P R O T O C O L

A 72-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA ( Pseudokirchneriella subcapitata )

OECD Proposal for Updating Guideline 201

EU Directive 92/69/EEC, Method C.3.- 2 -

A 72- HOUR TOXI CI TY TEST WI TH THE FRESHWATER AL GA ( Ps e udok i r c hne r i e l l a s ubc api t a t

a )

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 a rt 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 .: S p on sor A p p r oval D a te: 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 a

ppl i c a b 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 n a l g a l to xic ity te s t w ith the fre s h w a te r a l g a , Ps e udok i r c hne r i e l l a s ubc api t a t

a , 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 ou tlin ed in th e workin g
dr a f t o f OECD Pr o p o s a l f o r

Upda t i n g Gui d e l i n e 201: Fr e s hwat e r Al ga and Cy anobac t e r i a, Gr owt h

I nhi bi t i on Te s t (1 ) an d O fficial J o u r n a l of th e E u rop ean C o mmu n ities N o

. L383 C.3. Al gal I

nhi bi t i on Te s t (2 ), wh en p o ssib l e. R a w d a ta for all work p e rform ed at an d a cop y of t h e f i na 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 tox i city of a test m a terial to th e fresh water green alga, Ps e udok i r c hne r i e l l a s ubc api t a t

a , dur i n g a 72- ho ur e xpo s u r e pe r i o d

.

EX PERI MENTAL DESI GN

T h e green alga, Ps e udok i r c hne r i e l l a s ubc api t a t

a , will b e ex p o sed to a geom etric series of a t l e a s t
five water accommod a ted fraction (W A F ) load in g rates an d a n e gative (cu ltu re med i u m ) con trol for 9 6

h o u r 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 on
in form ation su ch as a resu lts of ran g efin 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

pro p erties o f the tes t s ubs tance o r o ther relev a nt info rm atio n. Targ et lo ading ra tes w ill no t exceed 1000

m g /L. 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 th e d ilu tion

water volu m e (mg/L) to ach ieve th e resp ective W A F solu tion

. G e n e rally
, each con cen tration of test

m a terial u s ed in th e d e fin itive test will b e at least 5 0 % of th e n e x t h i gh er treatm e n t , u n l ess in form ation

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 is more ap p r op riate.
Water samp les from each test con cen tration will b e collected for an aly s is of To t a l Or g a ni c Ca r b o n ( T OC)

.

Te s t s o lutio ns w ill be ino c ula t e d w ith a ppro x im a t e l y 10,000 Ps e udok i r c hne r i e l l a cells/m L. S i x

rep licates in th e con trol an d th ree rep licates p e r treatm e n t will b e tested

. I n ord e r to con trol b i as, th e

p o sition of th e flasks will b e d e term in ed b y in d i scrim i n a te d r aw at th e b e gin n i n g of th e ex p e rim e n t an d
d a i l y d u r i n g th e e x p o s u r e p e r i o d

. N o o th e r p o t e n t i a l s o u r c e s of b i as are ex p ected to affect th e resu lts of th e

s t

udy

.- 4 -

The re s p o n s e o f the a l g a e w ill be m e a s ure d in te rm s o f c h a n g e s in a r e a unde r the g r o w th c u rv e (b iom a ss) an d growth rate. E b L 50 a nd E r L5 0 valu es (b ased on th e th eoretical load in g rate th at p r od uc e s a
50% re duc tio n in a r e a unde r the g r o w th c u rv e (bio m a s s ) a nd g r o w th ra te , re s p e c tiv e l y) w ill be c a l c u la te d

at 2 4
, 4 8
, an d 7 2 h o u r s. T h e 7 2

- h o u r E L 1 0 an d E L 2 0 valu es will also b e calcu lated for b i om ass an d growth rate, if ap p licab le. T h e n o ob served effect con cen tration (N O E L) may b e selected b a sed u p o n
evalu a tion of th e statistical resu lts an d th e d o se- r esp o n s e p a ttern of growth in h i b ition

.

