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# 6)11-1-18 4 Docket Number: 

# A-83-2 2

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Sheet 6 of 198P. 2

J .k Epstei n rotatin g dive s hidings nth iisalem
. (Jun e A
. J
. (1974) ,i l process— 232-13 3 (Ma

y ic w approac h nighl y concen Waste Conf. chi c treatmen t natio n i n th e J. Wat. Pnllut. W

. L

. (1975 ) • i her n Nichiga n <mf. Water

Pol- ,975)
. •I
. & Jank

B F
. •lion s fo r sup - •:/ i Ann. Conf. lOctobe r 1975)
. >-> v tricklin g fillul y 1973)
. 

# o i n I J rea l BnUin

. 

# |<?7& -

< * 

# •v f CHRONI C TOXICIT Y O F WATER-BORN E AN D

DIETAR Y LEA D T O RAINBO W TROU T (SALMO GAIRDNERI) I N LAK

E ONTARI O WATE

R
PETE RV
. HODSON , BEVERLEY R
. BLUN T an dDOUGLA S J
. SPR Y Grea t Lake s Biolimnology Laboratory
. Canad aCentre fo r Inlan dWaters
. P.O
. Bo

x 5050
. BufiingtgfflD

A AI D Hrtf^KP T Ontario , L7 R 4A6

. Canad a • W«Mi n UUUI\C » {Received 6 January 1978

) Abstract—Rainbo w trou t

{Salmo gairdneri) exposedto lea di n Lak e Ontari o wate

r demonstrate da 21-da y LC 3 0 o f 2.4mgl" ' lead
. A

t lea dconcentration s rangin g fro m3 t o 120ugl" 1 , log, 0 o f lea d concentration s in mos t

tissue s o f expose dfish appeare dlinearit y relate dt o log, 0 o

f lea dconcentration s i n water . Highes t

concentration s occurre di n opercula

r bone followe

db y gil l an dkidney
. Lea

daccumu latio n b y brai n wa

s no t clearly demonstrated
. Exposur e to lea di n wate r a i mnrpniratjop t

a s lo w a s 1 3 ugl" ' cause dsignifican t increase s i

n redblo~o

dcel l (RBC ) numbers , decrease s i n RB C volumes , decrease s i

n KB L ceuuia r iro n conten t

an ddecrease s I

n RBI : <>-nnun

o levuhni c nci

ddehydratas e uc

tiviTyTN o change s wer

e observe di

n hematocri t

or,whol e bloo diron content . Th e change s indicate d increase derythropoiesi s to compensate fo r inhibitio n o

f hemoglobin productio n an dincreasedmortalit y o f matur e re dbloo dcells
. Afte r 3 2 weeks exposure t o 120ugl" ' lea din water , 30 % o

f remainin g fistkCxhibitedblack

tails , a n early indicatio n ol spina

l deformities
. Lea

dadde dt o foo

dwas no t availabl e <ro r lea duptake b

v fish. Lea

dconten t o f fis h exposedt o dietar

y lea dwa s no t elevate dabov e contro l level s an dthe majorit

y o f lea dconsume dcoul db

e accounte dfo

r i n th e faeces
. Dietar

y lea dma y hav e slightly inhibite duptake o l dietar y iron
. "


## P 

# .^ff^ INTRODUCTION Thi s pape r

demonstrate s tha t

water-born e lea di s readil y take n u p b y fish resultin g i n subtl e subletha l physiologica l responses . Dietar y lead , b y contrast , i s not "take n u p an ddoe s no t affec t fish. " Metal s ar e normall y ptesen l i n al t surfac e water s a t ver y lo w concentrations
. Offshor e i n Lake s Super

ior an dHuron , lea di s foun dmos t frequentl y a t

con centration s belo w lftgl" ' with 95 % o f al l lea d measurement s les s tha n 3/tgl" ' (I.J.C. , 1976)
. in off

shore water s o f

Lak e Ontario , averag e lea dconcen tration s rang e fro mles s tha n Ifigl" 1 i n summe r u p t o 2/igl" 1 i n th e sprin g (Cha u et al, 1970)
. I n con trast , u p t o 5 5 /j g 1" ' ha s bee n measure di n municipa l •$m wate

r intake s i n area s o f

th e Grea t

Lake s clos e t o industrial an durba n developmen t (Kop p & Kroner , 1967)
. Lea dconcentration s a s lo w a s 8 ji g I" ' hav e bee n ^ yttioym t o b e detrimenta l t o fish durin g prolonge d ^r exposure , th e harmfu l concentratio n varyin g with hardness , alkalinity , an dtes t specie s (Davie s et

al., 1976 ; Holcomb e et

al., 1976)
. Hodso n (1976 ) esti

matedb y a n indirec t

metho dtha t

13/igl" ' wa s harmfu l t o rainbo w trou t

(Salmo gairdneri) i n Lak e Ontari o wate r

bu t th e actua l concentratio n wa s un -

- known
. Th e Internationa l Join t Commissio n ha s pro pose dawate r

qualit y objectiv e fo

r lea din Lak e ;,? , Ontari o water s o f

25/tgl" ' (I.J.C. , 1976 ) base do n [
. a n interpolatio n o f

th e alkalinit y effec

t observe db y

, £ Davie s et

al. (1976)
. k*5 c Sinc e th e harmfu l concentratio n o f lea dfo r fish i n Lak e Ontari o ha s onl y bee n estimated , an dsinc e n o effect s o f dietar y leado n fish hav e bee n reported , thi s researc h wa s undertake n t o evaluat e th e physiologica l effect s o n fish o f

prolonge dlea dexposur e i n Lak e Ontari o water .

MATERI A U S \N D METHOD S Experimental

design Thre e experiment s wer

e conducte dt o determin e acute letha l andchroni c subletha

l toxicit y o f lea dadde dt o wate

r andchroni c subletha

l toxicit y o f dietar y lea dt o rainbo w trout . Th e letha

l toxicit y o f lea dwa s measure di

n 3 con tinuous-flo w bioassay s a

t 1T C followin

g th e methodolog y recommende db

y Spragu e (1969)
. Exposur e time s wer

e 3 weeks ; 2 0 fis h pe r tan k wer e testeda t 5 lea dconcentration s rangin g fro

m0. 6 t o 8mgl"'
. plu s control ; an dLC 50 ' s wer e calculate db

y probi t analysi s (Finney , 1971 ) base d on mea n measure dlea

dconcentrations
. The secondexperimen

t examinedrainbo w trou t i n group s o f 23 exposedt o nomina

l concentration s o

f 0 (con

trol) , 10 , 18 , 32
. 5 6 *n d100ugl" 1 lea dadde dt o wate

r b y acontinuous-flo w syste

mstartin g Marc h 25
. 1975
. N o lea dwa s adde dt o food
. Th e lea

dtreate dwate

r a t eac h o f th e concentration s wa

s divide d5 way s t o supply 3 0 tanks
. Th e si

x concentration s wer

e distribute da

t rando m within a3 x 1 0 tan

k grid
. J"is h wer

e sample dafte

r 1
. 2
. ,4
. 8 , 1 6 and3 2 week

s o f exnosure . an dth e followin g measurement s wer

e taken : hejriatocxiL ; bloo diron : number o f redbloo dcell s (RBC's ) pe r mm^erythrocyt e <5-amin o levuhni c aciddehydratas e activit y (ALA-D) ; length ; weight ; andlea

dconcentratio n i

n blood , gills , liver , kidney , remainin g viscera , opercula r bones , carcas s and , in th e large r fistirTo"na3s an

dcentral nervou s syste

m(C.N.S.)
. Result s wer

e analyzedb

y split-plo t

analyse s o

f varianc e (Stee l & Torrie , 1960

) wher e lea dconcentratio n wa

s th e whole plo t facto r andtim e o f exposur e th e sub-plo t

factor
. - Statistica l significanc e o

f difference s betwee

n mean s wa

s 869 IV



### i 4 

# I f 

# wP. 3

S/i . PETE R V

. HODSON , BEVERLE Y R

. BLUN T an d DOUGLA S J

. SPRY ! 

