P.1 

OAR Box 1128 

Prepped by Ollie Stewart 

Document Number: 

6)11-1-184 

Docket Number: 

A-83-22 

Printed 4/21/2008 11:07:10 AM Sheet 6 of 198 
P.2 

J .k Epstein 

rotating dives 

hidings nth 

iisalem. (June 

A. J. (1974) 

,il process— 

232-133 (May 

icw approach 

nighly concenWaste Conf. 

chic treatment 

nation in the 

J. Wat. Pnllut. 

W. L. (1975) 

• i hern Nichigan 

<mf. Water Pol-

,975). 

•I. & Jank B F. 

•lions for sup-

•:/i Ann. Conf. 

lOctober 1975). 

>->v trickling filluly 1973). 

o in I J real BnUin. |<?7&-

< * •vf 

CHRONIC TOXICITY OF WATER-BORNE AND 

DIETARY LEAD TO RAINBOW TROUT (SALMO 

GAIRDNERI) IN LAKE ONTARIO WATER 

PETER V. HODSON, BEVERLEY R. BLUNT and DOUGLAS J. SPRY 

Great Lakes Biolimnology Laboratory. Canada Centre for Inland Waters. P.O. Box 5050. BufiingtgfflDA AI D Hrtf^KP T 

Ontario, L7R 4A6. Canada • W«Min UUUI\C» 

{Received 6 January 1978) 

Abstract—Rainbow trout {Salmo gairdneri) exposed to lead in Lake Ontario water demonstrated a 

21-day LC30 of 2.4mgl"' lead. At lead concentrations ranging from 3 to 120ugl"1, log,0 of lead 

concentrations in most tissues of exposed fish appeared linearity related to log,0 of lead concentrations 

in water. Highest concentrations occurred in opercular bone followed by gill and kidney. Lead accumulation by brain was not clearly demonstrated. Exposure to lead in water ai mnrpniratjopt as low 

as 13 ugl"' caused significant increases in red blo~od cell (RBC) numbers, decreases in RBC volumes, 

decreases in KBL ceuuiar iron content and decreases In RBI: <>-nnuno levuhnic ncid dehydratase uctiviTyTNo changes were observed in hematocrit or,whole blood iron content. The changes indicated 

increased erythropoiesis to compensate for inhibition of hemoglobin production and increased mortality 

of mature red blood cells. After 32 weeks exposure to 120ugl"' lead in water, 30% of remaining 

fistkCxhibited black tails, an early indication ol spinal deformities. Lead added to food was not available 

<ror lead uptake bv fish. Lead content of fish exposed to dietary lead was not elevated above control 

levels and the majority of lead consumed could be accounted for in the faeces. Dietary lead may 

have slightly inhibited uptake ol dietary iron. " 

P .^ff^ INTRODUCTION 

This paper demonstrates that water-borne lead is 

readily taken up by fish resulting in subtle sublethal 

physiological responses. Dietary lead, by contrast, is 

not "taken up and does not affect fish. " 

Metals are normally ptesenl in alt surface waters 

at very low concentrations. Offshore in Lakes Superior and Huron, lead is found most frequently at concentrations below lftgl" ' with 95% of all lead 

measurements less than 3/tgl"' (I.J.C., 1976). in offshore waters of Lake Ontario, average lead concentrations range from less than Ifigl" 1

 in summer up 

to 2/igl"1 in the spring (Chau et al, 1970). In contrast, up to 55 /jg 1"' has been measured in municipal 

•$m water intakes in areas of the Great Lakes close to 

industrial and urban development (Kopp & Kroner, 

1967). 

Lead concentrations as low as 8 jig I" ' have been 

^ yttioym to be detrimental to fish during prolonged 

^r exposure, the harmful concentration varying with 

hardness, alkalinity, and test species (Davies et al., 

1976; Holcombe et al., 1976). Hodson (1976) estimated by an indirect method that 13/igl"' was 

harmful to rainbow trout (Salmo gairdneri) in Lake 

Ontario water but the actual concentration was un-

- known. The International Joint Commission has proposed a water quality objective for lead in Lake 

;,?, Ontario waters of 25/tgl"' (I.J.C., 1976) based on 

[. an interpolation of the alkalinity effect observed by 

,£ Davies et al. (1976). 

k*5c Since the harmful concentration of lead for fish in 

Lake Ontario has only been estimated, and since no 

effects of dietary lead on fish have been reported, this 

research was undertaken to evaluate the physiological 

effects on fish of prolonged lead exposure in Lake 

Ontario water. 

MATERIA US \ND METHODS 

Experimental design 

Three experiments were conducted to determine acute 

lethal and chronic sublethal toxicity of lead added to water 

and chronic sublethal toxicity of dietary lead to rainbow 

trout. The lethal toxicity of lead was measured in 3 continuous-flow bioassays at 1TC following the methodology 

recommended by Sprague (1969). Exposure times were 3 

weeks; 20 fish per tank were tested at 5 lead concentrations 

ranging from 0.6 to 8mgl"'. plus control; and LC50's 

were calculated by probit analysis (Finney, 1971) based 

on mean measured lead concentrations. 

The second experiment examined rainbow trout in 

groups of 23 exposed to nominal concentrations of 0 (control), 10, 18, 32. 56 *nd 100ugl"1

 lead added to water 

by a continuous-flow system starting March 25. 1975. No 

lead was added to food. The lead treated water at each 

of the concentrations was divided 5 ways to supply 30 

tanks. The six concentrations were distributed at random 

within a 3 x 10 tank grid. J"ish were sampled after 1. 2. 

,4. 8, 16 and 32 weeks of exnosure. and the following 

measurements were taken: hejriatocxiL; blood iron: number 

of red blood cells (RBC's) per mm^erythrocyte <5-amino 

levuhnic acid dehydratase activity (ALA-D); length; 

weight; and lead concentration in blood, gills, liver, kidney, 

remaining viscera, opercular bones, carcass and, in the 

larger fistirTo"na3s and central nervous system (C.N.S.). 

Results were analyzed by split-plot analyses of variance 

(Steel & Torrie, 1960) where lead concentration was the 

whole plot factor and time of exposure the sub-plot factor. -

Statistical significance of differences between means was 

869 

IV 

i4 

If 

w 
P.3 

S/i. PETER V. HODSON, BEVERLEY R. BLUNT and DOUGLAS J. SPRY 

!

» ; 

f; 

i:: 

f ;• 

tested at the 95% confidence level by partitioning the treatment sums of squares. Standard deviations and standard 

errors were calculated from the whole-plot error mean 

squares with error degrees of freedom of 24 and 5 repeats. 

