n y unus e d t e s t s ubs t a nc e a nd/ o r t e s t s ubs t a nc e c o nt a i ne r s r e m a i n i n g a t t h e e nd o f t h e s t udy

.

Pr e p ar at i o n of Te s t Loadi n g r a t e s T h e test su b s tan ce will b e m i x e d d i rectly with d ilu tion water on a weigh t:volu m e b a sis. E ach WAF w ill be pre p ar e d indiv i dua lly in a 13.2 L Pyre x® a s pira to r bo ttle w ith tubula tio n by m i xing a n

am ou n t of th e test su b s tan ce in ap p r ox im ately 1 2 L of d ilu tion water u s in g a T e flon ®- coated stir b ar on a

m a gn etic stir p l ate. C are will b e taken to m a in tain a vortex d e p th of ap p r ox im ately 3 0 % of th e test solu tion h e igh t

. F o llowin g th e m i x i n g p e riod , th e WA F solu tion s will th en b e allowed to settle for
ap p r ox imately 1 h o u r an d th e test solu tion will b e d ecan ted off th e top (or b o ttom, as ap p r op riate).- 5 -
Te s t Or gani s m T h e fath ead min n o w ( Pi me phal e s pr ome l as ) h a s b een selected as th e test sp ecies for th is stu d y
. F a th ead min n o ws are rep r esen tative of an imp o rtan t grou p of aq u a tic verteb rates, an d h a ve b een selected f o r

us e i n t h e t e s t ba s e d upo n pa s t us e hi s t o r y i n th e l abo r a t o r y . Fi s h w i l l b e f r o m t h e s am e s o u r c e an d y e a r class, an d th e stan d ard len g th of th e lon g est fish m easu r ed will b e n o m o re th an twice th at of th e sh ortest. F i sh will weigh b e tween 0

. 1 g an d 3

. 0 g each an d th e total weigh t in each test ch amb e r will n o t e x ceed0

. 8
gram s fish /L of solu tion

. T o tal len g th s an d wet weigh t s of th e in d i vid u a l fish in on e n e gative con trol
rep licate will b e m easu r ed at th e en d of th e test an d will b e con s id ered rep r esen tative of th e len g th an d

w e ig ht o f a ll fis h us e d in the s tudy . Fis h w ill be o b ta ine d fro m a

c o m m e rc ia l s upplie r o r ha tc he ry , a nd t h e ide n tity o f the s p e c i e s w ill be v e rifi e d by the s upplie r o r by pe rs o nne l us ing
ap p r op riate tax o n o mic key s , su ch as E d d y (6 ).

F a th ead m i n n o ws will b e h e ld for at least 1 4 d a y s p r ior to th e test in water from th e sam e sou r ce

a n d a t ap p r o x i m a t e l y th e s a m e t e m p e r a tu r e a s u s e d d u r i n g th e t e s t

. A n y c h an ges in water tem p eratu r e will

n o t ex ceed 3 ° C p e r d a y
. F a th ead min n o ws will b e h e ld at th e test temp eratu r e for a min i mu m of 7 d a y s p r ior to test in itiation

. F a th ead m i n n o ws will n o t b e u s ed in th e test if th ey sh ow sign s of d i sease or stress or if m o re th an 5 % d i e d u r in g th e 4 8 h o u r s p r ior to th e test. A t test in itiation , th e fath ead m i n n o ws will b e collected from h o ld in g or acclim ation tan k s an d tran sferred to th e test ch am b e rs.

D u rin g th e h o ld in g p e riod th e test fish will b e fed at least on ce d a ily

. T h e d i et will con s ist of live or frozen b r in e sh rim p n a u p lii ( Ar t e mi a s p
. ) , an d / or com m e rcial food

. F i sh will n o t b e fed for at least two d a y s p r ior to th e test or d u r in g th e test. S p ecification s for accep tab l e levels of con t amin an ts in fish d i ets h a v e n o t b e e n e s t ab l i s h e d

. H o w e v e r , th e r e ar e n o k n o w n l e v e l s of con t amin an ts reason ab ly ex p ected to b e pr e s e n t i n t h e di e t t h a t ar e c o ns i d e r e d t o i n t e r f e r e w i t h t h e pur po s e o r c o nduc t o f t h e t e s t

.