### » ;



## f ;

i: : f ; • teste d a tth e 95 % confidenc e leve l by partitionin g th e treat mentsum s o f

squares . Standar d deviation s and standar d error s were calculate d fro m th e whole-plo terro rmea n square s with erro rdegree s o f freedo m o f

2 4 an d 5 repeats . A log l 0 transformatio n wa s applie d t o th e tissu e lea d dat a t o avoi d non-homogenou s variance . Th e thir d experimen tinvolve d addin g lea d t o th e die t t o giv e a dail y dos e o f

0 (control) , 100 . 180 , 320 . 56 0 an d 100 0 u g lea d kg" ' o f

fish startin g Ma

y 20 . 1975 . N o lead wa s adde d t o th e water . Eac h o f th e si x dos e level s wa s teste d a t5 ratio n level s o f

1 . 1.5. 2.0 . 2. 5 an d 3.0 % o f th e bod y weigh tperday . Fis h wer e sample d a t2 . 4 . 8 , 16 an d 3 2 week s o f

exposure and th e measurement s liste d abov e were made . Th e results were agai n analyze d a tth e 95 % confidenc e leve l by split-plo tanalyse s o f varianc e with lea d a s th e whole-plo tfactor , tim e a s th e sub-plo tfacto r an d ratio n level s as replicates . Statistica l significanc e o f difference s betwee n mean s wa s teste d as indicate d above . Erro rdegree s o f

freedo m were 20 .
Test fish Rainbo w trou twere purchase d a s underyearling s fro m Eri n Conservatio n Holding s Lid. , crin . Ontario . Initial weight s i n th e wate ran d foo d experiment s wer e 6. 5 an d 10. 5 g . respectively . In th e wate rexperiment , fish were fe d a dry-pelletize d trou tcho w ("Ewos" . Astr a Chemical s Ltd. ) ad libitum onc e daily . In th e foo d experiment , fish wer e fe d th e sam e die tt o whic h lea d ha d bee n added . Feedin g wa s onc e dail y excep ta thig h ratio n level s where fish wer e fe d as ofte n as require d t o consum e th e dail y amoun to f food . Durin g thes e experiments , ther e were n o mortalitie s attribute d t o disease . However , in th e chroni c subletha l wate r an d foo d experiments , there wa s 2 5 and 7 % morta lit y respectively , du e t o th e effect s o f aggression , an d 2 an d 4% . respectively , probabl y du e t o a I-wee k perio d durin g whic h chlorin e ros e t o mor e tha n 40ugl"' . Death s du e t o aggressio n di d no tvar y with lea d exposur e an d mos toccurre d durin g th e first 4 week s o f exposure , Th e effect s o f aggressio n were reduce d after4 week s by placin g a vertica l Plexiglas s barrie rin th e for m o f a n "X " i n eac h tank . Th e barrier s had hole s drille d throug h eac h ar m t o permi twate rcirculatio n an d th e escap e o f smalle rfish. Th e barrier s were adde d t o tank s durin g th e firstweek o f th e lead-in-foo d experiment . Waterquality Th e wate rforthes e experiment s wa s draw n a s a munici pa l wate rsuppl y fro

m Lak e Ontario . Treatmen tinvolve d coars e filtration, chlorination . alum flocculation. san d filtratio n an d chlorin e supplementatio n t o a 0. 3 mgl" 1 tota l residual . Chlorin e dioxid e wa s adde d t o contro l tast e an d odou ran d sodiu m silic a fluoride forfluoridation. Prio r t o use , th e wate rwa s filtered throug h activate d charcoa l an d th e temperatur e adjuste d t o 11°C . Th e charcoa l usuall y reduce d chlorin e concentration s t o les s tha n o r equa l t o lOugl" 1 , th e limi to f detectio n by th e orthotoli din e method . However , "breakthrough " occurre d durin g th e experimen tan d chlorin e exceede d 40ugl~ ' forabou t 1 week . Th e mea n ( + 1 standar d deviation ) chemica l character istic s o f th e water , base d o n weekl y measurements , were : tota l hardnes s (a s CaCOQ : 13 5 ± 2 m g I"' ; tota l alkalinit y (a s CaCOO : 9 0 + 4 mgl"' ; conductivity : 24 5 ± 9umho s cm" 2 : an d j)H : 7.7 ± 1.8. Dissolve d oxyge n fluctuated from 6. 1 t o 12 mgl"'"an d mean s range d fro m 7. 1 t o 10.

7 with a standar d deviatio n o f 2.0mgl" 1
. Th e mea n tem perature s i n th e tank s range d from 10.3 t o 11.2° C wit h a standar d deviatio n o f 1.3°C . Th e photoperio d wa s 16 h light . 8 h dark . Fo rchemica l characteristic s othe rthan lead , ther e wa s n o significan tdifferenc e betwee n treat ments . Lead exposures Lead wa s adde d t o wate rby a dose rmodifie d fro m Hod - j so n (1974) . Thi s dose ruse d th e pressur e develope d withi n , a n intermitten tsipho n bottl e t o dispens e 1. 0 m l o f lea d % nitrat e stoc k solutio n durin g eac h cycle . Th e sipho n bottl e j provide d 13.5 1 o f

wate rpercycl e t o a proportiona l diluter ; (Moun t& Brungs . 1967) . Durin g eac h cycl e i n th e chroni c ! experiment , 1.

0 m l o f lea d stoc k wa s dilute d t o a nomina l j 100ug!" ' o f

lead by th e firstchambe ro f th e diluter . The } dilute rsubsequentl y provide d 2.25 1 pe rcycl e o f 6 concen-' l tration s o f

lea d (0 , 10 . 18 , 32 . 5 6 an d 100ugl" 1 ) every. " 1. 5 min . Th e 2.25 1 wa s divide d int o 5 portion s b y flow-^ splitter s givin g a tota l flow t o eac h tank o f 45 0 m l cycle" 1 o r300m l min" 1
. Th e experimenta l tank s wer e polyethy lene , dar k green , measure d 3 0 x 3 0 x 3 0 c m an d containe d 20 1 o f water . Th e 95 % molecula rreplacemen ttim e was . 3- 4 h an d th e flow represente d 1- 2 l/(g'o f fish day"') . Lea

d concentration s wer

e measure d dail y t o allo w comparison s ] o f result s t o measure d concentrations . In th e feedin g experiment , lea d wa s adde d t o th e com - i mercia l trou tdie tboun d t o beef live rprotei a A solutio n o f 2 4 g lead nitrate in 10 0 m l o f distille d wate rwa s adde d t o abou tI k g of

fresh , homogenize d beef liver . Afte

r thoroug h mixing , th e live rwa s freeze-dried . powdered , an d , th e lea d conten to f

th e powde rassaye d a t470 0 m g kg" ' Contro l freeze-drie d live rwith no adde d lea d contained • 17mgkg" ' lead . Appropriat e amount s o f th e powdered 1 live rpreparation s wer

e adde d t o powdere d trou tfood t o giv e 3 0 diet s ( 6 dose s x 5 rations ) o f sufficien tvolum e fo

r th e duratio n o f

th e experiment . Th e maximu m amoun tof ' drie d live radde d t o an y die twa s les s tha n 1 % b y weigh tJ an d th e maximu m nomina l lea d concentratio n wa s lOOmgkg" 1
. Th e diet s wer e pelletized . frozen , an d sub sample s take n fo rlea d analysis . Durin g th e experiment. ; th e fish in eac h tank were weighe d every 2 week s an d • th e dail y amoun to f

foo d wa s adjuste d fo rgrowth . Die t^ moistur e conten twa s monitore d t o compensat e fo r diet * moistur e change s induce d by col d storage . Lead uptak e by fish fro m foo d wa s measure d b y lea d analysi s o f

food , faeces , wate ran d th e fish . T o minimiz e lea d uptak e b

y fish o f lead leache d fro

m th e food , all faece

) an d uneate n foo d wer

e remove d fro

m eac h tank withi

n 3 h o f feeding . Th e leachability of lea d fro m foo d was teste

d by allowin g i

tt o stan d for2 4 h i n wate rbuffered t o severa l pH's . Th e suspension s wer e the n centrifuge d an d th e supernatan tanalyze d forlead . A secon d experimen tallowe d bacterial decompositio n fo r2 week s prio rt o analysis . Th e i | exposur e tank s i

n th e feedin g experimen twere identica l t o thos e i

n th e firstexperiment . Wate rflo w rat e wa s a minimu m 40 0 m

l min" 1
. ' I Sampling and

physiological

methods Tank s wer

e sample d i n a rando m orde ro n eac h samplin g day . Tw o fish wer e remove d an d th e weights an d i standar d length s recorded . Bloo d wa s collecte d i n microcapillary tube s fro

m th e cauda l arter

y afterseverin g th e taf

l a tth e cauda l peduncle . Th e tube s were heparinize d b y rins- 1 in g with a solutio n o f