A logl0 transformation was applied to the tissue lead data 

to avoid non-homogenous variance. 

The third experiment involved adding lead to the diet 

to give a daily dose of 0 (control), 100. 180, 320. 560 and 

1000 ug lead kg"' of fish starting May 20. 1975. No lead 

was added to the water. Each of the six dose levels was 

tested at 5 ration levels of 1. 1.5. 2.0. 2.5 and 3.0% of 

the body weight per day. Fish were sampled at 2. 4. 8, 

16 and 32 weeks of exposure and the measurements listed 

above were made. The results were again analyzed at the 

95% confidence level by split-plot analyses of variance with 

lead as the whole-plot factor, time as the sub-plot factor 

and ration levels as replicates. Statistical significance of 

differences between means was tested as indicated above. 

Error degrees of freedom were 20. 

Test fish 

Rainbow trout were purchased as underyearlings from 

Erin Conservation Holdings Lid., crin. Ontario. Initial 

weights in the water and food experiments were 6.5 and 

10.5 g. respectively. In the water experiment, fish were fed 

a dry-pelletized trout chow ("Ewos". Astra Chemicals Ltd.) 

ad libitum once daily. In the food experiment, fish were 

fed the same diet to which lead had been added. Feeding 

was once daily except at high ration levels where fish were 

fed as often as required to consume the daily amount of 

food. During these experiments, there were no mortalities 

attributed to disease. However, in the chronic sublethal 

water and food experiments, there was 25 and 7% mortality respectively, due to the effects of aggression, and 2 

and 4%. respectively, probably due to a I-week period 

during which chlorine rose to more than 40ugl"'. Deaths 

due to aggression did not vary with lead exposure and 

most occurred during the first 4 weeks of exposure, The 

effects of aggression were reduced after 4 weeks by placing 

a vertical Plexiglass barrier in the form of an "X" in each 

tank. The barriers had holes drilled through each arm to 

permit water circulation and the escape of smaller fish. 

The barriers were added to tanks during the first week 

of the lead-in-food experiment. 

Water quality 

The water for these experiments was drawn as a municipal water supply from Lake Ontario. Treatment involved 

coarse filtration, chlorination. alum flocculation. sand filtration and chlorine supplementation to a 0.3 mgl"1

 total 

residual. Chlorine dioxide was added to control taste and 

odour and sodium silica fluoride for fluoridation. Prior 

to use, the water was filtered through activated charcoal 

and the temperature adjusted to 11°C. The charcoal 

usually reduced chlorine concentrations to less than or 

equal to lOugl"1, the limit of detection by the orthotolidine method. However, "breakthrough" occurred during 

the experiment and chlorine exceeded 40ugl~' for about 

1 week. 

The mean (+ 1 standard deviation) chemical characteristics of the water, based on weekly measurements, were: 

total hardness (as CaCOQ: 135 ± 2 mg I"'; total alkalinity 

(as CaCOO: 90 + 4 mgl"'; conductivity: 245 ± 9umhos 

cm"2: and j)H: 7.7 ± 1.8. Dissolved oxygen fluctuated 

from 6.1 to 12 mgl"'"and means ranged from 7.1 to 10.7 

with a standard deviation of 2.0mgl"1. The mean temperatures in the tanks ranged from 10.3 to 11.2°C with 

a standard deviation of 1.3°C. The photoperiod was 16 h 

light. 8h dark. For chemical characteristics other than 

lead, there was no significant difference between treatments. 

Lead exposures 

Lead was added to water by a doser modified from Hod- j 

son (1974). This doser used the pressure developed within , 

an intermittent siphon bottle to dispense 1.0 ml of lead % 

nitrate stock solution during each cycle. The siphon bottle j 

provided 13.51 of water per cycle to a proportional diluter; 

(Mount & Brungs. 1967). During each cycle in the chronic ! 

experiment, 1.0 ml of lead stock was diluted to a nominal j 

100ug!"' of lead by the first chamber of the diluter. The} 

diluter subsequently provided 2.251 per cycle of 6 concen-'l 

trations of lead (0, 10. 18, 32. 56 and 100ugl"1) every." 

1.5 min. The 2.251 was divided into 5 portions by flow-^ 

splitters giving a total flow to each tank of 450 ml cycle"1 

or 300ml min"1. The experimental tanks were polyethylene, dark green, measured 30 x 30 x 30 cm and contained 

201 of water. The 95% molecular replacement time was. 

3-4 h and the flow represented 1-2 l/(g'of fish day"'). Lead 

concentrations were measured daily to allow comparisons ] 

of results to measured concentrations. 

In the feeding experiment, lead was added to the com- i 

mercial trout diet bound to beef liver proteia A solution 

of 24 g lead nitrate in 100 ml of distilled water was added 

to about I kg of fresh, homogenized beef liver. After 

thorough mixing, the liver was freeze-dried. powdered, and , 

the lead content of the powder assayed at 4700 mg kg"' 

Control freeze-dried liver with no added lead contained • 

17mgkg"' lead. Appropriate amounts of the powdered1 

liver preparations were added to powdered trout food to 

give 30 diets (6 doses x 5 rations) of sufficient volume for 

the duration of the experiment. The maximum amount of' 

dried liver added to any diet was less than 1% by weight J 

and the maximum nominal lead concentration was 

lOOmgkg"1. The diets were pelletized. frozen, and subsamples taken for lead analysis. During the experiment.; 

the fish in each tank were weighed every 2 weeks and • 

the daily amount of food was adjusted for growth. Diet ^ 

moisture content was monitored to compensate for diet* 

moisture changes induced by cold storage. 

Lead uptake by fish from food was measured by lead 

analysis of food, faeces, water and the fish. To minimize 

lead uptake by fish of lead leached from the food, all faece) 

and uneaten food were removed from each tank within 

3 h of feeding. The leachability of lead from food was tested 

by allowing it to stand for 24 h in water buffered to several 

pH's. The suspensions were then centrifuged and the supernatant analyzed for lead. A second experiment allowed 

bacterial decomposition for 2 weeks prior to analysis. The i| 

exposure tanks in the feeding experiment were identical 

to those in the first experiment. Water flow rate was a 

minimum 400 ml min"1. 

' I 

Sampling and physiological methods 

Tanks were sampled in a random order on each sampling day. Two fish were removed and the weights and i 

standard lengths recorded. Blood was collected in microcapillary tubes from the caudal artery after severing the tafl 

at the caudal peduncle. The tubes were heparinized by rins-1 

ing with a solution of 5000 ml"1 of sodium heparin followed by oven drying at 60°C. 

