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This is to certify that the
thesis entitled
The Impact of Naturally Weathered North
SIOpe Crude Oil on Reproduction and Survival
in Mallards
presented by

Gregg Allan Hancock

has been accepted towards fulfillment

of the requirements for
Natural

Science (“j-degree in Fisheries & Wildlife

 

 

131mm H. pm

Major professor

Date July 20, 1992

 

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THE IMPACT OF NATURALLY WEATHERED NORTH SLOPE CRUDE OIL
ON REPRODUCTION AND SURVIVAL IN MALLARDS

By
Gregg Allan Hancock

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife
1991

ABSTRACT

THE IMPACT OF NATURALLY WEA'I'HERED NORTH SLOPE CRUDE OIL
ON REPRODUCTION AND SURVIVAL IN MALLARDS

By
Gregg Allan Hancock

A series of toxicological tests were conducted to assess the impact of ingestion
of naturally weathered North Slope crude oil (WNSCO,) from the Exxon Valdez
accident, on survival and reproduction in mallards. The studies performed were a
LD50, a LC50, a food avoidance, a 14-day dietary feeding study, a 22-week one-
generation reproduction study and an eggshell application study. N o outward signs of
toxicity were noted in any of the birds in any study. The LD50 was > 5,000 mg
WNSCO / kg body weight, the L050 was > 50,000 ppm in the diet and the median food
avoidance concentration (FAC50) was > 20,000 ppm in the diet. The only effects noted
in birds fed up to 100,000 ppm WNSCO in the diet in the 14-day dietary feeding study
were significantly (P _<_ 0.05) increased livers in males when normalized on the basis
of body and brain weights. Hens in the high dose (20,000 ppm) group in the
reproduction study laid eggs with significantly reduced (P _<_ 0.05) shell thickness and
strength. Application of WNSCO to the eggshell showed no toxicity related affects on
developing embryos. WNSCO does not appear to be a highly toxic compound and
outward physical contact should be of more concern than ingestion.

ACKNOWLEDGEMENTS

I would like to thank Dr. Harold Prince, Dr. Robert Ringer and Dr. Scott
Winterstein for serving on my committee. Thanks especially to Dr. Prince for serving
as my major professor and giving me guidance and advise and to Dr. Ringer for all the
extra time he took to help me on the project.

I would like to especially thank Cathy Cook for all her fantastic help on my
project as well as in my classes. Her encouragement and guidance were above and
beyond the call of duty and I am deeply in her debt.

Thanks also go to Sara Thompson, whose hard work and dedication was greatly
appreciated and will not be forgotten.

Finally, I would like to thank my wife Tammi for putting up with me over the
last 3 years. I know it has not been easy but I hope it has been worth it. Thank you

from the bottom of my heart and remember that I love you more than anything.

iii

TABLE OF CONTENTS

LIST OF TABLES ................................................. v
INTRODUCTION ................................................. 1
MATERIALS AND METHODS ....................................... 2
Acute Oral LD50 ............................................ 2
Subacute Dietary LC50 ....................................... 3
Food Avoidance ............................................. 4
14-day Dietary Feeding ....................................... 5
Reproduction Study .......................................... 6
Eggshell Application Study .................................... 9
RESULTS AND DISCUSSION ...................................... 10
Acute Oral LD50 ........................................... 10
Subacute Dietary LC50 ....................................... 12
Food Avoidance ............................................ 14
14-day Dietary Feeding ...................................... 14
Reproduction Study ......................................... 17
Eggshell Application Study ................................... 31
CONCLUSION .................................................. 35
APPENDIX ..................................................... 39
LIST OF REFERENCES .......................................... 79

iv

LIST OF TABLES

Table 1. Survival, feed consumption and body weight (if 1 S. E. ) m male and
female mallards orally dosed with WNSCO. ............................ 11

Table 2. Survival, feed consumption and growth rate (i 1 SE.) in mallard
ducklings fed WNSCO in the diet for 5 days. ........................... 13

Table 3. Mean daily feed consumption and growth rate (R 1 SE.) of mallard
ducklings given a choice of food treated with WNSCO and untreated feed in the
diet for 5 days. ................................................... 15

Table 4. Mean daily feed consumption, body weight and organ parameters (32
1 SE.) in adult mallards fed WNSCO in the diet for 14 days ................ 16

Table 5. Mean daily feed consumption and body weights (i 1 SE.) in mallards
fed WNSCO in the diet for 22 weeks. ................................. 18

Table 6. Egg production, percent fertility, percent hatchability and percent
survival of ducklings from eggs laid by female mallards fed WNSCO in the diet

for 22 weeks. .................................................... 23
Table 7. Mean eggshell strength and thickness (3? 1 SE.) in eggs laid by hen
mallards fed WNSCO in the diet for 22 weeks. .......................... 25
Table 8. Mean initial body weight and growth rate (R 1 SE.) of hatchlings of
hen mallards fed WNSCO in the diet for 22 weeks. ...................... 26
Table 9. Mean organ weights (g) in mallards fed WNSCO in the diet for 22
weeks in a reproduction study (1': 1 SE.) ............................... 28
Table 10. Mean clinical blood parameters (3? 1 SE.) in adult mallards fed
WNSCO in the diet for 22 weeks. .................................... 29

Table 11. Number hatched, number 14-day survivors and hatchling growth rate
(g/bird/ day) from mallard eggs externally treated with various amounts of
WNS CO

....................................................... 32
Table 12. Mortalities observed in adult mallards orally dosed with weathered
North Slope crude oil. ............................................. 39
Table 13. Body weights (g) of adult mallards orally dosed with weathered
North Slope crude oil (mean 1 SE). ................................. 40
Table 14. Food consumption of adult mallards orally dosed with weathered
North Slope crude oil (mean 1 SE.) ................................... 41

V

Table 15. Mortalities observed in mallard ducklings fed weathered North Slope

crude oil in the diet for 5 days. ...................................... 42
Table 16. Food consumption of mallard ducklings fed weathered North Slope
crude oil in the diet for 5 days (mean 1 SE.) ............................ 43
Table 17. Body weights (g) of mallard ducklings fed weathered North Slope
crude oil in the diet for 5 days (mean 1 SE.) ............................ 44

Table 18. Mortalities observed in mallard ducklings given a choice of food
treated with weathered North Slope crude oil and untreated food in the diet
for 5 days. ...................................................... 45

Table 19. Feed consumption (g) in mallard ducklings given a choice between
feed treated with weathered North Slope crude oil and untreated feed in the
diet for 5 days (mean 1 SE.) ........................................ 46

Table 20. Body weights (g) of mallard ducklings given the choice of feed
treated with weathered North Slope crude oil or untreated feed in the diet for

5 days (mean SE.) ................................................ 47
Table 21. Mortalities observed in adult mallards fed weathered North Slope
crude oil in the diet for 14 days. ..................................... 48
Table 22. Mean daily feed consumption (g) in adult mallards fed weathered
North Slope crude oil in the diet for 14 days (mean 1 SE.) ................. 49
Table 23. Body weights (g) of adult mallards fed weathered North Slope crude
oil in the diet for 14 days (mean 1 SE.) ................................ 50
Table 24. Hematology results of adult mallards fed weathered North Slope
crude oil in the diet for 14 days (mean 1 SE.) ........................... 51
Table 25. Clinical chemistry results of adult mallards fed weathered North
Slope crude oil in the diet for 14 days (mean 1 SE.) ...................... 53
Table 26. Mortalities observed in adult mallards fed weathered North Slope
crude oil in the diet for 22 weeks. .................................... 55

Table 27. Mean feed consumption (g/b/d‘) by adult mallards fed weathered
North Slope crude oil in the diet for 22 weeks (mean 1 SE.) ............... 56

Table 28. Body weights (g) by sex of adult mallards fed weathered North Slope
crude oil in the diet for 22 weeks (mean 1 SE.) ......................... 57

Table 29. Organzbrain weight ratios of adult female mallards fed weathered
North Slope crude oil in the diet for 22 weeks (mean 1 SE.) ............... 58

Table 30. Organzbrain weight ratios 'of adult male mallards fed weathered
North Slope crude oil in the diet for 22 weeks (mean 1 SE.) ............... 59

Table 31. Organ:body weight ratios of adult female mallards fed weathered

North Slope crude oil in the diet for 22 weeks (mean 1 SE.) ............... 60
Table 32. Organ:body weight ratios of adult male mallards fed weathered North
Slope crude oil in the diet for 22 weeks (mean 1 SE.) ..................... 61
Table 33. Clinical chemistry results. of adult female mallards fed weathered
North Slope crude oil in the diet for 22 weeks (mean 1 SE.) ............... 62
Table 34. Clinical chemistry results of adult male mallards fed weathered
North Slope crude oil in the diet for 22 weeks (mean 1 SE.) ............... 65
Table 35. Hematology results of adult female mallards fed weathered North
Slope crude oil in the diet for 22 weeks (mean 1 SE.) ..................... 68
Table 36. Hematology results of adult male mallards fed weathered North
Slope crude oil in the diet for 22 weeks (mean 1 SE.) ..................... 70

Table 37. Reproductive data from mallard hens fed weathered North Slope
crude oil in the diet for 22 weeks. .................................... 72

Table 38. Reproductive data from mallard hens fed weathered North Slope
crude oil in the diet for 22 weeks - eggs set. ............................ 73

Table 39. Reproductive data from female mallards fed weathered North Slope
crude oil in the diet for 22 weeks - number hatched. ..................... 74

Table 40. Mean eggshell strength, in Newtons (N), of eggs laid by female
mallarS E ds fed weathered North Slope crude oil in the diet for 22 weeks (mean
1 . .). ........................................................ 75

Table 41. Mean eggshell thickness (mm) of eggs laid by female mallards fed
weathered North Slope crude oil in the diet for 22 weeks (mean 1
SE.) ........................................................... 76

Table 42. Mean initial and 14-day survivor body weights of mallard hatchlings
of hen fed weathered North Slope crude oil in the diet for 22 weeks (mean 1

SE.) ........................................................... 77
Table 43. Mortalities observed in hatchlings of mallard hens fed weathered
North Slope crude oil in the diet for 22 weeks. ......................... 78

vii

INTRODUCTION

Although seepage of petroleum through parts of the ocean floor has occurred
for thousands of years, an ever increasing consumption of petroleum products
worldwide since World War II has resulted in discharge of large quantities of
petroleum into the environment (Bourne 1968, Prince 1983). Oil pollution is the
inevitable consequence of dependence on a largely oil-based technology.
Approximately 1012 g of oil is released into the oceans each year from shipping alone.
Petroleum pollution has been implicated as a significant factor in the demise of some
seabird populations during the 20th century (Holmes 1984).

Oil spills pose a threat to many forms of aquatic wildlife, especially birds.
Bourne (1968) states that oil pollution affects birds sooner and more lethally than any
other form of wildlife. Direct physical contact with oil can soil the feathers resulting
in loss of insulation and buoyancy, thereby causing mortality. Ingestion of
contaminated food and water, as well as from preening oil covered feathers has been
shown to affect metabolic rates, reproductive success, and survivability under stress.
Incubating adults that have been exposed to oil may transfer it to the egg shells which
can result in reduced hatchability from embryo mortality due to toxicity or from
suffocation due to blockage of pores.

Shortly after midnight on 24 March 1989 the oil tanker Exxon Valdez, carrying
53 million gallons of crude oil, ran aground on Bligh Reef in Prince William Sound in
the Gulf of Alaska. Within 8 hours of the accident an estimated 10 million gallons of
Alaskan North Slope crude oil had spilled into the Sound and the slick measured 1000
feet wide by 4 miles long. Within 24 hours of the accident efforts were underway to
pump the remaining 43 million gallons of crude oil off the Valdez. Equipment
shortages, logistic problems and weather conditions confounded containment and

2

clean-up. The spill was not contained, quickly spreading and soiling hundreds of miles
of shore in and around the Sound. The latitude and currents in the semi-enclosed
Prince William Sound accentuated the severity, resulting in reduced rates of
weathering and biodegradation. Spring migration and large numbers of waterbirds and
shorebirds would potentially be exposed to the spilled oil since the spill preceded
spring migration. The initial impact of the oil on wildlife was directly observable on
waterbirds and sea otters (Enhydra lutris). Concern was expressed over the effects of
the spill on the fisheries of the area, so the herring and roe fishery was closed that
year.

Approximately three months (1 July 1989) after the spill occurred, weathered
oil lost from the Exxon Valdez was recovered by a skimmer for a series of tests on its
relative toxicity.

