P R O T O C O L

A 48-HOUR STATIC ACUTE TOXICITY TEST WITH THE CLADOCERAN ( Daphnia magna )

OECD Guideline 202

U.S. EPA OPPTS Number 850.1010- 2 -

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 a c u te to xic ity te s t w ith the c l a d o c e r a n , Daphni a

magna , for th e S p on sor at th e aq u a tic tox i cology facility in E a s t o n , M a r y l and.

T h e stu d y will b e p e rformed b a sed on p r oced u r es in th e OECD Gui d e l i n e f o r Te s ti n g o f Che m i c a l s , 202: Daphnia s p . Ac ute I mmobiliz ation Te s t and Re pr oduc tion Te s t ( 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 850.1010: A q u a tic In verteb r a t e A c u t e T o xicity T e st (2 ); an d

ASTM St a ndar d E- 729- 96, St andar d Gui d e f o r

Conduc t i ng Ac ut e Tox i c i t y Te s t s on Te s t Mat e r i al s wi t h

Fi s h e s , Mac r oi nv e r t e br at e s and Amphi bi ans ( 3 )

. R a w d a ta for all work p e rform edat

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.

OBJECTI V E

T h e ob j ective of th is stu d y is to d e termin e th e acu te effects of a test su b s tan ce on th e clad oceran , Daphni a magna , unde r s t a tic te s t c o nditio ns fo r a pe rio d o f 48 ho urs

.

EX PERI MENTAL DESI GN Daphni a magna 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 4 8 h o u r s. T w o rep licate

test ch amb e rs will b e main tain ed for each treatmen t an d th e con trol grou p , with 1 0 Daphni a magna in

each test ch amb e r for a total of 2 0 Daphni a magna p e r treatm e n t level.

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
in d i cates th at a d i fferen t d ilu tion factor wou l d b e more ap p r op riate. W ater samp les from each test

con cen tration will b e collected for an aly s is of T o tal O r gan i c C arb on (T O C ).- 4 -

To c o ntro l bia s , ne o n a t e daphnids w ill be im partia lly a s s i g n e d to e xpo s u re c h a m be rs a t te s t

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 /im m o b ility 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 4 8

- h o u r test p e riod

. C u mu lative p e rcen t m o rtality /im m o b ilty ob served in th e treatm e n t grou p s will b e u s ed to calcu late, wh e n

p o ssib l e, E L 5 0 valu es at 2 4 an d 4 8 h o u r s. T h 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

, im m o b ility 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).

Te s t Or gani s m

T h e clad oceran , Daphni a magna , h a s b een s e l e c t e da s t h et e s t s p e c i e s f o r

t

h i s s t udy . Da phni ds a r e

rep r esen tative of an imp o rtan t grou p of aq u a tic in verteb rates, an d h a ve b een selected for u s e in th e test

b a sed u p o n p a st u se h i story an d ease of cu ltu rin g in th e lab o ratory

. Da phni d ne o n a t e s t o be us e d i n t h e t e s t

will b e less th an 2 4 h o u r s old an d will b e ob tain ed from cu ltu res m ai n t ai n e d a t- 5 -

Ea s t o n , Mary la nd. The ide n tity o f the s p e c i e s w ill be v e rifie d by the s upplie r o f th e o r ig ina l c u lture o r by

pe r s o nne l us i n g appr o p r i a t e t a xo no m i c k e y s s u c h a s Pe nnak ( 4 )

.

Daphnids w ill be c u lture d in w a te r fro m the s a m e s o urc e a nd a t appro x im a t e l y the s a m e

te m p e r a t ure a s w ill be us e d during the te s t

. Daphnids in the c u lture s pro duc ing ne o n a t e s fo r the te s t w ill

be he l d f o r

a t l e a s t 10 da y s pr i o r t o c o l l e c t i o n o f t h e ne o n a t e s f o r t e s t i n g

. Adul t daphni ds i n t h e c u l tu r e will p r od u ce an average of at least 3 y o u n g p e r ad u lt p e r d a y over th e 7 d a y p e riod p r ior to th e test.

Ne o n a t e s fro m daphnids tha t s h o w s i g n s o f dis e a s e o r s tre s s w ill no t be us e d a s te s t o r g a nis m s

. Daphnids

in ho lding tha t pro duc e e phippia a l s o w ill no t be us e d to s upply ne o n a t e s fo r te s ting

.

