~:·.·

'

,;- ·

. -.,.

DEVELOPMENTAL DELAYS TN

EXPLORATION AND LOCOMOTOR ACTIVITY

IN tvlALE RATS EXPOSED TO LOW

LEVEL LEAD

A Thesis

Submitted to t he

Faculty of Miami University

in partial fulfillment of

the requirements for the degree of

Mas.ter of Science ·-~·

Department o£ Zoology

by

Kevin H. Cr-ofto.n

Miami University

Oxford, Ohio

1'980

APPROVE

ABS1''RACT

DEVELOFMENTAL DELAYS IN EXPLORATION AND LOCOMOTOR ACTIVITY IN MALE R..i\TS

EXPOSED TO LOW LEVEL LEAD

by

Kevin M. Crofton

Delays in the development of exploratory and locomotor

behavior in neonatal male rats (up to 21 days of age) are shown

to accrue as a consequence of low l evel lead (Pb) exposure.

Cross-fostering experiments indicate that these delays are pri-

mar:Lly due to prenatal exposure. Circadian activity analys is

demonstra-res that Pb exposure chiefly effects nocturnal activity .

These Pb induced behavioral modifications appear to be associ-

a-red t..;ith de-lays in synaptogenesis and biochemical development

of ·the cereb.ral c ort ex. A new behavioral bioassay for detecting

de Lays in brain development is described.

··.' ···

CONTENTS

LIST OF TABLES • • • • • • • • • • • • • • • • • 0 • • • • • iii

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . iv

INTRODUCTION • • • • • l

METHODS AND MATERIALS 6

RESULTS 0 0 • • • 0 • • • • • • • • • • • • • • • • • • • • ll

DISCUSSION 29

ACKNOWLEDGEMENTS 33

APPENDIX I : TIME COURSE BLOOD Pb ANALYSIS 34

APPENDIX II : BODY WEIGHTS ••• . . . . . . . . . . . . . . 43

APP.ENDD< I II: WATER CONSUMPTION 49

APPENDIX IV : FOOD CONSUMPTION 52

APPENDIX V: EYE OPENINGS 55

LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . 56

ii

·· .. :

.... · .-~ ..

1 .

-~· .. _.

LIST .OF TABLES

TABLE

l.

2.

3.

4.

... :J.

Duncan 1 s New Multiple Range _ Test for 30 mg/1 Pb tre'ated vs. con.trol groups . . . . . . . . . . . . . . . . .

Durte.an 1 s New Hultiple Range Test f ·or 200 mg/l Pb tre·ated vs. control groups . . . . . . . . . . . ~ . . . . .

Duncan 1 s New Mu l t:ip le Range Test f.or trOD mg/1 Pb · treated VS -. contr.ol groups . . . . . . . . . . . . . . . . .

Dunc an 1 s New Multiple Range Test for cross-fos'teri-hg e:~p.eriment . . . . . . . .. . . . . . . . . . .. .

Blood Pb foi' all. exposure regimes· . . . .

iii

PAGE

. 14

. 17

.. 20

2-4

25

LIST OF FIGURES

FIGURE PAGE

l. Schematic of Home Cage Apparatus 8- 9

2. Activity vs . Age Plot; control vs . 30 mg/l Pb e~posed litters • . . • • • • • . •.••• • •.• 12-13

3 . Activity vs . Age Plot; control vs. 200 mg/l Pb exposed litters . • . . • • . • . . . • • 15- 16

4 . Activity vs . Age Plot; control vs . 400 mg/l Pb exposed litters • • . • . . • • • • • • • • • •••• 18- 19

5. Activity vs. Age Plot; cross-foster ing experiment . . . . 22 - ·23

6 . Circadian Activity Analys is Plot ••••• • •.• ..• • 27- 28

iv

,,?,'

INTRODUCTION

Chronic lead e~pos.ure, in the .a.b·s.errce of gross symptoms of

in·toxica.tion, has been linked to. ment.al retardation and hyper­

ac·tiv.i cy in <Children (Beattie et al., 19 75.; David, 19 74; navid

et al. , 1978; de la B'urde & Choat·e, 19'75; Moore e.t .al., 1975).

Lead (Pb} exposure remains a per-sistent and widespr:e-ad health

problem-. In 1978 c:tpproximat-ely 7-, 55·0, ,00.0 children ·in the, United

States were . e·stimat-ed t ·o hav·e mildly elevat-ed blood l.ead 'levels

i.e.; betwee'n 25-55 ug/dl (D'avid ~ al., l9i8} . 'Low sucio­

economic status has been associat.ed \vith borderline. (30 - 40 · u.g/dl)

and higher blood lead levels (Stark et a.l,~, 1978) .

Settle and Patterson (19·80) have recently questioned the

ex<tent to which average Ame·rican lead intake of 29 , 000 ng/day

affec-ts health. They argue· that these levels are an .increase· of

2 tn 3 orders of· magnitude above the exposure of hurriaps to nat~ral

lead in prehistoric times. They also point out tha-t · this · repre­

sents a l:OO fo.ld increas.e above n·atural levels, while only a

five-fold incre·ase from the present i .nta·ke reache s toxic levels .

ThE:!Y also conclude· that i;lt current 11 normaln leve ls it is probable

that s ·ome b.i.oc'hemical proce'sses' within cells have bee·!). altered.,

pe1::-haps deleteriously .

The toxic ef"fects of inorganic lead are due mainly to two

of its proper-ties (B.~yc-e-Smith & vvaldron., 19 7'+). First, it has

a str..ong affinity for sulphydryl groups, which a re importan-t

functioh.al g!!.oups in many enzymes . Se-cond, metabo lism -of lead

closely resembles that o£ caLcium in many situations, par.ticu­

lax'ly at receptor sites on membranes. Toxic e£fect .s are

manife-sted in two m·ajor wa:ys,. heme syn:t~he·sis <:lisrupt.ioh and

neurot·o:xici ty (Damstra, 19 77) .

Although rec-ent s·tudie·s on .a Vqr.±ety o£ an;imals $tlggest that

subclihi·c al lead levels can produ.ce Short and long term effects

on behf(vior and various. physio1ogic.al p·roe:e·sses (Bull et ~ •. _,

1979; Fox et al., 1977; McCauley et al., 1979; 0\terrnan, 197?;

Zenick et al., 1'979), the evidence for a c·ausa1 relationship

betwe·en lead , ill,gestibn and behavioral dys_function in experiment'al

animals is s .c'ant (Shih and Hanin, 1978.) •

. In rats, neon,atal lead exposure, via an addition of. lt% lead

carbonate to the maternal d-iet, leads to ens lesions . in pups

similar to those found in children w.ith lead encepha lopathy

(Pen'tshaw & G·arro, 196.6). Such an exposure re:g_ime c auses

2

hypomye lination in e·x;posed suckling ra-ts and . the hy pomye ~,±nation

appea·rs to b,e due to- reta:rded grGwth and maturati on of the neurons

(Krigman e.t al., 19 74). Eff.ects resulting from exp osu;re·s of these

magni·t11d-es, howeve-r-, are confounding since the ~ffec ts o_f Pb

cann·ot be separat.ed from undernutrition of pups. , due t 'o ·curtailed

consump;t:ion of food and wate'r by the darn (Bornsch'e i n e~ al., 1977;

Micha·elson &. Sauerhoff., 1974).