D ilu tio n Wa ter Water u s ed for th e h o ld in g an d testin g of fath ead m i n n o ws will b e ob tain ed from a well
ap p r ox im ately 4 0 m e ters d eep located on th e t T h e water will b e p a ssed thro ug h a s a nd filte r a nd pum pe d into a 37,800- L s t o r a g e ta nk w h e r e the w a te r w ill be a e r a t e d w ith s p ra y

n o zzles. P r ior to u s e th e water will b e filtered to 0

. 4 5 μm in ord e r to rem ove fin e pa r ti c l e s
. W a t e r us e d f o r- 6 -
h o ld in g an d testin g is ch aracterized as m o d e rately h ard

. T yp i cal valu es for h ard n e ss, alkalin ity , p H an d

s pe c i f i c c o nduc t a nc e ar e appr o xi m a t e l y :

Har dne s s , m g / L a s Ca CO 3 145 A l kalin ity , m g /L as C a C O 3 190 pH 8.1 Spe c i f i c Co nduc t a nc e , μ mho s / c m 330

Hardne s s , a l k a linity , pH a nd s p e c i fic c o nduc ta n c e w ill be m e a s ure d w e e k ly to m o nito r the

con s isten cy of th e well water. M ean s an d ran g es of th e measu r ed p arameters for th e fou r
- w eek p eriod
p r eced in g th e test will b e p r ovid e d in th e fin a l rep o rt. A n aly s es will b e pe rf o r

m e d a t l e a s t o n c e annua l l y t o d e t e r m i n e th e c o n c e n tr a t i o n s o f s e l e c t e d o r g a n i c a n d i n o r g a n i c con s titu en ts of th e well water an dresu lts of th e most recen t G L P

- comp lian t an aly s es will b e su mmarized in th e fin al rep o rt.

T e st A p p a ra tu s T e st ch am b e rs will b e T e flon ®- lin ed , 2 5

- L, p o ly eth y len e aq u aria filled with ap p r ox im ately 1 5 L of water. A ltern atively , test ch am b e rs m a y b e all glass aq u aria filled to ap p r op riate volu m es to m eet load in g gu id elin es. T e st ch am b e rs will b e p o sition e d in an en viron m en tal ch am b e r or tem p eratu r econ trolled water b a th to m a in tain a tem p eratu r e of 2 2 ±1 ° C

. T e st ch am b e rs will b e lab e lled with th e p r oj ect n u m b e r, test con cen tration an d rep licate.

Envi r o nm e n t a l Condi t i ons Ligh tin g u s ed to illu m i n ate th e cu ltu res and t e s t c h am be r s dur i n g ho l d i n g , acclim ation , an dtestin g will b e p r ovid e d b y flu o rescen t tu b e s th at em it wavelen g th s sim ilar to n a tu ral su n ligh t (e. g
. , C o lorton e® 5 0 )

. A p h o top e riod of 1 6 h o u r s of ligh t an d 8 h o u r s of d ark will b e con trolled with an au tom a tic tim er. A 3 0

- m i n u t e tran sition p e riod of low ligh t in ten s ity will b e p r ovid e d wh en ligh t s go on an d off to avoid

s udde n c h a n g e s in lig ht inte ns ity . L i g h t inte ns ity w ill be m e a s ure d a t t e s t i n itiation with a S P E RS c ien tific L t d. l i g ht m e t e r o r e qui v a l e nt

.

T h e target test tem p eratu r e will b e 2 2 ± 1 ° C

. T e m p eratu r e will b e m eas ur e d i n a l l r e pl i c a t e s a t t

h e be g i nni ng o f the t e s t a nd a t appr o xi m a t e l y 24- ho ur i nt e r v a l s the r e a f t e r ( i nc l udi ng pr i o r t o a nd a f t e r

ren e wal) u s in g a liq u i d

- in

- g lass th erm o m e ter. T e m p eratu r e also will b e m easu r ed with a con tin u o u s- 7 -
record er in on e n e gative con trol rep licate. R ecord er m easu r em en ts will b e verified with a liq u i d

- in

- g lass th erm o m e ter p r ior to test in itiation

.