Samples of blood from the first fish were used fori 

measuring hematocrit, blood lead, and erythrocyte ALA-D < 

activity. Samples from the second fish were used for determinations of hematocrit, numbers of red blood cells per !j 

mm3 of blood and blood iron. 

To measure hematocrit, as many blood samples as po«-

ible were collected in microcapillary tubes pre-chilled bf| 

liquid nitrogen. They were immediately sealed and stored] 

in ice water to prevent anoxia and cellular swelling] 

(Snieszko. 1960). Within 5 min of sampling, they were cen- i 

trifuged for 5 min at 7000 rpm in a clinical centrifuge wilh^ 

a microhematocrit head. The head temperature watj 

reduced by pre-chilling the lid. Hematocrits were measured <j 

with a 

was ti' 

Bi, 

its pi 

samp1 

afilm 

'.'• crucit,: 

i' solved 

10.0 n 

;; water 

fused t<-

; .• Iron w 

'TRBCs, 

fivivRBC"' 

;!.ml blc 

"i. Tot:.' 

-

;

:'countci 

diluted 

mm" •' 

' * Eryll 

from (. 

calcul:. 

scribed 

Bio. 

1

 measi 

..„ accord i: 

7i for tist.i 

f storago 

sample 

piemen' 

sis an,: 

tissue v 

cible a 

at 80'(. 

The ten 

and m." 

ultrap' 

added . 

hot pla 

solved 

nitric t< 

conccr.' 

tion 

(Hods 

?r.. 

*p 

- Th.. 

LCJ0V 

2.3(1.. 

LC5 0 

In i! 

fluctu ..• 

the c;n 

were 0 

Nominal 1-

concenlr:-. 

0*l"'> 

. 0<Clll 

10 

'IS 

32 

"w'S* 

t.sw.pu 12/1'. 
P.4 

! f'om Hod-

\>ped within 

' nil of lead 

,:ion bottle . 

ional diluter 

the chronic 

> a nominal 

diluter. The 

of 6 concena 1" •) every 

ns by flow-

'il ml cycle"1 

r

e polyethy-

'.d contained 

nt time was 

'ay"1). Lead 

comparisons 

in the com-

: A solution 

IT was added 

liver. After 

Adered. and 

i'00 mg kg"1. 

:l contained 

lie powdered 

•rout food to 

: volume for 

••-. amount of 

'.. by weight 

: -ation was 

. n. and subexperiment, 

i weeks and 

growth. Diet 

;ate for diet 

•:L-J by lead 

To minimize 

< I. all faeces 

,nk within 

nl was tested 

.. '. to several 

•••••< the super-

'cnt allowed 

.nalysis. The 

. ,e identical 

i rate was a 

each sam-

«eights and 

! in microcaring the tail' 

nized by rins-

• heparin folate used for 

<cyte ALA-D 

cd for deterH>d cells per 

uplos as poss-

>rc-chilled by 

d and stored 

:i:ir swelling : 

ie) were cen-

;ntrifuge with 

pcrature was 

'.TC measured' 

4t 

with a microcapillary reader and the mean for each fish 

was used in all subsequent calculations. 

Blood iron was measured to indicate total hemoglobin, 

its precursors, and its breakdown products. Blood was 

sampled as described and immediately dispensed onto Parafilm*. A 50/d aliquot was transferred to an acid-washed 

crucible and ashed at 460°C for 8 h. The ash was redissolved in 2.0 ml BDH "Aristar" nitric acid and diluted to 

10.0 ml in acid-washed glassware rinsed with glass-distilled 

water. Blanks and the method of standard additions were 

used to check contamination and the accuracy of results. 

Iron was expressed as (ugml"1 whole blood), as (ugml"1 

RBC's) by dividing by hematocrit, and as (ug x 10"' 

RBC"1) by dividing by the number of red blood cells per 

ml blood. 

Total red blood cells were enumerated with a particle 

counter (Coulter Electronics) using a 5 ul blood sample 

diluted to 100 ml with 1% saline. Results, as number 

mm " \ were the means of 3 readings per sample. 

Erythrocyte ALA-D was assayed by a method modified 

from Granick et al. (1972) by Hodson (1976). Activity was 

calculated as (nM porphobilinogen/(ml RBC's-h)) as described by Hodson et al. (1977). 

Blood lead concentrations in 50 ul samples were 

measured by furnace atomic absorption spectroscopy 

according to the methods of Hodson et al. (1977). Fish 

for tissue lead analysis were frozen in plastic bags. After 

storage for up to 12 months, fish from the 16 and 32 week 

samples were thawed and dissected with stainless steel implements. Food was cleaned from the viscera before analysis and the skin was removed from opercular bones. Each 

tissue was placed in an acid-washed, dry, preweighed crucible and the wet weight recorded. The samples were dried 

at 80°C for 48 h and then transferred to a muffle furnace. 

The temperature was raised over several hours to 460°C 

and maintained for a further 24 h. After ashing, 2.0 ml of 

ultrapure concentrated nitric acid (BDH "Aristar") was 

added to each crucible and evaporated to dryness on a 

hot plate. The samples were re-ashed for 24 h, cooled, dissolved in 20% ultrapure nitric acid, and diluted with 20% 

nitric to a volume appropriate to the tissue weight. Lead 

concentrations were measured by furnace atomic absorption spectroscopy using the method of standard additions 

(Hodson et al., 1977). 

RESULTS 

The three short-term lethality tests gave 21-day 

LC50's (with 95% fiducial intervals) of 2.6 (1.9-3.4), 

'2.3 (1.6-3.3) and 2.3 (1.7-3.2) mgl"1 lead. The mean 

LC5o was 2.4mgl"1. 

In the sublethal tests, lead concentrations in water 

fluctuated with time. Concentrations were highest in 

the earliest weeks of exposure and the overall means 

were 0-20% higher than nominal (Table 1). 

The lead concentrations in food were similar to the 

desired concentrations except for a few diets at high 

concentrations and low ration levels (Table 2). Up 

to 100% of lead added to a number of diets was found 

in the faeces. Only 0.2-1.2% of the lead in the food 

could be leached by standing it in water. Acidification 

or long-term leaching with bacterial decomposition 

did not increase the release of lead (Table 3)., 

Mean hematocrits of fish exposed to water-borne 

or dietary lead were unaffected by lead exposure. 

However, in both experiments hematocrits decreased 

significantly with time from an initial 32.8 to 26% 

after 4 wneks exposure (Fig. 1) after which values 

slowly returned to the initially observed levels. 