This project was undertaken to evaluate the potential of impact of naturally
weathered North Slope crude oil (WNSCO) on survival and reproduction of waterbirds
using the mallard (Anas platyrhyncos) as the test subject. All studies were conducted

under Good Laboratory Practices guidelines.

MATERIALS AND METHODS

Acute Oral LD50

The acute toxicity test (LD50) is designed to determine the single oral dose level
which kills fifty percent of the treatment population. Prior to test initiation, food was
withheld from all test groups for 17 hours. Weathered North Slope crude oil was
administered as a single oral gavage in gelatin capsules. Preliminary range-finding
studies indicated no mortalities through 5000 mg/ kg body weight dosages. Treatments
included a male and female treatment group (5000 mg/ kg body weight) and a male and
female control group (0 mg/kg body wt.). Doses were administered in 5 capsules per

3

bird and control birds received empty capsules.

Adult mallards were purchased from a commercial source (Whistling Wings,
Hanover, IL). Birds were housed in 70 x 68 x 28 cm (L x W x H) galvanized steel cages
(2 males or 2 females per cage) and photoperiod was set at 8:16 hr light:dark. Test
subjects were randomly assigned to treatments. Birds were dosed following a 2 week
acclimation period. Food (duck maintenance) and water were provided ad libitum.
Food consumption, mortality and clinical signs of toxicity were recorded daily
throughout a 14-day post-treatment observation period. Body weights were taken at
initiation, day 7 and termination of the study. Gross necropsies were performed on
all birds at study termination.

Body weights and food consumption were analyzed by t-test using TOXSTAT
Version 3.2 software (Gulley et al. 1990).

Subacute Dietary LC50

The dietary concentration of weathered crude oil at which fifty percent of the
treatment population dies (LC50) was evaluated. Preliminary studies indicated no
mortalities when ducklings were fed up to 20,000 ppm WNSCO mixed in the diet.
Treatments included a control group given untreated food and a high dose group
exposed to food containing 20,000 ppm WNSCO in the diet, consisting of 20 and 10
birds, respectively. The control group in this study was fed a diet with 2% mineral oil.
A supplemental test was also conducted consisting of a new control group given
untreated feed and one dose group given feed containing 50,000 ppm WNSCO, each
containing 12 birds.

One-day old ducklings were purchased from a commercial source (Whistling
Wings, Hanover, IL). Ducklings were housed in an animal room with a 16:8 hr
light:dark photoperiod in 91 X 71 x 23 (Lx W x H) cm galvanized steel brooders with

4

a temperature gradient from approximately 36°C at the rear to room temperature (26
to 29°C) at the front of the broader. Ducklings were acclimated to duck starter ration
and cages for three days prior to test initiation and were 5 days old at test initiation.
The appropriate dietary concentrations were presented for 5 days after acclimation.
A test material-free diet was presented for 3 additional days after the 5 day test, prior
to sacrifice. Body weights were recorded at test initiation, day 5 and at termination
(day 8). Food consumption, mortality and clinical signs of toxicity were recorded daily
throughout the 8 day test period. Food and water were provided ad libitum.

Body weights, mean growth and food consumption were analyzed by t-test using

TOXSTAT Version 3.2 software (Gulley et al. 1990).

Food Avoidance

The purpose of this experiment was to determine if mallard ducklings avoided
feed containing various concentrations of WNSCO. Treatment levels were set at 0,
1,250, 2,500, 5,000, 10,000 and 20,000 ppm WNSCO in the diet. Mineral oil was used
as a carrier in the treatments. Each test group was given the choice of untreated food
and food treated with WNSCO. Feed hoppers were rotated daily to adjust for position
effect Kononen et al (1986). Food and water was provided ad libitum.

One-day old ducklings were purchased from a commercial source (Whistling
Wings, Hanover, IL). Ducklings were housed in an animal room with a 16:8 hr.
light:dark photoperiod in 91 X 71 x 23 (L x W x H) cm galvanized steel brooders with
a temperature gradient from approximately 36°C at the rear to room temperature (26
to 29°C) at the front of the brooder. Ducklings were acclimated to duck starter ration
and cages for three days prior to test initiation and were 5 days old at test initiation.
The appropriate dietary concentrations were presented for 5 days after acclimation.
A material-free diet was presented for 3 additional days after the 5 day test, prior to

5
sacrifice. Body weights were recorded at test initiation, day 5 and at termination (day
8). Food consumption, mortality and clinical signs of toxicity were recorded daily
throughout the 8 day test period.
Body weight, mean growth and food consumption were analyzed by one-way
AN OVA followed by Dunnett’s test or the Bonferroni t-test (for an unequal number
of replicates) using TOXSTAT Version 3.2 software (Gulley et al. 1990).

l4-day Dietary Feeding
This experiment evaluated the toxicity of weathered crude oil to adult mallards
during a 14-day exposure. These data were helpful in the determination of
experimental concentrations of WNSCO in the food given to mallards in an experiment
designed to evaluate the effect of WNSCO on egg production and survival of mallard
ducklings. Food (duck breeder) treated with 0, 10,000, 30,000 and 100,000 ppm
WNSCO was fed to 4 groups of 10 birds each (5 per sex per group). 'IVventy birds (5
per treatment level) were randomly chosen for evaluation of clinical blood parameters
at study initiation and termination. Parameters evaluated include: red cell count,
white cell count, differential count, total hemoglobin, mean cell volume, packed cell
volume, mean cell hemoglobin, mean cell hemoglobin content, reticulocyte count, AST
(aspartate aminotransferase), uric acid, total protein, albumin, CPK, ALT (alanine
aminotransferase), alkaline phosphatase, calcium, glucose, potassium, and sodium.
Upon termination of the study all surviving birds were euthanized by CO2 asphyxiation
and post mortem examinations were performed. Wet weights were taken for liver,
kidney, adrenal, spleen and brain. In addition to these tissues, lung, bone marrow, eye,
skin, salt gland, heart, skeletal muscle, gastrointestinal tract, reproductive organs and
all gross lesions were taken for histopathological evaluation.
Adult mallards were purchased from a commercial source (Whistling Wings,

6

Hanover, IL) and housed in an animal room with an 8: 16 hour light:dark cycle in 70
x 68 x 28 cm (L x W x H) galvanized steel cages containing 1 male and 1 female. There
was a 2 week acclimation period prior to test administration. Food and water were
provided ad libitum. Food consumption was recorded every other day and body
weights were recorded at initiation, day 7 and termination of the study. Mortality and
clinical signs were recorded daily during the 14-day exposure period. Photoperiod was
set at 8: 16 hours light:dark.

Body weight, organ weight, organ:brain weight ratios, organ:body weight ratios,
food consumption, and blood chemistry parameters and hematology were analyzed by
one-way analysis of variance followed by Dunnett’s test or a Bonferroni t-test. Data
that did not meet assumptions of homogeneity of variance or normality were
transformed and analyzed as above. Data that did not meet these assumptions
through transformation were analyzed by the Kruskal-Wallis test followed by Dunn’s
multiple comparison. All analyses were done using TOXSTAT Version 3.2 software
(Culley et al. 1990).

Reproduction Study

This study assessed the impact of an extended dietary exposure of mallards on
egg production, fertility, oviposition, hatchability, eggshell thickness and strength, and
survivability of ducklings. Clinical blood parameters of adult birds were evaluated as
indicators of any physiological changes resulting from the oil consumption.

One-hundred twenty mallards at least 16-weeks old were obtained from a
commercial source (Whistling Wings, Hanover, IL) and housed in 70 x 68 x 28 cm (L
x W x H) galvanized steel cages. There was a 2 week acclimation period prior to test
initiation. Photoperiod was set at 7:17 hr. light:dark for the first eight weeks of the
study and shifted to 17:7 hr light:dark for the remainder of the study to sexually

7

stimulate the birds. Treatment levels of WNSCO in duck breeder-layer were set at 0,
200, 2,000 and 20,000 ppm based on the results from the 14-day dietary feeding study.
As up to 100,000 ppm was readily consumed in the 14 day study without apparent
adverse affects the upper limit for this study was set at 20,000 ppm as any higher
concentration might have resulted in caloric imbalances. Food and water were
provided ad libitum. Body weights were recorded at weeks 0, 2, 4, 6, 8 (prior to egg
production) and at termination of the study. Forty birds (10 per treatment level, 5 per
sex) were randomly chosen for evaluation of clinical blood parameters at study
initiation, weeks 5 and 10 (prior to egg production), and at study termination (at the
end of the ten week egg production period). Blood was drawn from the area of the
brachial vein. Parameters evaluated included: red cell count, white cell count,
differential count, total hemoglobin, mean cell volume, packed cell volume, mean cell
hemoglobin, mean cell hemoglobin content, reticulocyte count AST (aspartate
aminotransferase), uric acid, total protein, albumin, CPK, ALT (alanine
aminotransferase), alkaline phosphatase, calcium, glucose, potassium, sodium and total
bilirubin.

When at least 50% of the hens in the control group (0 ppm) had begun to lay
eggs, the 10 week period of egg production was begun. Eggs were collected daily and
stored at 50°F until set. All eggs were inspected for cracks prior to incubation. Eggs
were set in a Petersime incubator at 37.5°C and 60 to 70% humidity. Eggs were
misted with distilled water daily and candled on days 14 and 21 of incubation. Eggs
were transferred from the incubator to a Petersime hatcher, set at 37 .5°C and 70 to
80% humidity, on day 24 of incubation.

Eggshell thickness and strength measurements were made on eggs laid the first
day of weeks 1, 3, 5, 7 and 9 of the experiment. Eggshell strength was measured on
an Instron Testing Machine (Model No. 4202). Force was applied to the egg until the

8
shell fractured. Egg shell thickness (including the shell membrane) was then

measured with a micrometer at 4 points around the circumference of the shell after
the contents of the shell had been rinsed out with tap water and the shell dried for
at least 24 hours at room temperature.

Ducklings were housed in 91 x 71 x 23 (L x W x H) cm galvanized steel brooders
(Petersime Brood Unit, Model No. 2.S.D.). with a temperature gradient from
approximately 36 to 22°C for 14 days. Photoperiod was set on a 14:10 hour light:dark
cycle. Food (duck starter) and water were provided ad libitum and health checks were
made daily. At the end of the two week observation period all surviving ducklings
were euthanized by C02 asphyxiation.

All surviving adult birds were euthanized by CO2 asphyxiation upon termination
of the study and post mortem examinations were performed. Wet weights were taken
for liver, kidney, adrenal, spleen and brain. In addition to these tissues lung, bone
marrow, eye, skin, salt gland, heart, skeletal muscle, gastrointestinal tract,
reproductive organs and all gross lesions were taken and stored in 10% buffered
formalin for histopathological evaluation.

Body weight, organ weight, organ:brain/body weight ratios, food consumption,
egg shell strength and thickness, and blood clinical chemistry and hematology
parameters and reproductive parameters were analyzed using the one-way analysis of
variance procedure followed by a Dunnett’s test or a Bonferroni t-test to test for
significant differences between treatments and control. Data that did not meet
assumptions of homogeneity of variance or normality were transformed and analyzed
as above. Data that did not meet these assumptions through transformation were
analyzed by the Kruskal-Wallis test followed by Dunn’s multiple comparison. All
analyses were done using TOXSTAT Version 3.2 software (Gulley et al. 1990). Analysis

of percentage of hens laying eggs was conducted using the chi-square test.

Eggshell Application Study

WNSCO was applied externally to mallard egg shells and its effects on embryo
mortality and duckling survival were assessed. Freshly laid eggs were ordered from
a commercial supplier (Whistling Wings, Hanover, IL) and inspected for cracks upon
receipt. Each egg was divided into six approximately equal application zones by using
a template and marking with a lead pencil (the air sac was not included) and set in a
Petersime incubator at 37 .5° C and 70-80% humidity. Treatment with petrolatum or
WNSCO occurred on day 7 of incubation. All dead and infertile embryos, as
determined by candling, were removed at this time. Petrolatum was used as a positive
control to test for gas exchange effect. Preliminary experiments using petrolatum as
a pore blocker demonstrated that covering more than 1/3 of the egg resulted in almost
total mortality of embryos. Based on these results no more than 1 / 3 of the egg was
covered with weathered crude oil in this experiment. There were 4 treatment levels
of petrolatum or WNSCO: coverage of the top 1/6 of the egg; the top 1/3; the middle
1/6; and the middle 1/3. The crude oil was a thick, brown mousse in consistency and
appearance so it was applied with a watercolor paintbrush by covering the appropriate
zones. Eggs were weighed prior to and after treatment to determine dose amount.
Eggs were not candled during the course of the incubation as the oil made the eggs
slippery and the possibility of spreading the oil to larger portions of the egg.