D i ssolved ox y g en will b e m easu r ed in all rep licates of th e treatm e n t an d con trol grou p s at test in itiation an d at ap p r ox im ately 2 4

- h o u r in tervals th ereafter (in c lu d i n g p r ior to an d after ren e wal) u s in g a The r m o Or i o n Mo de l 850Apl us di s s o l v e d o x y g e n m e t e r , o r e q u i v a l e n t

. I n t h ee v e n t t

h a t d i s s o l v e do x y g e n levels ap p r oach or fall b e low 6 0 % satu ration , th e S p on sor will b e n o tified , an d ap p r op riate corrective action s will b e taken , if n ecessary , to main tain d i ssolved ox y g en levels ab ove 6 0 % satu ration

. Measu r emen ts of p H will b e mad e in all rep licates of each tr e a tm e nt and c o nt r o l g r o up a t t e s t i n itiation an d

a t

appr o x i m a t e l y 24- ho ur i n t e r v a l s th e r e a f t e r ( i nc l udi ng pr i o r t o a nd a f t e r r e ne w a l ) us i n g a The r m o Or i o n Mo del 525Aplus pH m e ter, o r equiv a lent. I f a tr eatm e nt replicate reaches 100% m o rt ality , dis s o lv ed

ox y g en , p H , an d tem p eratu r e m easu r em en ts will b e taken in th at rep licate at th at tim e, an d th en di s c o n t i nue d.

Hardne s s , a l k a linity , a nd s p e c i fic c o nduc ta nc e w ill be m e a s ure d in the d ilutio n w a te r a t te s t in itiation

. H ard n e ss an d alkalin ity m easu r em en ts will b e m a d e b y titration u s in g p r oced u r es b a sed on

m e th od s in St andar d Me t hods f o r

t

h e Ex ami nat i on of W a t e r and W a s t e w at e r ( 7 )

. Spe c i f i c c o nduc t a nc e

w ill be m e a s ure d us ing a Ye llo w Spring s I n s trum e nt Mo de l 33 Sa linity

- C o nduc ti v i t y
- T e m p e r a t u r e m e t e r ,
or eq u i valen t

. A d d ition a l water q u a lity measu r emen ts may b e taken as d eemed n ecessary b y stu d y p e rson n e l in con s u ltation with th e S t u d y D i rector. T h e reason for th e ad d ition a l m easu r em en ts will b e do c u m e nt e d i n t h e r a w da t a a nd s u m m ar i z e d i n t h e f i na l r e po r t

.

Bi ol ogi c a l Me as ur e m e nt s O b servation s of m o rtality an d clin ical sign s of tox i city will b e m a d e b e tween 0

- 2 4 h o u r s, an d at 2 4 , 4 8 , 7 2 an d 9 6 h o u r s ± 1 h o u r
. Leth ality is d e fin e d as th e lack of visib l e m ovem e n t (e. g
. lack of fin or op ercu lu m m ovem e n t ) in th e fish

. A ll clin ical ob servation s in clu d i n g ab n o rm al b e h a vior will b e n o ted

.

Sam p l i n g f o r Anal yt i c al Me as ur e m e n t s Water samp les will b e collected from th e WA F p r ep aration vessels for th e con trol an d each test

con cen tration at th e b e gin n i n g of th e test an d at th e b e gin n i n g of each ren e wal, an d from p ooled (old )- 8 -
solu tion s from each test con cen tration s at th e end o f each ren e wal p e riod

. T o tal organ i c carb o n (T O C ) will be m eas ured in each s a m p le us ing a Shim adzu Mo del 5000 TOC analy zer, o r e quiv a lent.

Condi t i ons f o r t h e Val i d i t y of t h e Te s t T h e followin g criteria will b e u s ed to j u d g e th e valid ity of th e test: 1 ) m o rtality of th e fish in t h ene g a ti v ec o nt r o l g r o up w ill n o t ex ceed10% by the end of th e test, an d 2 ) th e d i ssolved ox y g en con cen tration will b e at least 6 0 % th e air- satu ration valu e th r o ug ho ut th e t e s t

.

Dat a Anal ys i s T h e m o rtality p a ttern in th e d e fin itive mu ltip le con cen tration test will b e u s ed to calcu late, if
p o ssib l e, th e 2 4 , 4 8 , 7 2 an d 9 6

- h o u r LL5 0 con cen tration s
. T h e LL5 0 valu es will b e b ased on n o m i n al load in g rates an d calcu lated u sin g th e comp u t er software of C

. E

. S t ep h a n (8 ). T h e p r ogram wa s de s i g n e d to calcu late th e LL5 0 valu e an d th e 9 5 % con f id en ce in terval b y p r ob it an aly s is, th e movin g average

m e th od , an d th e b i n o m i al p r ob ab ility m e th od (9 , 1 0 , 1 1 )

