P.1 

OAR Box 1128 

Prepped by Ollie Stewart 

Document Number: 

8)11-1-186 

Docket Number: 

A-83-22 

Printed 4/21/2008 11:07:10 AM Sheet 8 of 198 
VJ. Great Lakes Res., 1979 

Internat. Assoc. Great Lakes Res. 5(l):84-89. 

Pr-f*r3Z>-

i 

EFFECT OF FISH AGE ON PREDICTED AND OBSERVED CHRONIC TOXICITY OF \ 

LEAD TO RAINBOW TROUT IN LAKE ONTARIO WATER $ 

I 

V. Hodson, B. R. Blunt, D. Jensen, and S. Morgan 

Great Lakes Biolimnology Laboratory 

Canada Centre for Inland Waters 

867 Lakeshore Rd., P.O. Box 5050 

Burlington, Ontario L7R 4A6 

ABSTRACT. Exposure of rainbow trout /"Salmo gairdneri^ starting at the egg stage to water-borne lead in 

Lake Ontario water (hardness of 135 mg CaC03/L) resulted in black tails (early symptoms of spinal deformities) at lead concentrations as low as 22 yg/L. No black tails were observed at 11 ng/L. Previous publications demonstrated that the lowest lead concentration causing black tails in trout exposed from the fingerling stage in Lake Ontario water was 120 (ig/L, suggesting that fish exposed from the egg stage are 5 times 

as sensitive as those exposed from the fingerling stage. Compared to previously published data on fish exposed from the egg stage, blood lead offish exposed from the egg stage was greater for a similar lead exposure. 

However, the relationship between blood lead and enzyme inhibition (erythrocyte 5 -amino levulinic acid 

dehydratase) was unchanged. Therefore the increased sensitivity of trout exposed from the egg stage was 

probably due to enhanced lead accumulation rather than to an enhanced response to the lead taken up. 

The incidence of black tails at this hardness corresponded well to a prediction based on published lead 

toxicity at extremes of water hardness. 

This paper demonstrates important sublethal 

effects of lead on rainbow trout (Salmo gairdneri) 

at lead concentrations of 22 Mg/L or greater. The 

sublethal toxicity of lead is shown to vary with the 

life stage exposed and can be predicted on the basis 

of water hardness. 

Chronic exposure of rainbow trout to waterborne lead resulted in spinal deformities and black 

tails, an early symptom of spinal deformities, at 

concentrations as low as 8 Mg/L in soft water (28 

mg/L as CaC03) (Davies et al. 1976). The duration 

of exposure before development of symptoms was 

longer when the exposure started in the fingerling 

stage (average weight = 6 g) as compared to an 

exposure started in the egg stage. Hardness also 

reduced toxicity since 360 Mg/L total lead was 

required to produce the same symptoms in water 

of 353 mg/L hardness (Davies et al. 1976). A linear 

interpolation of the relationship between hardness 

and logio lead concentration suggested that the 

lowest lead concentration causing these effects 

should be about 25 Mg/L in Lake Ontario water 

(135 mg/L hardness). An attempt to verify this 

prediction using fingerling rainbow trout (average 

weight =L6.5 g) demonstrated that only fish at 120 

Mg/L. lead exhibited symptoms after 32 weeks 

exposure (Hodson, Blunt, and Spry 1978a). The 

present experiment was conducted to determine 

whether trout exposed to lead from the egg stage 

in Lake Ontario water would develop black tails 

at a concentration closer to that predicted from 

Davies ef al. (1976). 

MATERIALS AND METHODS 

Rainbow trout eggs were exposed to nominal lead 

concentrations of 0 (Control), 10, 32, and 100 Mg/ 

L. After hatch, exposure of the fry continued for 

a further 30 weeks. Egg mortality, median time to 

hatch, hatching success, and fry mortality were 

measured. Incidence of black tails, the first sign of 

developing spinal deformities, was recorded at 

weekly intervals. After 30 weeks post-hatch (mean 

weight = 29 g), blood lead, erythrocyte 6-amino 

levulinic acid dehydratase (ALA-D), hematocrit, 

and condition factor were measured according to 

the methods of Hodson et al. (1977). All lead concentrations were replicated four times for a total of 

16 tanks. Results were subjected to analyses of variance at the 95% confidence level (Steel and Torrie 

* 

1960). 

of cor 

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lead c 

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P.3 

EFFECT OF FISH AGE ON LEAD TOXICITY 85 

120 

-eks 

The 

tine 

:age 

:ails 

om 

ead 

Mg/ 

for 

3 to 

/ere 

i o f 

at 

ean 

.ino 

;rit, 

5 to 

;onof 

varrrie 

i960). Significance of differences between means 

0f control and treated fish were tested by partitioning the treatment sums of squares. Slopes of 

regression lines were compared by t-tests. 