500 0 ml" 1 o f

sodiu m hepari n fol

lowe d by ove n dryin g a t60°C . Sample s o f

bloo d from th e first fish wer e use d for i measurin g hematocrit , bloo d lead , an d erythrocyt e ALA- D < activity . Sample s fro

m th e secon d fish were use d for determination s o f

hematocrit , number s o f red bloo d cell s pe

r ! j mm 3 of bloo d and bloo d iron . T o measur e hematocrit , as many bloo d sample s a s po« - ibl e wer e collecte d i n microcapillar y tube s pre-chille d bf | liqui d nitrogen . The y were immediatel y seale d an d stored ] i n ic e wate rt o preven tanoxi a an d cellula r swelling ] (Snieszko . 1960) . Withi n 5 mi n o f sampling , the y wer

e cen - i trifuge d for5 mi n a

t700 0 rpm i n a clinica l centrifug e wilh ^ a microhematocri thead . Th e hea d temperatur e wat j reduce d b y pre-chillin g th e lid . Hematocrit s wer e measure d < j with a

wa s ti ' Bi , its p i samp 1 afilm '.'• crucit, : i ' solve d 10. 0 n ;; wate r fuse d t< - ; . • Iro n w ' T RBCs , fivi v RBC"' ;!.m l bl c "i . Tot:. '

- ; :'countc i dilute d mm " • ' ' * Eryl l fro m ( . calcul: . scribe d Bio . 1 meas i

.. „ accor d i : 7i for tist. i f storag o sampl e piemen ' si s an, : tissu e v cibl e a a t80'( . Th e ten an d m. " ultrap ' adde d . ho tpla solve d nitri c t < conccr. ' tio n (Hod s 

## ?r. .

* p

- Th. . LC J0 V 2.3(1. . LC 5 0 I n i ! fluctu .. • th e c; n wer e 0

Nomina l 1

- concenlr:- . 0*l"' >

. 0<Cll l 1 0 'I S 3 2 "w'S *
t.s w.p u 12/1' .P. 4

! f'o m Hod - \>pe d withi n ' nil o f lea d ,:io n bottl e . ional dilute r th e chroni c > a nomina l diluter . Th e of 6 concen a 1 " • ) every n s by flow- 'i l m l cycle" 1 r e polyethy - '. d containe d n ttim e wa s 'ay" 1 ) . Lea d comparison s i n th e com - : A solutio n I T wa s adde d liver . After Adered . an d i'0 0 m g kg" 1
. : l containe d lie powdere d •routfoo d t o : volum e fo

r ••-. amoun tof '. . b y weigh t : -atio n wa s

. n . an d sub experiment , i week s an d growth . Die t ;at e for die t •:L- J by lea d T o minimiz e < I . al l faece s ,n k withi n n l wa s teste d

. . '. t o severa l •••••< th e super - 'cntallowe d .nalysis . Th e

. , e identica l i rate wa s a

eac h sam - «eight s and ! i n microca rin g th e tail ' nize d b y rins - • hepari n fol ate use d fo

r <cyt e ALA- D c d for deter H> d cell s pe

r uplo s a s poss - >rc-chilled b

y d an d store d :i:i r swellin g : ie ) wer e cen - ;ntrifug e wit

h pcratur e wa s '.T C measured ' 

## 4 t wit

h a microcapillar y reade ran d th e mea n fo reac h fish was use d i n all subsequen tcalculations . Bloo d iro n wa s measure d t o indicat e tota l hemoglobin , it s precursors , an d its breakdow n products . Bloo d wa s sample d a s describe d an d immediatel y dispense d ont o Par afilm* . A 50/ d aliquo twa s transferre d t o a n acid-washe d crucibl e an d ashe d a

t460° C for8 h . Th e as h wa s redis solve d i n 2. 0 m l BD H "Aristar " nitri c aci d an d dilute d t o 10. 0 m l i n acid-washe d glasswar e rinse d with glass-distille d water . Blank s an d th e metho d o f standar d addition s were used t o chec k contaminatio n an d th e accurac y o f results . Iro n wa s expresse d as (ugml" 1 whol e blood) , a s (ugml" 1 RBC's ) b y dividin g by hematocrit , an d a s (u g x 10" ' RBC" 1 ) by dividin g by th e numbe ro f re d bloo d cell s pe r m l blood . Tota l re d bloo d cell s were enumerate d wit h a particl e counte r (Coulte rElectronics ) usin g a 5 u l bloo d sampl e dilute d t o 10 0 m

l with 1 % saline . Results , as numbe r m m " \ wer

e th e mean s o f

3 reading s persample . Erythrocyt e ALA- D wa s assaye d by a metho d modifie d fro m Granic k et

al. (1972 ) by Hodso n (1976) . Activity wa s calculate d as (nM porphobilinogen/(m l RBC's-h) ) a s de scribe d by Hodso n et

al. (1977) . Bloo d lea d concentration s i n 5 0 u l sample s were measure d by furnac e atomi c absorptio n spectroscop y accordin g t o th e method s o f Hodso n et

al. (1977) . Fis h fo r tissu e lea d analysi s were froze n in plasti c bags . After storag e foru p t o 12 months , fish from th e 16 an d 3 2 week sample s were thawe d an d dissecte d with stainles s stee l im plements . Foo d wa s cleane d from th e viscer a befor e analy si s an d th e ski n wa s remove d fro m opercula rbones . Eac h tissu e wa s place d i n a

n acid-washed , dry , preweighe d cru cibl e and th e wetweigh trecorded . Th e sample s were drie d a t80° C fo r4 8 h an d then transferre d t o a muffle furnace . Th e temperatur e wa s raise d ove rsevera l hour s t o 460° C an d maintaine d fo r a further2 4 h. Afterashing , 2. 0 ml o f ultrapure concentrate d nitri c aci d (BD H "Aristar" ) wa s added t o eac h crucibl e an d evaporate d t o drynes s o n a ho tplate . Th e sample s were re-ashe d for2 4 h , cooled , dis solve d in 20 % ultrapur e nitri c acid , an d dilute d with 20 % nitri c t o a volum e appropriat e t o th e tissu e weight . Lead concentration s were measure d by furnac e atomi c absorp tio n spectroscop y usin g th e metho d o f standar d addition s (Hodso n et al., 1977) .
RESULT S Th e thre e short-ter m lethalit y test s gav e 21-da y LC 50 ' s (wit h 95 % fiducial intervals ) o f 2. 6 (1.9-3.4) , '2. 3 (1.6-3.3 ) an d 2. 3 (1.7-3.2 ) mgl" 1 lead . Th e mea n LC 5 o wa s 2.4mgl" 1
. I n th e subletha l tests , lea d concentration s i n wate r fluctuated wit h time . Concentration s wer e highes ti n th e earlies tweek s o f

exposur e an d th e overal l mean s wer e 0-20 % highe rtha n nomina l (Tabl e 1) . Th e lea d concentration s i n foo d wer e simila rt o th e desire d concentration s excep tfo ra fe w diet s a thig h concentration s an d lo w ratio n level s (Tabl e 2) . U p t o 100% o f

lea d adde d t o a numbe ro f diet s wa s foun d i n th e faeces . Onl y 0.2-1.2 % o f th e lea d i n th e foo d coul d b e leache d b y standin g i ti n water . Acidificatio n o rlong-ter m leachin g wit h bacteria l decompositio n di d no tincreas e th e releas e o f lea d (Tabl e 3). , Mea n hematocrit s o f fish expose d t o water-born e o rdietar y lea d wer e unaffecte d b y lea d exposure . However , i n bot h experiment s hematocrit s decrease d significantl y wit h tim e fro m a n initia l 32. 8 t o 26 % afte r4 wnek s exposur e (Fig . 1 ) afte rwhic h value s slowl y returne d t o th e initiall y observe d levels . Bloo d iro n concentrations , whethe rexpresse d a s (ugml" 1 whol e blood ) o r(/igml" 1 RBC's) , di d no t chang e du e t o exposur e o f fish t o water-born e lea d (Fig . 2) . However , cellula rbloo d iro n a s (u g RBC" 1 ) decreased-significantl y a tal l lea d concentration s b y a s muc h a s 17 % fro m contro l values . Bloo d iro n a s (ugml" 1 whol e blood ) an d (ugmr 1 RBC's ) wa s significantl y reduce d fro m contro l value s i n fish receivin g dietar y lea d dose s equa l t o o rabov e 26 0 an d 20 0 ug/(kg-day ) respectivel y (Fig . 2). Th e maximu m effec twa s a 10 % decrease . Cellula rbloo d iro n a s (u g RBC" 1 ) wa s no taffecte d b y dietar y lead . Iro n (ugml" 1 whol e blood ) increase d significantl y wit h tim e b y 25 % fro m week s 1 t o 3 2 i n th e water born e lea d experimen twhil e i tdecrease d 21 % i n th e dietar y lea d experiment . Th e numbe ro f

re d bloo d cell s m m " 3 increase d sig nificantl y wit h lea d i n wate rfro m 9./ 4 x iu J i n corr tro l tank s t o 1.1 5 x 10 6 a t^Ougl" 1 an d al l lea d concentration s ha d a significan teffect . Dietar y lea d di d no taffec tre d cel l number s bu tmean s wer e gener all y highe rtha n i n th e water-born e lea d experiment . Re d bloo d cel l volum e (th e invers e o f th e numbe r o f RBCTm l * divide d b y th e hematocrit ) fel l b y 14 % fro m contro l value s i n fish expose d t o 12 0 u g 1 " ' lea d (Fig . 3) . Cel l volume s wer e significantl y reduce d b y al l lea d concentration s excep t13ugl _ 1
. Re d bloo d cel l volume s di d no tvar y wit h lea d dos e i n th e dietar y lea d experiment . Re d bloo d cel l count s increase d (Fig . 4 ) whil e cellu la rvolume s decrease d durin g th e summe r months . Fis h fro m th e water-born e lea d experimen twer e sam ple d les s frequentl y durin g th e summe rtha n thos e 