Rat pups :whose mothers are given 200 mg/l Pb in their

drinking water from two weeks priGr to b'ree.dirtg up until .-weaning

at: 21 days of ag!2, exhibit a delay in t"he normal increases in

cerebr.a l corti€al cytoehromes (Bull et al., 19 7'9) . The same

exposure regime also results in a delay of synaptogenesis and

si.gns of metabolic uncoupling in cerebral cortical t .iss·ue taken

at: 15 days of -age (Mc~Cauley et al., 19 7'9) ~ The exposure regimes

in these latter two studies resulted in bl(}od Pb levels in the Pb

exposed · rats of 36 u·g/d'l at wean:i'ng-~ a leve-l in a range. ·not

uncommon in human populations (Kuhasik and Voloson, 19/2 ; Stark

et ~-, 1978). No signs of decreased body weights associated

with decr.e.ased food and water consumption wer.e observe9,.

Although Pb exposure has been reported to delay the time of

eye opening and righting reflex in rat pups (Reiter et ~., 1975;

Silberge l d & Goldberg~ 1974-), there have been few studies that

have attempted to dire·ctly corre-late the delays i n behavioral

development to delays in morphological and biochemical brain de­

velopment. Additional ly, there are no studies tha-c have specif­

ically linked behavioral deficits to prenatal exposure although

several i nvestigators have examined the behavioral effects pro­

duced by lead exposure during gestation and up to weaning i.e . ,

3

up to 21 days of age (Driscoll & Stegner, 19 76; Driscoll & Stegner,

19 7 8 ; Padich & Zenick~ 19 7 7 ; Zenick e t al. ~ 19 TS) • G rou,ps

exposed pre -nata lly and post -natally to Pb, 750 mg/kg daily via a

re!Stricted water intake of the dam (Zenick et ~- , 1979}, showed

no dif;fer.ence between exposed vs. control animals in terms of

fixed interval performance. These results, however, are subject

to question in that the conditioning treatment began approxi­

mately 4-0 days post- partum and 20 days after cessation of treat­

me!nt. I t is likely that this interval led to a decreased sensi­

tivity to developmental delays in behavior. Although circadian

activi ty analysis of resident ial maze activity of adult rats

exposed to lead from gestation until testing at 120 days of age

(Reiter, . 1977), showed no significant dif::erences, there has been

4

a to:t{a l ,lactk of . .deFt a on the eff~::ct:s of lead exposur,e on oircadiah

activ:;i. ty .bh,ythm.s. The ge;stati:0n peniod is known t 'o be .. more ::;ensi­

ti.ve to environment al te:ra'togens (Wilson~ 1975) .. Crqss-fo·s ·ter i ng

experimE;:!nts are required ih -order to address specifically the

effect-s of pre·-nata l exposure.

The pre$ent study had. two objecti-ves .in r'elat;,ionship to

previous --work . (Bw.ll et al . , 1.9.79; Mcc:auley . et a L., 19,79). :

a) to determine if such objeotive measu~res iFl brain dev elopment

were reflected i ·n delays in behavioral development an!i b) if

so, tq d.e-termine if these effe-c.t·s wer·e· a cons-equence ·of prenatal

(~:.!2, 4te,roo} or postnatal .exposure to Pb.

Tb f.acil.i tate these obj ectives, this study was sep arated

into three components.. The· fir;st expe-riment examined d ose

response effects on pre - weaning a,etivit;y. Dose levels (30,:200,

and 400 mg/1 PbCl2 in t ·he dri:nking water) were used· ~vhich were

kn.own- to result in bhood Pb le:ve.ls within the normaL range,

boord~::rl.ine bet.ween n-ormal and .elevated, and el-evated, ~ 29 ug/dl,

30-39 ug/dl, an,d ~40 ug/dl respe·ctively (St.ar'k e,t a·l. , 19 78).

The second, experime·nt, cros-s-fostering, examined the contrtbu­

ti.on of 2l! u.:tero vs. post~natal exposure on these . . ef£ects. The

third exp_e,r :iment·, circ.adian ac.t-ivi t y analys.is, exajnined ,the.

effe~t-s of l-ead exp:d·sure on the cir:<:!.adiap rhythm ef pos 'l;-_weaning

animals.

I t is here re,porte.d t .hat low level Pb exposed :ra t pups~

exhibit a delay in the development of expJ,..oration and l ocomotor

behav.i~or and that the m.odified behav.ior patt.e-rn iS·' due, ahnost

excll:.l?ive'ly, to prenatal Pb exposure. Also, that .a .c omparison

.. ·

of circadian activity ana lysis rhythms in Pb exposed and control

rat pups showed significant differences between the two. groups

from age 21 - 29 days, during the nocturnal phase of 'the .light

eye le •

5

. ...--'

., ... ~-

·'

All r ·esea.rch w.~s cor:tducte·d .at the Ehvirohment~.l I{es~arch

Cente.~, u.:s .. Ehv:ironmeh.t .a l Protection Agency, Cinc~n~ati, Ohio,

while on a Maste-r 1 s De-gree. Int!2,r.nshi.p Progr·am sponso-red· ·qy

Dr . Richard J~ Bull, Chief, Tox:icologi-c.al Asse·ssrrrent Branch,

Wate.r Qua1i:try Division •

-Sprague Dawley C.D. strain albino -rats, approx~mate' ly 70

days af age, were obtained f -rom Charles- River Breeding- Labora-

tories, Inc.~ and kep·t -on an ad- lib d·iet of Purina Lab Chow..

Incoming· animals- were held for a minimum O'f three days .in

quarantine,. t0 a.cc.limate the .animals, allow f.o-r visua l .observation,

and serological testing. 1'hr'oughout the e·xper imept ·the ra.ts

were ha.us_ed under a 12::12 light/dark cycle.

t3ibotl -samples were taken, from the d8JTlS by r etroorbi ta l .

sinus, bleeding of the dam at 18 da~s of gestation and-- when- the

pups were' we-aned. One pup per litter was sacrificed at 21 days;

and blpod samp;tes were obtained by carotid artery puncture . The

blood samples we.re analyzed f0r Pb content by anodic str ipping

voltammetry. Body weights .and food and water constimption for

both · the· dams and tb_e pups · we.r .e monitnre~¢1: - through.ot,it · :the 'exper·i-

ment at weekly intervals:.

:Eye 6pen;ing c:hecks were made for the 200 mg/l PbCJ2 dose

level, s:ta:rting at 13 d ays of age. ~ch- ptip t-J as checked eve-ry

four hours, until a:ll pups had opened -their eyes . The first

observab'le partitioning of the !2'.yelid was used a s a criterion

for open eye's.

j.

7

ln Exge.rirne:n·t l, f:e:·ma"les were e·xpos.ed to . 3'P~ . a'q~r or ·4.00 mg/l '

PbC1,2 in their drinkin·g _ water (22·, 24 and ·12 litters r.espec­

tively), whlle equal numbers of contrqls for each ·dos-e level

wE~re . provid~d with di·stilled .water . Ex·posure J:asted .fr .om '"hvo

weeks .prio·r to breeding<, t:tntil the offspri~g wer~ wecme4 at 21

days of age. At birth all litters we·re culled to ·eJ·gh.t pups.