Water 

The dilution water was from Lake Ontario as 

described by Hodson et al. (1977). Temperature in 

the egg incubator was 11.9 ± .68°C and the variation between trays was less than 0.9CC. The temperature in the exposure tanks followed a seasonal 

cycle, increasing from the incubation temperature 

of 11.9°C to a maximum of 22.5°C over 4 months 

and decreasing to 10°C over 3 months. The maximum of variation between tanks was 1.1 °C. The 

standard deviation of oxygen measurements across 

all 16 tanks ranged from 0.29 to 0.77 mg/L and 

overall oxygen concentrations were 8.5 ± 0.7 mg/ 

L. Average pH was 8.16 ± 0.25. 

Fish 

About 100 ml (700 eggs) of rainbow trout eggs 

from Norfolk Trout Fisheries, Simcoe, Ontario, 

were exposed to lead within 3 1 /2 hours of fertilization in each of sixteen 10-L egg incubator trays 

(Health Techna Corp.), modified for separate inlets 

and drains. No prophylactic tratments for fungus 

were used other than removal of dead eggs at regular intervals. During swim-up, 500 fry per tray were 

transferred to separate polyethylene tanks (30 x 30 

x 30 cm) containing 20 L of water with the same 

lead concentration as the original tray. They were 

fed a commercial trout feed at a ration and granule 

size adjusted weekly for average fish size and temperature. The fish were thinned when the biomass 

approached the loading limitations of the flow of 

each tank (see below). Consequently, sample size 

in each tank decreased with time but was a maximum for any given fish size. Results were expressed as a percentage of the fish at the time of observation. 

Lead Exposure System 

The 4-stage diluter (Mount and Brungs 1967) was 

fed by a doser described by Hodson (1979). At 

each concentration, it delivered 680 ml/min split 

four ways to give 170 mL/min per tank. The flow 

rate gave 3.5 L/day for every gram of eggs (about 

0.1 g each) and limited biomass of fish in each tank 

to 122 g at 2 L per g fish per day. The carrying 

capacity was increased to 160 g/tank when the fish 

averaged 1.6 g by increasing the flow to 222 mL/ 

min per tank. The 95% molecular replacement time 

was 3 h in the egg trays, and 6 and 4.8 h for the 

low and high flow rates, respectively, in the exposure tanks (Sprague 1969). Solvent extractable lead 

was measured daily on an unfiltered sample by 

atomic absorption spectrophotometry (IWD 1974) 

at each concentration of one replicate. Extractable 

lead in laboratory water was found to be the same 

as total lead measured after acid digestion of the 

water sample. 

RESULTS 

Lead concentrations in the egg trays were fairly 

constant during the egg incubation period, but 

mean measured concentrations were somewhat less 

than nominal (Table 1). In the exposure tanks, 

concentrations were fairly constant from month to 

month except for September when averages fell by 

about 20% from the overall average. Measured concentrations were within 15% of the measured values in the egg trays. Biological results are compared 

to the mean measured lead concentrations (Table 

2). 

Egg development was normal in the presence of 

lead and few eggs died before or during the eyed 

stage (Table 1). There was a slight but nonsignicant elevation in eyed egg mortality and a decrease 

in overall hatch at the highest lead concentration. 

After hatch there was no difference between treatments in sac fry, swim-up fry, or fingerling mortality. At 3 weeks post-hatch, a diluter malfunction 

caused extensive mortality in some reps. The surivors from unaffected tanks were redistributed 

within each treatment to equalize the number of 

fish per tank. 

By 30 weeks exposure, the incidence of black 

tails was 0% at 5.8 and 11 Mg/L, 5.5% at 22 M 

g/L, and 88% at 65 Mg/L (Table 2). Blood lead 

concentrations were significantly inhibited at all 

lead concentrations above control. The blood 

lead concentrations corresponding to the occurrence of black tails was 1700 Mg/L while the ALAD activity was 64 nM PBG/mL RBC's /h (nanomoles porphobilingen per millitre of red blood cells 

per hour) (equivalent to 74% inhibition). There 

was no effect of lead on condition factor and 

hematocrit. 

DISCUSSION 

This experiment demonstrated that symptoms of 

lead toxicity in rainbow trout occurred at concentrations of 22 Mg/L of higher after 30 weeks posthatch in contrast to a previous experiment in 

which similar toxic effects in rainbow trout occurred after 32 weeks exposure only at a concentra-
86 

TABLE 1. Summary of egg development data. 