# • ! 

# ; i 1

Tabl e 1. Dail y measurement s of lea d concentration s in wate r

ft". 

# 1 w %r* 

### •ifc 

### p Nomina l lea d concentratio n W I

OtCll ) 1 0 1 8 3 2 5 6 10 0 Interva l

(Wecks l 0- 4 X - 4 1 3 2 1 3 8 6 2 13 0 s * 3. 7 3. 5 10 0 7:8 9. 1 3 3 5 X 4 1 3 22 4 0 7 1 14 0 - 8 s 3.6 3.6 4. 5 7. 2 11. 8 3 3 9 - X 2 1 2 2 2 3 8 6 7 13 0 1 2 s 2. 7 2. 0 3. 1 4. 6 6. 1 1 4 13

- X 2 1 3 2 2 3 9 6 2 14 0 1 6 s 1.5 . 4.2 6 5 104 1 8 8 1 3 Measure d lea d concentration s (** g 1

17 - X 2 I I 1 9 2 9 5 1 9 7 2 0 S 2.4 1. 9 3. 1 3 5 5. 8 4. 4 2 1 X 2 1 2 1 9 3 2 5 2 10 0 -2 4 S 2. 0 1. 7 2. 8 3. 9 3. 5 1 3 25 -

X 3 1 0 1 8 29 5 0 9 5 "' 1 2 8 S 2. 1 2. 2 2. 9 2.6 6. 5 5.0 29 X 4 1 0 1 6 2 7 4 6 9 1 3 2 S 6. 0 2.7 4. 2 4. 6 5.8 5. 7 Overal l

X S 3 3. 8 1 3 6. 3 2 1 5. 9 3 5 8. 0 6 0 11. 2 12 0 3 0 ( 1 , ' i

Jr' ft



# fo l( * I* . 

# t 

# i 

# IUf . P. 5

Tabl e 2 . Measure d lea d concentration s i n experimenta l diet s an d calculate d lea d dosag e rate s

• ; tt i 

# r ';3 > 

## I P

, j | • •

. « 5 Nomina l lea d dos e l/i g Ikg-day l ' 1 Ratio n (". . hod y w l dav"' l 1. 0 1 5 2. 0 2. 5 3. 0 X con e

1. 1 2. 4 J. 2 1. 0 2. 0 OlCll l

dos e

1 0 2 0 3 0 1 0 2 0 2 0 M

cone . 1 0 X 7. : 4. 4 7. 2 6. 0



### - casurc d lea d 10 0

dos e 11 0 n o 9 0 18 0 18 0 13 0 concen t ratio n

cone . 14. 4 12. 0 9. 6 11. 2 7. 6



### - 18 0 l/igg "

dos e 14 0 18 0 19 0 28 0 23 0 20 0 we t weight ) an d dose s

cone . 13. 1 18. 0 16. 8 9. 6 11. 0 

### — 32 0

dos e

13 0 28 0 30 0 24 0 33 0 26 0 l/i g (kg- d

cone . 2 4 29. 9 2 6 2 0 15. 6



### - av r 56 0 

### ')

dos e 24 0 45 0 51 0 50 0 47 0 43 0 con e

73. 7 51. 7 40. 0 31. 9 26. 0



### - 100 0
j

dos e 74 0 78 0
• 80 0 80 0 78 0 " 78 0 

# w

Tabl e 3 . Availabilit y o f lea d fro m selecte d experimenta l diet s
Nomina l dos e (p g lead/(k g ris h day' 1 ) ) Measure d lea d concentratio n 0igg~' )

—we t weigh t —dr y weigh t Ratio n | 0 o o f bod y wt./day ) Dr y wt . o f too d eate n ove r 2 week s (g )
Tota l amoun t o t lea d i n foo d (/ig ) Concentratio n o t lea d i n faece s

—dr y weigh t t^gg"' ) Dr y weigh t o f collecte d faece s Ig ) Tota l amoun t o f lea d recovere d utg )
Pe r cen t o f foo d lea d i n faece s Pe r cen t leache d i n wate r 4 day s p H 7. 6 4. 6 2 week s p H 7. 6 0 2. 4 3. 8 1.5 4 9
186

17. 7 9 8 17 3
9 3



### — — 32 0

13. 1 20. 2 1. 0 1 3
26 3

14 2 1. 9 27 0
10 3



### — —

- 56 0

24. 0 38. 0 1. 0 2 5

• J 5 0

20 1 4. 5 90 5
9 5



### _ ' —

- 56 0

26. 0 38. 7 2. 0 5 0
l<J.I <

8 5 13. 0 110 5 5 7



### — —

- 56 0

20. 0 30. 3 2. 5 7 2
218 2

8 8 11. 2 98 6
4 5



### _ —

- 100 0

73. 7 117. 9 1. 0 2 0
235 8

40 7 3. 1 126 2 5 4

0. 5 1. 2 0. 5 0. 2 100 0

40. 0 61. 0 2. 0 6 0
366 0

16 6 12. 6 209 2 5 7



### — —

-
fro m th e dietary lea d experiment . Consequently , mea n contro l re

d bloo d cel l count s and volumes , take n wit h lea d acros s time , diffe

rbetwee n experiments . Erythrocyte ALA- D activit y was significantl y depresse d fro

m contro l activit

y a tal l lea d concen tration s in water (Fig . 5) . Per cen tinhibitio n increase d fro m a n averag e o

f 25 % a t1 3 u g 1" ' t o a

n averag e 60 % a

t120ugl" 1
. Dietary lead cause d onl y a sligh t(4% ) bu tnon-significan tinhibitio n o f ALA- D a

tth e two highes tlead doses . There was a significan tfluctuation o f ALA- D activit

y wit h tim e in bot h experiments bu t n o consisten tincreas e or decreas e relate d t o season' ! o rduratio n o f

exposure . Bloo d lead level s o f

fish expose d t o lea d in wate r Fig . 2 . in e <:

o n blood ir<

" (dietary, s - RBC s (u g i water-borne

. F e ml' 1 : water-borne

, treat -P. 6

dos e 74 0 78 0 80 0 80 0 78 0 78 0

u t (4% ) 'h e tw o 'uatio n Tits bu t reaso n i wate r 

# m Chroni c toxicit y o f rainbo w trou t 87 3



### « 1>i


# l 4 

# s "S 

# 4.



# n LEA D 0OS E I N FOOOlMftlkg-dayl ) IC O 100 0 LEA D DOS E I N FOO O (ug/(kg-day » KX ) VJO O 1000 0

a 2 ? * w o 

### ^ » E 

### s - • m • - - •—



### ~°~ \ E El3 0 vt' 

### | I IK )

O -I . o g o / ^ P b i n wate r

-—— H •«•—• • •^3 ,

\ o U J C C ! <

IEA O CONCENTRATIO N I N WATE R (ug/^ > Fig
. 2
. m e effec

t o i exposure o

t rainbo * trou t

t o lea d o n bloo diron , expresse da s A : ^gFeml" 1 whol e bloo d (dietary , s = 35 ; water-borne , s = 38) ; B : fig Feml" 1 RBC' s (u g Feml" 1 -

r hematocrit/100 ) (dietary , 5 = 90 ; water-borne , 5 = 125) : an dC : u g Fe x 10" 9 RBC" 1 (u

g F e ml" 1 H

- No
. o f RBC' s mmJ x 1000)

) (dietary , .