. T h e m e th od u s ed will d e p e n d u p o n t h e m o r t ality pattern and its s u itability fo r each m e tho d . Additio na l analy s is o f data m a y be co nducted if deem ed
ap p r op riate b y th e S tu d y D i rector. T h e resu lts of th e an aly s is will b e d o cu m e n t ed in th e raw d a ta an d

su m m arized in th e fin a l rep o rt. A n o
- o b s erved

- effect level (N O E L) will b e rep o rted if it is d e fin e d b y th e d o se- r esp o n s e p a ttern

. T h e m a x i mu m WA F level cau sin g 0 % m o rtality an d th e m i n i mu m WA F level

c a u s i ng 100% m o rta lity w ill be pre s e n te d, if po s s i ble

.

RECORDS TO BE MAI N TAI N ED R e c o r d s t o b e m a i n t a i n e d f o r d a t a g e n e r a t e db y will in clu d e , b u t n o t b e lim ited to: 1

. A cop y of th e sign ed p r otocol. 2

. I d en tification an d ch aracterization of th e test su b s tan ce, if p r ovid e d b y th e S p on sor. 3

. D a tes of in itiation an d term in ation of th e test. 4. Te s t o r g a ni s m hi s t o r y , ho l d i n g a nd a c c l i m a t i o n r e c o r d s
. 5. Re s u l t s o f r a ng e f i ndi ng t e s t s , i f pe r f o r m e d

. 6

. S t ock solu tion calcu lation an d p r ep aration , if ap p licab le.- 9 -
7. Obs e r v a t i o ns
. 8. Wa t e r c h e m i s tr y r e s u l t s ( e .g ., a l k a l i n i t y a nd c o nduc t a nc e )

. 9

. T h e meth od s u s ed to an aly ze test su b s tan ce con cen tration s an d th e resu lts of an aly tical

measu r emen ts.
1 0

. S t atistical calcu lation s , if ap p licab le. 1 1

. T e st con d ition s (ligh t in ten s ity , p h o top e riod , etc. ). 12. Ca l c ul a t i o n a nd pr e p ar a t i o n o f t e s t c o nc e n tr a t i o ns
. 13. Co py o f f

i na l r e po r t

.

FI NAL REPORT A fin al rep o rt of th e resu lts of th e stu d y will b e pr e p a r e d by W i l d l i f e I n t e r n a ti o na l , L t d. The r e po r t will in clu d e , b u t n o t b e lim ited to th e followin g , wh en ap p licab le: 1

. N a m e an d ad d r ess of th e facility p e rform in g th e stu d y
. 2

. D ates u p o n wh ich th e stu d y was in itiated an d com p leted , an d t h e de f i n itive ex p e rim e n t al start an d term in ation d a tes. 3

. A statemen t of comp lian ce sign ed b y th e S t u d y D i rector ad d r essin g an y ex cep tion s to G ood Lab o ratory P r actice S t an d ard s. 4

. O b j ectives an d p r oced u r es, as stated in th e ap p r oved p r otocol, in clu d i n g all ch an ges to th e p r otocol. 5. The t e s t s ubs t a nc e i d e n t i f i c a t i o n i n c l udi ng na m e , c h e m i c a l ab s tr a c t num be r o r c o de num be r , stren g th , p u r ity , com p osition , an d oth e r in form ation p r ovid e d b y th e S p on sor. 6. Stab ility a nd s o lubility o f the te s t s ubs ta nc e unde r the c o nditio ns o f a d m i n i s t r a

ti o n , i f p r o v i d e d b y th e S p on sor. 7. A de s c r i pt i o n o f t h e m e t h o d s us e d t o c o nduc t th e t e s t

. 8. A de s c r i pt i o n o f t h e t e s t o r g a ni s m s , i n c l udi ng th e s o ur c e , s c i e n ti f i c n am e , a g e o r l i f e s t a g e , l e n g t h s
a nd w e i g ht s o f a r e pr e s e n t a t i v e g r o up o f t e s t o r g a ni s m s , f e e d t ype s , l i g ht i n t e ns i t y a nd pho t o pe
riod

. 9

. A d e scrip tion of th e p r ep aration of th e test solu tion s
. 10. The m e th o d s us e d t o a l l o c a t e o r g a ni s m s t o t e s t c h a m be r s a nd be g i n th e t e s t , th e num be r o f

o r g a ni s m s a nd c h a m be r s pe r tr e a t m e nt , a nd t h e dur a t i o n o f t h e t e s t