Blood iron concentrations, whether expressed as 

(ugml"1 whole blood) or (/igml"1 RBC's), did not 

change due to exposure of fish to water-borne lead 

(Fig. 2). However, cellular blood iron as (ug RBC"1) 

decreased-significantly at all lead concentrations by 

as much as 17% from control values. 

Blood iron as (ugml"1 whole blood) and (ugmr 1 

RBC's) was significantly reduced from control values 

in fish receiving dietary lead doses equal to or above 

260 and 200 ug/(kg-day) respectively (Fig. 2). The 

maximum effect was a 10% decrease. Cellular blood 

iron as (ug RBC"1) was not affected by dietary lead. 

Iron (ugml"1 whole blood) increased significantly 

with time by 25% from weeks 1 to 32 in the waterborne lead experiment while it decreased 21% in the 

dietary lead experiment. 

The number of red blood cells mm "3 increased significantly with lead in water from 9./4 x iuJ

 in corrtrol tanks to 1.15 x 106

 at ^Ougl" 1 and all lead 

concentrations had a significant effect. Dietary lead 

did not affect red cell numbers but means were generally higher than in the water-borne lead experiment. 

Red blood cell volume (the inverse of the number 

of RBCTml * divided by the hematocrit) fell by 14% 

from control values in fish exposed to 120 ug 1" ' lead 

(Fig. 3). Cell volumes were significantly reduced by 

all lead concentrations except 13ugl _ 1. Red blood 

cell volumes did not vary with lead dose in the dietary 

lead experiment. 

Red blood cell counts increased (Fig. 4) while cellular volumes decreased during the summer months. 

Fish from the water-borne lead experiment were sampled less frequently during the summer than those 

•

!

;i 1 

Table 1. Daily measurements of lead concentrations in water 

ft". 

1 

w 

%r* 

•ifc 

pNominal lead 

concentration 

W I 

OtCll) 

10 

18 

32 

56 

100 

Interval 

(Wecksl 0-4 

X

-

4 

13 

21 

38 

62 

130 

s* 

3.7 

3.5 

100 

7:8 

9.1 

33 

5 

X 

4 

13 

22 

40 

71 

140 

-8 

s 

3.6 

3.6 

4.5 

7.2 

11.8 

33 

9-

X 

2 

12 

22 

38 

67 

130 

12 

s 

2.7 

2.0 

3.1 

4.6 

6.1 

14 

13-

X 

2 

13 

22 

39 

62 

140 

16 

s 

1.5 . 

4.2 

6 5 

104 

18 8 

13 

Measured lead concentrations (**g 1 

17-

X 

2 

II 

19 

29 

51 

97 

20 

S 

2.4 

1.9 

3.1 

35 

5.8 

4.4 

21 

X 

2 

12 

19 

32 

52 

100 

-24 

S 

2.0 

1.7 

2.8 

3.9 

3.5 

13 

25-

X 

3 

10 

18 

29 

50 

95 

"'1 

28 

S 

2.1 

2.2 

2.9 

2.6 

6.5 

5.0 

29 

X 

4 

10 

16 

27 

46 

91 

32 

S 

6.0 

2.7 

4.2 

4.6 

5.8 

5.7 

Overall 

X S 

3 3.8 

13 6.3 

21 5.9 

35 8.0 

60 11.2 

120 30 

( 1 

, ' i 

Jr' 

ft 

fo 

l(* 

I*. 

t 

i I 
Uf. 

P.5 

Table 2. Measured lead concentrations in experimental diets and calculated lead dosage rates 

•; tti 

r 

';3> 

IP 

, j | •• 

. «5 

Nominal lead 

dose 

l/ig Ikg-dayl '1 

Ration 

(".. hody 

wl dav"' l 

1.0 

15 

2.0 

2.5 

3.0 

X 

cone 

1.1 

2.4 

J.2 

1.0 

2.0 

OlClll 

dose 

10 

20 

30 

10 

20 

20 

M 

cone. 

10 X 

7.: 

4.4 

7.2 

6.0 

-

casurcd lead 

100 

dose 

110 

no 

90 

180 

180 

130 

concent ration 

cone. 

14.4 

12.0 

9.6 

11.2 

7.6 

-

180 

l/igg" 

dose 

140 

180 

190 

280 

230 

200 

wet weight) and doses 

cone. 

13.1 

18.0 

16.8 

9.6 

11.0 

— 

320 

dose 

130 

280 

300 

240 

330 

260 

l/ig (kg-d 

cone. 

24 

29.9 

26 

20 

15.6 

-

avr 

560 

') 

dose 

240 

450 

510 

500 

470 

430 

cone 

73.7 

51.7 

40.0 

31.9 

26.0 

-

1000 

j 

dose 

740 

780 

• 800 

800 

780 " 

780 w 

Table 3. Availability of lead from selected experimental diets 

Nominal dose (pg lead/(kg rish day' 1)) 

Measured lead concentration 0igg~') 

—wet weight 

—dry weight 

Ration | 0

o of body wt./day) 

Dry wt. of tood eaten over 2 weeks (g) 

Total amount ot lead in food (/ig) 

Concentration ot lead in faeces 

—dry weight t^gg"') 

Dry weight of collected faeces Ig) 

Total amount of lead recovered utg) 

Per cent of food lead in faeces 

Per cent leached in water 

4 days pH 7.6 

4.6 

2 weeks pH 7.6 

0 

2.4 

3.8 

1.5 

49 

186 

17.7 

98 

173 

93 

— 

— 

320 

13.1 

20.2 

1.0 

13 

263 

142 

1.9 

270 

103 

— 

— 

-

560 

24.0 

38.0 

1.0 

25 

•J 5 0 

201 

4.5 

905 

95 

_' 

— 

-

560 

26.0 

38.7 

2.0 

50 

l<J.I< 

85 

13.0 

1105 

57 

— 

— 

-

560 

20.0 

30.3 

2.5 

72 

2182 

88 

11.2 

986 

45 

_ 

— 

-

1000 

73.7 

117.9 

1.0 

20 

2358 

407 

3.1 

1262 

54 

0.5 

1.2 

0.5 

0.2 

1000 

40.0 

61.0 

2.0 

60 

3660 

166 

12.6 

2092 

57 

— 

— 

-

from the dietary lead experiment. Consequently, mean 

control red blood cell counts and volumes, taken with 

lead across time, differ between experiments. 