Whenever possible only ma·le. ra-ts were used. Qn oc.casiqn, how­

ever, 1 -2 ·females had to be kept within t he litter to maihta.in

equal l it:ter sizes. I~ Experiment . 2, pr.,e-.par tur:ition treatment

w<~s the. s?ffie .as. in Exper:i:'J)lent l w.i th a dose leve'l of 200 mgJl

PbCl'z· A,t p·arturitiGn a ero·ss-fos:tt:rihg proce·ss· ' I.A{a·s 'ini'tia"ted.:

(1) Pups born to Pb-exgosed dams were placed 9n non.: Pb exposed

dams for rea·ring (6 litters) ; (2) pups horn to. non-Pb exposed

dams were· placed on Pb dams for. rearing (6 litters); (3) a third

group ·of . pups .born to . non- Pb exp.osed dam·s were left. with their

m10thers (6 li.tters) ;. (4) .a f.ourth group. consisted of litters bo~n

to Pb expos.ed mothers which remained with their mothers (6

litters}.

Start·ing 5 days . post..-part.um and la-Sting until iAie ·aning

(21 days of age) each litter was placed i .n Ct:lges des.igned to

moni t :pr the lit't :er activity levels of ' the pups (Fig.- l). · The

cag~s cons,i:sted .of two sec,ti·ons, a. home. c·age se·ction (~4 x 30 x

18 cnr) which had e.xploration ho·les (3 . 2 em diamete:r). that

a llowed-d novemerit of the. pups back and f -orth from. a se_cond' s~ction.,

the .exploration cag~ (28 x 1.8 x 15 em}, while · rest.Cicting move­

ment of the d·arri to the home cage. Infrar·ed photpbeams ·

(Auto:tron, I nc.) were p-laced 7 em fi'pm the. wall of' the home cage

Fig. l. Schematic of Home Cage Apparatus; A = Home Cage , B = Exploration Cage, C = Exploration Holes , D = I nfra- red Photobeam

conta:Lning.' the· exp:ioration f1pl~·s. In this loc.ation tne darns

could not break t he beams. The infrared apparatus was c onnected

to an event c ounter . . (Durant Digital Inst., Inc.} which 'was used

to record the movements of the pups. · Total counts were recorded

from the counters dai ly a t 0900 hours. All data were subjected

to a three way ANOVA with repeated measures andto Duncan 1 s

New Multiple Range Test (28). Interactions across day·s and

runong groups we re tested. All statisti~s were based on litters

as the sma·llest unit of measure.

In Experimen~ 3, n~o groups of rat pups (control and Pb

treated) were moni t :ored continuously from 21 days of. age through

37 days of age . The Ph- treated group consisted of four pups

from different lit ters whose dams were expos-ed to 200 mg/1 Pb

a.s previously described. A control group consisted of four

contro l pups taken from non-Pb treated dams . At 21 days of age

animals -f~om each group were taken from the ir dams and placed in

individua l cages which were then placed on individual activity

monitors (Columbus Instruments t-'loni toring Systems). All pups

tvere given food and wate r ad . libituni and were kept on a .g:l2

~photoperiod (09 Q.0-2 l00). The data were compiled separately

for the .control group and the Pb-expqs:ed group. Data from each

group were combined for four c onsecutive 4-day intervals i.e.,

2.1-25, 26~29, 30-33 and 34- 3 7 days old. The combined data for

E~ach group were then plotted for each three hour i nterval · of a

24 hour composi t e of each four day period and subjected to ANOVA .

10

RESULTS

Data from Experiment 1 shows no significant differences

(F (1~ 26) = 0. 79, p ~0. 7301), in te,rms· of t.reatment over ciays·

f ·or the 30 mg/1 group (Fig. 2 and Tab.le 1). Data from the 200

mg/1 group c learly indicates that there is a significant delay

(F 1, 27) = l0 . 31, p ~ 0. 0001) in ~he level of explora tion and

locomotor activity of the litters exposed to 200 mg/1 of Pb via

their mother, in utero and during lactation (Fig. 2). .This

difference betWeen control litters and Pb exposed litters mani­

fE2Sts itself from day 16 through day 21~ the last t ime point in

the experiment (Table I.). There are no significan~ differences

bE:tween the cont·roL and Pb exposed litters until they reach 16

days of age (Tabl-e I). There is an a!? proxi mate one day de lay in

the rate of development of exploratory and locomotor activity

(Fig. 2). The 400 mg/l group, parallels the 200 mg/l group.

There is a significant delay (F (l, 8) = 4. 01, p ~ . 0008) in

ti1e level of exploration and locomotor activity (Fig ~ ~). This

d:Lfference manife:sts itself from day 17' through day ~0 ,(Table 3).

The behavioral delays observed in both the 200 mg/1 and ~00 mg/1

gJ~oups parallel the delay in the normal increase in cerebral

cortical cytochromes and synaptogenesis seen in r at pups simi­

larly exposed to Pb (Bull et E·, 1979; .McCauley et al., 1979) .

The results ef Experiment 2, cros.s-fostering, indica te that

the altered behavior patterns seen during days 16-21 of the Pb

exposed litters is a consequence of events that t ake plc:~ce during

prenatal exposure. An ANOVA (repeated measure) i ndicated that

there were significant differences between 1:he treat:Jnents ove.t'

Fig. 2. Daily activity per l itter plotted against age: 0 = Control Litters, • = 30 mg/l Pb exposed litters,

· activity was measured as the numb~r of infra-red photobeam interruptions p~r day.

..... 2

..... Q) c. >

>

v <0

:>.

<0 a

2000

1000

0

11

o Cont.rol

• 30 mg Pb/1

1 6

Postnatal age, days

•··

.· TABLE I · Dunc;:lrt 1s t{e'tv Multip:1e Range .. T-es t

for. 30 P.H-t P'n T.re'ate·a , \Js Coi+tro·l Grdup$

TRE.Z\-TMENT DAY N

Ct 21

f.b 21

20

Pb 2'0

·Ct 19

19

C.t 18

Pb 18

-C.t 17

Ph 17

16

Pb 16

Ct 15

Pb .. 15

4-.0;65• + l. 3

36.43 + l.l

37 . 17 + 1.2

. 3L 23 + 1. 2

31 .. 15 + 1. 2

25. 6·6 + L. l

18~ 06- + l. :2

16.-00 + 1.3

11. 70 + 1. 2

lO. 81 + l. 3

6. 49· + o. 8

6.70+ 0.8

19

20

21

21

21

22

22

21

22

21

22

21

GROUPING**

·A,B

B,C

c

D

D

:£

E

f

F

G

G H . '

H,i

H,I

*Sqt1ar¢ Root _ Trans£orma:tio:h "~*Means wi:fh the same iet'te,r are npt si_"gnif-icantly , differ~nt,

. p s .. 05~

' . '

fig. 3 -~ Daily .ac'tivi ty pe·r Litter p1o:t:ted aga·in.st ·age: · 0 = Cdn1:rol Li t t e_r$, e' ,; ·20'0 mg/l Ph e::{po_sed littefrs, .at: ti:v.j.'ty was .meastwed. a.s th~e rill)11be.r of ill.fr'a_:red · photobe·ain irrterrupt.i ·ons per · day.