Ducklings were housed in 91 x 71 x 23 (L x W x H) cm galvanized steel brooders
(Petersime Brood Unit, Model No. 2.S.D.). with a temperature gradient from
approximately 36 to 22°C for 14 days to evaluate survivability. Food (duck starter) and
water were provided ad libitum.

Initial duckling body weight and 14-day growth rate were analyzed by one-way
AN OVA followed by Dunnett’s test or the Bonferonni t-test (Gulley et al. 1990).

Analysis of hatching success was conducted using the chi-square test.

RESULTS AND DISCUSSION
Acute Oral LD50

No mortalities or signs of toxicity were observed in any of the test groups
(Table 1). Boersma et al. (1988) found no effect on gross morphology or immediate
survival in fork-tailed storm-petrels (Oceanodroma furcata) orally dosed with artificially
weathered Prudhoe Bay crude oil (PBCO). A single dose of artificially weathered
South Louisiana crude oil (SLCO) resulted in reduced survival in herring gull (Larus
argentatus) chicks (Butler and Lukasiewicz 1979). No oil related mortalities were
reported in adult sandhill cranes (Grus canadensis) repeatedly dosed with raw PBCO,
however the high dose group was observed to be weaker and more lethargic as well as
maintaining a different standing and walking posture (Fleming et al. 1982). Using
single oral doses of crude oil on both young herring gulls and adult black guillemots
(Cepphus grylle), other researchers report no significant mortalities (Peakall et a1.
1980, Peakall et al. 1982, Peakall et al. 1983, Peakall et al. 1985). Peakall et a1. (1980)
used weathered SLCO. Even multiple doses of up to 20 mg crude oil/kg
bodyweight/ day for as long as to 6 days does not significantly increase mortality in
nestling herring gulls and Atlantic puffins (Leighton et al. 1983, Lee et a1. 1985,
Leighton 1985, Leighton et al. 1985).

Male mallards consumed an average of 34 g more food than females which is
consistent with the 13% differential in weight between sexes (Table 1). A significant
reduction of 18 g (17%) (P g 0.05) was noted for males consuming treated food. This
pattern was not observed in mean daily food consumption of females. Engel et al.
(1978) noted reduced food intake on day 1 following dosing in Japanese quail orally
dosed with PBCO, but consumption returned to normal the following day.

10

11

Table 1. Survival, feed consumption and body weight (if 1 SE.) in male and female

mallards orally dosed with WNSCO.

 

 

 

 

 

 

Treatments (mg/kg)
Male Female

Parameter 0 5000 0 5000
Survival

Lived 6 6 6 6

Died 0 0 O 0
Feed
Consumption
(g/b/d)‘ 127 1 4.0 109:: 5.6 93 1 3.5 94 1 2.3
Body weight (g)

Initial 1123 1 44 1166 1 30 994 1 22 1008 1 34

Terminal 1166 1 30 1224 1 33 1059 1 40 1069 1 26

 

‘'Significantly different from male control (P _<_ 0.05)
'g/b/d = grams per bird per day

12

Postmortem examination of all ducks at study termination showed no
compound-related lesions. The single dose acute LD50 of mallard ducks orally dosed
with WNSCO is greater than 5,000 mg/kg.

Subacute Dietary LC50

Consumption of duck starter treated with 20,000 and 50,000 ppm WNSCO in
the diet resulted in no compound-related mortalities or grossly observable signs of
toxicity in ducklings during the course of the study (Table 2). Some previous studies
report no significant treatment related mortalities in young birds fed crude oil in the
diet (Szaro et al. 1978b, Eastin and Murray 1981, Eastin and Rattner 1982). Rattner
and Eastin (1981) reported 2 mortalities in mallard ducklings fed 1.5% PBCO, but this
was not statistically significant. Pullet chicks fed 2, 4, and 6% artificially weathered
Nigerian light crude oil in the diet for 42 days exhibited significant treatment related
mortality (Nwokolo and Ohale 1986). Up to 5% weathered crude oil in our tests
showed no observable effects on the ducklings.

Ducklings between the ages of 5 to 10 days ate 21.0 to 23.9 g of food per day.
Mean food consumption between treatment and control groups was not significant (P
S 0.05). The mean daily growth rates ranged from 15.1 grams per bird in the 50,000
ppm treatment group to 16.3 grams per bird in the control group. Postmortem
examination of all ducklings at study termination showed no compound-related effects.
Eastin and Murray (1981) fed mallard ducklings 2.5% Prudhoe Bay crude oil and found
no significant difference in mean body weights or growth. Mallard ducklings fed SLCO
up to 5% from hatching to 8 weeks showed a significantly depressed growth rate

compared to controls (Szaro et al. 1978b).

13

Table 2. Survival, feed consumption and growth rate (7: 1 SE) in mallard ducklings fed

 

 

 

 

WNSCO in the diet for 5 days.
Treatment (ppm)
Parameter 0 20,000 0 50,000
Survival
Lived 20 10 12 12
Died 0 0 0 0
Mean Feed
Consumption“
(g/bird/day) 21.8 1 0.9 23.9 1 1.5 22.7 1 1.0 21.0 1 0.8
Growthb rate
(g/bird/day) 16.3 1 0.8 16.0 1 1.2 15.8 1 1.3 15.1 1 1.1
ll“Day 0 to day 5

I’Day 0 to day 8

14
Food Avoidance

Ducklings that were given a free choice of food treated with up to 20,000 ppm
WNSCO versus non-treated food did not display a preference (Table 3). Food
consumption averaged from 24.1 to 27.3 grams per bird per day. Postmortem
examination of all ducklings at study termination showed no compound-related effects.
Nwokala and Ohale (1986) noted a significant decrease in food consumption for pullet
chicks fed 2, 3 and 6% artificially weathered crude oil in thediet. They found that the
contaminated feed was initially rejected and feel that the degree of rejection was
related to the level of contamination, but by the end of the second week their
consumption was back to normal. Eastin and Murray (1981) noted an initial drop in
weight of mallard ducklings fed 2.5% PBCO but thereafter weight gain was similar to
controls. They felt this probably reflected food avoidance.

The average mean daily growth rate of 16.9 grams per duckling in this
experiment was similar to the growth rates of 15.8 grams per duckling found in the
LC50 experiment where there was no choice of food and treatment concentrations were
as high or higher. N 0 significant differences (P > 0.05) in growth were noted between

any of the dietary exposure groups.

l4-day Dietary Feeding

Mallards consumed an average of 100 grams of food per bird per day treated
with as much as 10,000 ppm WNSCO (Table 4). Mean food consumption of test group
and control mallards was not significantly different (P > 0.05). Consumption of
WNSCO treated food resulted in no significant changes (P > 0.05) in body weight
compared with controls.

Consumption of food treated with WNSCO resulted in no significant change in

organ weight parameters compared with controls (P > 0.05) with the exception of high

15

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16

Table 4. Mean daily feed consumption, body weight and organ parameters (2 1 SE.) in adult
mallards fed WNSCO in the diet for 14 days.

 

Treatments (ppm)

 

 

Parameter 0 10,000 30,000 100,000
Feed Consumption
(g/bird/day) 100.3 1 5.9 103.2 1 6.1 98.8 1 5.7 97.5 1 5.6
Body Weight (8)
Male: initial 1227 1 45 1208 1 61 1265 1 65 1210 1 61
terminal 1247 1 69 1219 1 70 1278 1 73 1204 1 67
Female: initial 1101 1 51 1091 1 23 1107 1 67 1096 1 52
terminal 1120 1 51 1105 1 35 1105 1 70 1096 1 57
Organ Weights (3)
Liver
male 27.0 1 2.5 30.3 1 2.2 30.4 1 2.8 34.7 1 1.7
female 27.4 1 2.7 28.5 1 0.7 30.5 1 2.6 31.9 1 0.9
Kidney
male 9.5 1 1.1 9.4 1 0.6 9.6 1 1.0 9.6 1 0.7
female 8.5 1 0.8 9.1 1 0.2 9.0 1 0.7 9.1 1 0.9
Spleen
male 0.64 1 0.07 0.75 1 0.13 0.67 1 0.08 0.55 1 0.07
female 0.88 1 0.17 0.86 1 0.07 0.73 1 0.12 0.58 1 0.03
Adrenal
male 0.17 1 0.02 0.20 1 0.02 0.19 1 0.05 0.21 1 0.02
female 0.14 1 0.02 0.11 1 0.02 0.21 1 0.01 0.17 1 0.03
Brain
male 5.1 1 0.2 5.1 1 0.1 4.8 1 0.2 4.8 1 0.1
female 4.7 1 0.1 4.7 1 0.1 4.7 1 0.1 4.5 1 0.2
Liver:Brain Weight
(male) 5.4 1 0.6 5.9 1 0.5 6.3 1 0.4 7.3' 1 0.4
Liver:Body Weight
(male) 0.022 1 0.001 0.025 1 0.002 0.024 1 0.002 0.029' 1 0.002

 

‘Significantly difi’erent from control (P _<_ 0.05)

17
dose male liver weights normalized on the basis of either brain or body weights. Male
liver weights normalized on brain weight and body weight were 35 and 32% larger,
respectively, in the high dose group when compared with control birds.

Liver enlargement can be an indicator of increased function associated with
detoxification (Szaro 1977). Consumption of crude oil has been shown to result in an
increase in liver size (Miller et al. 1977, Patton and Dieter 1980, Gorsline et al. 1981).
The ability of birds to withstand oil pollution is thought to lie in the ability of the liver
to metabolize the hydrocarbons that are ingested. The mixed function oxidase (MFO)
system is the primary means by which the liver metabolizes these hydrocarbons
(Gorsline et al. 1981). Gorsline et al. (1981) found an increase in specific and total
liver microsomal enzyme activity in mallards fed five different crude oils. Gorsline and
Holmes (1982a) attempted to determine if crude all could in turn induce the MFO
system in the ducklings of hens consuming. crude oil. They found more than a
two-fold increase in specific liver activity of ducklings hatched from hens consuming
1 and 3% SLCO and 3% PBCO.

Control and high dose tissues were examined histopathologically. Due to the
lack of consistent and significant difference in the appearance of these tissues the
treatments in between were not examined. The only differences noted were that
spleens showed a small increase in the amount of white pulp in the birds from the

high dose group. The high dose livers had lower numbers of focal accumulations of

lymphocytes.

Reproduction Study

The 22 week experimental period included a 12 week prelaying and a 10 week
laying period. Prior to the breeding period, food consumption averaged about 97 grams
per bird per day (Table 5). During the period of egg production, food consumption

Table 5. Mean daily feed consumption and body weights (i 1 SE.) in mallards fed WNSCO in

18

 

 

 

the diet for 22 weeks.
Treatment (ppm)

Parameter 0 200 2,000 20,000
Feed Consumption
(ab/d) ,
Pre-breeding 99.3 1 2.6 101.4 1 2.4 95.2 1 2.1 93.2 1 2.0
Breeding 177.3 1 11.5 153.2 1 7.0 169.7 1 9.6 166.9 1 12.3
Adult Body Weight
(8)
Male :initial 1150 1 25 1177 1 26 1188 1 32 1203 1 26

:terminal 1228 1 28 1276 1 23 1233 1 24 1304 1 33
Female :initial 1007 1 24 1006 1 22 1046 1 22 1033 1 26

:terminal 1250 1 21 1254 1 45 1251 1 46 1200 1 17

 

19
increased markedly in all groups, averaging about 167 grams per bird per day. N 0
significant differences (P > 0.05) were noted between treatment groups and controls,
either before or during the breeding period.

The effect of crude oil on food consumption is a variable parameter in the
literature. Compared to control birds receiving no crude oil, there have been
increases, decreases and no difference in feed consumption reported. N 0 significant
differences in food consumption were noted in mallards consuming SLCO
contaminated food up to 5% of the diet (Szaro et al. 1978b, Gorsline and Holmes
1982b, Gorsline and Holmes 1982c, Vangilder and Peterle 1982). Adult mallards fed
1.5% PBCO for 7 days showed decreased food consumption the first day but not the
remainder of the time period (Rattner 1981). Domestic mallards fed North Sea crude
oil suffered reduced intake for the first 2 weeks but increased to control levels by the
third week (Harvey et al. 1982). This reduction was accompanied by a fall in body
weight which was not regained until the ninth week of oil ingestion. Most studies
note an increase in food consumption in controls after photoperiod was increased but
not in treatment birds. All treatment groups in our study showed increased food
consumption after photoperiod was increased.