Daphnids in the c u lture s w ill be fe d o n c e da ily . The die t w ill be a m i xture o f y e a s t, Ce ro phy ll®,

a nd tr o u t c h o w ( Y CT)

, s uppl e m e n t e d w i t h a s u s p e n s i o n o f t h e f r e s h w a t e r g r e e n a l g a Se l e nas t r um

c apr i c or nut um

. A d u lts are fed d u r in g th e 2 4

- h o u r p e riod p r ior to test in itiation

, b u t n e on ates will n o t b e fed during the tes t . Specifi catio ns fo r acceptable lev e ls o f co ntam in ants in daphnid diet s hav e no t been

estab lish ed

. H o wever, th ere are n o kn own levels of con t amin an ts reason ab ly ex p ected to b e p r ese 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

.

N e on ates will b e ob tain ed for testin g from at least th ree in d i vid u a l ad u lts th at h a ve alread y

p r od u ced at least on e p r eviou s b r ood

. P r ior to test in itiation

, th e n e on ates will b e collected from cu ltu res

a nd tra n s f e rre d to s m a ll c o nta i ne rs . The daphnids w ill be re le a s e d into the te s t c o m p ar tm e n ts be lo w the

water su rface u s in g a wid e- b ore p i p ette.

D ilu tio n Wa ter

T h e water u s ed for cu ltu rin g an d testin g 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 h e water will b e p a ssed th rou g h a san d filter an d

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 no z z l e s

. Prio r to us e the

water will b e filtered to 0

. 4 5 μm in ord e r to rem ove fin e p articles, an d m a y b e U V

- sterilized

. Water u s ed

for cu ltu rin g an d testin g is ch aracterized as mod e rately h ard

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

, p H

a nd 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 145 3 A l kalin ity

, m g /L as C a C O 190 3 pH 8.1- 6 -
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 term in e th e con cen tration s of selected organ i c an d in organ i c con s titu en ts in th e water an d resu lts of th e

most recen t an aly s es will b e su mmarized in th e fin a l rep o rt.

T e st A p p a ra tu s

T e st ch am b e rs will con s ist of glass b eakers or glass aq u aria. A ltern atively

, test ch am b e rs will

con sist of test comp artmen ts immersed in glass b eakers or glass aq u aria. W h en test comp artmen ts are

us e d , the y w ill be 300 m L g l a s s be ak e r s w ith ny lo n- c o v e re d ho le s o n o ppo s ite s i de s o f the be ak e r . The

size of th e test ch amb e r an d th e volu m e of test solu tion in each ch amb e r will b e su fficien t to meet th e

req u i rem e n t s of sam p lin g for an aly tical verification of test su b s tan ce con cen tration s an d rou tin e water

ch em istry an aly s es. T e st ch am b e rs will b e in d i scrim i n a tely p o sition e d in a tem p eratu r e- con trolled water

b a th or an en viron m en tal ch am b e r. T h e water b a th will b e en closed in a p l ex igass ven tilation h ood

. 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 a te th e cu ltu res an d test ch am b e rs d u r in g cu ltu rin g an d testin 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 arkn e ss will b e con trolled with an au tom a tic tim e r

. A 30-

m i nute tra n s itio n pe rio d o f lo w lig ht inte ns ity w ill be pro v i de d w h e n lig hts g o o n a nd o ff to a v o i d s udde n
ch an ges in ligh t in ten s ity

. Ligh t in ten s ity will b e m easu r ed at test in itiation with a S P E R S c ien tific Ltd

. ligh t m e ter or eq u i valen t

.