< "

2000

a.. cu o Control /~ .... .... • 200 mg Pb/ 1 ·-- # , .. ,

I cu 1000

, , c.

If' ~ I .... I

I ·- I

> I

I ·- , .... .~f' u

C'CI , , , ,

~ ,,

I , - ,if ·-C'CI , , Q , ,

. ..---+'' .... 0

I I I 1 1 16 21

Postnatal age·, days

·TABLE II Dunc~nts New Multiple Range Test

for 200 .PPM Pb Treated Vs Control Groups

TRANSFORMED* TREATMENT DAY MEAN + S. E. N GROUPING**

Ct ·21 43 . 47 + 1.4 16 A

Pb 21 36.49 + l.l 17 B

· . . . , Ct 20 3~. 20 + 1.1 18 . B

Pb 20 3 0. 2.0 ;f, 1.-1 18 D

Ct 19 33 . 65 + 0.9 18 c

Pb 19 2.5. 02 + 1.0 17 E

Ct 18 26 . 48 + 1.2 18 E

Pb 18 18 . 46 + 1.1 17 F

Ct 17 19 . 13 + 1.3 18 F

Pb 17 11. 15 + 1.0 17 · G

Ct 16 10. 23 + 1.1 18 G

Pb 16 6.54 + 0.8 18 . H

Ct 15 5. 73 + 0.5 18 f{

Pb · 15 4. 76 + 0. 8 18 H

*Square R·o.ot Tr-ansformi:l tibn **Means with the same . letter are not significant ly diffe rent,

p ~ . 05.

Fig. 4. Daily activity per litter plotted against age : 0= Control "Litters, • = 400 mg/l Pb exposed litters, activity was measured as the number of i nfra-red phot.obeam interruptions per day .

.... Cl)

a. .·>-

> -"'' u ro

ro Q

2000

1000

0

11

o Control

·• 400 mg Pb/1

16

I

Rl I

I

Postnatal age , days

I I

I I

I

I

I

I I

21

TABLE III DuncaJ1 1 s:·· New Multiple Range Test

for 400 PPM' Pb Treated Vs Control Groups

TR.fu\lSFORMED* TREA TlV!Et\l T DAY MEAN + S.E. N GROUPING**

Ct 21 45 . 22 + l.l 5 A -Pb 21 41.33 + o.s s A, B-

Ct 20 40. 78 + 1.4 5 B

Pb 20 . 37 . 12 + 1.6 5 c

Ct 19 35.48 + 1.6 5 c

Pb 1.9 28. 77 + 1.2 r-:;) D

Ct 18 26. 78 + l.3 r :;) D

Pb 18 16. 39 + 1.6 5 F

Ct 17 22.04 + 0.6 5 E

Pb 17 9 . 08 + 3.3 5 G,H

Ct 16 9.41 + 1.4 5 G,H,I

Pb l-6 11. 34 + 2.2 5 G

Ct 15 7.63 + 1.7 5 . G,H,I

Pb 15. 5.69 + 0. 7 5 H..; I

.. *Squar~· Rbot Transformation **Heans sharing tne sam·e grouping letters are riot significantly

different, p ~ o.os.

days (F (3, ~4} = 2 . ~9 , p S 0.003). The lev~ls of exp loration

and locomotor activ i ty in litters t re ated only prenatally with

Pb (reared on non- Pb· exposed dams) were not significantl~ dif­

ferlmt from the litters which received Pb exposure during

gestation . and lactati·o:ri (Table I V) . Mo$t i mportantly there were

no signif;icant differences , with ·respect to the levels. of

activity, between control litters and litters born to contro l

mothers and cross -:foster.ed to Pb exposed dams (Fig . 5, Tab l e IV).

There were, however, significant differences (p· S ·O. 05) between

the firs t two groups and these latter two grQups with respect

to the ra t e of developmen t of exploration an.d locomotor act i vity

(Table I:V). There . were also significant differences (p ~ 0. 05}

between those litters treated only prenatally. and those treated

only postnatally during days 17-21 (TableiV). The prenatally

exposed .litters exhibi ted the same l - 2 day delay in the level of

exploration as seeh i n litters exposed to Pb during gestation

and through weaning.

Table V shows the blood Pb l evels for all exposure regimes.

Data from time course ana lysis of blood· Pb levels (Bull, 1980),

can be found in Appendix I .

Figiire 6 shows the combined circadian activity data for both

the con~rol and Pb.,. t reated gr~mps in Experiment 3. Fr.om 21 days

of age through to 29 days of age significant differences ·

(P ~ .05) were observed between t:he control and Ph-treated groups

during -the dark phase of the light cycle but not during t he

light period . The dif£erences were manifes.t ed as dec'reased

activity continued into the postweaning period as compared to

.21

· .. -··

... ' .

• I .;

Fig;. 5-• . ·_Daily . a~ .. t ,iv.ity o;f' crpss-:-fps-tered li:tt~:rs 'pLotted ag~lnst., postnaital' age·: b = co:ritrol-_ pups r~ared· on . . coritr·ol · dams, ; :e~·-= :fiilp~- .born to 'pb : ·tt-eat~c( :dams reared

· ,ori fb,. ~:r.e .~te·a·,~·arn~; -:a. =· p~+,ps ,b~rn _i;:o i?b tr.ee1t~_d ·dams . 'reared -· bn contr·o~ .dams; · • ~ '= pup_s ·· born to .c.onfrol dams

-rear~d. --c)ir· Pb · treat~d dams·; a·qtivi'ty ~/as .me.asured as the tota·l . :numi:>e'r o:f'· ±rift-a.:.ped. :ph_otob~am '.iri:terruptions per day~ See·"Tabi~ tv · f .or 5-tandardB.r-ro.rs' · of the . me·Em·. . .

2000

0 :Ct/ Ct

·- e Ply"Pb F Q) o P1V'ct -_, • Ct/Pb -Q)

c. ~ -> -u ca >.

·-· ca. Cl

0

u 16 21

Postnatal ag.e; da ys

. , .

TREATMENT

Ct' Ct/Pb Pb/~t

Pp Ct

C't/Pb. Ph/Ct.

Pb Ct

C t/Pb. Pb/Ct

Ph C.t

C:t/Pb Pb/Ct.