HODSON et al. 

*S = pooled standard deviation-calculated from the error mean square of the analysis of variance 

Sj^= pooled standard error of the mean 

R = number of replicate exposure trays 

TABLE 2. Summary of physiological measurements at week 30. 

Parameter 

Measured lead in water 

G*g/L±S) 

No. of eggs tested 

Median time to hatch (h) 

Percent eggs not developing 

to the eyed stage 

Percent eggs dying in the 

eyed stage 

Percent hatch 

Percent mortality of sac fry 

Percent mortality of 

swim-up fry 

0 (Control) 

4.2+1.7 

2646 

673 

3.03 

1.26 

95.72 

2.06 

2.92 

10 

12±5 

2428 

686 

3.46 

1.07 

95.48 

1.82 

3.75 

Nominal lead concentration (Mg/L) 

32 100 S* 

26±8 

2465 

676 

3.86. 

1.24 

94.93 

2.06 

1.42 

76±23 

2066 

670 

3.18 

2.14 

94.67 

2.17 

1.67 

8 

0.92 

0.87 

1.48 

0.46 

1.53 

SX 

4 

0.46 

0.44 

0.74 

0.23 

0.77 

R| 

J 

1 i 

<; 

4 

4J 

4J 4

I 

4 ] 

•i 

Parameter 

Measured lead concentration 

in water (jug/LtS) 

Condition factor 

Hematocrit (%) 

Blood lead (Mg/L) 

Whole blood 

ALA-D! (nM PBG/mL 

RBCs/h) 

Percent inhibition of ALA-D 

Percent black tails 

0 (Control) 

5.8±4.8 

1.583 

31.4 

150 

242 

0 

0 

10 

11±7 

1.564 

29.2 

420* 

166* 

32 

0 

Nominal lead concentration 

32 

22+9 

1.495 . 

32.4 

1700* 

64* 

74 

5.5 

100 

65±26 

-

 : 1.506 

31.2 

1800* 

87* 

64 

88.0 

(Mg/L) 

S 

0.240 

3.9 

52 

52 

— 

-

SX 

0.12 

2.0 

26 

26 

— 

-

- i l 

RJ 

4 1 

4 1 

" 4 | 

4 1 

-i 

4 1 

R = number of replicate exposure tanks 

*Significantly different from controls at the 95% confidence level 

•ALA-D = erythrocyte delta amino levulinic acid dehydratase (nanoMoles Porphobilinogen synthesized per mL red blood cells 

per hour) 

tion of 120 Mg/L (Hodson et al. 1978a). Since the 

dilution water, species and exposure time were the 

same, the difference was due either to the fluctuating temperature regime of the present experiment 

or the life stage at which the exposure was started. 

Since Davies et al. (1976) observed that trout 

exposed as sac fry were more sensitive than those 

exposed starting as fingerlings, and since both their 

tests employed fluctuating temerature regimes, the 

life stage hypothesis is probably correct. Sac fry 

are probably the stage most sensitive to lead since 

only the chorions of eggs take up lead and not the 

embryo (Holcombe et al. 1976). 

There are two possible explanations for the 

increased sensitivity of fingerlings exposed from 

the egg stage. Either these fish take up more lead 

for a given lead exposure, or a given amount of 

lead taken up exerts a greater toxicity; i.e., the 
P.5 

EFFECT OF FISH AGE ON LEAD TOXICITY 87 

4 

4 

4 

4 

toooo^ 

logro BL= 1.9730+ .6099 tog,0 WL 

log*, BL = 2.0639 + .4434 logw WL 

log*, BL* 1.6407+5935 logioWL 

logro BL' 1.4745+1.0805log,0WL 

I I I I I 11 

10OO 

Lead Concentration in Water (u.g/L) 

ft 

flG. 1. Blood lead (BL) of rainbow trout exposed to waterborne lead (WL). *The slope for line D was significantly 

different from that of the other lines at the 95% confidence 

level 

dose-response relationship of the target site has 

changed. In the first case, the rate of uptake or excretion has changed, resulting in a higher equilibrium concentration at the target site and hence a 

greater response. In the second case, lead uptake 

dynamics have remained constant but the response 

to a given level of lead taken up is much greater. 

fi These hypotheses were tested by examining the 

relationship between lead in water and blood lead 

(uptake) and between lead taken up and response 

(toxicity) for trout exposed only as fingerlings 

(published data) and for trout exposed from the 

egg stage (this experiment) (Figures 1, 2, and 3). 