T h e target test temp eratu r e will b e 2 0 ± 1 ° C

. T emp eratu r e will b e measu r ed in each test ch ambe r

a t t h e be g i nni ng a nd e nd o f t h e t e s t , a nd a t 24- ho ur i n t e r v a l s dur i n g t h e t e s t , us i n g a ha nd- he l d

th erm o m e ter. T e m p eratu r e also will b e m e a s ur e dw i t h a c o nti nuo us r e c o r d e r i n a c o nt ai ne r o f w a t e r pl aced

ad

j acen t t o t h e t e st ch amb e r s

. R ecor d er measu r em e n ts w ill bev e r i f

i e dw ith a hand- he ld the r m o m e te r pr io r
to test in itiation

.- 7 -

D i ssolved ox y g en an d p H will b e m easu r ed i n each treatmen t an dcon t rol rep licate at test in itiation

an d at ap p r ox imately 2 4

- h o u r in tervals d u r in g th e test. I n th e even t th at d i ssolved ox y g e n l e v e l s a ppr o a c h

or fall b elow 6 0 % satu ration

, th e S p on sor will b e n o tified

, and a ppr o p r i a

t e c o r r e c ti v e a c ti o ns w ill b etaken

, if n ecessary , to m ain tain d i ssolved ox y g en levels ab ove 6 0 % satu ration

. Di s s o l v e d o x y g e n c o nc e n t r a

ti o ns

w ill be m e a s ure d w ith a The r m o Orio n Mo de l 850Aplus dis s o lv e d o x y g e n m e te r, o r e quiv a le nt a nd pH

w ill be m e a s ure d w ith a The r m o Orio n Mo de l 720Aplus pH m e te r, o r e quiv a le nt. I f a

tre a t m e nt re plic a t e
reaches 100% m o rtality , di s s o lv ed o x y g en, pH and tem p erature m eas urem ents w ill be tak e n in that

rep licate at th at time, an d th en d i scon tin u e d

.

Har dne s s , a l k a l i n i t y , s p e c i f i c c o nduc t a nc e a nd t o t a l o r g an i c c a r b o n ( T O C ) w ill b em easu r edin th e
d i l u t i o n w a t e r a t

t h e b e g i n n i n g o f t h e t e s t

. H ar d n e s s a n d a l k a l i n i t y m e asu r em en ts will b e m a d e b y titration

us i ng pr o c e dur e s ba s e d o n m e t ho ds i n 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 (5). Spe c i fic c o nduc ta nc e w ill be m e a s ure d us in g a Ye llo w Spring s I n s trum e nt Mo de l 33 Sa linity

Co nduc tiv ity

- T e m pe ra ture m e te r, o r e quiv a le nt. To ta l o r g a nic c ar bo n w ill be m e a s ure d o n a Shim a d z u
Mo de l 5000 TOC a n a l y z e r , o r e quiv a le nt. Additio na l w a te r qua lity m e a s ure m e n ts m a y be tak e n a s
d eem ed n ecessary b y stu d y p e rson n e l. T h e reason for th e ad d ition a l m easu r em e n t s w ill b ed o cu m e n t edin

th e raw d a ta an d su m m arized in th e fin a l rep o rt.

Bi ol ogi c a l Obs e r vat i o ns
O b servation s of m o rtality

, im m o b ili z a ti o n and c l i n i c a l s i g n s o f t o xi c i t y

w ill b em a d eb e tween 0

- 2 4

ho ur s , a nd a t 24 a nd 48 ho ur s ±1 ho ur

. I m m o bi l i z a t i o n i s de f i ne d a s a l a c k o f m o v e m e nt by t h e t e s t

organ i sm ex cep t for m i n o r activity of th e ap p e n d a ges.

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 from p ooled (old ) solu tion s from each test con cen tration s at

th e en d of th e test. T o tal organ i c carb o n (T O C ) will b e measu r ed in each samp l e us i n g a Shi m a d z u M o de l

5000 TOC a n a l y z e r , o r e qui v a l e nt

.

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:- 8 -

1) im m o bility o f the daphnids in the neg a tiv e co ntro l g r o up w ill no t exceed 10% by the end o f

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 rou g h o u t

th e test.

Dat a Anal ys i s
Wh en th e d o se- r esp o n s e p a ttern allows calcu lation of an E C 5 0 valu e, th e m o rtality /im m o b ility

p a ttern will b e u s ed to calcu late th e 2 4 an d 4 8

- h o u r E L 5 0 con cen tration s

. T h e E L 5 0 valu es will b e b a sed

o n no m i na l l o a d i n g r a t e s a nd c a l c u l a t e d us i n g t h e c o m put e r s o f t w ar e o f C.E. St e pha n ( 6 )

. The pr o g r a m
was d e sign ed to calcu late th e E L 5 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 (7