Pb Cf

Ct/Pb Pb/Ct

Pb c't

c t/P.b Pb/Ct

I?JJ· Ct

Ct/.Pb Pb/Ct :

Ph

.· . :fABL{ IV '·' .. t>unc ap :. ~ N§w :-~lctlt ii:r:L~ · .R.:~ng·e '';'te,~ t' ~ ·for :cr6sl3·.:.:Fost.er.ingExper:lment

·DA:Y - --21 21 21 21 ~0 2p .20 2o 19

.. 19 19 19• 18. is 18 1~· ).. 7 1'7 1.7 17 i6 l-6 16 16 15 15 15 ·15;

TRAl'JSFORMED**·· MEAN :··± ..S:.r:E •· . ·

45,~' 63' ' + . 0 •. 8 42 . 18 :+ l. 9 3 7 • 5'3- ,:; r. 3 36 •. Z-2 + 1.9. 41. ·7.9 + L. 9 . . 36 ~53-- + 2. 3 3 2 ~ 21 + i .. ~-3 2.lr + -o .. -8 3 4-~:8$. + o., 4 3 0~.58 +· -2.0 25 . 38 .+ l.. 6· 26 .49 + l~- 0 2 6 . z:z + 0 ~ 9' 24~ 9.0' + 2 • 3 19 . Lf4 + L 1 19_~ 9'? 1 z:. 4 18 ·~ 7.2 + 1.; 1 17 '"'9'9 -+ 2. ?· 10;8.7 ' +: o. :5 ti~o7 + . z.·o

7 • /8 +. . 0'~ -5 9 .80 + l. .• ~ T. 7fl + L -.0 h .2.2 .+ o.6 6 •. as ·F o.s 6·. 63 + cr. 7 4 .9·6 + L.t. 4 . 84- + 0 •. 3 ·-

!i 5·. 6 6 5 5 6 6 5 5 6 6 s s 6 6

s 6 6 5 5 6 6 ,.. :::J ·

s s .-6 5

GROUPING**

.A ·A';B C' G B c E,D E,D C,D E F F F F G G

·G G R

. H, I I ,J,K

··. H, I , J I, J,K I,J,K

.J ,K J ' K

' K .K

*Squa·:re ' Root · :rr·ansfor:mat,ion **Me arts ::sharl:ng the-·s;arrte grouping lett~rs are. not signif:icantly

:cti:ffe'r'~,nt, -' I> ~ o.os~ · ·

....

:: ..

\..,'

· ... ·'

f-ABLE V . ' Blo·od Pb Conceht;at:Lorts' of .Pups and

Dams f~~ ;All E).(p6'~u~e·':Regirties::· ' tx: . .!: s .t. ),. *

Cont r'ols- .

3 0 mg/;L ·.-Pb . ,,

b.OSE '.-RESPONSE .ExrERIMEN'rS , ,pups .. ,. . . . DAMS' . .

. At ·w~ilg·::· .. .18 _ Day''Gest·ati~yAt, Weaning

J. 7 ::t J. a (N:::l8)·

21- 0 + 2 1 ·(N:::8) . •

3 •. 7 ± ·o.4 (N=6) .

25 7 + ' 3 2 . • (N-;5).· .

' 3.. 4" .:!: .l.-0 (N=8)

li 8 '-f· l -3 . ' (N;;8) •

2 0 0 mg;/,1 · Pb 3'8 ... 2 ± 3.~· 2 (N=Z3)

37 • .8 · + 1.9 (N=t=1) . ·

3 s~ .. s. + .3 • 1 (N-;,7)

40 0 m~/1 Ph 49;. .9 +; 5.0 . ·.(N=2l).

Ct/Ct ,·

Ct/ Pb* ~*·

Pb/Ct* *

Pb/PJJ .

*ug/ d'l ··

· CRGS'S-FOS-TE~ING' ~E!{PERIMEN:T . PUES · . DAMS* ·.

PJ. t : _w:eatnpg;~ . At· .. wean irtg ..

'4. B + LS . · CN'=6j .

3.6. 0 + Lt. 0 . (N="&) .

14.5 ± 6.·8 tN=6J-

29 9 + 4 0 ~ . . ...,...' .. . (N=o) ·

s·. s. ± . 1:1: . (N=4).

.27.5 .±); 1 (N- 5) '' - -; · . ,

.<.

**For ' dams,; ctl~b = DainS ' which were· e~po·se.d :to 200 ·:ing/1 Pb for . the ·durat{iori of the expet:"im.ent; ·Eb/Ct' dams ' re.Celved distilled f;.iafer · fo~; tb.e du'r~:ti:oh ·6£· th-e ·experj,i'hent. . ·

' ~ . '·· ,. . . ,, . ··

thE~ -c~rh~:oi , anifuE!lS·. N·o·.$:{f?;ri~f·:ldan:t: ~~~~~re\~~es ... r.?_~·re r?t,ed during

thE! ' ligh~ pf1:as·e. ·Begirin·ing· at"J9 .days : o:f _age ·an'd,. la'stiJ:lg. ~hrough

37 days . of $ge the acti:itity c"S-;ele of-. Pp.~·tr.eated .pups· was indistin­

guish.abl.e S.tatistically, fr_'.Offi. ... tl:+e c ontrOl eni:,mal,.s.

None of ,the · expo'sure .:regimes . ,resulted ii1 significant , dif­

ferences- 'in body wei·gh:~s ·of ei'th$r. tl:i_e·· d:ams o:r. puP.s (see Ap.pendix

II) a_I}d there ;,;ere ··no .s~grii.fic·a'n:t .dif.fe'ren~es ~D- t.:_ood cons'umptiort

or i n -. w.~ter ·co!fsuinptl.~IJ {s,e~, Appendix I.IT :a;t:1d · IV) . between. th~

contr.ol .and· Pb J'treated·. dams. ·No signif:ic'ant differE¥nc.es ·. iri .tiine

26 .

of ey'e . opening, were., fp'UJ1d ~bet'f1een the. ' 2QO;.,mg/l group and t he controls . , . ' "' ;·. '

Fig; • .6. Circadian ae.tiV.i ty analys is plot"; Cumulative time . ·(combined ccm.secutive 4-day inter:ti als from 21- 3,7 days of age) vs. ac-tivity measured as, ,'the number of conductive fields activated; 200 mg/1 Pb expos'ed •·••••• •••, vs. distilled water contro is • . _e.-

. ·.·· -,

·f ' 0 0 <">

•.. .. ·:•

', . .:. , ..... ~'· .......................... . ..

.. ;:_:: :::::: _··. ·····. ... . ~ ···!··· ··••···•·· ...

::::: ::~:~:::~ ..... .. ··~

.. ··• ~:· · ·· ·.

~---------~---. ~~. -·~ . ' ·:-··:~ .......... , ....•. , .....• ..

'j

······ ·•···· .. ' .. .. ....... •... ....

, ..... . . ··· , .

.. . •:\r······· ·· ·· ~ .. ..

~ · :~· ..... ..:. I , I

~·

DISCUSSION

Th~ .results of ··the present study 'point to the prenatal

period a's · the .cri t;Lcai fi.me for subclinical Pb exposure . in the

induction of a behavioral deficit (de:lay ·in the rats of explora-

tion and locomotor activity) that only manifes1:s itself approxi -

mately 16 days after birth. Note that . these findings relate to

a delay in the dev~loi:Jrnent of exp loratory-:-lacomotor activity and

do not relate to findi.,ng$ ·Of hyperactivity and . hypoactivfty of

older animals by other inves.tigator.s (Drisc·oll and Stegner, 1976;

Driscoll and Stegner; 1978; Golter and Michaelson, 1975; Hastings

et al. , 1977; Kostas et al ., 1976; Overman, 1977; Reiter . et al ., -- -- --- -- -- --1975 ; Reiter, 1977; Wirmeke et al., l$77) . In the present study

the~ observed effects. can be attribu·ted primarily to . prenatal

exposure to lead, (Tab le IV~ in contradistinction to other studies

which have failed to discriminate be"tw.een prenatal and postnatal

effects . Contradictory to the present finding, delays have been

shown in the development of the Visual. Evoked Response in

neonatal rats . exposed .only: during l act-ation (Fox et al., 19 77).