Erythrocyte ALA-D activity was significantly 

depressed from control activity at all lead concentrations in water (Fig. 5). Per cent inhibition increased 

from an average of 25% at 13 ug 1"' to an average 60% 

at 120ugl"1. Dietary lead caused only a slight (4%) 

but non-significant inhibition of ALA-D at the two 

highest lead doses. There was a significant fluctuation 

of ALA-D activity with time in both experiments but 

no consistent increase or decrease related to season'! 

or duration of exposure. 

Blood lead levels of fish exposed to lead in water 

Fig. 2. ine <: 

on blood ir<" 

(dietary, s -

RBCs (ug i 

water-borne. 

Fe ml'1 : 

water-borne, 

treat-
P.6 

dose 

740 

780 

800 

800 

780 

780 

ut (4%) 

'he two 

'uation 

Tits but 

reason 

i water 

m 

Chronic toxicity of rainbow trout 873 

« 

1>i 

l 

4 

s 

"S 

4. 

n 

LEAD 0OSE IN FOOOlMftlkg-dayl) 

ICO 1000 

LEAD DOSE IN FOOO (ug/(kg-day» 

KX) VJOO 10000 

a 

2? 

* wo ^ » 

E 

s 

- • m • - - •— 

~°~ \ 

E 

El30 

vt' | 

I IK) 

O -I. 

o g 

o / ^ Pb in water 

-—— H •«•—• • 

•^3 , 

\ 

o 

UJ 

CC !< 

IEAO CONCENTRATION IN WATER (ug/^> 

Fig. 2. me effect oi exposure ot rainbo* trout to lead 

on blood iron, expressed as A: ^gFeml"1 whole blood 

(dietary, s = 35; water-borne, s = 38); B: fig Feml"1 

RBC's (ug Feml"1 -r hematocrit/100) (dietary, 5 = 90; 

water-borne, 5 = 125): and C: ug Fe x 10"9 RBC"1 (ug 

Fe ml"1 H- No. of RBC's mmJ x 1000)) (dietary, .v = 36; 

water-borne, s = 52). The blood iron values are the lead 

treatment means over all sampling times. 

increased significantly from control concentrations of 

220ugl"' to an average of 1040ugl- 1 at the highest 

lead concentration in water (Fig. 6). There was no 

significant effect of lead dose in food on lead concentrations in blood. Average blood lead concentrations 

LEAD CONCENTRATION IN WATER lug/fl 

Fig. 3. The effect of exposure of rainbow trout to lead 

on the red blood cell count (dietary, s = 0.176 x 106; 

water-borne, s = 0.143 x I06) and red blood cell volume 

(1/No. RBC's mm"J -f- hematocrit/100)(dietary, s = 0.048; 

water-borne, s = 0.050). 

were within the range of the control values from both 

experiments. ALA-D activity was significantly inversely correlated to (log10 blood lead) (Fig. 7). 

Lead concentrations in other tissues increased directly with lead concentrations in water (Fig. 6). The 

slopes of lines relating lead in each tissue to lead in 

water appear similar, except for liver and C.N.S. tissue. C.N.S. tissue (principally brain) of lead exposed 

fish contained more lead than control fish but concentrations in C.N.S. did not increase with increasing 

lead exposure. 

Opercular bones demonstrated the highest concen-

.411! 

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•i 

-it

 :!L '-:: Hi 

I 
P.7 

I , 

.liiK . ntiosoN, BEVLRLEV H. BLUNT and L/UUULAS J. .SI'K. 

LEAD DOSE IN FOOD <ug/(kg-day)) 

100 1000 

o m <r 

2 

o m 

a. 

Q 

< 

Pb in locxl 

Pb in water 

10 too.. 

LEAD CONCENTRATIO N IN WATER (M0.^ l 

Fig. 5. Effect of exposure of rainbow trout to lead on erythrocyte ^-aminolevulinic acid dehydratase 

(ALA-D) activity (dietary, s = 69; water-borne, s = 48). Each point is the lead treatment mean over 

all sampling times. 

trations of lead while gill and kidney showed the next 

highest. .Fish exposed to dietary lead rlemnnstrated 

no lead accumulation in any tissue and fluctuations 

in concentration appear random Concentrations of 

lead in the C.N.S. of fish exposed to dietary lead at 

all levels were equal to or greater than those of 

treated fish in the water-borne lead experiment. 

The ration levels in the dietary lead experiment 

affected neither the measured physiological parameters nor lead accumulations. The major effect of 

I

2 

LEAD IN WATER 

3 

CO 

CO 

z 

o 

< 

or. 

u 

z 

o 

o 

Q < 

LJ 

ration size was on growth and there were no significant lead-ration interactio'ns evident for any parameter, including growth. ,. 

"Black-tails" appeared in 3 fish of 10 remaining £";" 

ajter 32 weeks exposure to 120ugl"' lead in water 

The skin darkened on the left side only, Irof 

the dorsal to caudal fin, and it ended in a sharp 

line underneath the dorsal fin. Netting the fish 

caused the temporary disappearance of the skin 

darkening. 

LEAD IN FOOD 

Blood Lg/'m/l i.o -

Ooe'cul*. 0O"« 

LEAD CONCENTRATION IN 

100 

WATE R 

100 

LEAD DOSE 

1000 

(u.g/(kg-rjay)) 

Fig. 6. The concentration of lead in tissues of rainbow trout exposed to lead. With the exception 

of lead in blood, each point is the mean of all measurements taken at weeks 16 and 32. For blood, 

each point is the mean for all sample times. The standard deviations for lead in tissues of fish exposed 

to lead in water were: opercular 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; and blood 0.20ugml"1. 

Fig. 7. The * 

activity of i.' 

curved lines -

following "l 

\ [leak 

W$ -"'•""• 

1

..A> ---^These exp 

fc> ii readily a 

£;",_. accumulate: 

^changes. L<-

unavailable 

' effects on tl 

• Relative! 

in water wt. 

: of 2.4 mg I 

i for rainbi' 

Brown's (V 

Davies et <. 

in 28 mgi 

j . lead respec 

'£. 96-h LC50 

,

:

ii solved lead 

';. solved lea' 

' •••• Pickering 

96-h LC50 

t\. to 45.4 mg: 

and from 

; (360 mgl" ' 

bioassays : 

"absorption. 

| ^ It is unl: 

'.observed ii. 

fbility of lc. 

accumulate 

'some dept^ 

(probably tt 

phates, etc 

fe#;» probi. 

/rapidly (3 

he lethal ' 
P.8 

i-iiiiu -v Ircui 61: 

.use 

ver 

no signifi-

•r any pari remaining 

•d in water. 

nly, from 

in a sharp 

; the fish 

I the skin 

•- oney 

tvef 

-.•is 

fi.ood (..g/mi) 

:,MCasa 

1 

i 

'.on 

•od. 