, 8

, 9 ). T h e m e th od u s ed will d e p e n d u p o n th e
im m o bility pattern and its s u itability fo r each m e tho d . Additio nal an aly s is o f data m a y be co nduc ted if

d eem ed ap p r op riate b y th e S t u 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 i s de f i ne d by

th e con cen tration

- resp on se p a ttern

. T h e m a x i mu m WA F level cau sin g 0 % im m o b ility an d th e m i n i mu m
WAF le v e l c a u s i ng 100% im m o bility 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 Wild life I n tern ation a l, Ltd

. 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

. H i story of th e test organ i sm , cu ltu re an d h o ld in g record s.
5

. S t ock solu tion calcu lation an d p r ep aration

. 6

. D a ily ob servation s

. 7

. Water ch em istry resu lts (e. g

. , h ard n e ss an d alkalin ity ).

8

. I f ap p licab le, th 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.

9

. S t atistical calcu lation s

. 1 0

. T e st con d ition s (ligh t in ten s ity

, p h o top e riod

, etc. ).- 9 -

11. 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

. 12. 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 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 t h 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 t h e s o ur c e , s c i e nt i f i c na m e , a g e o r l i f e s t a g e a nd

feed typ e s.
9

. A d e scrip tion of th e p r ep aration of th e test solu tion s

. 10. The m e t h 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 , t h 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

con c lu sion s d r awn from th e an aly s es. A grap h p l ottin g th e con cen tration resp on se cu rve at 4 8

ho ur s , w h e n s u f f i c i e n t da t a e x i s t s . I f t h e da t a i s c o nduc i v e t o e v a l ua t i o n by pr o b i t a n a l y s i s , t h e slop e of th e con cen tration

- resp on se cu rve will b e rep o rted

.- 1 0 -

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

t h e s t udy

. 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 t h 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 b y th e S t u d y D i rector. T h e am en d m en 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 t u 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 ith Go o d L abo rato ry Practi ce Standards fo r EPA (40

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 t u 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 t udy 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 t h 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 -

REFERENCES

1 Or gani z ati o n f o r

Ec onom i c Coope r a ti on and De ve l o pm e n t

. 1984. Gui d e l i n e 202: Daphni a s p

. Ac ute Immobiliz ation Te s t and Re pr oduc tion Te s t

. O E C D G u id elin e for T e stin g of C h em icals. Upda t e d Gui d e l i n e , a d o p t e d Apr i l

, 1984.

2 U

. S

. E n v i ro n m en ta l P r o t ectio n A g en cy . 1996. A q u a tic In verteb r a t e A c u t e T o xicity T e st, Fre s hwat e r Daphni ds

. 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 ( dr af

t )

, OPPTS Num b e r 850.1010.

3 ASTM St andar d E729- 96. 1996. St andar d Gui d e f o r

Conduc t i ng Ac ut e Tox i c i t y Te s t s on Te s t Mat e r i al s wi t h Fi s h e s , Mac r oi nv e r t e br at e s , and Amphi bi ans

. A m erican S o ciety for T e stin g an d Ma te ria l s

.

4 Pe nnak , R.W. 1978. Fr e s hw a t e r I n v e r t e b r a t e s o f t h e Uni t e d St a t e s . 2nd Ed. 365 p.

5 APHA, AWWA, WPCF. 1998. St andar d Me t hods f o r t h e Ex ami nat i on of W a t e r and Wa stew a t er

. 2 0 t h E d ition

, A m erican P u b lic H ealth A ssociation

. A m erican Water Works A ssociation

. Water P o llu tion C o n trol F e d e ration

, N e w Y o rk.

6 St e phan, C.E. 1978. U.S. EPA, Env i r o nm e n t a l Re s e ar c h L abo r a t o r y , Dul u th

, Mi nne s o t a

. P e rson al commu n i cation

.

7 Fi nne y, D.J. 1971. St at i s t i c al Me t hods i n Bi ol ogi c a l As s a y

. S econ d ed ition

. G r iffin P r ess, L o ndo n.

8 Thom ps on, W

. R. 1947. B a cterio lo g i ca l R eview s . Vo l

. I I
, No . 2. Pp. 115- 145.

9 St e phan, C.E. 1977. Me t h o d s f o r

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

. A m erican S o ciety for T e stin g an d M a terials. P u b licati o n Num b e r STP 634. Pp 65- 84.