TM~s postnatal .eff·ect might be explained by the much h igher dose

of Pb to the dam (0.2% lead acetate in drinking water) during

thE! postnatal period which resulted in · blood Pb concentrations

of 65 .! 9 ug/dl. A'ddi tionally, no deh~y h.as been observed in the

development of the Vi.sual Evoked Respo_nse in animals f rom the

200 mg/dl dose group df this study (Laurie, 1979) . Exposure

to Pb during gestatidn, lactation and post-weaning up to 42 days

of age resulted in deficits in both fixed ratio schedule of

reinforcement and fixed- interval performance (Padich and Zenick,

19 i'7 ;<'Zenic}<~_~et ·<al.;t ~9.~7~·) !·: A')...ih?ugl). , ·exp:?~ure j us(:'during g _es:.. .

tat:ion and l actation or }).i~t -.ip ~the post.:.-W.e.£ining st~g~ fa1.led to

result ).n, ·d.efic.it:s · in pe;r.forrhajlce~. ~.6 atteinp.t W?S made . tb ·differ.­

ent:iat~ ·b~.tween -~pr·e .:..: v·S .. post·-:-;rtatal e~'Po.§u·pe; .and pos.sl.bly,

testing.'i-yas ca~'r±ed out too f ar ·'tJ_as•:t . a stage ~here a :deve-lopment

de lay .. c,au l d have b.e·en dhs·e~ed in those· just, treat ed ;during the . • ¥ • • ••

prE! - and ·post-natal · per:fod.

Ih _.:Cppb:Si:t;iqh t o ' t)Je present study 3 .a delay in . eye opening .

has b~en - sr.O~n ·a..t · dose . .Levels (S'Ei' mg/lJ lower tlian · those used in

thj.s s:'tudy _(Reite;r: et al~. :~ 19753. I n the -Rei-ter et <al. · (1975)

study· 9-~Tay_,s i n - th~ . r-i'ght.ing·.'r.~flex ·ip:, neonatal r?ts exposed .

throughout :· .ges t :at"ior_i. ·and:· lactation w;ere reporte·tf~ :·a'1 though no , : • <. . -_·, .- • • •

att:empt-:}..vas made · t .o separ~at.~ · i ·n utero vs·. · ppstnata l effects. . . -.- ·· . · -·

Lat:er '. \v6rk has fai~led tCt r .eplicate th.es·e ~indirig~ (Reiter, 1980).

Gestational- exposube to ,.b~_ad .is ... N:p orted· to; result in slowed · . ,. . ~ . .

learn~ng 'i ·n · lanilis' (Ca:r :s·on et al., 19.75}. . -.- .-

Tne ,.results of. tl}e dose response·_ experiment of this study

arE! iii:· agr~·emen:t · with o·th~rp . i;1 ·t e rms of' the trend of ah in­

creased effect wi'th irtcreased ·.dose l e'v.els·. · A decrease in loco-~ ·' • > J • • ·' ,• •

moi:or activ,i ty ,in 'both 5 .. mg/1 a.rtd 50 . . ~g/l· .Pb· exposed r~ts has

beEm r~port~d (Ref te.r et :al. ,, 1975), w{th the SO '. mV.l exposure

group have a greater: d:epressiort i n ac.tivi:tY than the .S. mg/l

exposy.r~~, group. Al:though effe-cts are se_el) ' both .at l.eve ls which

are lf2w~r · ·cs mg/l .. Pb) and hi~:Per (S d mg/L:PbJ than ··the lowest

expos!-J.~~· l~vel li$e.d in this ,.s±udy (30 ' J}lg/J Pb}, the-. an_imals used

were .-:f es'ted as. aaults and t he Pb expost.tpe' '¢ortti'nued ' throughout

g_estat,~b~, ,,· l ac tcftion· -and up tb .the time of testing- a.t .120 days of.

30

~· :.

ag~. / Ail . inc~~-~-~·~t.i~·ly _:,g~:~d~d. .i;_t~jla,·:?f-;:~~~f:g·~.a~c'e deficit in · post­

wea,nfng· ·PJ:t ci<?sed. ' ra~ts . (0, < 9_,: z·7; --''clnd Si.·:rnykg .Pb . acetate intubated

daily:·- for· ·thr.ee weeks p .Q.st ~~§ni~g0 - h~s -<be.en sho~n . (Sobotka .and

Cook, 19.74)\ .a} though .in adul;t-s the sam~ methodo1qgy _ .resuited in

a slightl y ·: skewe'd responsE! in ~~h;!.ch the. 81 mg/kg group shpwed

bet:ter:· pe·r:-formance th<m :i:he ~ 2'7 mg;/kg grmip. A .dose . e·ffect in . the .. . . '.

ext:inct;,io~ of ... active shock . avo_idance .. ijl< i'ats Pb , expo.sed· ·co, 10,

3 0, 9'0 ·mg;J<g .inwb·ati.bl'\ ·fr'om '3'::.21 .q;~y.s ,of age.) . has beefi shown only . ,. . - , . . . •. . .

at th~ · Q.ighest :¢.:b§e le:vel ·cover:man; 19.77}; .ai though· there was a

dose res·pon_se effect ·qetv;>'e~n the hi~he&.1=' two ;:lose.:. leve ls and the . ~ ._,. -· ·• . . - . . "' . .... '

control ·fq'r. mo,tor ac~iv.i,ty levels:. ... . , ·

·The ·.results· f r,.01:n .£-x,~periment 3 {Fi :g. ·6), .are · evidence that

the! 2Qb.!Tig/l Pb e:X.posea :.rats had decr·eased le_vels of- locomotor

act:ivi ty, dtlrirtg .. days _2'1...:29. · . Howev.er., -~igriif'icarft differences

were ~fo_und··, ortl~ during · .:the :np,ct):lrna:l period · of t hese· 'eight days • . .;• .. ' ' .. · . , . .. . .(. . . . .

In c·ontras't to ,thi-s a~ r'~pbrt of. a: de.cre~ase in both .tot.al and .- . ,- ' ' . . . ~: '

noct:urnal .. acti:VJty \vas ·opse.rve<i . :i,n -Pb tre ated rats·· (SO mg/1 Ph . . ,• . . . ,_ .. . . _, ·'' . .

acetate .in .d~inking< W,'a.t~r frgm gest:atiqn until tes.t~ng_, at, 120

days of age) (Rei ter,et al., 1975}, although a· .subsequent . - .- ... . . ., ~

report :.sljows no ,... s .ignific·ant .,di;f.f~ ·rences (Re.iter,' 19)7)'. · It is

als;o obvious-- from these . ct.ata that .irive?tigators. mttst . employ ·' 4 •¥ . . •

foretfiought , ln, J?lann:lng ;:J:)ehavio.r:al tests of t1}e. effects of chronic

ex:pos,ure :,to tbxic .. ants, ip. fi:-at t he . time period of te~ting may be of . . ..

crucJ:.al ' .in]port'anc'E~. A me-asurem~nt. t aken midw.ay t hrough ·the

lighf peribd , (£lg ... ,6}, would sho~ no difference bet:Wee'n t he •• . • _,. . • • -:J.