The data for these figures were taken or calculated 

from Hodson et al. (1977) for line A in each figure, 

Hodson et al. (1978a) for line B in each figure, 

Hodson, Blunt, and Spry (1978b) at pH 8.0 for 

Knee C in Figure 1, and this experiment for line 

D in each figure. Equations for each line are given 

I i cells on each figure and the statistics for the regression 

lines are given in Table 3. 

•£ The slope of a line relating log10 [blood lead] 

to log! 0 [lead in water] is relatively constant over 

; fry three separate experiments with rainbow trout 

•ince 'exposed only as fingerlings (lines, A, B, and C, FigII the ure 1). In contrast, the slope for trout exposed 

from the egg stage is much greater (line D, Figure 

the 1), indicating a higher than expected blood lead 

rom for a given lead exposure above 10 Mg/L lead. The 

lead water temperature at the time of sampling in this 

t of experiment was 10°C. The experimental temperathe tures of lines A, B, and C were 11, 11, and 15°C 

320-t 

.c 

> 

O 

03 OAnrr 

240-

o 

00 

0- 160-

c 

I 80H 

(0 

Q 

I 

< 

_l 

< „ 

ALA-D = 320-102log1 0WL 

10 WL 

log1 0WL 

1 — I I I I I | 

1 0 

I I I I I I 

100 

Lead Concentration in Water (u.g/L) 

FIG. 2,Erythrocyte 8-amino levulinic acid dehydratase activity (ALA-D) of rainbow trout exposed to water-borne 

lead (WL). Line B is an unpublished analysis from Hodson 

et al. (1978a). *The slope of line D was significantly 

different from that of line A but not from that of line B 

at the 95% confidence level. 

320- 1 

JO 

O 

CO 

QC 240-

o 

co 

Q. 160 -

c 

'_> 

o 

TO 

a 

i 

< 

8 0 -

ALA-D = 547- 128 log10 BL 

ALA-D= 590-146 log10 BL 

BL 

T | 

100 

I I I I I | — 

1000 

Lead Concentration in Blood (u.g/L) 

FIG. 3. The relationship between erythrocyte 8-amino 

levulinic acid dehydratase activity (ALA-D) and blood lead 

(BL) for rainbow trout exposed to water-borne lead. Line 

B is an unpublished analysis from Hodson et al. (1978a). 

*There were no statistically significant differences between 

the slopes of these lines at the 95% confidence level. 

respectively. 

These results are supported by the relationship 

between ALA-D activity and waterborne lead. The 

negative slope relating ALA-D to waterborne lead 
88 HODSON et al. 

TABLE 3. Statistics describing the regression lines shown in 

Figures 1, 2 and 3. 

Figure 

1 

2 

3 

Line 

A 

B 

C 

D 

A 

B 

D 

A 

B 

D 

s

 a *y.x 

0.2350 

0.1817 

0.1017 

0.2651 

33 

41 

46 

37 

70 

54 

%

b 

0.0838 

0.0612 

0.0388 

0.1703 

13.4 

13.8 

29.4 

12.1 

15.7 

14.4 

r

c 

0.84 

0.78 

0.97 

0.86 

-0.85 

-0.82 

-0.83 

. -0.85 

-0.58 

-0.83 

a

Standard deviation of the regression of y on x 

"Standard deviation of the slope 

Correlation coefficient 

is similar in two previous experiments (lines A and 

B, Figure 2) starting with fingerlings. In the present experiment starting with eggs, the slope is 

again greater (line D, Figure 2), indicating a greater 

than expected lead uptake. The consistency of 

response shown by lines A, B, and C in Figure 1 

and 2, developed with different lots of fish, suggests that the difference shown by line D is experimental and not due to random fluctuation. 

Therefore, Figures 1 and 2 support the first 

hypothesis that the rates of uptake or excretion of 

lead vary between fish exposed as fingerlings and 

fish exposed as eggs. The net result is an increase in 

the apparent premeability of lead by fish exposed 

from the egg stage. 

In contrast, the enzymatic response to lead 

taken up (ALA-D inhibition) indicates that this 

dose-response relationship remains unchanged. 

Figure 3 illustrates that the slopes of lines relating 

ALA-D activity to blood lead are the same for fish 

exposed only as fingerhngs and fish exposed from 

the egg stage; ie., lead taken up exerts the same 

effect in both groups. 