. 1 1

. A d e scrip tion of circu m stan ces th at m a y h a ve affected th e q u a lity or in tegrity of th e d a ta. 12. The na m e o f t h e St udy Di r e c t o r a nd t h e na m e s o f o t he r s c i e nt i s t s , pr o f e s s i o n a l s , a nd s upe r v i s o r y pe r s o nne l i n v o l v e d i n t h e s t udy

. 1 3

. A d e scrip tion of th e tran sform a tion s , calcu lation s , an d op eration s p e rform ed on th e d a ta, a
su mmary an d an aly s is of th e b i ological d a ta an d an aly tical ch emistry d a ta, an d a statemen t of th e- 1 0 -

con c lu sion s d r awn from th e an aly s es. A grap h of th e con cen tration

- m o rtality cu rve at th e en d of th e test will b e p r ovid e d

. 1 4

. S t atistical m e th od s u s ed to evalu a te th e d a ta. 1 5

. T h e sign ed an d d a ted rep o rts of each of th e in d i vid u a l scien tists or o t he r pr o f e s s i o n a l s i n v o l v e d i n th e s tudy

. 16. The l o c a t i o n w h e r e r a w da t a a nd f i na l r e po r t ar e t o be s t o r e d

. 1 7

. A statem en t p r ep ared b y th e Q u ality A ssu ran ce U n it listin g th e d a tes th at stu d y in sp ection s an d

a udi t s w e r e m a de a nd th e da t e s o f a n y f

i ndi ng s r e po r t e d t o th e Study Di r e c t o r a nd Ma na g e m e nt

. 1 8

. I f it is n ecessary to make correction s or ad d ition s to a fin a l rep o rt after it h a s b een accep ted , su ch ch an ges will b e m a d e in th e form of an am en d m en t issu ed by t h e St udy Di r e c t o r
. The am e ndm e n t will clearly id en tify th e p art of th e fin a l rep o rt th at is b e in g am en d e d an d th e reason s for th e
am en d m en t, an d will b e sign ed b y th e S tu d y D i rector.

CHANGES TO PROTOCOL P l an n e d ch an ges to th e p r otocol will b e in th e form of written am en d m en ts sign ed b y th e S tu d y D i rector an d ap p r oved b y th e S p on sor’ s R e p r esen tative. A m en d m en ts will b e con s id ered as p art of th e p r otocol an d will b e attach ed to th e fin a l p r otocol. A n y oth e r ch an ges will b e in th e form of written d e viation s sign ed b y th e S t u d y D i rector an d filed with th e raw d a ta. A ll ch an ges to th e p r otocol will b e in d i cated in th e fin a l rep o rt.

GOOD LABORATORY PRACTI CES This s t udy w ill be co nducted in acco rdance w i t h G o o dL a b o r a t o r y

P r a c ti c eS t an d a r d s f o r

E P A ( 4 0 CFR Par t 160 a nd/ o r Par t 792) ; OECD Pr i n c i pl e s o f Go o d L abo r a t o r y Pr a c t i c e s ( E NV/ MC/ C HEM ( 98) 17) ; a nd Japa n MAFF ( 11 No hSa n , No t i f i c a t i o n No . 6283, Ag r i c u l tu r a l Pr o duc t i o n Bur e a u , 1 Oc t o be r 1999) . Ea c h s tudy c o nduc t e d by i s r o ut i n e l y e x a m i n e d by th e Q u ality A ssu ran ce U n it for com p lian ce with G ood Lab o ratory P r actices, S t an d ard O p eratin g P r oced u r es an d th e sp ecified p r otocol. A statemen t of comp lian ce with G ood Lab o ratory Pra c tic e s w ill be pre p ar e d fo r a ll po rtio ns o f the s tudy c o nduc te d by The S p on sor will b e resp on sib l e for com p lian ce with G ood Lab o ratory P r actices for p r oced u r es p e rform ed b y oth e r lab o ratories (e. g
. , resid u e an aly s es or p a th ology ). R a w d a ta for all work p e rform ed at

an d a cop y of th e fin a l rep o rt will b e filed b y p r oj ect n u m b e r in arch ives located on th e site, or at an altern ative location to b e sp ecified in th e fin a l rep o rt.- 1 1 -
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Ca l c ul a t i n g a n L C 50,” Aquat i c Tox i c o l ogy and Haz a r d Ev al uat i ons
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