·:-- .

31

This study, and- others' 'conducted. .by this laboratory (Bull .

et al., 1979 ; McCauley et aL~, 1979), indicate a definite cor-- - - - .

relation between CNS developmental aberrations and be havior due

to low level lead exposure . · To our knowledge this is the first

work to unequivocal ly document the greater sensitivity fo Pb of

a I?arc1rrieter of brain deve loprnent during gestation. The impli­

ca1:i0ns of these dat·a are disquieting in that significant

behavioral differences in e~posed animals are shown to result

from blood lead levels currently .ac.cepted as beirig within the

upper range of normal in humans (Kubasik and Volson, 1972 ;

Stark ,et al. , 19 78) ~ · Furthermore, · recent evidence (Settle and

Pat:terson, 1980) implies that these. normal levels are actually

highly elevated -when compared to natural prehistoric lead levels

in humans, and that these ttnormaln _levels ·could possibly be . ..

respons:Cble for subtle" perh.lfbations . of cellular b:C6chemical

processes .

In surrunary, this study provides riew information on behav-

ioral .modifications induced by Pb exposure; delayed development

of explorationand locomotor activity due primarily to prenatal

exposure and chiefly affecting nocturnal activity. Additionally,

the b.ehavior~Ltest described in th:is study has _prbven to be a

rapid, sensitive, r eliable and relatively _inexpensive behavioral

biaas~ay for detecting delays in bra·in development (Crofton

et al., 1978) . At least in the case of Pb, delayed behavioral

development . appears to be directly re.lated to de.layed synapto-

genesis and biochemical development of the cerebral cortex

(Bull et al. , .19 79; McCauley et al._, 19 79).

32

. . /.

ACI<NOWLEDGEtvlENTS

I thank Dr. Douglas H. Taylor, without whose. help and

support this ·would never have been accompl i shed. I thank

Dr. Rich~rd Jw Bull, who g ave me the· opportunity , s upport artd

f lli!ding to conduct my rese arch. I thank J . Morrow and R. Radigan

for their assistance i n constructing the test apparatus. I am

indebted to S. Se'ill<op .for his statisti'cal assistance. I am

also indebted to D. Sivulka and s .. Lutkenhoff .fo r help with t he

data coll ection and animal care. I a lso thank R. Miller , for

performi·ng the blood-Pb analysis . This . study was . conducted at

the .Hea lth Effects Research Laboratory , U. S. Environmental

Protection Agency , Cincinnati, Ohio •

. '

APPENDIX I

TIME GOURSE BLOOD Pb CONCENTRATIONS

Time course. analys~s of blood Pb concentrations ·of dams

exposed throughout gestation and lactation to either 30 or 200 mg/ 1

Pb (Fig. A), indicate ·a sharp rise in Pb levels late in gestation.

Throughout lactation, blood Pb levels decrease s teadily (Fig. A).

Th«?se changes are reflected in pup blood Pb concentrations

(F:ig. BL Time course analyses .of blood Pb concentrations during

lactation show no difference between the Pb/ Pb group and the Ct/Pb

group. This indicates no effect ·of cross - fostering pups on the

blood Pb concentrations of the dam (Fig;. C) . Blood Pb concentra­

tions of the cross-fostered pups (Fig. D), also reflect the sharp

decrease in blood Pb levels during lactation. ·

The findings of Experiment 2 in this study, in which the be­

hav ioral deficits are attributed primarily to pre·nata l exposure

to Pb, are substantiated by these time course blood Pb concen­

trations. Both the higher exposure levels found prenatally

(Fig. A; - D), and t.l:ie fact that the prenatal period is more

susceptible to ·environmental toxins (Wilson, 1975), support these

cone lusi.ons.

Fig. A. Time course dam blood Pb concentrations during pregnancy and lactation.

',

, , .. - ·

,~···

. ~·· ..

DAM BLOOD LEAD .CONCENTRATIONS

DURING PREGNANCY AND LACTATION

J; X -:fll'lTRO-S . :!- ...:. -1: :; 3lil r-G PB/1... ..

10 ,, 15

GESTATIONAL AGE /DAYS POSTNA T ~ AGE /DAYS

. : . · ...

,_;.

21

~ .

Fig. B. Time course pup blood Pb concentrations.

-', .. :·.··

·.-

'.:J' 0 ;:o oo. u I:

.- ..

. 70. -~

.................... .. .....,.,...........,. .. .. ~,.~,;..; ;..;.. :..:;t . -~ M6 'pB/.C'

::'; . . .· .. ; · . . ]: ... ':: ; .. \ :[ '-~ ·I'G :~ :

AGE: /DAYS

·.·

· . · r· :

~ ...

.. : ... ···1

15 21

!

I I I ! I I

I

Fig. C. · Time course dam blood Pb concentrations in the ct6ss - fostering expet~ment.

··' ..

. ~-·

z .o . H 1-

~ ~ 1-z w u -a .u CD a_

0 0 0

a:l

l i ME COURSE ;OF .DAM \ 5 4 ..... •

: ... .- y•

IN CROSS_:FOSTERING . EXPERIMENT . . :·

~ -· ·:t' .-- · r c+) ci·'

: ~' <· <~". . " ~- ~ :;~ -4: PS/PS , . >- ~i,_:7:·-;..-i;ea/6i:"

C+/P.B

.: ~ '

15 .. . .

'····.·

I

I

I I

I i

-·-·-·----.4 21

I I

I

Fig. D. Time course _pup blood Pb concentrations in the ·cross - fostering experiment.

-' ·'

_J 0

' (D u I:

z 0 H ...... ~ ...... z w u a u Ol a.. § 0

al

'• .•·

100.

oo. z....-..-.;: C+/C+

70. ~·-·-·~ PB/C+

60 . . ;r ... •• •• ;,.J: C+/f'B

51'.!'.

40 ·

30.

20. .·

10.

0 .01~----------~--~------~~~~----~r-------------~

0 > 5 . ~ ·. 15 21

FOSTNATAL AGE /DAYS

·.

j

I I I I I I

APPENDIX . II

BODY WEIGHTS

Table :A. Dam .body .weights (g); _}_0 mg/1 PbCl vs • . distil·led water controls (X + S.E. ) . 2

Controls 30 mg/1

Day 0 (hegin expp~ure) 232 ·_::: '+. 2 (N=l6) 231+ + 3. l

Partu.ri t i on 306 + 10. 2 (N=l2) 305 + 4. 6

Weaning* 326 + 7. 9 (N= l2) 333 + ~.8

*21 days postpartum

Pb

(N.=16)

(N= ll)

(N= ll)

' .

Table B~ Neonate body weights (g); 30 mg/l · PbC12 vs •. dis'tilled water ' contro.ls (X _! .S. E.). *

Controls 30 mg/l Pb

Birth 7 . 04 + .20 (N= l2) 6 . 33 + .12 (N= ll)

7 Days of A~e 18.42 + . 51 (N;::l2) 16 . 58 + • 33 (N~ll)

21 Days of Age . 53. 73 + l. 84 . (N'=l2) 47.15 + 1.18 (N=ll)

*Neonate weights, N = number of litters with 8 neonates per litter

Table C. Dam body weigh ts (g); 200 mg/1 PbCl2 vs. distilled water controls (X+ S.E . ).