BLOOD LEAD CONCENTRATION (no//) 

Fig 7. The effect of blood lead on erythrocyte ALA-D 

activity of rainbow trout exposed to lead in water. The 

curved lines represent the 95% confidence belt about the 

following "best fit" line: ALA-D = 590 - 146 (log,0 

[lead]) (r = 0.578; Syx = 70; Sb = 15.7). 

DISCUSSION 

These experiments HpmnnstratpH that lead, in watpr 

is readily available to fish for uptake, and that lead 

accumulated by fish caused sublethal physiological 

changes. Lead in food, by contrast, appeared to be 

unavailable lor uptake and consequently had no 

etlects on the tish. 

Relatively low concentrations (1-10 mg 1"l) of lead 

in water were lethal to fish with a mean 21-day LC30 

of 2.4mgl- 1. This value is close to the 48-h LC50 

for rainbow trout of 1.5mgl"1 projected from 

Brown's (1968) studies at lower water hardnesses. 

Davies et al. (1976) observed 96-h and 14-day LC50's 

in 28mgl"1 hardness water of 1.32 and 0.2mgl"1 

lead respectively. At a hardness of 353 mgl"', two 

s 96-h LCjo's were 471 mgl"1 total or 1.47mgl"1 dis-

, ' solved lead, and 542mgl"1 total or 1.32mgl"1 dis-

• solved lead. 

Pickering & Henderson (1966) demonstrated that 

96-h LC50's for four species of fish ranged from 5.6 

;K to45.4mgl"1 in soft water (20mgl' 1 total hardness) 

'•h and from 410 to 482mgl"1 in hard water 

' (360mgl"1). These results were derived from static 

bioassays in which lead content could vary due to 

absorption, adsorption and precipitation. 

\ It is unlikely that acute lethality will be frequently 

observed in natural conditions due to the low solubility"of lead. Considerable quantities of white salts 

accumulated in tanks at 5 mg 1"' lead or above and 

some deposits were visible at lrngl"1. These were 

|& probably insoluble lead carbonates, hydroxides, phosphates, etc. Unless spilled at very high concentrations, 

it is probable that the formation of these salts would 

;: rapidly (3-4 h) reduce lead concentrations to below 

Uhe lethal level (Lam et al., 1976). 

g

^Hfthal """"""^aticm—of' Iparl (less, than 

1000 Ugl" 1) are, far mnrp liVply tn orrur since they 

appear to be at or below the concentration that precipiTareg in Lake Ontario water. I tie three principal 

ertects of prolonged sublethal lead exposure on fish 

are lead uptake and contamination (this study; Holcombe et al., 1976), changes in hematological parameters (this study) and development of spinal curvatures (lordosis and scoliosis) (Davies et al., 1976; Holcombe et ai. 1976). 

Lead uptake appears linearly related to lead concentrations in Watei su that any iuutase in Concentration irom background levels will result in increased 

lead content of fish. This correlates well with the 

observations of Pagenkopf & Neuman (1974) that 

lead content of bone was higher in fish from a river 

compared to fish from a hatchery. The river water 

contained 2.56-2.93 ug 1"' lead, twice that of hatchery 

water. In addition. Brown & Chow (1975) report-' 

that fish from Baie du Dore, Lake Huron contained 

only 0.19 ugg"' lead in muscle while those from Toronto Harbour contained 1.78 ugg"'.The lower limit 

is close to the mean value of 0.14 and 0.10/tgg"' 

observed in carcasses (mostly muscle) of control fish 

from the water-borne lead and dietary lead experiments respectively. The Toronto harbour fish contained a lead concentration characteristic of exposure 

to 20-30 ug 1"' lead in water (Fig. 6), a range bracketing the IJC recommended objective. This suggests 

local lead contamination, perhaps due to urban 

runoff. It is interesting to note that the mean lead 

content of carcasses did not exceed the Canadian 

Food and Drug Directorate (NHW, 1952/53) guidelines of lOmgkg"1

 for lead in marine and freshwater 

animal products. Exposure of fish to 200 ug 1"' lead 

or more on a continuous basis might lead to contamination in excess of the guideline. 

High concentrations of lead in bone suggests that 

growing bone may provide a site for lead storage as 

a detoxifying mechanism. In mammals, up to 90% 

of the body burden of lead is found in bone (Goyer 

& Mahaffey, 1972). The accessibility of the opercular 

bone might make it suitable for monitoring lead 

exposure and estimating body lead content. 

The high concentrations of lead in gills and kidneys 

probably reflect the direct contact of gills with lead 

contaminated water and possible excretion of lead via 

urine. The lower accumulation rate of lead in liver 

suggests that lead may be cleared to other sites of 

accumulation more rapidly than in other tissues. This 

may be the result of the relatively high blood flow 

and metabolic rate of the liver. 

Lead in C.N.S. did not correlate well with lead 

exposure in water. All lead-exposed-fish demonstrated 

elevated C.N.S. lead concentrations but the amount 

of the increase was not related to lead concentration 

in water. In the dietary lead experiment, C.N.S. lead 

concentrations, including those of control fish, were 

the same or higher than those of fish exposed to 

water-borne lead. Therefore, it is quite possible that 

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the low C.N.S. lead concentrations of control fish in 

the water-borne lead experiment were incorrect and 

the apparent lead accumulation was anomalous. This 

would agree with the observed lack of C.N.S. lead 

accumulation in lead-exposed brook trout (Holcombe 

et at., 1976). 

Dietary lead was not taken up by exposed fish. The 

majority appeared in the taeces and the portion unaccounted for may have been in small faecal particles 

washed down the drain. High lead concentrations in 

faeces but not in tissue (especially viscera) indicates 

that the lead was not taken up. It implies either a 

failure to release lead from the liver protein, or a 

higher binding capacity for lead of other faecal material compared to gut walls. The former may be. correct 

since lead was not leached from the food by water 

or acid solutions although liver protein is digestible 

and should release lead. These results correspond to 

the observations by Leland & McNurney (1974) that 

lead is not transmitted through fond chains In fact, 

the higher the trophic level examined, the lower the 

concentration of lead. Lead appears to contaminate 

by adsorbing to surfaces so that small animals and 

plants at lower trophic levels, with a higher surface 

to volume ratio, have higher lead levels. 