Hyperphagia, increased food consumption normally in response to nutritional
deficit, appears to be somewhat common in birds fed crude oil contaminated feed
(Holmes et al. 1978b, Holmes et a1 1979, Gorsline and Holmes 1981, Gorsline et al.
1981). Mallards fed 1% and 3% SLCO in the diet for 50 days exhibited no significant
difference in food consumption during the first week, but by the end of the 50 day test
period food consumption was significantly higher in the treatment groups (Gorsline
and Holmes 1981). Harvey et al. (1982) feel that differences in the amount of food
eaten by ducks fed different types of crude oil appear to reflect differences in their

chemical composition. Of the studies discussed here, SLCO is the predominant crude

20

oil used and all studies where no significant change in feed consumption occurred used
this oil. However, in all three studies with significantly increased food consumption,
SLCO was used. PBCO resulted in reduced feed consumption in one study and
increased in another. These results may very well reflect the differences in
composition in crude oils. Crude oils from different locations are known to differ in
chemical composition, but Peakall et al. (1983) have shown that oils from the same
location collected at separate times can also differ. The oils used were South Louisiana
crude oils collected in 1976 and 1978. The batch from 197 6 caused significant effects
in parameters they were evaluating while the 1978 batch exhibited no such effects.

There is a sexual dimorphism in body weight between males and female
mallards. This is apparent in the initial body weight data where average male body
weight is 1180 grams while average female body weight is 1023 (Table 5). However,
during the breeding period the body weights of males and females are similar. Male
body weights have increased about 7% over initial body weight while females have
increased over 20% of initial body weight. Most of this is due to increased weight of
organs involved with reproduction. Females put more into the reproductive effort,
therefore they exhibit a greater increase in weight.

' A number of dietary feeding studies with adult mallards fed up to 3% SLCO,
KCO or PBCO in the diet report no significant difference in body weights for oil
treatment groups (Holmes et al. 1978a, Coon and Dieter 1981, Gorsline and Holmes
1981, Rattner 1981, Gorsline and Holmes 1982a, Gorsline and Holmes 1982b, Gorsline
and Holmes 1982c, Vangilder and Peterle 1982). Mallards fed SLCO in the diet for 26
weeks showed no significant reduction in body weight (Coon and Dieter 1981). Pattee
and Franson (1982) fed naturally weathered Ixtoc I crude oil to American kestrels and
noted that birds fed 3% crude in the diet lost significantly more weight than the

controls. In separate experiments, Vangilder (1981) noted a significant decrease in

21
body weight in mallard hens fed 2% SLCO compared to controls, but a significant

increase in hens fed 0.5% SLCO. When Gorsline et al. ( 1981) examined the effect of
50 days exposure of 5 different crude oils in mallards they found significant losses in
body weight in some groups, even in those that consumed significantly more food than
controls. Both 1 and 3% PBCO resulted in a significant loss in body weight by the
termination of the experiment when compared to initial body weight, while controls
did not.

The four experimental groups of mallards in this study laid nearly 3000 eggs
during the 70 day test period (Table 6). The rate of oviposition was similar in all
groups (P > 0.05). Fertility and hatchability averaged 97.4% and 83.8%, respectively,
and was similar in all groups (P > 0.05). Survivability of 14 day ducklings was very
similar in all treatment groups (P > 0.05).

Numerous investigators have reported various effects on these reproductive
parameters in birds ingesting crude oil. Ainley et al. (1981) force fed wild Cassin’s
auklets a single oral dose of 1000'mg PBCO in gelatin capsules and reported a marked
decrease in percentage of eggs laid from controls, but the eggs hatched normally.
Mallards fed SLCO at 0.25 and 2.5% in the diet for 26 weeks showed a significant
reduction in egg production, but fertility and hatchability were not affected (Coon and
Dieter 1981). Egg production in Japanese quail fed single oral doses of up to 800 mg
PBCO was significantly reduced on day 2 following dosing, but by day 4 was back to
normal (Engel et al. 1978). Hartung (1965) reported that 3 ducks fed 2g/kg crude oil
by oral intubation stopped laying immediately and didn’t resume for 2 weeks while
sham-dosed birds continued laying throughout that period. Vangilder (1981) found
that SLCO in the diet significantly reduces reproductive performance in mallards,
lowering egg production by 36.6%, but did not affect fertility. Egg production was
significantly reduced in mallards fed SLCO (V angilder and Peterle 1982). Vangilder

22

and Peterle (1983) noted that egg production, onset of laying and egg fertility were
unaffected by dietary ingestion of 0.5% SLCO, however, hatchability of SLCO hens was
significantly reduced compared to controls. Cavanaugh et al. (1983) determined that
mallards ingesting 3% SLCO took significantly longer to complete the reproductive
cycle than control birds, 71 days as opposed to 56 days for controls. Cavanaugh and
Holmes (1982, 1987) found that ovarian maturation was delayed significantly in
mallards ingesting 3% SLCO in the diet. In both instances, mean plasma estradiol
concentrations were significantly lower in contaminated birds after increase in
photoperiod. They believe that the crude oil in some way interferes with the
transmission of information from the photoreceptor to hormonally-regulated events.
Domestic mallards fed 5% North Sea crude oil in the diet exhibited a significantly
reduced oviposition rate and the onset of laying was delayed by at least 4 weeks
(Harvey et. al 1981, Harvey et al. 1982). Holmes et al. (1978a) found that mallards
consuming 1% SLCO or Kuwait crude oil (KCO) in the diet had no effect on
oviposition but at 3% SLCO caused significant inhibition of oviposition while 3% KCO
caused complete inhibition.

Survivorship to two weeks is a key measure of the number of young that will
be recruited into the population (V angilder and Peterle 1982). Survivorship of
hatchlings from treated birds in our study was not significantly different (P > 0.05)
from hatchlings of control birds (Table 6). Survival data in the literature deals mainly
with hatchling survival under stress conditions. Vangilder (1981) observed that
hatchlings of mallards fed 0.5% SLCO in the diet had a significantly reduced survival
time compared to controlswhen one-hour old hatchlings were placed at 20°C but the
same did not occur in a separate experiment with hatchlings of mallards fed 2% SLCO
in the diet. Survival in SLCO ducklings was significantly less under stress conditions
(V angilder and Peterle 1982).

23

Table 6. Egg production, percent fertility, percent hatchability and percent survival
of ducklings from eggs laid by female mallards fed WNSCO in the diet for 22 weeks.

 

 

 

Treatment (ppm)
Parameters 0“ 200° 2,000a 20,000”
Eggs laid (N) 730 711 778 616
Rate (eggs/hen/day) .695 .703 .741 .625
Fertility (%) 97.4 99.4 94.4 98.4
Hatchability (%) 84.5 82.2 84.6 84.0
14-Day Survivability (%) 98.7 97.9 ' 98.0 98.6

 

° Based on 15 hens
° Based on 14 hens

24

Eggshell thickness and strength are important measures of the effects of
environmental contaminants on reproductive performance in birds. The eggshell
provides a physical barrier that protects the embryo from desiccation and infection,
and this protection is compromised in the event of eggshell thinning. Both total
eggshell thickness and egg shell strength in the 20,000 ppm WNSCO treatment group
were significantly reduced (P _g 0.05) by approximately 8% when compared to the
control group (Table 7). Some investigators have recorded similar reductions in
eggshell thickness in birds exposed to crude oil while others have noted no effect.
Harvey et al. (1981) found significant eggshell thinning in eggs from domestic mallard
hens fed 5% North Slope crude oil in the diet. Refeeding uncontaminated diet to
these hens increased egg shell thickness, but not to control levels. Eggs from mallards
ingesting 2% SLCO had shells that were significantly thinner than control eggs
(Vangilder 1981). Holmes et al. (1978a) reported that ingestion of SLCO caused
significant thinning of eggshells at 1 and 3% while KCO did not at either
concentration. When these birds were put back on uncontaminated feed, eggshell
thickness became similar to the control group. Vangilder and Peterle (1982) also
found significant eggshell thinning in mallards ingesting SLCO. Coon and Dieter
( 1981), on the other hand, fed mallards up to 2.5% SLCO for 26 weeks and found no
reduction in eggshell thickness in treatment birds. A single oral dose of PBCO in
Japanese quail resulted in significantly reduced eggshell thickness on day 1 following
dosing, but was back to normal the next day (Engel et al. 1978).

Initial body weights for ducklings of hens consuming 200 and 2,000 ppm
WNSCO were significantly higher (P g 0.05) than controls over the 10 week period
(Table 8). However, ducklings of hens consuming 20,000 ppm WNSCO in the diet
were not significantly different (P > 0.05) than controls. On the contrary, the mean
daily growth rate in the 20,000 ppm ducklings was significantly higher (P _<_ 0.05) than

25

Table 7. Mean eggshell strength and thickness (if 1 SE.) of eggs laid by hen mallards fed
WNSCO in the diet for 22 weeks.

 

Treatment (ppm)

 

Parameter 0 200 2,000 20,000
Egg shell

thickness (mm) 0.417 1 0.004 0.417 1 0.004 0.405 1 0.004 0.386' 1 0.006
Egg shell

strength

(Newtons) 32.3 1 0.5 31.6 1 0.7 32.1 1 0.6 29.5‘ 1 0.8

 

‘Significantly different from control (P g 0.05)

26

Table 8. Mean initial body weight and growth rate (It 1 SE.) of hatchlings of hen mallards fed

 

 

 

WNSCO in the diet for 22 weeks.
Treatment (ppm)
Parameter 0 200 2,000 20,000
Initial Body
Weight (g) 35.3 1 0.2 36.2‘ 1 0.2 36.3' 1 0.2 35.8 1 0.2
Growth rate
(g/b/d)” 9.7 1 0.1 10.0 1 0.1 9.6 1 0.1 10.5: 1 0.1

 

‘Significantly different from control (P _<_ 0.05)

t"For 14 days

27
controls while the 200 and 2,000 ppm ducklings were not different from the control.

No significant differences (P > 0.05) were found between treatment and control
mallards by sex for mean organ weights (Table 9) or organ weights normalized on the
basis of body weight or brain weight. Liver weights of males were 24 g less than
females and similar to liver weights of non-reproductive males in the 14-day study.
The large liver weights of females in this study can be associated with it being a
storage site for fat going to yolk deposition.

The females consuming 20,000 ppm WNSCO in the diet suffered a 26%
reduction in serum albumin and a 17% decrease in serum total protein (Table 10).
Calcium levels were reduced by over half (54%) in the 20,000 ppm females compared
to controls. Ingestion of crude oil did not significantly impact other clinical blood
parameters.

The decrease in plasma calcium and protein in the high dose group appears to
be related to the reduced eggshell thickness and strength of eggs observed for this
group. Calcium and protein are important components in shell structure and if a hen
suffers from reduced levels, this could compromise shell thickness and strength. The
lower calcium level in the high dose birds may be the result of reduced absorption in
the intestinal mucosa as the amount of food consumed by the high dose group is not
significantly less than the controls. Harvey et al. (1982) suggest that contamination
of the diet with crude oil may irritate the gastrointestinal tract and it is possible
therefore that petroleum may have a direct effect on the absorptive properties of the
mucosa cells in the small intestine which might indirectly lead to a reduction in egg
production and egg quality. They found that domestic mallards fed North Sea crude
oil exhibited a significantly lower plasma calcium level compared to control. This
reduction remained even after oil ‘had been removed from the diet and food

consumption increased, and they feel this provides evidence that petroleum ingestion

Table 9. Mean organ weights (3) in mallards fed WNSCO in the diet for 22 weeks in a

reproduction study (E 1 S.E.).