v = 36 ; water-borne , s = 52)
. Th e bloo diro n value s ar e th e lea d treatment mean s ove

r al l samplin g times
. increase dsignificantl y fro mcontro l concentration s of 220ugl" ' t o a n averag e o f 1040ugl - 1 a t

th e highes t lea dconcentratio n i n wate r (Fig
. 6)
. Ther e was n o significant effec t of lea ddos e i n foo do n lea dconcen tration s i n blood
. Averag e bloo dlea dconcentration s LEA D CONCENTRATIO N I N WATE R lug/f l Fig
. 3
. Th e effec

t o f exposur e o f rainbo w trou t

t o lea d o n th e redbloo dcel

l count (dietary , s = 0.17 6 x 10 6 ; water-borne , s = 0.14 3 x I0 6 ) an dre dbloo dcel l volum e (1/No
. RBC' s mm" J -f

- hematocrit/100)(dietary , s = 0.048 ; water-borne , s = 0.050)
. wer e withi n th e rang e o f th e contro l value s fro mbot h experiments
. ALA- D activit y wa s significantl y in

versel y correlate dt o (log 1 0 bloo dlead ) (Fig
. 7)
. Lea dconcentration s i n othe r tissue s increase ddi

rectl y wit h lea dconcentration s i n wate r (Fig
. 6)
. Th e slope s of line s relatin g lea din eac h tissu e t o lea di n wate r appea r similar , excep t

for live r an dC.N.S
. tis

sue . C.N.S
. tissu e (principall y brain ) of lea dexposed fish containe dmor e lea dtha n contro l fish bu t concen tration s i n C.N.S
. di dno t increas e with increasin g lea dexposure . Opercula r

bone s demonstrate dth e highes t concen - 

### .411 !

! 

### 4 •i

-i t: ! L '-: : H i

IP. 7

I , .lii K
. ntiosoN , BEVLRLE

V H
. BLUN T an d L/UUULAS

J
. .SI'K
. LEA D DOS E I N FOO D <ug/(kg-day) ) 10 0 100 0

o m <r 

# 2 o m a .

Q < P b i n locx l

P b i n wate r

1 0 too. . LEA D CONCENTRATIO N I N WATE R (M0.^ l Fig
. 5
. Effec

t o f exposur e o f rainbo w trou t

to lea don erythrocyte ^-aminolevulini c aciddehydratas e (ALA-D ) activit

y (dietary , s = 69 ; water-borne , s = 48)
. Each point i s th e lea

dtreatmen t

mea n ove r al l samplin g times
. tration s o f

lea dwhil e gil l an dkidne y showe dth e nex t highest . .Fis h expose d t o dietar y lea d rlemnnstrate d n o lea daccumulatio n i n an y tissu e an d fluctuations i n concentratio n appea r rando m Concentration s o f lea di n th e C.N.S
. o f

fish expose dt o dietar y lea da t al l level s wer e equa l t o o r greate r tha n thos e o f treate dfish i n th e water-born e lea dexperiment
. Th e ratio n level s in th e dietar y lea dexperimen t affecte dneithe r

th e measure dphysiologica l par ameter s no r

lea daccumulations
. Th e majo r effec t o f

I 2 LEA D I N WATE R

3 C O C O

z o < or .
u z o o Q < L J ratio n siz e was o n growt h an d ther e wer e n o signifi

can t lead-ratio n interactio'n s eviden t for an y par ameter , includin g growth
. , . "Black-tails " appeare di n 3 fish o f 1 0 remainin g £";

" ajte r 3 2 week s exposur e t o 120ugl" ' lea di n wate r Th e ski n darkene do n th e lef t sid e only , Iro f th e dorsa l t o cauda l fin, an d i t ende din ashar p lin e underneat h th e dorsa l fin. Nettin g th e fis h cause dth e temporar y disappearanc e o f th e ski n darkening
.

LEA D I N FOO D

Bloo d Lg/'m/ l i. o - Ooe'cul* . 0O" «

LEA D CONCENTRATIO N I N 10 0 WATE R 10 0 LEA D DOS E 100 0

(u.g/(kg-rjay) ) Fig
. 6
. Th e concentratio n o f lea di n tissue s o

f rainbo w trou t

exposedto lead
. Wit

h th e exceptio n o f lea di n blood , each point

i s the mea

n o f al l measurement s taken a

t week s 1 6 an d 32
. Fo r

blood , eac h poin t

i s th e mea

n fo r al l sampl e times
. Th e standar ddeviation s fo r lea din tissue s o

f fish expose d t o lea di n wate r were : opercula

r bone : 1. 3 ugg" 1 ; gills : 1.5 ; kidney : 1.5 ; carcass : 1.4 ; liver : 1.1 ; viscera : 2.1 ; C.N.S. : 1.7 ; an dbloo d0.20ugml" 1

. Fig . 7 . Th e * activit y o f i. ' curve dline s - followin g " l \ [lea

k 

## W$-"'•""• 1 ..A > ---^Thes e exp fc> i i readil y a £;",_
. accumulate : ^changes
. L<

- unavailabl e ' effect s o n tl
• Relative ! i n wate r wt . : o f 2. 4 m g I i fo r rainbi ' Brown' s (V Davie s et <. i n 2 8 mg i j
. lea drespe c '£
. 96- h LC 5 0

, :i i solve dlea d ';
. solve dlea '
' •••• Pickerin g 96- h LC 5 0 t\. t o 45. 4 mg : an dfro m ; (36 0 mgl" ' bioassay s : "absorption
. | ^ I t i s unl : '.observe dii
. fbilit y o f lc
. accumulat e 'som e dept

^ (probabl y t

t phates , et c fe#;» probi
. /rapidl y ( 3 h e letha l 'P. 8

i-iiii u - v Ircu i 61:

.us e ve r

n o signifi - • r an y par i remainin g
• d in water . nly , fro m i n a shar p ; th e fish I th e ski n

• - one y tve f -.•i s fi.oo d (..g/mi ) :,MCas a 

# 1


# i

'.o n •od . BLOO D LEA D CONCENTRATIO N (no// )

Fi g 7 . Th e effec to f blood lea

d o n erythrocyt

e ALA- D activit y o f rainbo w trou texpose d t o lea d i n water . Th e curve d line s represen tth e 95 % confidenc

e bel tabou tth e followin g "bes tfit" line : ALA- D = 59 0 - 14 6 (log, 0 [lead] ) ( r = 0.578 ; Syx = 70 ; Sb = 15.7). DISCUSSIO

N Thes e experiment s Hpmnnstratp H tha t lead , i n watp r i s readily availabl e t o fish foruptake , and thatlea d accumulate d by fish cause d subletha l physiologica l changes . Lead i n food , by contrast , appeare d t o b e unavailabl e lo r uptak e and consequentl y had n o etlect s o n th e tish . Relativel y lo w concentration s (1-1 0 m g 1" l ) o f lead i n water were lethal t o fish wit h a mea n 21-da y LC 3 0 o f 2.4mgl - 1
. Thi s valu e i s clos e t o th e 48- h LC 5 0 fo r rainbo w trou to f

1.5mgl" 1 projecte d fro m Brown' s (1968 ) studie s a tlowe rwate r hardnesses . Davie s et al. (1976 ) observe d 96- h and 14-da y LC 50 ' s i n 28mgl" 1 hardnes s wate ro f 1.3 2 an d 0.2mgl" 1 lea d respectively . A

ta hardnes s o f

353 mgl"' , tw o s 96- h LCjo' s were 47 1 mgl" 1 tota l o r1.47mgl" 1 dis - , ' solved lead , an d 542mgl" 1 tota l o r 1.32mgl" 1 dis - • solved lead . Pickerin g & Henderso n (1966 ) demonstrate d tha t 96- h LC 50 ' s fo

r fou rspecie s o f

fish ranged fro

m 5. 6 ; Kto45.4mgl" 1 in sof

twate r(20mgl' 1 tota l hardness ) '•h an d fro m 41 0 t o 482mgl" 1 i n har d water ' (360mgl" 1 ) . Thes e results wer e derive d fro

m stati c bioassay s in which lead conten tcoul d var y du e t o absorption , adsorptio n an d precipitation . \ I ti s unlikel y thatacute lethalit y will b e frequently observe d in natura

l condition s du e t o th e lo w solu bility"o f lead . Considerabl e quantitie s o f white salt s accumulated in tank s a

t5 m g 1" ' lea d o r abov e and some deposit s were visibl e a tlrngl" 1
. Thes e were | & probabl

y insolubl e lea d carbonates , hydroxides , phos phates , etc . Unles s spille d a tver y high concentrations , i ti s probabl e thatth e formatio n o f thes e salts woul d ; : rapidly (3- 4 h