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 a rd 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 th a t th e t e s t s u b s t a n c e h a s b e e n c h a r 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 th e e nd o f t h e s t udy

.

Te s

t Sol u t i on Pr e p ar at

i o n

T h e test su b s tan ce will b e m i x ed d i rectly with cu ltu re m ed i u m on a wei g ht : v o l um e ba s i s

. Ea c h WAF w ill be pre p a r e d indiv i dua lly in a 4 L Py re 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 4 L of d ilu tion water u s in g a T e flon ®- coated stir b a r on a

m a gn etic stir p l ate. C a re 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 fresh water green alga, Ps e udok i r c hne r i e l l a s ubc api t a t

a will b e u sed i n t

h i s t e s t

. Thi s s p e c i e s is rep r esen tative of an imp o rtan t grou p of algae an d was selected for u s e in th e test b a sed u p o n p a st u s e

an d ease of h a n d lin g in th e lab o ratory

. S t ock cu ltu res, ob tain ed from th e C u ltu re C o llection of A l gae at
th e U n iversity of T e x a s at A u stin or an oth e r su p p lier, will b e m a in tain ed in cu ltu re m e d i u m at
for a m i n i m u m of two weeks p r ior to u s e in a tox i city test. A l gae u s ed in tox i city tests- 5 -

will b e in ex p o n e n tial growth p h a se, wh ich is d e fin e d as th e p e riod of gr o wt h w h e n a l g a l c e l l s a r e di v i di ng
at a con s tan t rate.

J u st p r ior to b e gin n i n g th e test, an in ocu l u m of th e stock cu ltu re will b e p r ep ared so th at each

m illilite r o f ino c ulum c o nta i ns e n o u g h c e lls to pro v i de a n initia l c e ll de ns ity o f a

ppro x im a t e l y 10,000
cells/mL in each rep licate.

Cul t ur e Me di

um

C u l t u r e m e d i u m p r e p a r e d accord in g to S t an d a rd

O p eratin g P r oced ur es
will b e u s ed as d ilu tion water. T h e m e d i u m com p on en ts an d c o nc e n t r a

t i o ns a r e pr e s e n t e d i n Ta bl e 1 ( 1
, 3 )

. S t ock n u trien t solu tion s will b e p r ep ared b y ad d i n g reagen t- grad e or b e tter ch em icals to well water p u r ified b y reverse- osm o sis. A p p r op riate volu m es of th e stock n u trien t
solu tion s will th en b e d ilu ted with p u r ified well water to p r ep are th e m e d i u m

. T h e m e d i u m will b e filter

sterilized (0

. 2 μm) or au toclaved p r ior to u s e. T h e fin a l p H of th e med i u m sh ou ld b e 8

. 0 ± 0

. 1

. A n aly s es

w ill be pe rfo rm e d a t le a s t o n c e a nnua lly to de te rm ine the c o nc e n tra tio ns o f s e le 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 d resu 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 a l rep o rt.

S p ecification s for accep ta bl e l e v e l s o f c o nt a m i n a n t s ha v e no t be e n e s t a bl i s he d f o r

c u l t ur e m e di um

. H o wever, th ere are n o levels of con t amin an ts reason ab ly ex p ected to b e p r esen t in th e med i u m th at are

c o ns i d e r e d t o i n t e r f e r e w i th th e pur po s e o r c o nduc t o f th e s t udy

.