28

 

Treatment (ppm)

 

Parameter 0 200 2,000 20,000
Liver
male 27.8 1 1.2 27.5 1 0.9 27.3 1 1.2 30.5 1 1.1
female 49.1 1 4.0 53.2 1 5.1 53.8 1 4.8 51.6 1 4.5
Kidney
male 8.5 1 0.3 8.8 1 0.3 8.5 1 0.3 8.9 1 0.4
female 10.7 1 0.4 10.5 1 0.4 10.4 1 0.5 10.6 1 0.3
Spleen
male 0.74 1 0.05 0.69 1 0.04 0.69 1 0.05 0.57 1 0.03
female 0.91 1 0.11 0.79 1 0.06 1.52 1 0.79 0.62 1 0.06
Adrenal
male 0.28 1 0.02 0.37 1 0.03 0.29 1 0.02 0.29 1 0.03
female 0.42 1 0.04 0.33 1 0.03 0.30 1 0.03 0.31 1 0.03
Brain
male 5.0 1 0.1 5.1 1 0.1 5.0 1 0.1 5.0 1 0.1
female 4.6 1 0.1 4.5 1 0.1 4.6 1 0.1 4.5 1 0.1

 

29

Table 10. Mean clinical blood parameters (i 1 SE.) in adult mallards fed WNSCO in the diet

 

 

 

for 22 weeks.
Treatment (ppm)
Parameter 0 200 2,000 20,000
Female
Albumin (g/dl) 2.3 1 0.1 2.3 1 0.1 2.2 1 0.1 1.7' 1 0.2
Total Protein
(g/dl) 5.7 1 0.2 5.9 1 0.5 5.8 1 0.1 4.4: 1 0.6
Calcium (mg/d1) 33.4 1 3.6 32.7 1 2.5 27.4 1 1.3 15.5‘ 1 4.2
Basophils (%) 4.3 1 0.6 4.3 1 1.3 2.0' 1 0.4 1.8' 1 0.5
Male
Albumin (g/dl) 1.9 1 0.1 1.5‘ 1 0.1 1.8 1 0.1 1.9 1 0.1
RBC
(10° cell/mm") 3.1 1 0.21 3.0 1 0.03 2.7: 1 0.04 2.7: 1 0.07
Packed Cell 9
Volume (%) 48.2 1 0.4 48.6 1 0.9 44.2' 1 1.1 47.4 1 1.0

 

'Significantly different from control (P g 0.05)

30
inhibits calcium uptake.

Two adult females died during the 10-week egg production period. An adult
female in the high dose treatment group died during the first week of egg production
and an adult female in the 200 ppm treatment group was found dead 4 weeks after egg
production began. Upon autopsy, neither death was determined to be treatment
related. No birds exhibited signs of gross toxicity. Our mortality results are consistent
with other studies of adult mallards fed crude oils in the diet at concentrations up to
5% have found no treatment related mortality even after 30 weeks of continuous
ingestion (Holmes et al. 1978a, Holmes et al. 1978b, Holmes et al. 1979, Coon and
Dieter 1981, Gorsline and Holmes 1981, Gorsline et al. 1981, Rattner 1981, Cavanaugh
and Holmes 1982, Gorsline and Holmes 1982c, Harvey et al. 1982, Cavanaugh and
Holmes 1983, Cavanaugh et al. 1983, Vangilder and Peterle 1983). Vangilder (1981)
reported 5 mortalities in an experiment where mallards were fed SLCO but these were
not treatment related.

No treatment related gross abnormalities or lesions were observed at necropsy
in this study. Histopathological examination revealed no consistent differences in
tissues of high dose birds compared to control birds, by sex, except in the number of
circumscribed nodules of lymphoid tissue in the spleens of females. There was a
higher average number of circumscribed 'nodules in the spleens of high dose females
than in controls. Coon and Dieter (1981) reported no histopathological differences
between birds fed 2.5% SLCO in the diet and control birds. Pathological responses of
birds from oil spill sites or of birds given single oral doses of oil as noted by Leighton
et al. (1983) have consistently included hepatic fatty changes, lipid pneumonia,

gastrointestinal irritation and renal tubular nephrosis.

3 1
Eggshell Application Study

Hatching success was significantly reduced (P g 0.05) in only the WNSCO
treatment that covered the middle 1/3 of the egg (Table 11). On the contrary,
hatching success was significantly reduced (P g 0.05) from the control group in all
petrolatum treatment groups (Table 11). As petrolatum is not known to be toxic, this
is most likely an effect of pore blockage resulting in inefficient gas and water
exchange. Embryos from unhatched eggs in the petrolatum application group were
examined and most showed signs of being hydrocephalic, a sign of insufficient water
exchange. Many embryos from unhatched eggs in the WNSCO treatment groups also
showed signs of being hydrocephalic. The apparent differences between treatments
is most likely due to the efficiency of the treatment in blocking pores. The
petrolatum, being smooth and applying evenly, proved an efficient pore blocker. The
WNSCO was of a firmer consistency and did not spread as evenly and was probably not
an efficient pore blocker, so more WNSCO was needed to significantly reduce
hatchability. This suggests that the weathered crude is not toxic to the embryos at
the levels applied and they would be impacted by pore blockage before toxicity.

Crude oil toxicity to developing embryos has been demonstrated in the
literature and is a viable concern. Typically, only microliter quantities of crude oils (5
50 ul) applied directly to the egg shell of bird eggs are needed to result in significant
embryo mortality (Albers 1978, Hoffman 1978, Szaro et al. 1978a, Hoffman 1979a,
Hoffman 1979b, Macko and King 1980, Lambert et al. 1982, Couillard and Leighton
1989, Couillard and Leighton 1990). Hartung (1965) demonstrated that oil can be
transferred from feathers to eggs. Albers (1980) has taken this one step further and
shown that an incubating bird will enter oiled water, become oiled, and subsequently
transfer the oil to the eggs. He also showed that at 100 ml of PBCO/m2 of water
surface there was a significant reduction in hatchability in mallard eggs. Hallet et al.

32

Table 11. Number hatched, number 14-day survivors, and hatchling growth rate
(g/b/ d) from mallard eggs externally treated with various amounts of WNSCO.

 

 

Treatment Number Number 14-day Hatchling growth
hatched ~ survivors rate 6 1 SE.)
Control“ 43 42 11.2 1 0.4
Petrolatum
Top 1/6 32° 31 12.4 1 0.6
Top 1/3 3" 3 13.4 1 2.8
Middle 1/6 34° 32 12.6 1 0.6
Middle 1/3 4° 4 9.6 1 2.5
WNSCO
Top 1/6 42 42 11.3 1 0.4
Top 1/3 38 38 11.2 1 0.4
Middle 1/6 39 39 11.4 1 0.4
Middle 1/3 37° 37 11.1 1 0.4

 

*All test groups N=45
I’Significantly different from control (P _<_ 0.05)

33

(1983) have demonstrated that aromatic hydrocarbons from petroleum pass through
the shell and membrane and into the developing embryo.

The age of the developing embryo at which exposure to oil takes place is an
important factor as well. Albers (197 8) treated mallard eggs with 5 ul of SLCO on days
2, 6, 10, 14, 18, and 22 of a 26 day incubation period and found that hatchability of
eggs decreased as the age of the embryo decreased. His results show that after day
10 the impact on the embryo is reduced to a large extent. Hoffman (1979b)
demonstrated two periods within the first ten days of incubation in which mallard
embryos were most susceptible to petroleum toxicity. Mallard eggs were treated with
crude oil on day one of development, and three and seven days after treatment there
were significant embryo mortalities while there was little mortality after 13 days.
Mallard eggs on day 3 of development exhibited a significant decline in embryo
mortality by day 6 and then a rapid decline on days 8 and 9 (Hoffman 1978). Using
chicken embryos, Hoffman (1978) found little mortality within four days after
treatment on day 3 but on the seventh day there was a sudden increase in embryo
mortality.

Two studies have looked at the impact of weathered crude oils on bird eggs.
Szaro et al. (1980) treated mallard eggs with 0 to 50 ul of raw PBCO or PBCO that had
been artificially weathered for 10 days, either indoors or outdoors. All treatment
groups caused significant embryo mortality and weathering had no effect on toxicity
of the PBCO. They then weathered it outdoors for 4 weeks. PBCO did not show a
decrease in toxicity until the third week, and even at 4 weeks as little as 20 11] caused
significant mortality. The weathering process that our PBCO underwent occurred
under natural conditions and had weathered for over 3 months and the physical state
it was in probably did not allow for it to leach into the egg in large quantities. Ten

microliters of naturally weathered Libyan crude oil collected after a pipeline rupture

34
caused significant embryo mortality in heron eggs but surprisingly the raw Libyan
crude oil did not (Macko and King 1980). This naturally weathered crude was collected
from bottom sediments which may account for the difference in toxicity. The crude
oil in bottom sediments may contain concentrated aromatic hydrocarbons as these can
enter the water column through dissolution and may salt out into the bottom
sediments.

Ellenton (1982) has tried to elucidate the fraction of crude oil which is
responsible for the embryotoxicity. Vangilder and Peterle (1983) also looked at PBCO
fractions and their effects on chicken embryos. They found that the aromatic fraction
of PBCO was the most toxic fraction of the oil. Ellenton (1982) split PBCO into
aliphatic, 2 or 3 ring (F3) aromatic, and 4 or 5 ring (F4)aromatic fractions, and applied
these to chicken embryos. The aliphatic portion caused no decrease in survival or
increase in developmental abnormalities, but did result in significant decrease in
weight, crown-rump length and head length. The F3 and F4 fractions had no effect
on weight or length and only the F3 fraction caused a significant increase in
developmental abnormalities. Their results led them to the conclusion that the
embryotoxic activities reside in the F3 fraction.

Survivability of the hatchlings from any of the treatment groups was not
different (P > 0.05) from the control group (Table 10). Albers (1980) found no
significant difference in mallard hatchling survival up to one week, nor did they find
any gross behavioral or physical abnormalities, in hatchlings of hens exposed to PBCO.
Under similar conditions, Albers and Gay (1982) found no significant difference in one
week survivability of one week old hatchlings. Growth rate of the hatchlings did not
differ significantly (P > 0.05) for any treatment level for either treatment when

compared to the controls (Table 11).

 

 

 

 

CONCLUSION

The results of this series of studies seem to be in agreement with the majority
of the literature, which shows that crude oils, in general, appear to be of low toxicity.
At levels of up to 5000 mg/ kg body weight in a single oral dose there was no mortality
or loss of body weight, but the male birds did eat significantly less food than controls
over the two week observation period. No mortalities, adverse behavioral reactions,
or effects on growth or food consumption were noted in mallard ducklings fed up to
50,000 ppm weathered crude oil in the diet for 5 days. No food avoidance behavior was
noted in mallard ducklings given a choice of clean feed and contaminated feed up to
20,000 ppm in the diet. So we can assume that feed contaminated with weathered
crude oil is not a deterrent to ingestion upto at least 2% of the diet. The only
pathological changes noted in the organs was in the significant increase in liver
weights of birds consuming 100,000 ppm in the 14-day dietary feeding study. No
difference in food consumption or body weights was found in the 14-day dietary
feeding or reproduction studies. N 0 significant differences were noted in the
reproductive parameters except for a decrease in eggshell thickness and strength of
hens consuming 20,000 ppm in the diet. Reduced concentrations of plasma calcium,
total protein and albumin may have led to this condition. Hens from this group also
laid fewer eggs and in a more erratic fashion than control hens, but this was not
statistically significant. The results from the eggshell application experiment show
that the main concern of transfer of crude oil, at this stage of weathering, from the
hen to the eggs would be physical covering, which blocks the pores and results in
reduced gas and water exchange.

Weathering is a complicated process involving a complex interaction of

35

36

environmental variables that is not likely to be accurately duplicated in a laboratory
situation. Results from artificial weathering experiments have been variable at best
and show that artificial weathering does not always reduce the toxicity of crude oil
(McEwan and Whitehead 197 8, Butler and Lukasiewicz 197 9, Peakall et al. 1980, Szaro
et al. 1980, Peakall et al. 1983, Leighton 1985, Fry et al. 1986, Nwokolo and Ohale
1986, Boersma et a1. 1988). McEwan and Whitehead (1978) and Szaro et al. (1980)
have shown slight decreases in toxicity of crude oil after weathering, and in the case
of Szaro et al. it still only took microliter quantities to cause significant embryo
mortality. In the other studies the effects of weathered crudes were similar to raw
crudes. There have been only two studies that I am aware of that have looked at
naturally weathered crude oil (Pattee and Franson 1982, Macko and King 1980). Ixtoc
I crude oil from the Ixtoc well blowout in the Gulf of Mexico had little effect on
American kestrels ingesting it up to 3% in the diet (Pattee and Franson 1982). .Macko
and King (1980) found naturally weathered Libyan crude oil collected from bottom
sediments at the spill site to be highly toxic to Louisiana heron embryos while raw
Libyan crude was not. In this case; weathering actually enhanced the toxicity of the
oil. This oil had only weathered for 4 weeks and might be different in composition to
weathered oil skimmed from the surface. There are many opportunities giving access
to naturally weathered crude oils and other petroleum products resulting from
accidental spills, and these need to be taken advantage of and studied.