Exposure of trout to lead in water produced subtle 

hematological effects. Increased numbers of red cells 

without a corresponding increase in hematocrit indicated a shift in the red cell population from mature 

to_ newly formed, immature cells. This suggests accelerated mortality and removal of mature red cells 

from the blood and acceleration of hemopoiesis to 

compensate for the loss in oxygen carrying capacity. 

This would also explain why the iron content of 

whole blood (or of the total RBC fraction) did not 

change, but iron per RBC was reduced. The reduction 

of iron per RBC may indicate a reduction in cellular 

hemoglobin, assuming that the ratio between hemoglobin iron and ferritin iron (the storage form) did 

not increase. Since the activity of ALA-D, an enzyme 

jnvolved in hemoglobin formation, was reduced, it is 

possible that reduction of cellular hemoglobin 

occurred. A reduction of cellular hemoglobin would 

reduce maximum oxygen carrying capacity of the 

blood and also stimulate erythropoiesis. Therefore, 

erythropoiesis probably maintained a constant total 

hemoglobin and oxygen carrying capacity despite a 

reduction in cellular hemoglobin and numbers of 

mature cells. 

Mean erythrocyte ALA-D activity, while reduced 

by up to 60%, did not decline to zero. The negative 

linear relationship of activity to log10 of lead in water 

describes an asymptotic relationship, or in other 

words, a reserve capacity of activity. The result is that 

hemoglobinemia may never reach serious levels in fish 

at these levels of lead exposure. This is supported 

by the relatively small decrease of 17% in RBC content and increase of 18% in RBC numbers. It appears 

u»»ii u/jihjp 'h*

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D'Amelio et ai, (1974) also noted reduction of 

erythrocyte ALA-D in goldfish (Carassius auratus) 

exposed to 200ugl" ' lead for 5 days. The change 

was accompanied by reduction in hemoglobin synthesis and red blood cell density. The effect of lead 

on red cell density was probably due to mortality 

of mature cells with no compensating erythropoiesis 

since five days is a relatively short time for increased 

red cell production (Schalm et ai, 1975). Anemia was 

also induced in brown bullheads, Ictalurus nebulosus, 

by exposure to 28mgl" 1 lead for 16-183 days 

(Dawson, 1935). The anemia was characterized by 

deformed erythrocytes and accumulation of heme pigments in the spleen and liver. Peguignot et al. (1975) 

observed a decreased hematocrit from 38 to 23%, depletion of red blood cells in the "red pulp" of the 

spleen, and accumulation of brown pigmented, leadcontaining cells in the "white pulp" of the spleen of 

• lead poisoned carp (Cyprinus atr'pio). Therefore, a jj 

general feature of exposure to sub-lethal lead concen-t 

trations in fish, as in mammals (Goyer & Mahaffey, 

1972), appears to be mortality of mature red blood 

cells, compensated for by release of red cells front" 

the spleen or, at low exposure levels, by increased 

red blood cell production. 

Therefore, the severity and significance of the 

. observed hematological chances is dependent on the 

seventy of the lead exposure. At the low concentrations of this experiment, compensation indicates a * 

low biological significance. The principal importance * 

of hematological changes is to indicate the occurrence 

of lead exposure and the possibility of other harmful 

effects (Hodson, 1976). 

In contrast, the hematological '.-ffects of dietary lead 

did not fit the above pattern. While small decreases 

(maximum 10%) in blood iron occurred at the whole 

blood level, cellular iron, hematocrits, numbers of 

RBC's, RBC volumes, and erythrocyte ALA-D 

remained unchanged. Since lead was not taken up 

from the food,, it may somehow have inhibited iron i 

uptake from food rather than directly affecting some 

hematological parameter. , 

The most important aspect of lead toxicity to fish 

is development of spinal deformities that increase J 

IIIOI tality~rates and prevent successful reproduction 

(Davies et al., 1976; Holcombe et ai, 1976). The probf V 

lem is characterized by the darkening of the skin in J| 

the area caudal to the dorsal fin. At first, this black j 

tail disappears when the animal is frightened or:3 

anaesthetized. Black tails are followed by dorsc-jf 

lateral spinal curvatures (lordoscoliosis—lordosis plusj 

scoliosis) which are reversible by anaesthetics in the! 

early stage. Later spinal calcification prevents reversal] 

(Holcombe et ai, 1976). The ultimate effects are atrc-1 

phy of the tail region, interference with movement,! 

and death. 

In the present study, rainbow trout exposed to lead! 

demonstrated black tails in 3 of 10 fish remaining] 

after 32 weeks at a measured concentration of.-j 

120ng I"1. Davies et ai (1976) observed similar! 

syi 

as 

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symptoms in rainbow trout at concentrations of lead 

as low as 8 ugl" 1 in soft water (20mgl"' hardness) 

and severe lordoscoliosis at higher concentrations. 

These effects were observed at 32 ug I" ' free lead in 

water of 353 mg I" ' hardness. Similarly, Holcombe et 

al. (1976) observed these symptom's in brook trout 

(Salvelinus fontinalis) at concentrations of total lead 

of 119ugl"' (84ugl" 1 dissolved lead) in water of 

44mgl" 1 hardness. These authors demonstrated that 

the development of symptoms required 26 weeks of 

exposure when the experiment was started with 

fingerlings and 6-7 weeks when it was started with 

eggs or fry. Therefore, the low incidence of these 

effects at a relatively high lead concentration is probably not due to a lack of lead toxicity, but rather 

to an inadequate exposure time*. While it is impossible to state the minimum concentration causing these 

effects from this experiment. Hodson (1976) hypothesized a relationship between lead concentrations causing lordoscoliosis and those causing significant inhibition of erythrocyte ALA-D. ALA-D is closely related 

to lead exposure in several species of fish and indicates lead uptake (Hodson et ai, 1977). Assuming the 

hypothesis correct, the lowest lead concentration inhibiting ALA-D was about 13 ugl" 1

 and hence this 

concentration should cause lordoscoliosis. 

The time effect on hematocrit (Fig. 1) was related 

to the time spent in the experimental tanks and suggests a stress due to confinement with a gradual 

physiological adaptation. However, the split-plot experiments allowed the testing of lead effects without 

confounding by these time effects, although there was 

some loss in precision compared to standard factorial 

experiments. The range of observed mean hematocrits 

corresponded to the values reported by Barnhart 

(1969) for hatchery-reared trout. 

The numbers of RBC's ml"1, and hence volume, 

also varied with time, but in relation to season rather 

than to exposure time (Fig. 3). The sharp early summer peak might be due to an endogenous response 

• to rapid summer growth. The observed red blood cell 

counts fell within the range of mean values 

(0.9-1.9 x 106mm"3) reported for rainbow trout 

(Bouck & Ball, 1966; Snieszko. 1960; McCarthy et 

ai, 1973, 1975; Barnhart, 1969). 