T e st A p p ara tu s

Te s t c h a m be rs w ill be s t e r ile , 250- m L Erle nm e y e r fla s k s plug g e d w ith fo a m s t o ppe rs a nd

c o nta i ning 100 m L o f te s t s o lutio n. Te s t c h a m be rs w ill be indis c rim i na te ly po s itio ne d o n a m e c h a n ic a l

s h a k e r in a n e n v i ro nm e n ta l c h a m be r a nd w ill be s h a k e n c o ntinuo us ly a t a

ppro x im a t e l y 100 rpm

. Te s t
ch am b e rs will b e lab e led 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

Te s t fla s k s w ill be he ld a t 24 ± 2° C unde r c o ntinuo us c o o l

- w hite fluo re s c e n t lig hting a t a n
inte ns ity o f a

ppro x im a t e l y 6000 lux +/- 20%. L i g h t inte ns ity w ill be m e a s ure d a t fiv e lo c a tio ns o n the- 6 -

sh aker tab l e at test in itiation

. T e m p eratu r e will b e m easu r ed twice d a ily in th e en viron m en tal ch am b e r

u s in g a calib rated
, h a n d

- h e ld th erm o m e ter.

T h e p H of each treatmen t an d th e con trol will b e measu r ed at test in itiation an d termin ati o n us i n g

a

Fis h e r Ac c u m e t Mo de l 915 pH m e te r o r e quiv a le nt. Sa m p le s w ill be c o lle c t e d fro m the ba tc h o f te s t
solu tion p r ep ared at test in itiation for each treatmen t an d con trol grou p f o r t h e m e a s ur e m e n t o f pH. At t e s t
termin ation
, p H will b e measu r ed in p ooled test solu tion samp les collected from th e rep licates of each

tr e a t m e nt a nd c o nt r o l g r o up.

Bi ol ogi c a l

Me as

ur e m e nt s

Ce ll de ns itie s w ill be m o nito re d during the te s t by c o nduc ting c e ll c o unts us ing a n e l e c tro nic

p a rt i c l e c o u n t e r ( C o u l t e r C o r p o r a t i o n M o d e l Z M ) o r a

h e m a c y t o m e t e r an d a m i croscop e. O n e sam p le will

b e collected from each rep licate of th e treatmen t an d con trol grou p s at ap p r ox imate 2 4

- h o u r in tervals

d u r i n g th e e x p o s u r e p e r i o d

. A p p r o p r i a t e l a b o ratory S t an d a rd

O p eratin g P r oced ur es will b e followed wh en

p e rform in g algal cell d e n s ity evalu a tion s

. I f test solu tion s sh ow sign s of p r ecip itates, cell d e n s ity

en u m eration will b e p e rform ed with a h e m acy tom e ter an d m i croscop e

. I f p r ecip itate s a r e no t e v i d e n t , c e l l

de ns i ty e num e r a t i o n m a y be m a de us i n g th e e l e c tr o ni c pa rt i c l e c o unt e r . Expo ne nt i a l g r o w th i n th e

n e gative con trol an d solven t con trol will b e con f irm e d at 7 2 h o u r s.

S amp les of test solu tion s will b e collected from each rep licate p er treatmen t an d con trol grou p at

th e e nd o f th e t e s t . Whe n c e l l c o unt s a r e c o nduc t e d us i n g a n e l e c tr o n i c p a r t i c l e c o un t e r , t h e s e s a m

p l e s w ill

b e p ooled with in th eir resp ective treatm e n t s, an d su b s am p l es will b e rem oved an d ex am in ed microscop i cally for aty p ical cell morp h o logy (e. g

. , ch an ges in sh ap e, size or color). W h en cell cou n t s are

co nducted us ing a he m acy to m e ter a nd a m i cro s co pe, each replicate s a m p le w ill be exam ined

m i croscop i cally for aty p ical cell m o rp h o logy

. G r owth of cells in th e flasks also will b e assessed for aggregation s or floccu lation s of cells an d ad h e ren ce of th e cells to th e test ch amb e r.

Sam p l i n g f o r Anal yt i c

al

Me as

ur e m e n t

s

S a mp 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- 7 -

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 zu 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:

1 ) th e mean cell d e n sity in th e con trol flasks mu st h a ve in creased b y a factor of at least 1 6

with in 3 d a y s , 2 ) th e p H of th e con trol sh ou ld n o t n o rm ally d e viate b y m o re th an 1

. 5 u n its d u r in g th e test,

a nd

3 ) th e coefficien t of variation of average sp ecific growth rates in th e con trol rep licates

d u r in g th e wh ole test p eriod sh ou ld n o t ex ceed 7 %

.