Therefore, increased sensitivity to lead in rainbow trout exposed as eggs appears to be a function 

of increased lead accumulation rather than increased toxicity of the lead taken up. This change in 

lead accumulation may represent an alteration of 

gill membrane structures across which lead is taken 

up, or an alteration of the rate of some depuration 

mechanism. Since blood lead is in equilibrium with 

lead in water within 48 h (Hodson et al. 1978b), 

it is not possible to distinguish between the two 

mechanisms without further uptake-depuration 

studies. The difference in permeability to lead 

between fingerling-exposed and egg-exposed fish" 

is probably long lasting since the half-time for 

depuration from blood is about 4 weeks (Hodson 

et al 1977). Consequently if permeability was 

affected only temporarily in very young fish^ 

blood lead would be the same as that of fisf 

exposed as fingerhngs by 30 weeks post-hatch. 

These results agree well with, those predicte" 

for Lake Ontario water from Davies et al. (197 $ 

A linear interpolation of lead toxicity, based on 

total lead, against water hardness, indicates that IS 

Mg/L lead should cause no development of black 

tails while about 25 Mg/L should. This experiment 

demonstrated that 22 Mg/L caused 5.5% black tails 

while 11 Mg/L caused none. Recent studies in our 

lab suggest that organic particulates in water may 

reduce lead accumulation as does increased pH 

(Hodson et al 1978b). Consequently predictions 

of the variation of lead toxicity with hardness wiD 

be most valid where pH and particulates are constant. ' '% 

Davies et al. (1976) measured total lead in a 

settled water sample by atomic absorption spectrophotometry and "free" lead by pulse polarography. They felt that "free" lead more closely 

described lead toxicity; but they did not specify 

the form of lead included in the measurement 

However, monitoring "free" lead by pulse polan* 

graphy is not yet practical on a surveillance basis 

(IJC 1976). The successful prediction of sublethal 

toxicity based on total lead indicates that total 

lead, after filtration to remove particulates, ma£ 

provide a practical basis for water pollution stand: 

ards. j§ 

In summary, four important results of this study 

are that sensitivity of fish to lead is related to the 

life stage exposed to lead, that increased sensitivity 

to lead results from a greater apparent permeability 

to lead rather than from an enhanced response to 

the lead taken up, that prediction of hardness 

effects on sublethal lead toxicity between me* 

sured extremes appears valid, and that measure; 

ments of total lead may provide a reasonable bask 

for predicting lead toxicity.

 i? 

ACKNOWLEDGMENTS 

The authors thank the Water QuaUty LaboratoO' 

Network, Inland Waters Directorate, Canada Centre for Inland Waters, for lead analyses of water 

and fish tissues. 4 
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.ndEFFECT OF FISH AGE ON LEAD TOXICITY 89 

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REFERENCES 

„ „ , - - H-, Goettl, J. P. Jr., Sinley, J. R., and Smith, N. 

c 1976. Acute and chronic toxicity of lead to rainbow 

i/out, Salmo gairdneri, in hard and soft water. Water 

Res. 10:199-206. 

Ilodson, P- V. 1979. A metering device for toxicants used 

in bi'oassays with aquatic organisms. Prog. Fish Cult. In 

press. 

, Blunt, B. R., Spry, D. J., and Austen, K. 1977. 

—Evaluation of erythrocyte 5-amino levulinic acid dehydratase activity as a short-term indicator in fish of a 

harmful exposure to lead./ Fish. Res. Board Can. 34: 

501-508. 

, Blunt, B. R., and Spry, D. J. 1978a. Chronic toxicity of water-borne and dietary lead to rainbow trout 

(Salmo gairdneri) in Lake Ontario water. Water Res. 

12:869-878. 

^ 1978b. pH-induced changes in blood 

lead of lead-exposed rainbow trout (Salmo gairdneri). 

J.Fish. Res. Board Can. 35:437445. 

Holcome, G. W., Benoit, D. A., Leonard, E. N., and McKim, 

J. M. 1976. Long-term effects of lead exposure on three 

generations of brook trout (Salvelinus fontinalis). J. 

Fish. Res. Board Can. 33:1731-1741. 

IJC. 1976. Toxicity to Biota of Metal Forms in Natural 

Water. Proceedings of a workshop held in Duluth, Minnesota, Oct. 7-8, 1976. Edited by R. W. Andrew, P. V. 

Hodson, and D. E. Konasewich. International Joint 

Commission, Windsor, Ontario. 

IWD. 1974. Analytical Methods Manual. Water Quality 

Branch, Inland Waters Directorate, Environment Canada, 

Ottawa, Canada. 

Mount, D. I., and Brungs, W. A. 1967. A simplified dosing 

apparatus for fish toxicology studies. Water Res. 1:21-

29: 

Sprague, J. B. 1969. Measurement of pollutant toxicity to 

fish. 1. Bioassay methods for acute toxicity. Water Res. 

3:793-821. 

Steel, R. G. D., and Torrie, J. H. 1960. Principles and procedures of statistics. McGraw-Hill, New York. 

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