) reduc e lea d concentration s t o belo w Uh e letha l level (La

m et al., 1976) . g ^Hftha l """"""^aticm—of ' Iparl (less , tha n 100 0 Ugl" 1 ) are , fa r mnr p liVpl y t n orru r sinc e the y appea rt o b e a

to rbelo w th e concentratio n tha tpre cipiTare g i n Lake Ontari o water . I tie thre e principal ertect s o f prolonge d sublethal lea d exposur e o n fish ar e lead uptak e and contaminatio n (thi s study ; Hol comb e et al., 1976) , change s in hematologica l par ameters (thi s study ) and developmen to f spinal curva ture s (lordosi s and scoliosis ) (Davie s et al., 1976 ; Hol comb e et ai. 1976) . Lea d uptak e appears linearly relate d t o lead con centration s in Wate

i s u tha tan y iuutas e i

n Concen tratio n irom backgroun d level s will resul ti n increase d lead conten to f

fish. Thi s correlate s well wit h th e observation s o f

Pagenkop f

& Neuma n (1974 ) that lead conten to f

bon e was highe r in fish fro m a river compare d t o fish fro

m a hatchery . Th e river wate r containe d 2.56-2.9 3 u g 1" ' lead , twic e tha to f hatcher y water . I n addition . Brown & Cho w (1975 ) report- ' thatfish fro

m Bai e d u Dore , Lake Huro n containe d onl y 0.1 9 ugg" ' lead in muscl e whil e thos e fro m Tor ont o Harbou rcontaine d 1.7 8 ugg"'.Th e lowe r limi t i s clos e t o th e mean valu e o f 0.1 4 and 0.10/tgg" ' observe d i n carcasse s (mostl y muscle ) o f contro l fish fro m th e water-born e lead an d dietary lead experi ments respectively . Th e Toront o harbou rfish con taine d a lead concentratio n characteristi c o f exposur e t o 20-3 0 ug 1" ' lea

d i n wate r (Fig . 6) , a rang e bracket in g th e IJ C recommende d objective . Thi s suggest s local lea d contamination , perhap s du e t o urba n runoff . I ti s interestin g t o not e tha tth e mean lead conten to f

carcasse s di d no texcee d th e Canadia n Foo d and Dru g Directorat e (NHW , 1952/53 ) guide line s o f lOmgkg" 1 fo

r lea d i n marin e an

d freshwate r animal products . Exposur e o f fish t o 20 0 u g 1" ' lea d ormor e o n a continuou s basi s migh tlea d t o contami natio n in exces s o

f th e guideline . Hig h concentration s o f

lead i n bon e suggest s tha t growin g bon e ma

y provid e a sit e fo

r lea d storag e a

s a detoxifyin g mechanism . I n mammals , u

p t o 90 % o f th e bod y burden o f

lea d i s foun d i n bon e (Goye r & Mahaffey , 1972) . Th e accessibility o f th e opercula r bon e migh tmak e i

tsuitabl e fo

rmonitorin g lea d exposur e and estimatin g bod y lea d content . Th e high concentration s o f lead i n gill s and kidney s probabl y reflec

tth e directcontac to f

gill s wit h lead contaminate d wate rand possibl e excretio n o f lea d vi a urine . Th e lowe r accumulatio n rat e o f lea d in live r suggests tha

tlead ma

y b e cleare d t o othe rsite s o f accumulatio n more rapidly than i n othe r tissues . Thi s ma y b e th e resul to f

th e relativel y high bloo d flow an d metaboli c rat

e o f th e liver . Lead i n C.N.S . di d no tcorrelate wel l wit h lea d exposur e in water . Al

l lead-exposed-fis h demonstrate d elevate d C.N.S . lead concentration s butth e amoun t o f th e increas e was no trelate d t o lea d concentratio n i n water . I

n th e dietar

y lead experiment , C.N.S . lead concentrations , includin g thos e o f contro l fish, were th e sam e o rhighe r than thos e o f fis h expose d t o water-born e lead . Therefore , i ti s quite possibl e tha

t 

### : 

# M'\\<

t \ i.i. f •

ii: i t l
II;- ; h 

# n

3 1 , 

# i

; «.*' • 

# m It !



# I M 

# IP. 9

i f

r. ' •

5
.



# Hi '

i s . 

# Hi ! > i ; a -r th e lo w C.N.S
. lea dconcentration s of contro l fish in th e water-born e lea dexperimen t wer e incorrec t

an d th e apparen t

lea daccumulatio n was anomalous
. Thi s woul dagre e with th e observe dlac k o f C.N.S
. lea d accumulatio n i n lead-expose dbroo k trou t (Holcomb e et at., 1976)
. Dietar y lea dwas no t

take n u p b y exposedfish. Th e majorit y appeare di n th e taece s an dth e portio n unac counte dfor

ma y hav e bee n i n smal l faeca l particle s washe ddow n th e drain
. Hig h lea dconcentration s i n faece s bu t

not i n tissu e (especiall y viscera ) indicate s tha t th e lea dwa s no t

take n up . It

implie s eithe r

a failur e t o release lea dfro mth e liver protein , o r

a highe r bindin g capacit y fo

r lea do f othe r faeca l mater
ia l compare dt o gut

walls
. Th e forme r

may be
. correc t sinc e lea dwa s not

leache dfro mth e foo db y wate r o r aci dsolution s althoug h live r protei n i s digestibl e an dshoul drelease lead
. Thes e result s correspon dt o th e observation s b y Lelan d& McNurne y (1974 ) tha t lea di s no t

transmitte dthroug h fon dchain s I n fact , th e highe r

th e trophi c leve l examined , th e lowe r th e concentratio n o f

lead
. Lea dappear s t o contaminat e by adsorbin g t o surface s s o tha t smal l animal s an d plant s a t lowe r trophi c levels , wit h ahighe r surfac e t o volum e ratio , hav e highe r lea dlevels
. Exposur e of

trou t t o lea di n wate r

produce dsubtl e hematologica l effects
. Increase dnumber s o f re dcell s withou t acorrespondin g increas e i n hematocri t indi

cate dashif

t i n th e re dcel l populatio n frommatur e to

_newl y formed , immatur e cells
. Thi s suggest s acce lerate dmortalit y an dremova l of matur e re dcell s fro mth e bloo dan dacceleratio n o f hemopoiesi s t o compensat e fo

r th e los s i n oxyge n carryin g capacity
. Thi s woul dals o explai n wh y th e iro n conten t

of whol e bloo d(o r

o f th e tota l RB C fraction ) di dno t change , bu t

iro n pe r RB C wa s reduced
. Th e reductio n of iro n pe r RB C may indicat e areductio n in cellular hemoglobin , assumin g tha t th e rati o betwee n hemo

globi n iro n an dferriti n iro n (th e storag e form) di d not increase
. Sinc e th e activit y o f ALA-D , a n enzym e jnvolve di n hemoglobi n formation , wa s reduced , i t i s possibl e tha t

reductio n of

cellula r

hemoglobi n occurred
. A reductio n of cellula r hemoglobi n woul d reduc e maximu moxyge n carryin g capacit y of th e bloo dan dals o stimulat e erythropoiesis
. Therefore , erythropoiesi s probabl y maintaine daconstan t tota l hemoglobi n an doxyge n carryin g capacit y despit e a reductio n i n cellula r

hemoglobi n an dnumber s of matur e cells
. Mea n erythrocyt e ALA- D activity , whil e reduce d b y u p t o 60% , di dno t

declin e t o zero . Th e negativ e linear relationshi p of

activit y t o log 1 0 of

lea di n wate r describe s a n asymptoti c relationship , o r in othe r words , areserv e capacit y of activity
. Th e resul t

i s tha t hemoglobinemi amay neve r reac h seriou s level s i n fish a t thes e level s of

lea dexposure . Thi s i s supporte d b y th e relativel y smal l decreas e of 17 % i n RB C con ten t an dincreas e of