Most of the chronic tests have been done in the laboratory under ’ideal’
conditions where all environmental variables remain relatively constant. Studies have
shown that when birds ingesting oil are removed from the ’ideal’ laboratory conditions
and put under some type of environmental stressor they are unable to adapt to the
new conditions as well as the control birds, and suffer greater mortality (Holmes et al.
1978b, Holmes et. al 1979). The physiological response of oiled birds to adaption to

37

sea water, as well as the effect of temperature stress, was examined. This would be
a major concern in the area at the Valdez spill. Holmes et al. (1978b) adapted
domestic mallards to artificial seawater and then exposed them to SLCO or KCO in
the diet while maintaining them at 27°C. They found that after exposing them to mild
cold stress at 3°C, birds that consumed crude oil died earlier and in greater numbers
than seawater- or fresh-water maintained control birds. Holmes et a1 (1979), in a
similar study, looked at the effects of five different crude oils. The results obtained
were similar, and they also felt that PBCO to be the most toxic oil tested, as the birds
ingesting PBCO consumed the least oil and suffered the highest mortality under cold
stress.

Crocker et al. (1974) suggest that ingested oil might hinder intestinal
absorption in birds and be an important factor in seabird mortality. When exposed to
hypertonic saline solution, equivalent to ocean water, there was an increase in
exchange rates of water and sodium in the mucosal lining of the intestine. After
giving mallard ducklings a single oral dose of crude oil, Crocker et al. (1974) found that
birds given oil at the start of maintenance on hypertenic seawater never developed
high mucosal rate transfers, and in birds adapted to hypertonic saline solution prior
to dosing the high mucosal rate transfer that had been established was lost within 24
hours of treatment. In a follow up experiment, Crocker et al. (1975) looked at the
effects of different crude oils on mucosal transfer rate. They found that of the three
crudes tested, PBCO had the least effect while SLCO and KCO had the greatest effect.
They also artificially weathered crude oil and found enhanced suppression of mucosal
rate transfer. Eastin and Murray ( 1981) looked at the effects of dietary ingestion of
PBCO on intestinal absorption on sodium, chloride, potassium and water in
freshwater-adapted mallards. They found no effect on intestinal absorption and

suggest that the effect of crude oil on transport might be related to mechanisms

38
stimulated by saltwater-adaption. From these results, and as suggested by Harvey et

al. (1982), it could also be postulated that the nutrient absorptive qualities of the
intestine may be compromised by crude oil ingestion, which may explain reduced
reproductive potential, i.e. depressed egg production, slowed ovarian maturation,
delayed oviposition, thinned egg shells, reduced hatchability, etc. This becomes
critically important, especially in an arctic or subarctic environment in which the
breeding season is short and the timing of the reproductive cycle is critical. If young
are not fledged and flying on their own within this critical time period then
recruitment into that population could be severely reduced.

From the data presented in this series of experiments, it appears that naturally
weathered crude oil is not highly toxic when fed to birds under controlled laboratory
conditions. There is a great need to look at the effects of naturally weathered crude
oil as there is a severe lack in the literature. We need to be able to determine
whether this oil poses a threat to seabird populations after going through the
weathering process, including looking at the effects on birds ingesting oil and then put
under environmental stresses. It is hard to extrapolate results from controlled
laboratory experiments to wild populations and there needs to be an effort to assess
its impact from this angle. At this point, it seems that the greatest threat to seabird
populations is from physical covering with oil resulting in loss of buoyancy and
insulation, and inhalation of toxic fumes in the first hours of a spill, both resulting in
death.

APPENDDK

39

Table 12. Mortalities observed in adult mallards orally dosed with weathered North
Slope crude oil.

 

Diet Concentration (mg/kg) #Dead/#Exposed/#Exhibiting Toxic Signs
0 0/12/0
5,000 0/ 12 / 0

 

 

Table 13. Body weights (g) of adult mallards orally dosed with weathered North Slope

crude oil (mean 1 SE).

40

 

Mean Body Weight (g) 1 SE

 

 

Diet
Concentration
(mg/ kg) Sex Initiation Day 7 Termination
0 M 1123 1 44 1189 1 36 1191 1 36
0 F 994 1 22 1042 1 33 1059 1 40
5,000 M 1166 1 30 1217 1 35 1224 1 32
5,000 F 1008 1 34 1055 1 33 1069 1 26

 

41

Table 14. Food consumption (g) of adult mallards orally dosed with weathered North
Slope crude oil (mean 1 SE).

 

Mean Food Consumption (g) 1 SE

 

 

Concgitifation Initiation - Day 7 - Initiation -
(mg/kg) Sex Day 7 Termination Termination
0 M 135 1 5.8 118 1 4.9 127 1 4.0
0 F 99 1 4.7 86 1 4.9 93 1 3.5
5,000 M 122 1 9.5 96 1 4.4 109° 1 5.6
5,000 F 102 1 2.8 87 1 2.9 94 1 2.3

 

‘Signiflcantly different from control males (P _<_ 0.05)

42

Table 15. Mortalities observed in mallard ducklings fed weathered North Slope crude
oil in the diet for 5 Days.

 

 

Diet Concentration (ppm) #Dead/#Exposed/#Exhibiting Toxic Signs
0 0/20/0
20,000 0/ 10 / 0
0 0/12/0

50,000 0/12/0

 

43

Table 16. Food consumption (g) of mallards ducklings fed weathered North Slope
crude oil in the diet for 5 Days (mean 1 SE).

 

 

Study Days
Diet
Concentration : Initiation - . ' Day 5 -
(ppm) Day 5 Termination
0 c 21.8 1 0.9 33.9 1 1.4
20,000 23.9 1 1.5 35.7 1 1.8
0 22.7 1 1.0 34.5 1 2.4

50,000 21.0 1 0.8 31.6 1 1.7

44

Table 17 . Body weights (g) of mallard ducklings fed weathered North Slope crude oil
in the diet for 5 days (mean 1 SE).

 

 

 

Study Day
Diet
Concentration
(ppm) N Initiation 5 Termination
0 20 66.5 1 1.9 137.4 1 5.7 197.3 1 7.5
20,000 10 68.3 1 2.1 140.8 1 9.4 196.0 1 13.5
0 12 61.9 1 4.0 130.5 1 9.6 188.3 1 13.2

50,000 12 65.4 1 4.4 130.1 1 9.0 185.9 1 12.5

45

Table 18. Mortalities observed in mallard ducklings given a choice of food treated with
weathered North Slope crude oil and untreated food in the diet for 5 days.

 

 

Diet Concentration (ppm) #Dead/#Exposed/#Exhibiting Toxic Signs
0 0/20/0
1.250 0/ 10 / 0
2,500 0/ 10 / 0
5,000 1/ 10 / 0
10,000 0/ 10 / 0

20,000 0/10/0

 

46

Table 19. Feed consumption (g) in mallard ducklings given a choice between feed
treated with weathered North Slope crude oil and untreated feed in the diet for 5 days
(mean 1 SE).

 

Feed Consumption (g) 1 SE

 

 

Diet
Concentration
(ppm) N Treated Feed Control Feed Total
0 20 11.9 1 1.3 12.9 1 1.4 24.3 1 1.5
1,250 10 13.1 1 1.7 11.8 1 1.4 24.9 1 2.0
2,500 10 12.2 1 1.8 12.0 1 2.1 24.2 1 1.8
5,000 10 15.2 1 2.0 12.1 1 1.9 27.3 1 2.4
10,000 10 13.7 1 2.0 13.8 1 2.2 27.5 1 2.3
20,000 10 13.2 1 2.0 12.3 1 1.5 25.4 1 2.6

 

47

Table 20. Body weights (g) of mallard ducklings given the choice of feed treated with
weathered North Slope crude oil or untreated feed in the diet for 5 days (mean 1 SE).

 

 

 

Study Day
Diet
Concentration

(ppm) N Initiation Day 5 Termination
0 20 67.6 1 2.2 141.0 1 5.2 202.7 1 7.4
1,250 10 67.9 1 3.7 141.8 1 9.1 202.1 1 12.9
2,500 10 64.5 1 4.1 135.1 1 12.2 193.6 1 18.5
5,000 10 64.9 1 4.9 144.9 1 9.0 212.2 1 12.2
10,000 10 67.9 i 3.9 146.6 1*. 8.4 209.6 _+_ 10.3
20,000 10 65.9 i 3.0 139.3 :L 8.6 196.7 1 11.3

 

48

Table 21. Mortalities observed in adult mallards fed weathered North Slope crude oil in the
diet for 14 Days.

 

 

Diet Concentration (ppm) #Dead/#Exposed/#Exhibiting Toxic Signs
0 0 / 10 / 0
10,000 0/ 10 / 0
30,000 0/ 10 / 0

100,000 0/10/0

 

49

Table 22. Mean daily feed consumption (g) in adult mallards fed weathered North
Slope crude oil in the diet for 14 days (mean 1 SE).

 

Feed Consumption (g) 1 SE

 

 

Diet
Concentration
(ppm) N Day 0-8 Day 814 Day 0- 14
0 10 108.9 1 7.9 88.8 1 8.0 100.3 1 5.9
1,250 10 103.5 1 7.8 102.9 1 10.0 103.2 1 6.1
2,500 . 10 92.1 1 7.7 107.6 1 8.2 98.8 1 5.7

5,000 10 94.1 1 8.2 102.1 1 7.3 97.5 1 5.6

50

Table 23. Body weights (g) of adult mallards fed weathered North Slope crude oil in
the diet for 14 days (mean 1 SE).

 

 

 

Study Day
Diet
Concentration
(ppm) Sex Initiation Day 7 Termination
0 M 1227 1 45 1259 1 61 1247 1 69
10,000 M 1208 1 61 1237 1 70 1219 1 70
30,000 M 1265 1 65 1266 1 76 1278 1 73
100,000 M 1210 1 61 1225 1 71 1204 1 67
0 F 1101 1 51 1146 1 53 1120 1 51
10,000 F 1091 1 23 1119 1 27 1105 1 35
30,000 F 1107 1 67 1096 1 59 1105 1 70
100,000 F 1096 1 52 1116 1 52 1096 1 57

 

51

Table 24. Hematology results of adult mallards fed weathered North Slope crude oil in the diet
for 14 days (mean 1 SE).

 

Exposure Concentration (ppm)

 

Parameter Day 0 10,000 30,000 100,000
Red Blood 0 2.5 1 0.13 2.6 1 0.09 2.5 1 0.08 2.4 1 0.06
Count 14 2.71 0.11 2.61 0.09 2.61 0.10 2.51 0.07
(10°cell/mm3)

White Blood 0 9467 1 865 12500 1 2191 9505 1 1603 8521 1 1210
Count 14 10145 1 1434 8747 1 699 8322 1 1793 8873 1 2093
(cells/mms)

Lymphocytes 0 25.4 i 5.4 36.2 i 6.4 25.2 1. 5.0 25.2 _+_ 7.5
(%) 14 28.2 i 4.3 34.4 _4; 8.8 34.8 -_+_ 3.6 45.4 _1; 6.1
Monocytes 0 6.8 5; 1.9 7.0 i 2.4 12.4 1 3.1 3.8 i 1.4
(%) 14 9.2 _t 3.2 11.6 1; 5.9 5.4 i 1.6 3.8 i 1.0
Heterophils 0 63.0 i 4.4 55.4 i 5.0 59.8 i 6.6 68.8 _+_ 7.0
+ Eosinophils 14 58.2 i. 4.2 51.2 .t. 10.9 56.0 :42 48.2 i. 5.9
(%)

Basophils 0 4.8 .1“. 0.8 1.4 .t 0.7 2.6 i 0.9 2.2 _+_ 1.0
(%) 14 4.4 :L 1.3 2.8 _+_ 1.4 3.8 _+_ 1.0 2.6 i 1.3
HGB 0 15.6 1 0.3 15.8 1 0.4 15.1 1 0.4 14.8 1 0.3
(g/dl) 14 16.0 1 0.6 15.9 1 0.4 14.6 1 0.5 14.5 1 0.2
Mean Cell 0 186.8 i 6.1 181.3 1 2.9 190.4 .1 3.6 192.0 i 3.6
Volume 14 170.8 _4; 5.8 177.8 1 5.1 171.4 i 3.8 176.8 :1. 4.5

(fL)

Table 24 (cont’d).