Controls 200 mg/1 Pb

Day 0 (begl.n exposure) 216 + 2 .-s (N=l2) 216 + 2. 2 (N=l2)

Parturition 276 + 6.2 (N=lO) 271 + 6.2 (N=8)

Weaning* 287 + 3.9 (N=lO) 299 + S. 6 (N=8)

*21 days postpartU;ID

· .. . ·. ·, .

Table D. Neonate body weights (g); 200 mg/l PbCl2 vs . distilled water controls (X 2:. S_.E.).*

Birth

7 Days of Age

21 Days of Age : . . .

Controls

6'. ·12 + • 05 (N=td)

15.4.: + .51 (N=lO)

200 mg/l Pb

6.27 :+ .15 (N=8)

16.1 · + • 6 (N=S)

47.6 + l.l3 (N=8)

*Neonate weights, N = Nuinbe:r of li tter with 8 neonates per litter

Table E. Dam body weights (g) ; 400 mg/1 PbCl2 vs. distilled water conrrols (X~ S. E. ) .

Controls 400 mg/1 Pb

Day 0 (bE:!gin exposure) 205 + 1.3 (N=l2) 204 + l.7 (N=l2)

Parturition 332 + 7. 5 (N=6) 318 + 5.6 (N=7)

Weaning* 337 + 3.7 (N=6) 347 + 4.6 (N=7)

*21 Days postpartum

·-

Table .F. Neonate body ~eight~. (g) ; 400 mg/l . · PbC i2 vs. .dis'ti iled water 'contra ls . (X .:.:_ S. E . ) .*

Bir-rh

7 Days of Age

21 Days of Age

Controls

6. 5 l .: • .2 8 (N=6)

15 .5 + • 85 (N=S)

47.09 + 2~ 02 (N=6)

400 mg/1 Pb

6 . 83 .:.:_ . 26 (N=7)

17.01 + l.03 (N=7)

50.72 + 1 . 10 (N= 7)

*Neonate weights , N = Number of litters with 8 neonates per litter.

APPEND~X I I r .. WATER CONSUMPTION

- l - l Table .A. Dam water con~umption (nil rat day ) ;

30. mg/~ PbCl2_.vs_. dist,illed water .qontrols . (X_! S.E. ).

Controls 30 mg/l Pb

Day 7* . . 35.1 + L8 . - (N=8} 30 . 1 ~ + l. 6 (N=8)

Day 21 36.9 + 2. 1+ (N=8) 31+. 6 + l. 2 (N=8)

Day 35 1+7 . 9 + 2.9 (N= l6) 38.8+ 2. 4 (N=l6)

Day 56 91+.0 + 8.1 (N= l2) 95.0 + 6 . 6 (N= ll)

*Days recorded starting with .. the f irst day of exposure, parturition is approximately day 35, weaning is approxi-mately day 56 . . · ·

' • . ...

T~ble B. .Dam water ~onsumption . (ml rat~ 1 day-

1);

200 mg/1 PbCl2 vs . -distilled water controls (X _2:. S . E.).

Controls 200 'mg/l Pb

Day 7* 31.9 + l.O (N=6) 32.3 + 1.3 (N=6)

Day 21 39.9 + 1.2 (N=l2) 3 7 .• 1 + 2.1 (N=l2)

Day 35 4-5 . 9 + 3 . 3 (N= l2) 4-l. 4 + ~.7 (N=12)

Day 56 .. 128.6 + 8.1 (N=8) 109·. 8 + 7.2 (N= 7)

*Days recorded star~ing with the first day of exposure , parturition is . appr:oximate ly d,ay 35, we·aning is approxi ­mately day 56~ .

Table C. Dam water consumption (ml ·rat- 1 day- 1); 4-00 mg/1 PbCl2 vs . distilled •..11ater controls (X _2: S. E. ) .

Controls ·.· 400 mg/1 Pb

Day 7* 34.0 + 2. 9 (N=6) 30:J.. + l.S (N=6)

Day 21 40.0 + 1.3 (N=l2) 36 + 1..6 (N=l2)

Day 35 50. 6 + . 2. 9 (N=l2) 48 05 + 2. 9 (N=l2)

Day 56 94-. 9 + 4-.2 (N=7) 9 7. 9 + 3. 8 (N=6)

*Days recorded starting with the firs t day of exposure, parturition is ' approximately day ' 35, weaning is approxi­mately day 56 •

. . . . ·~

APPENDI X IV

' ' " ' · fOOD .CONSuMPTION

Table A.- bam fpod consumption (g rat- l d~y- 1); . 30 mg/1 PbCl 2 vs. disti lled water controls (X _::: S. E.).

Controls 30 m~/1 Pb

Daty 7* ' 17.7 + . 49 .(N=8) 17.0 + .25 (N=8)

Daty 21 29. 7 + l.l8 (N=l6) 33 . 1 + 3 . 23 (N=lS)

Daty 35 23.5 + .95 (N=l6) 22.9 + l. 03 (N=l6)

Daty 56 . s7.7 + 3 .2 (!;-l=12) 62.5 + 2. 53 (N=ll)

*Days recorded star.ting with the. first day of exposure~ , parturition is', approximate1y ' day 35, , ·weaning is apprc)xi-mate1y day 56. · · · · ·

·~ ...

' .. .

Table B. Dam food conswnption (g , rat-1 day-1 ) ; 200 mg/1 Pb.Cl2 vs • . distilled water controls (X _:: S. E. ) .

Controls · 200 m~/1 Pb

Day 7* 15. 5 + .28 (N=.6) 15.6 + . 53 (N=6)

Daty 21 19. 4 + • 69 (N=6) 19.6 + . 57 (N=6)

Da1y 35 '20-. 9 + • 72 (N=t2) 20 . 2 + ~87 (N= l2)

Da.y 56 55.2 + 1. 71 (N=10) 56 . 1 + - l. 45 (N=8)

*Days recorded starting with the first ' day of exposure, ·part uri tiori is . approx imately · day 35, we aning i s approxi­

. mately _qay 56;

Table C. Dam food constimption (g rat :- ~ day- 1); 400 mg/1 PbCl2 vs. distilled water controls (X+ S.E . ) .

Controls 400 mg/1 Pb

Day 7* .<·' 19 . 4 ·,+ • 48 {N=6) 22.4 + 2~86 (N=6) .

Day 21 ' 24. 1 + l.O (N=l2) 24.0 + . 95 (N= l2)

Day 35 ·. 27.0 + 1 . 23' . (N=l2) 2 8 •. 8 + 1.63 (N=l2)

Day 56 58.7 + 2. 45 . (N=6) 62.3 + 2.03 (N=7)

*Days .recorded star•tl.ng with the first day of exposure, · parturition is · approximately· day ·35, .weaning is approxi­mately day 56 .

APPENDIX V

. EYE OPENING OBSERVATIONS-

Table A. Effect of 200 mg/lPbCl2 on eye opening in neonate ma'le rats*

Group

Distilled Water C.on.trols

. 200 mg/1 Pb

Mean Day of Development + S.E.

15.4 + . 24

15 . 5 + .31

*No significant. di£ferences, Students t - Test

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