18 % i n RB C numbers
. I t appear s u»»i i u/jihj p 'h * fi 'h(?s capacit y t o compensat e fo r th e hematologica l fffprt

c nf IPI H . . . . - : .if' d IJci t . , .- , , j , Sl' W , 

### ti D'Ameli o et

ai, (1974 ) als o note dreductio n o f erythrocyt e ALA- D in goldfis h (Carassius auratus) exposedt o 200ugl" ' lea dfor 5 days
. Th e chang e wa s accompanie db y reductio n i n hemoglobi n syn thesi s an dre dbloo dcel l density
. Th e effec t o f lea d o n re dcel l densit y wa s probabl y du e t o mortality of matur e cell s wit

h n o compensatin g erythropoiesi s sinc e five day s i s arelativel y shor t tim e fo r increase d re dcel l productio n (Schal met ai, 1975)
. Anemi awa

s als o induce di n brow n bullheads , Ictalurus nebulosus, b y exposur e t o 28mgl" 1 lea dfor 16-18 3 day s (Dawson , 1935)
. Th e anemi awa s characterize db y deforme derythrocyte s an daccumulatio n of hem e pig

ment s i n th e splee n an dliver
. Peguigno t et al. (1975 ) observe dadecrease dhematocri t fro m3 8 t o 23% , de

pletio n o f

redbloo dcell s in th e "re dpulp " of th e spleen , an daccumulatio n of brow n pigmented , lead

containin g cell s i n th e "whit e pulp " o f th e splee n o

f
• lea dpoisone dcar p (Cyprinus atr'pio). Therefore , aj j genera l featur e of

exposur e t o sub-letha l lea dconcen- t tration s i n fish, a s i n mammal s (Goye r & Mahaffey , 1972) , appear s t o b e mortalit y o f matur e redbloo d cells , compensate dfo

r b y release o f

redcell s front " th e splee n or , a t

lo w exposur e levels , b y increase d redbloo dcel l production
. Therefore , th e severit y an dsignificanc e o f th e

. observe dhematologica l chance s i s dependen t o n th e sevent y o f

th e lea dexposure . A t th e lo w concen tration s o f

thi s experiment , compensatio n indicate s a* lo w biologica l significance
. Th e principa l importanc e * of hematologica l change s i s t o indicat e th e occurrenc e of lea dexposur e an dth e possibilit y of othe r harmfu l effects (Hodson , 1976)
. I n contrast , th e hematologica l '.-ffect s o f dietar y lea

d di dno t fit th e abov e pattern . Whil e smal l decrease s (maximu m10% ) in bloo diro n occurre da t th e whol e bloo dlevel , cellula r

iron , hematocrits , number s o f RBC's , RB C volumes , an derythrocyt e ALA- D remaine dunchanged
. Sinc e lea dwa s no t take n u p fro mth e food, , i

t may someho w hav e inhibite diro n i uptak e fro mfoo drathe r tha n directl y affectin g som e hematologica l parameter . , Th e mos t importan t

aspec t of lea dtoxicit y t o fish i s developmen t

of spina l deformities tha t increas e J IIIO I tality~rate s an dpreven t successfu l reproductio n (Davie s et

al., 1976 ; Holcomb e et

ai, 1976)
. Th e probf

V lemi s characterize db y th e darkenin g of th e ski n i

n J | th e are acauda l t o th e dorsa l fin. A t first, thi s blac

k j tai l disappear s whe n th e anima l i s frightene dor: 3 anaesthetized
. Blac k

tail s ar e followe db y dorsc-j f latera l spina l curvature s (lordoscoliosis—lordosi s plus j scoliosis ) whic h ar e reversibl e b y anaesthetic s in the ! earl y stage . Late r

spina l calcificatio n prevent s reversal ] (Holcomb e et

ai, 1976)
. Th e ultimat e effect s ar e atrc- 1 ph y o f th e tai l region , interferenc e with movement, ! an ddeath
. I n th e presen t

study , rainbo w trou t

exposedt o lead ! demonstrate dblac k

tail s i n 3 o f

1 0 fish remaining ] after 3 2 week s a

t ameasure dconcentratio n of.- j 120 n g I" 1

. Davie s et

ai (1976 ) observe dsimilar ! syi a s an . T h %; > al.

• (S.. of 4 4 th i ex i
' fin cg j j el l

- ; ah : | to . ib !
• ell
• si. ' ir

tic
. t o

. . ca ' hyt ; b r c o I gl-.

- P i pei

e x i 

### (! ' S ' 

### &

# • • 

# ft tS gfc f a. th ; m t o ( 0 ( B al t h s o a t .si
. 

# a in


## u

l: t 8 1 n•luctto n o i
• • > auratus) li e chang e : <>Hi n syn -

. of lea d mortalit y hropoiesi s increase d lemi awa s nebulosus, 18 3 day s 'prize db y
• iem e pig

- ' ai (1975 )

• 23% , de

ip " of th e ;tted
. lead

-

. splee n o

f lierefore , a J concen Mahaffey , e dbloo d cell s fro m increase d

-e o f th e 'i t o n th e concen - 'iidicate s a
• iportanc e iccurrenc e r harmfu l utar y lea d Jecrease s th e whol e 'inber s o f ALA- D take n u p ne diro n lin g som e •jt o fish
'
. increas e iductio n I h e prob

i". ' ski n i n hi s blac k '•.tene do r
• v dorso

- • losi s plu s tic s i n th e ! : s reversa l ar e atro
ovement , ..'
. t o lea d

. mainin g .i t io n of.- ] simila r 4* 1 

# 1

f: S 

### $



## • p



# mz. symptom s i n rainbo w trou t a t concentration s of

lea d a s lo w a s 8 ugl" 1 i n sof t wate r (20mgl" ' hardness ) an dsever e lordoscoliosi s a t highe r concentrations
. Thes e effect s wer e observe da t 3 2 u g I" ' free lea din wate r o f 35 3 m g I" ' hardness
. Similarly , Holcomb e et al. (1976 ) observe dthes e symptom' s i n broo k trou t (Salvelinus fontinalis) a t

concentration s of

tota l lea d o f 119ugl" ' (84ugl" 1 dissolve dlead ) in wate r o f 44mgl" 1 hardness
. Thes e author s demonstrate dtha t th e developmen t

of symptom s require d2 6 week s of exposur e whe n th e experimen t wa s starte dwit

h fingerlings an d6- 7 week s whe n i t wa s starte dwit

h egg s o r fry
. Therefore , th e lo w incidenc e of thes e effects a t arelativel y hig h lea dconcentratio n i s prob

abl y no t du e t o alac k

of lea dtoxicity , bu t

rathe r t o a n inadequat e exposur e time*
. Whil e i t i s imposs

ibl e t o stat e th e minimu mconcentratio n causin g thes e effects fro mthi s experiment
. Hodso n (1976 ) hypothe

size darelationshi p betwee n lea dconcentration s caus in g lordoscoliosi s an dthos e causin g significan t inhibi

tio n of erythrocyt e ALA-D
. ALA- D i s closel y relate d t o lea dexposur e in severa l specie s o f fish an dindi

cate s lea duptak e (Hodso n et ai, 1977)
. Assumin g th e hypothesi s correct , th e lowes t lea dconcentratio n inhi

bitin g ALA- D wa s abou t 1 3 ugl" 1 an dhenc e thi s concentratio n shoul dcaus e lordoscoliosis
. Th e tim e effec

t o n hematocri t

(Fig
. 1 ) wa s relate d t o th e tim e spen t

i n th e experimenta l tank s an dsug gest s astres s du e t o confinemen t wit h agradua l physiologica l adaptation
. However , th e split-plo t ex periment s allowe dth e testin g of lea deffects withou t confoundin g b y thes e tim e effects , althoug h ther e wa s some los s i n precisio n compare dt o standar dfactoria l experiments
. Th e rang e of observe dmea n hematocrit s corresponde dt o th e value s reporte db y Barnhar t (1969 ) fo r hatchery-reare dtrout . Th e number s o f

RBC' s ml" 1 , an dhenc e volume , als o varie dwith time , bu t in relatio n t o seaso n rathe r tha n t o exposur e tim e (Fig
. 3)
. Th e shar p earl y sum me r peak migh t b e du e t o a n endogenou s respons e
• t o rapi dsumme r

growth
. Th e observe dre dbloo dcel l count s fel

l withi n th e rang e o f mea n value s (0.9-1. 9 x 10 6 mm" 3 ) reporte dfo r rainbo w trou t (Bouc k & Ball , 1966 ; Snieszko
. 1960 ; McCarth y et ai, 1973 , 1975 ; Barnhart , 1969)
. Variation s of

bloo diro n conten t

an dALA- D ac tivit y with tim e wer e no t

relate dt o samplin g o r sea so n but ma y hav e bee n rando mo r aresul t o f system
ati c erro r i n methodology
. Sinc e ALA- D activit y slowly increase s followin g adecrease dlea dexposure , (Hodso n et

ai, 1977) , th e variatio n with tim e may reflec t long-ter mfluctuations i n lea dconcentration s