52

 

 

Parameter Day 0 10,000 30,000 100,000
Mean Cell 0 62.0 1 2.5 62.0 1 0.6 59.9 1 1.4 62.0 1 1.4
Hemoglobin 14 59.0 1 2.1 61.0 1 2.0 56.6 1 1.8 57.5 1 1.3
(uug)

Mean Cell 0 33.2 1 0.5 33.4 1 0.7 31.4 1 0.3 32.2 1 0.3
Hemoglobin 14 34.4 1 0.4 34.2 1 0.3 33.0 1 0.5 32.7 1 0.3

Concentration
(%)

Packed Cell 0 47.2 1 1.5 47.4 1 1.5 48.2 1 1.6 45.8 1 0.5
Volume 14 46.4 1 1.7 46.2 1 1.1 44.4 1 1.5 44.4 1 0.5

(%)

Reticulocyte 0 3.8 1 0.7 3.8 1 0.6 4.3 1 0.3 4.3 1 0.6

Count 14 3.7 1 0.6 2.6 1 0.3 2.9 1 0.3 2.9 1 0.2

 

Table 25. Clinical chemistry results of adult mallards fed weathered North Slope crude oil in the

53

 

 

 

diet for 14 days (mean 1 SE).
Exposure Concentration (ppm)
Parameter Day 0 10,000 30,000 100,000
Na 0 144.2 1 0.5 146.2 1 0.8 145.4 1 0.5 144.2 1 0.7
(mEq/L) 14 144.4 1 1.1 144.0 1 1.4 145.2 1 0.7 143.2 1 0.9
K 0 2.7 1 0.2 2.7 1 0.3 2.4 1 0.2 2.3 1 0.1
(mEq/L) 14 2.6 1 0.1 2.6 1 0.2 2.7 1 0.2 2.7 1 0.2
Alk Phos 0 226.0 1 29.8 210.4 1 31.3 150.0 1 16.2 230.8 1 33.3
(U/L) 14 156.8 1 20.8 166.2 1 15.9 130.8 1 18.1 152.6 1 22.7
CPK 0 317.4 1 107.0 349.2 1 53.4 285.6 1 39.8 305.8 1 46.0
(U/L) 14 293.0 1 107.6 841.4 1 467.3 282.4 1 59.3 212.2 1 33.3
Glucose 0 188.2 1 6.4 191.4 1 8.7 181.4 1 7.2 181.6 1 7.3
(m/dl) 14 186.8 1 2.9 193.4 1 4.2 194.6 1 7.8 179.2 1 5.9
ALT 0 23.8 1 1.9 29.2 1 3.1 19.0 1 2.3 24.4 1 3.2
(SGPT) 14 22.2 1 3.2 33.8 1 3.1 28.4 1 0.9 32.6 1 2.5
(U/L)
AST 0 7.6 1 2.3 9.8 1 2.6 8.8 1 1.2 8.8 1 1.5
(SGOT) 14 9.2 1 2.1 25.2 1 11.3 10.6 1 2.0 10.0 1 1.6
(U/L)
Total 0 3.7 1 0.2 3.7 1 0.1 3.9 1 0.2 3.8 1 0.16
Protein 14 4.0 1 0.3 3.8 1 0.2 4.3 1 0.2 4.4 1 0.0

(g/dl)

 

 

Table 25 (cont’d).

Parameter Day 0 10,000 30,000 100,000
Albumin 0 1.6 1 0.1 1.5 1 0.0 1.6 1 0.1 1.7 1 0.1
(g/dl) 14 1.7 1 0.1 1.8 1 0.1 1.8 1 0.1 1.9 1 0.0
Globulin 0 2.1 1 0.1 2.2 1 0.1 2.3 1 0.1 2.1 1 0.1
(g/dl) 14 2.3 1 0.2 2.0 1 0.1 2.5 1 0.2 2.5 1 0.1
Uric Acid 0 8.7 1 1.5 7.6 + 0.8 7.4 1 1.5 8.1 1 1.6
(mg/d1) 14 4.6 1 0.6 4.0 1 0.5 6.2 1 1.2 4.4 1 0.2
Ca 0 10.0 1 0.3 10.1 1 0.2 10.4 1 0.3 10.4 1 0.2
(mg/dl) 14 10.3 1 0.3 10.3 1 0.2 11.0 1 0.4 11.1 1 0.4

 

55

Table 26. Mortalities observed in adult mallards fed weathered North Slope crude oil
in the diet for 22 weeks.

 

 

Diet Concentration (ppm) #Dead/#Exposed/#Exhibiting Toxic Signs
0 0/30/0
200 1 / 30 / 0
2,000 0 / 30 / 0

20,000 1/30/0

56

Table 27 . Mean feed consumption (g/b/d“) by adult mallards fed weathered North
Slope crude oil in the diet for 22 weeks (mean 1 S.E.).

 

Diet Concentration (ppm)

 

 

Week 0 200 2,000 20,000

1 8r 2 105.6 1 4.4 113 1 4.9 107.6 1 3.9 100.2 1 5.1
3 & 4 96.8 1 4.3 94.4 1 4.4 92.2 1 3.5 91.4 1 3.4
5 & 6 97.1 1 5.3 106.2 1 4.4 92.9 1 4.1 91.2 1 3.7
7 & 8 97.9 1 6.6 91.3 1 3.6 88.3 1 3.4 90.2 1 3.4
9 & 10 97.6 1 6.8 96.8 1 5.9 93.5 1 4.9 87.9 1 4.3
11 & 12 141.4 1 10.3 150.2 1 10.4 127.3 1 7.1 129.7 1 11.9
13 & 14 146.0 1 8.3 142.7 1 8.3 125.2 1 4.7 122.3 1 9.4
15 & 16 126.4 1 9.7 151.3 1 13.0 121.1 1 9.7 138.4 1 7.6
17 & 18 146.4 1 12.1 140.3 1 7.0 130.7 1 8.3 147.0 1 7.4
19 & 20 144.4 1 15.0 142.0 1 7.5 144.5 1 9.3 143.6 1 12.1
21 & 22 157.8 1 11.3 174.0 1 11.0 173.1 1 8.5 186.1 1 18.0

 

"' g/b/d = grams per bird per day

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58

Table 29. Organ:brain weight ratios of adult female mallards fed weathered North
Slope crude oil in the diet for 22 weeks (mean 1 SE).

 

 

Concentration
(ppm) Liver Kidney Spleen Adrenal
0 10.7 1 1.0 2.3 1 0.1 0.20 1 0.02 0.091 1 0.009
200 11.8 1 1.1 .3 1 0.1 0.17 1 0.01 0.073 1 0.005
2,000 11.8 1 1.2 2.3 1 0.1 0.34 1 0.18 0.066 1 0.006

20,000 11.6 1 1.1 2.4 1 0.1 0.14 1 0.01 0.069 1 0.007

 

59

Table 30. Organ:brain weight ratios of adult male mallards fed weathered North Slope
crude oil in the diet for 22 weeks (mean 1 SE).

 

Concentration

 

(ppm) Liver Kidney Spleen Adrenal
0 5.6 1 0.2 1.7 1 0.1 0.15 1 0.01 0.058 1 0.004
200 5.5 1 0.2 1.8 1 0.1 0.14 1 0.01 0.073 1 0.006
2,000 5.5 1 0.2 1.7 1 0.1 0.14 1 0.01 0.058 1 0.005

20,000 6.2 1 0.3 1.8 1 0.1 0.12 1 0.01 0.059 1 0.006

 

 

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62

Table 33. Clinical chemistry results of adult female mallards fed weathered North Slope crude
oil in the diet for 22 weeks (mean 1 SE).

 

Diet Concentration (ppm)

 

Parameter Week 0 200 2,000 20,000

K 0 2.7 1 0.46 2.8 1 0.18 3.0 1 0.23 2.8 1 0.3]
(mEq/L) 5 2.6 1 0.14 2.8 1 0.19 2.9 1 0.29 2.3 1 0.06
10 2.9 1 0.74 2.2 1 0.19 2.7 1 0.45 2.4 1 0.24
Term. 2.4 1 0.29 2.8 1 0.30 2.8 1 0.11 2.2 1 0.20

Na 0 147.8 1 1.9 148.2 1 1.4 147.0 1 1.2 147.0 1 1.0

(mEq/L) 5 145.8 1 1.1 145.6 1 0.4 143.6 1 0.8 144.0 1 0.9

10 145.4 1 1.5 144.4 1 1.8 146.0 1 1.5 148.0 1 1.6

Term. 134.6 1 2.2 135.8 1 1.7 136.4 1 1.2 136.2 1 1.2

Alk Phos 0 302.8 1 67.6 313.8 1 39.3 215.2 1 30.1 188.6 1 47.1

(U/L) 133.4 1 25.2 199.8 1 32.6 110.61 13.0 101.6 1 18.7

10 422.2 1 98.4 456.6 1 112.0 195.6 1 71.7 246.2 1 86.3

Term. 927.6 1 531.1 207.2 1 62.3 103.0 1 8.3 81.4 1 11.6

CPK 0 401.2 1 197.3 332.8 1 40.0 407.6 1 144.0 305.6 1 39.1
(U/L) 5 273.4 1 71.0 277.2 1 43.5 247.2 1 58.2 313.6 1 69.9
10 715.2 1 100.3 637.6 1 120.4 599.0 1 124.4 370.2 1 85.3

Term. 302.6 1 70.3 356.0 1 64.7 275.4 1 45.9 292.6 1 29.3

Glucose 0 212.6 1 6.1 240.6 1 10.5 240.0 1 11.1 232.7 1 6.8
(m/dl) 5 207.4 1 10.4 202.6 1 11.4 201.8 1 9.5 216.6 1 15.8

10 187.2 1 10.3 186.6 1 6.2 204.4 1 4.5 200.0 1 6.8

Term. 206.9 1 7.1 215.8 1 9.2 218.0 1 8.3 211.6 1 8.0

63

Table 33. (cont’d).

 

 

Parameter Week 0 200 2,000 20,000
ALT 0 18.4 1 1.4 21.6 1 7.3 26.4 1 6.1 21.6 1 2.0
(SGPT) 5 23.2 1 3.6 22.2 1 1.9 23.2 1 2.1 27.2 1 3.0
(U/L) 10 22.6 1 6.3 26.2 1 2.9 31.6 1 2.6 36.6 1 2.1
Term. 26.2 1 3.9 25.2 1 4.2 31.2 1 1.9 42.8 1 11.4
AST 0 11.2 1 2.5 13.4 1 1.6 15.0 1 3.9 11.2 1 1.7
(SGOT) 5 11.2 1 1.3 11.8 1 1.4 10.4 1 2.1 10.0 1 3.8
(U/L) 10 15.2 1 3.0 21.6 1 7.7 15.0 1 3.1 10.6 1 1.8
Term. 3.6 1 1.7 6.4 1 2.1 8.4 1 2.2 13.2 1 7.4
Total 0 4.1 1 0.40 3.8 1 0.13 3.5 1 0.17 3.8 1 0.15
Protein 5 3.6 1 0.21 3.6 1 0.10 3.9 1 0.16 3.9 1 0.11
(g/dl) 10 5.2 1 0.36 5.4 1 0.70 4.7 1 0.32 4.5 1 0.28
Term. 5.7 1 0.23 5.9 1 0.49 5.8 1 0.10 4.4' 1 0.55
Albumin 0 1.8 1 0.04 1.9 1 0.09 1.7 1 0.13 1.9 1 0.04
(g/dl) 5 1.6 1 0.08 1.6 1 0.06 1.7 1 0.10 1.7 1 0.08
10 2.2 1 0.10 2.2 1 0.20 2.0 1 0.12 2.0 1 0.10
Term. 2.3 1 0.12 2.3 1 0.14 2.2 1 0.14 1.7' 1 0.17
Globulin 0 2.3 1 0.40 1.7 1 0.22 1.8 1 0.13 1.9 1 0.12
(g/dl) 5 2.0 1 0.19 2.0 1 0.11 2.3 1 0.19 2.2 1 0.10
10 2.9 1 0.26 3.2 1 0.52 2.7 1 0.25 2.5 1 0.19
Term. 3.4 1 0.23 3.6 1 0.40 3.6 1 0.08 2.6 1 0.40
Uric Acid 0 6.2 1 0.7 4.5 1 0.8 4.7 1 1.0 6.4 1 0.9
(mg/dl) 5 4.8 1 0.9 3.9 1 0.3 4.5 1 0.7 5.1 1 1.0
10 6.0 1 0.5 4.5 1 0.8 5.9 1 0.9 5.1 1 0.6

Term. 6.01 0.5 5.31 0.9 6.51 0.9 5.21 1.1

 

 

Table 33 (cont’d).

Parameter Week 0 200 2,000 20,000
Ca 0 11.0 1 0.4 10.9 1 0.3 10.6 1 0.3 10.6 1 0.3
(mg/dl) 5 10.4 1 0.2 10.0 1 0.4 10.8 1 0.2 10.5 1 0.3
10 28.4 1 6.5 36.6 1 18.9 16.0 1 4.2 16.1 1 4.3
Term. 33.4 1 3.6 32.7 1 2.5 27.4 1 1.3 15.5: 1 4.2
Total 0 0.24 1 0.08 0.12 1 0.02 0.24 1 0.08 0.22 1 0.05
Bilirubin 5 0.34 1 0.04 0.32 1 0.02 0.36 1 0.04 0.32 1 0.04
(mg/d1) 10 0.42 1 0.15 0.66 1 0.27 0.60 1 0.45 0.24 1 0.10
Term. 0.40 1 0.10 0.38 1 0.07 0.34 1 0.04 0.16 1 0.02

 

‘Significantly difi‘erent from control (P g 0.05)

Table 34. Clinical chemistry results of adult male mallards fed weathered North Slope crude oil

in the diet for 22 weeks (mean 1 SE).