St

at

i s

t i c

a l

Anal ys e s

Are a unde r the g r o w th c u rv e w ill be c a l c u la te d fo r the tre a t m e nt a nd c o ntro l g r o ups us ing the

followin g form u l a:
A = ((N 1 -N o )/2 )(t 1 )+((N 1 +N 2 -2 N o )/2 )(t 2 -t 1 ) + ...+ ( ( N n- 1 +N n -2 N o )/2 )(t n -t

n- 1 )

W h ere:

A = Ar e a unde r th e g r o w th c u r v e N o = No m i na l num be r o f c e l l s / m L a t t o N 1 = M easu r ed n u m b er of cells/mL at t 1 N 2 = M easu r ed n u m b er of cells/mL at t 2 N n = M easu r ed n u m b er of cells/mL at t n t 1 = T i m e of first m easu r em en t after b e gin n i n g of test (h ou rs)
t 2 = T i me of secon d measu r emen t after b e gin n i n g of test (h ou rs)
t n = Ti m e o f nth m e a s ur e m e n t a f t e r be g i nni ng o f t e s t ( h o u r s )

A v erage sp ecific growth rates will b e calcu lated for th e treatm e n t an d con trol grou p s u s in g th e
followin g form u l a:

o n o n t

- t N ln

- N ln μ- 8 -

W h ere:
μ = A v erage sp ecific growth rate N 0 = N o m i n a l cell d e n s ity (cells/m L) at t o N n = Measu r ed cell d e n s ity (cells/m L) at t n t n = T i m e after b e gin n i n g of test (h ou rs)
t o = T i me of b e gin n i n g of test (h ou rs)

P e rcen t in h i b ition will b e calcu lated for each treatmen t grou p u s in g th e followin g formu l a:

100 x Response Control Mean Response Treatment Mean

- Response Control Mean Inhibition Percent

E b L 50 v a l ue s a nd E r L 5 0 v a l u es will b e d e term in ed
, wh en p o ssib l e, u s in g lin ear in terp olation (4 ) or

o the r s ui t a bl e t e c hni que s w i th tr e a t m e nt r e s po ns e ( bi o m a s s a nd g r o w th r a t e ) a nd e xpo s ur e c o nc e ntr a t i o n
d a ta. T h e 7 2

- h o u r E L 1 0 an d E L 2 0 valu es will also b e calcu lated for b i om ass an d growth rate, if

ap p licab le.

A n o ob served effect load in g rate (N O E L) may b e selected b ased on an evalu ation of th e d o seresp on se p a ttern an d th e resu lts of th e statistical an aly s is of th e b i om ass an d growth rate d a ta at 7 2 h o u r s.
S t atistically sign ifican t d i fferen ces b e tween th e con trol an d th e treatm e n t grou p s will b e id en tified u s in g

a n a l y s i s o f v a r i a n c e ( ANOVA) a nd a t e s t t o c o m p a r e tr e a t m e nt m e a n r e s p o n s e s t o th e c o ntr o l r e s p o n s e (e .g ., Dunne tt’ s te s t o r Bo nfe rro ni’ s t- te s t ). Da ta w ill be a s s e s s e d fo r no rm a lity a nd ho m o g e ne ity o f

v a rian ces p r io r to p e rfo rm in g th e ANOVA. Tran sfo r m a tio n s w ill b e u s ed , w h en p o ssib le, to co rrect an y

con d ition of n o n

- n o rm ality or u n e q u a l error varian ces. I f a solven t con trol is u s ed in ad d ition to th e

n e gative con trol, th eir resp on ses will b e com p a r e d us i n gSt ude nt ’s t

- t e s t

. Da t a

f

r o m t

h e t w o c o nt r o l g r o ups
will b e p ooled for treatm e n t level an aly s es if n o statistical d i fferen ces ex ist. O th e rwise, all com p arison s

s h o u l d be m a de t o th e s o l v e n t c o ntr o l g r o up. St a t i s t i c a l c o m p a r i s o n s f o r

th e ANOVA a n d t h e c o m p a r i s o n
of m ean resp on ses will b e carried ou t u s in g T O X S T A T V 3

. 5 software (4 ) or eq u i valen t

.