, i n water . I n conclusion , prolonge dexposur e of rainbo w trou t t o lea di n foo dha dlittleerfcr t n n tri p fis h wherea s P' V * A recen t confirmator

y experimen

t demonstrate dblac

k tail s i n alo w proportio n o

f trou t exposedt o 30ugl" 1 an di n 100 % o f trou t exposedt o 60-100ugl" 1 fo r 1 0 ' month s fro

mth e egg stage . exposur e t o lea di n wate r cause dlea daccumulatio n in tissues , significan t

change s in hematologica l par ameter s an dpreliminar y indication s of developin g spina l curvatures . Thes e thre e effect s occurre da t measure dwate r

lea dconcentration s of 13 , 13 , an d 120ugl" 1 lea drespectively
. Sinc e th e mos t seriou s effec t occurre da t

120ugl" 1 , th e propose dIJ C objec

tiv e o f 25ugl" 1 tota l lea di n Lak e Ontari o shoul d protec t rainbo w trout . Acknowledgements —We than k Mr
. A
. Sippcl fo r technica l assistanc e on severa

l aspect s o

f thi s project

an dth e Wate

r Qualit y Laborator

y Network , Inlan dWater s Directorate , fo r metal s analyse s o

f wate r an dfish tissues
. REFERENCE

S Barnhar t A
. (1969) Effec

t o f certai n variable s on hematolo

gica l characteristic s o

f rainbo w trout . Trans. Am. Fhh. Soc. 98
. ,411-418
. Bouc k G
. R
. & Bal

l R
. C
. (1966

) Influence o

f captur e method s o n bloo dcharacteristic s andmortalit y i n th e rainbo w trou t

(Salmo gairdneri). Trans. Am. Fish. Soc. 95 , 170-176
. Brow n V
. M
. (1968) The calculatio n o f th e acute toxicit

y o f mixture s o

f poison s t o rainbo w trout . Water Res. 2 , 723-733
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. R
. & Chow L
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mBai e d u Dore , Lak

e Huron , andToront o Harbour , Lak e Ontario
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ing s o f th e Internationa l Conferenc e o n Heav y Metal s i n th e Environment , Toronto, Canada
. October , 1975
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weide r R
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n Lak e Ontario
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. & Ferrar o D
. (1974 ) The effec

t o f heav y metal s on protei n synthesi s i n crustacean s and fisp«^Rei;. Intern. Oceanogr. Medicate 33 , 111-118
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. H. , Goettl J
. P
. Jr. , Sinle y J
. R
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h N
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n hardan dsof t water . Water

Res. 10 , 199-206
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fish Ameiurus nebulosus, t o chroni c lea dpoisoning
. Biol. Bull. 68 , 335-346
. Finne y D
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. (1971 ) Probit

analysis. 3r

dEdition
. Universit

y Press , Cambridge
. 33 3 p . Granic k S. , Sass aS. , Granic k

J
. L.
. Lever

e R
. D
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. (1972 ) Assay s fo

r porphyrins , r5-amin

o levulini c acid dehydratase , an dporphyrinoge n synthetas e i n microlite

r sample s o f whole blood : Application s t o metaboli c de

fect s involving the hem

e pathway
. Proc. Nat. Acad. Sci. U.S.A. 69 , 2381-238,5
. Goye r R
. A
. & Mahaffe

y K
. R
. (1972

) Susceptibilit

y t o lea dtoxicity
. Env. Health Perspectives 2, 73-80
. Hodso n P
. V
. (1974

) The effec t o f temperature on th e toxi

cit y o f zinc to fish o

f th e genu s Salmo. Ph.D
. thesis , Dept
. o f Zoology , U
. o

f Guelph , Guelph , Canada
. 18 9 p . Hodso n P
. V
. (1976

) <5-amin o levulini c aciddehydratas e activit y o f fish bloo da s a

n indicato r

o f aharmfu l expo sur e t o lead
. J. Fish. Res. Bd Can. 33 , 268-271
. Hodso n P
. V. , Blun

t B
. R. , Spr

y D
. J
. & Auste

n K
. (1977 ) Evaluatio n o

f erythrocyte <5-amin

o levulini c aciddehyd

ratas e activit

y as ashort-ter

mindicato r

i n fish o f a harmfu l exposur e t o lead
. J. Fish. Res. Bd Can. 34 , 501-508
. Holcomb e G
. W

„ Benoi t D
. A.
. Leonar

dE
. N
. & McKi

m J
. W
. (1976

) Long-ter meffect s o f lea dexposur e o n thre e generation s o

f brook trou t (Salvelinus fontinalis). J. Fish. Res. BdCan. 33 , 1731-1741
. 

# I P I m

»•-• . iiif'i t.k. 

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## \A

• : :
•!' i 

### *' ;



# S 1 ! ii > 

# if ? 

# m iiif' i 

# w

i "

;P.1 1

87 8 PETE RV . HODSON . BEVERLE Y R . BLUN T an d DOUGLA S J . SPR Y

Rl ; 

## !ll . f.' i i


# n*

lb

• t
.

' : •

. t; ' ; ltii : . • T : .: - • 

## i i i


# H I

: t > . ' • IJC . (1976 ) Wate r Qualit y Objective s Subcommitte e

Report . Appendi x A t o th e Wate r Qualit y Boar d Repor t t o th e IJC . 1975 . Internationa l Join l Commission . Windsor . Canada . 19 8 p . Kop p J . F . & Krone r R . C . (1967 ) Trac e metal s i n water s o f th e Unite d States : a five yea rsummar y o f trac e metal s i n river s an d lake s o r th e U.S . (Oct . I . 1962-Sept . 30 . 1967) . Federa l Wat . Pollut . Contro l Agency , Cincinnati , Ohio . La m D . C . L. . Minn s C . K. . Hodso n P . V. , Simon s T . J . & Won g P . (1975 ) Compute rmode l fo rtoxican tspill s i n Lak e Ontario . In . Environmental Biogeochemistry. Vol . 2 . Edite d b y J . O . Nriagu . An n Arbo rScienc e Publishers . An n Arbor . U.S.A . Leian d H . V . & McNurne y J . (1974 ) Transpor to f lea d i n a rive r ecosystem . Proceeding s o f th e Internationa l Conferenc e o n Transpor to f persisten tchemical s i n aquati c ecosystems . Universit y o f Ottawa . Ottawa . Ma y 1-3 . 1974 . McCarth y D . H. . Stevenso n J . P . & Robert s M . S . (1973 ) Som e bloo d parameter s o f th e rainbo w trou t(Salmogairdneri Richardson) . J. Fish. Biol. 5 , 1-8 . McCarth y D . H. . Stevenso n J . P . & Robert s M . S . (1975 ) Som e bloo d parameter s o f th e rainbo w trou t(Salmo gairdneri Richardson) . J. Fish. Biol. 7 , 215-219 . Moun tD . 1. & Brung s W . A . (1967 ) A simplifie d dosin g apparatu s forfish toxicolog y studies . WaterRes. I ; 21-29 . N . H . W . (1952/53 ) Divisio n 15 , Regulation s of th e Foo d an d Dru g Act . Dept . o f Nationa l Healt h an d Welfare . Ottawa . Canada . 35 pp . Pagenkop f

G . K . & Neuman n D . P . (1974 ) Lea d concen tration s i n nativ e trout . Bull. Env. Com. Tox. 12 , 70-75 . Peguigno tJ. , Gir e M.-P . & Mog a A , (1975 ) Intoxicatio n parl e nitrat e d e plomb e che z l a carp e (Cyprinus carpio L.). Influenc e su rl a structur e histologiqu e d e l a rat e e t 1'hematocrite . Europ. J. Toxicol. 8 , 165-168 . Pickerin g Q . H . & Henderso n C . (1966 ) Th e acut e toxicity o f som e heavy metal s t o differen tspecie s of

war m water, . fish. AirWat. Pollut. Inter. J. 10 , 453-463 . Schal m O . W. . Jai n N . C . & Carrol l E . J. (1965 ) Veterinary Hematology. 2n d Edn . Le a an d Febiger , Philadelphia . 80 5 pp . Snieszk o S . F . (1960 ) Microhematocri tas a too l in fishery research an

d management . Spec . Scient . Rept. , U.S . Fish,; . an d Wildl . Serv . No , 341 . ; ' .• Spragu c J. B . (1969 ) Measuremen to f pollutan ttoxicity t o "'

. fish. I. Bioassa y method s fo racut e toxicity . Wafer Res. 3 , 793-821 . Stee l R . G . D . & Torri e J

. H. (1960 ) Principles and Pro- , cedures of

Statistics. McGraw-Hill . Ne w York . 481pp .

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