Variations of blood iron content and ALA-D activity with time were not related to sampling or season but may have been random or a result of systematic error in methodology. Since ALA-D activity 

slowly increases following a decreased lead exposure, 

(Hodson et ai, 1977), the variation with time may 

reflect long-term fluctuations in lead concentrations 

, in water. 

In conclusion, prolonged exposure of rainbow trout 

to lead in food had littleerfcrt nn trip fish whereas 

P'V *A recent confirmatory experiment demonstrated black 

tails in a low proportion of trout exposed to 30ugl"1 

and in 100% of trout exposed to 60-100ugl"1 for 10 

' months from the egg stage. 

exposure to lead in water caused lead accumulation 

in tissues, significant changes in hematological parameters and preliminary indications of developing 

spinal curvatures. These three effects occurred at 

measured water lead concentrations of 13, 13, and 

120ugl"1 lead respectively. Since the most serious 

effect occurred at 120ugl"1, the proposed IJC objective of 25ugl" 1

 total lead in Lake Ontario should 

protect rainbow trout. 

Acknowledgements—We thank Mr. A. Sippcl for technical 

assistance on several aspects of this project and the Water 

Quality Laboratory Network, Inland Waters Directorate, 

for metals analyses of water and fish tissues. 

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Barnhart A. (1969) Effect of certain variables on hematological characteristics of rainbow trout. Trans. Am. Fhh. 

Soc. 98. ,411-418. 

Bouck G. R. & Ball R. C. (1966) Influence of capture 

methods on blood characteristics and mortality in the 

rainbow trout (Salmo gairdneri). Trans. Am. Fish. Soc. 

95, 170-176. 

Brown V. M. (1968) The calculation of the acute toxicity 

of mixtures of poisons to rainbow trout. Water Res. 2, 

723-733. 

Brown J. R. & Chow L. Y. (1975) The comparison of heavy 

metals in fish samples taken from Baie du Dore, Lake 

Huron, and Toronto Harbour, Lake Ontario. Proceedings of the International Conference on Heavy Metals 

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Chau Y. K., Chawla V. K., Nicholson H. F. & Vollenweider R. A. (1970) Distribution of trace elements and 

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D'Amelio V., Russo G. & Ferraro D. (1974) The effect 

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fisp«^Rei;. Intern. Oceanogr. Medicate 33, 111-118. 

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^ r . (1976) Acute and chronic toxicity of lead to rainbow 

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10, 199-206. 

Dawson A. B. (1935) The hemopoietic response in the catfish Ameiurus nebulosus, to chronic lead poisoning. Biol. 

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Finney D. J. (1971) Probit analysis. 3rd Edition. University 

Press, Cambridge. 333 p. 

Granick S., Sassa S., Granick J. L.. Levere R. D. & Kappas 

A. (1972) Assays for porphyrins, r5-amino levulinic acid 

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samples of whole blood: Applications to metabolic defects involving the heme pathway. Proc. Nat. Acad. Sci. 

U.S.A. 69, 2381-238,5. 

Goyer R. A. & Mahaffey K. R. (1972) Susceptibility to 

lead toxicity. Env. Health Perspectives 2, 73-80. 

Hodson P. V. (1974) The effect of temperature on the toxicity of zinc to fish of the genus Salmo. Ph.D. thesis, 

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Hodson P. V., Blunt B. R., Spry D. J. & Austen K. (1977) 

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501-508. 

Holcombe G. W„ Benoit D. A.. Leonard E. N. & McKim 

J. W. (1976) Long-term effects of lead exposure on three 

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I 

Pm I 

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iiif'i 

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878 PETER V. HODSON. BEVERLEY R. BLUNT and DOUGLAS J. SPRY 

Rl; 

!ll . 

f.'i 

i 

n* 

lb 

• t . 

': • 

. t;' ; 

ltii:. • 

T : .: - • 

i i i 

HI 

: t >. '• 

IJC . (1976) Water Quality Objectives Subcommittee 

Report. Appendix A to the Water Quality Board Report 

to the IJC. 1975. International Joinl Commission. 

Windsor. Canada. 198 p. 

Kopp J. F. & Kroner R. C. (1967) Trace metals in waters 

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in rivers and lakes or the U.S. (Oct. I. 1962-Sept. 30. 

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Lam D. C. L.. Minns C. K.. Hodson P. V., Simons T. J. 

& Wong P. (1975) Computer model for toxicant spills 

in Lake Ontario. In. Environmental Biogeochemistry. Vol. 

2. Edited by J. O. Nriagu. Ann Arbor Science Publishers. 

Ann Arbor. U.S.A. 

Leiand H. V. & McNurney J. (1974) Transport of lead 

in a river ecosystem. Proceedings of the International 

Conference on Transport of persistent chemicals in 

aquatic ecosystems. University of Ottawa. Ottawa. May 

1-3. 1974. 

McCarthy D. H.. Stevenson J. P. & Roberts M. S. (1973) 

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McCarthy D. H.. Stevenson J. P. & Roberts M. S. (1975) 

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21-29. 

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Ottawa. Canada. 35 pp. 

Pagenkopf G. K. & Neumann D. P. (1974) Lead concentrations in native trout. Bull. Env. Com. Tox. 12, 70-75. 

Peguignot J., Gire M.-P. & Moga A, (1975) Intoxication 

par le nitrate de plombe chez la carpe (Cyprinus carpio 

L.). Influence sur la structure histologique de la rate et 

1'hematocrite. Europ. J. Toxicol. 8, 165-168. 

Pickering Q. H. & Henderson C. (1966) The acute toxicity 

of some heavy metals to different species of warm water,. 

fish. Air Wat. Pollut. Inter. J. 10, 453-463. 

Schalm O. W.. Jain N. C. & Carroll E. J. (1965) Veterinary 

Hematology. 2nd Edn. Lea and Febiger, Philadelphia. 

805 pp. 

Snieszko S. F. (1960) Microhematocrit as a tool in fishery 

research and management. Spec. Scient. Rept., U.S. Fish,;. 

and Wildl. Serv. No, 341. ;' .• 

Spraguc J. B. (1969) Measurement of pollutant toxicity to "'. 

fish. I. Bioassay methods for acute toxicity. Wafer Res. 

3, 793-821. 

Steel R. G. D. & Torrie J. H. (1960) Principles and Pro- , 

cedures of Statistics. McGraw-Hill. New York. 481pp. 

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