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 d a t W i l d l i f e In t e r n a t i o n a l, Ltd

. will in clu d e , b u t n o t b e
lim ited to:- 9 -

1

. C o p y of 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 S p on sor.
3

. D a tes of in itiation an d term in ation of th e test.
4

. S o u r ce of algae.

5

. C u ltu re con d ition s

. 6

. G r owth measu r emen ts.

7. Ca l c ul a t i o n a nd pr e p a r a t i o n o f t e s t c o nc e n tr a t i o ns

. 8. Obs e r v a t i o ns

. 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

. 1 1

. T e st con d ition s an d p h y sical/ch e m i cal m easu r em en ts.
12. Co py o f f

i na l r e po rt

.

FI NAL REPORT

A fin a l rep o rt of th e resu lts of th e stu d y will b e p r ep ared b y Wild life I n tern ation a l, Ltd

. T h e rep o 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 a rd 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 a b 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. Sta b 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

t i 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 th e m e th o d s us e d t o c o nduc t th e t e s t

. 8

. A d e scrip tion of th e test organ i sm s, in clu d i n g th e sou r ce, scien tific n a m e , age or life stage, ligh t
i n t e ns i ty a nd pho t o pe r i o d

. 9

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

.- 1 0 -

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 th 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 th e St udy Di r e c t o r a nd t h e na m e s o f o the 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.
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 t udy

. 16. The l o c a t i o n w h e r e r a wda t a a nd f i na l r e po rt a r 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 were

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

i ndi ng s r e po rt e d t o th e St udy 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 a m e ndm e n t
will clearly id en tify th e p a rt 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.

CHANGI N G OF 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 t u 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 a rt 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 hG o o d L a b o r a t o r y

P r a c t i c e S t a n d a r d s f o r

E P A ( 4 0

CFR Pa rt 160 a nd/ o r Pa rt 792) ; OECD Pr i n c i pl e s o f Go o d L a bo r a t o r y Pr a c t i c e s ( E NV/ MC/ C HEM ( 98) 17) ; a nd Ja pa 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 th e- 1 1 -

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 a rd

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 a r e d fo r a ll po rtio ns o f the s t udy 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 2 -

REFERENCES

1 OECD. 2004. Wo r k i n g dr a f t o f OECD Pr o p o s a l f o r

Upda t i n g Gui d e l i n e 201: Fr e s hwat e r A l g a and Cy anobac te r i a, Gr owth Inhibition Te s t . Ci r c ul a t e d 9 Apr i l 2004.

2 Of f i c

i al Jour n al of t h e Eur o pe an Com m uni t i e

s

. 1992. No . L 383. Me th o d C.3. Al gal I nhi bi t i on Te s t

.

3 ASTM St andar d Gui d e 1218- 90E. 1990. St andar d Gui d e f o r

Conduc t i ng 96- Hour Tox i c i t y Te s t s wi t h Mi c r oal gae

.

4 West, In c. a n d D . D . G u lley . 1996. TOXSTAT Re l e as e 3.5. We s t e r n Ec o S y s t e m s Te c hno l o g y , I n c. C h ey en n e , W y omin g.- 1 3 -

TABL E 1

FRESHWATER AL GAL MEDI UM CONSTI TUENTS

Com p one n t Nom i nal Conc e n t

r

at

i o n

















T h e p H of th e med i u m will b e ad j u sted
, as n ecessary , to 8 ± 0

. 1 u s in g 0

. 1 N Na OH o r 10% HCl

.