65

 

Diet Concentration (ppm)

 

 

Parameter Week 0 200 2,000 20,000

K 0 2.3 1 0.20 2.6 1 0.31 2.8 1 0.18 2.8 1 0.43
(mEq/L) 5 2.2 1 0.20 2.1 1 0.07 2.9 1 0.25 3.4 1 1.16
10 2.3 1 0.22 2.2 1 0.11 . 2.9 1 0.19 4.4 1 1.66
Term. 2.5 1 0.39 2.3 1 0.19 2.6 1 0.15 3.9 1 1.28

Na 0 146.4 1 1.3 146.6 1 1.5 146.0 1 0.5 146.4 1 1.3
(mEq/L) 5 145.2 1 1.5 144.4 1 1.4 145.4 1 0.5 145.0 1 0.8
10 148.4 1 0.9 148.2 1 1.2 146.2 1 0.9 149.0 1 2.3

Term. 137.8 1 1.8 136.6 1 0.7 139.4 1 1.2 140.2 1 0.7

Alk Phos 0 131.8 1 30.0 198.6 1 19.1 218.4 1 44.7 177.8 1 22.4
(U/L) 5 89.8 1 27.4 109.6 1 17.9 102.4 1 11.1 98.8 1 11.9
10 63.6 1 11.6 66.0 1 7.1 56.2 1 4.0 84.8 1 15.8

Term. 50.8 1 9.6 57.2 1 7.0 45.8 1 2.4 67.0 1 10.6

CPK 0 466.0 1 197.6 281.4 1 38.6 384.0 1 37.6 279.8 1 77.2
(U/L) 5 551.0 1 95.9 255.4 1 49.2 482.0 1 152.5 393.4 1 76.9
10 641.4 1 181.7 605.2 1 78.4 550.2 1 115.8 432.4 1 161.4

Term. 418.4 1 22.8 341.0 1 83.2 375.2 1 83.7 413.6 1 93.8

Glucose 0 217.0 1 4.3 245.0 1 9.1 216.9 1 4.5 221.4 1 6.2
(m/dl) 5 200.8 1 13.0 208.4 1 12.5 225.6 1 10.5 213.0 1 11.0
10 188.8 1 7.5 198.2 1 6.7 193.2 1 5.3 183.4 1 3.5

Term. 196.8 1 8.3 199.6 1 6.6 201.6 1 6.5 205.2 1 5.2

Table 34. (cont’d).

66

 

 

Parameter Week 0 200 2,000 20,000
ALT 0 24.8 1 2.0 21.6 1 3.3 22.2 1 6.7 23.8 1 1.2
(SGPT) 5 24.0 1 4.3 16.4 1 4.1 26.8 1 5.2 29.8 1 2.7
(U/L) 10 31.2 1 3.6 26.6 1 3.2 31.6 1 4.8 33.8 1 4.2
Term. 53.8 1 27.1 28.4 1 5.0 32.2 1 5.4 43.2 1 3.9
AST 0 10.0 1 3.5 9.0 1 1.8 9.0 1 1.3 10.0 1 1.4
(SGOT) 14.2 1 2.4 10.0 1 1.5 12.6 1 5.1 13.2 1 3.2
(U/L) 10 18.6 1 5.9 12.4 1 1.4 13.4 1 2.8 14.0 1 3.1
Term. 10.6 1 1.7 7.8 1 0.4 8.8 1 2.6 9.8 1 0.9
Total 0 3.8 1 0.20 3.6 1 0.11 3.9 1 0.14 3.8 1 0.16
Protein 5 3.7 1 0.17 3.6 1 0.14 3.7 1 0.05 3.6 1 0.16
(g/dl) 10 4.2 1 0.23 3.7 1 0.15 4.0 1 0.15 4.2 1 0.07
Term. 4.1 1 0.26 3.8 1 0.25 4.2 1 0.13 4.0 1 0.11
Albumin 0 1.8 1 0.10 1.7 1 0.07 1.8 1 0.09 1.8 1 0.07
(g/dl) 5 1.5 1 0.13 1.5 1 0.02 1.6 1 0.07 1.6 1 0.11
10 1.9 1 0.11 1.5‘ 1 0.08 1.8 1 0.08 1.9 1 0.06
Term. 1.6 1 0.06 1.6 1 0.08 1.8 1 0.11 1.7 1 0.09
Globulin 0 2.0 1 0.12 1.9 1 0.11 2.1 1 0.12 2.0 1 0.11
(g/dl) 5 2.2 1 0.12 2.1 1 0.15 2.0 1 0.06 2.0 1 0.06
10 2.2 1 0.21 2.2 1 0.15 2.2 1 0.09 2.3 1 0.06
Term. 2.5 1 0.22 2.2 1 0.26 2.4 1 0.12 2.3 1 0.11
Uric Acid 0 5.1 1 0.8 6.2 1 1.0 4.6 1 0.3 6.0 1 0.6
(mg/dl) 5 4.6 1 0.6 4.0 1 0.5 6.2 1 1.2 4.4 1 0.2
10 6.7 1 2.0 5.8 1 0.3 6.1 1 1.4 5.6 1 0.4
Term. 7.4 1 1.4 7.8 1 0.7 6.7 1 0.9 5.1 1 0.9

Table 34 (cont’d).

67

 

Parameter Week 0 200 2,000 20,000
Ca 0 10.4 1 0.2 10.2 1 0.4 10.3 1 0.2 10.0 1 0.1
(mg/dl) 5 10.1 1 0.4 10.0 1 0.2 9.8 1 0.3 9.8 1 0.2
10 10.1 1 0.3 10.7 1 0.2 10.0 1 0.2 10.4 1 0.3
Term. 10.6 1 0.3 10.3 1 0.3 10.9 1 0.3 11.0 1 0.2
Total 0 0.18 1 0.04 0.14 1 0.04 0.14 1 0.04 0.22 1 0.06
Bilirubin 5 0.28 1 0.05 0.22 1 0.02 0.20 1 0.05 0.20 1 0.03
(mg/d1) 10 0.26 1 0.05 0.22 1 0.05 0.20 1 0.06 0.26 1 0.09
Term. 0.16 1 0.02 0.26 1 0.02 0.22 1 0.06 0.20 1 0.06

 

‘Significantly different from control (P g 0.05)

68

Table 35. Hematolog results of adult female mallards fed weathered North Slope crude oil in the diet
for 22 weeks (mean t SE).

 

Diet Concentration (ppm)

 

 

Parameter Week 0 200 2,000 20,000
Red Blood 0 2.6 a 0.17 2.7 a 0.14 2.7 2 0.15 2.7 1 0.13
Count 5 3.0: 0.23 2.8: 0.10 3.0: 0.11 2.8: 0.07
(loacell/mms) 10 2.7: 0.04 2.6: 0.15 2.8: 0.15 2.8: 0.09
Term. 2.32 0.14 2.4: 0.09 2.3: 0.13 2.5: 0.17
White Blood 0 13740 t 5110 17400 t 6164 10060 t 2470 10600 a 2844
Count 5 9520 t 1715 14880 1 4269 10740 1 2922 12780 1 3127
(cells/mm") 10 8475 , 844 10350 1 601 10620 1 1075 10556 a 3593
Term. 16340 1 3225 19020 1 4835 19700 t 2710 10720 1 2042
Heterophils 0 57.6 1. 14.1 40.4 i 10.8 65.2 1 8.2 71.8 1 5.8
+ EosinOphils 5 56.8 1 6.1 52.2 1 6.8 48.2 _+_ 5.1 53.6 1 6.7
(%) 10 62.8 _+_ 6.6 63.5 1 11.5 58.2 _+_ 11.4 60.0 _+_ 6.1
Term. 75.6 1 6.1 79.6 1 3.7 81.8 1 4.4 66.8 1 8.7
Lymphocytes 0 28.0 1 12.7 39.6 1 6.5 26.2 1 6.3 15.0 _+_ 5.2
(%) 5 27.8 .1 5.2 30.6 .1 6.0 32.0 1 5.3 30.2 1 5.1
10 20.0 _+_ 6.2 21.5 i 8.9 27.0 i 8.5 30.0 i 7.0
Term. 16.0 1 4.6 12.2 i 1.4 12.8 1 4.5 26.8 .1 9.1
Monocytes 0 11.8 1 2.6 16.0 1 4.6 5.4 _+_ 1.6 11.4 1 1.5
(%) 5 12.4 1 3.6 13.0 1 2.4 16.8 1 2.9 12.4 1 2.2
10 13.0 .1 3.3 10.8 1 4.5 12.8 1. 3.7 8.2 _+_; 1.9
Term. 6.8 i 1.7 4.0 _+_ 1.4 3.0 _+_ 0.6 3.6 1 2.7
Basophils 0 2.6 _+_ 1.2 4.0 .t 0.9 3.2 i 1.4 1.8 i. 0.6
(%) 5 3.0 1 1.3 4.2 1 1.1 3.0 _+_ 0.7 3.8 1 1.1
10 4.3 .t 0.6 4.3 _+_» 1.3 2.0‘I 1 0.4 1.8‘ 1 0.5
Term. 1.6 i 0.9 4.2 _+_ 2.6 2.4 i 0.7 2.8 i 0.9

Table 35 (cont’d).

69

 

 

Parameter Week 0 200 2,000 20,000
HGB 0 14.2 1 0.9 14.8 1 0.8 14.3 1 0.8 14.4 1 0.6
(g/dl) 5 14.6 1 0.4 14.3 1 0.4 15.0 1 0.2 14.8 1 0.3

10 15.9 1 0.6 14.7 1 0.8 14.8 1 0.8 14.4 1 0.5

Term. 13.2 1 0.9 13.0 1 0.5 12.1 1 0.9 13.7 1 1.3

Mean Cell 0 166.6 1 2.8 170.6 1 2.2 164.2 1 3.0 161.8 1 3.2
Volume 5 152.8 1 10.0 162.0 1 4.1 159.8 1 5.4 163.8 1 2.5
(11.) 10 164.3 1 3.1 166.0 1 3.7 163.6 1 3.2 168.5 1 5.1
Term. 150.6 1 5.2 147.2 1 9.6 139.4 1 6.4 152.2 1 4.5

Mean Cell 0 54.1 1 1.4 55.4 1 1.5 52.7 1 0.9 52.6 1 2.1
Hemoglobin 5 49.6 1 3.4 51.6 1 1.3 50.9 1 1.4 52.8 1 1.2
(1mg) 10 59.5 1 3.8 55.5 1 1.4 53.8 1 0.9 53.3 1 1.4
Term. 57.0 1 1.6 55.1 1 2.0 52.7 1 2.4 53.9 1 3.2

Mean Cell 0 32.6 1 0.8 32.5 1 0.5 32.2 1 0.9 32.5 1 0.4
Hemoglobin 5 32.4 1 0.4 31.9 1 0.3 31.8 1 0.5 32.2 1 0.4
Concentration 10 36.3 1 2.2 33.3 1 1.2 32.9 1 0.6 31.6 1 1.0
(%) Term. 38.0 1 1.6 38.1 1 2.9 38.0 1 2.0 36.2 1 1.8
Packed Cell 0 43.6 1 2.6 45.6 1 2.7 44.6 1 3.1 44.2 1 1.7
Volume 5 45.0 1 1.1 45.0 1 1.6 47.0 1 0.5 46.0 1 0.5
(%) 10 44.4 1 0.9 42.0 1 2.7 45.0 1 1.7 46.0 1 0.9
Term. 35.0 1 2.7 34.4 1 1.3 32.2 1 3.0 38.8 1 3.4

Reticulocyte 0 1.5 1 0.1 1.6 1 0.3 1.4 1 0.3 1.2 1 0.2
Count 5 1.7 1 0.3 1.2 1 0.2 1.3 1 0.4 1.6 1 0.2
(%) 10 2.5 1 0.4 2.0 1 0.4 1.9 1 0.6 2.8 1 1.0
Term. 4.0 1 0.5 2.7 1 0.5 4.3 1 0.7 3.9 1 1.8

 

'Significantly different from control (P _<_ 0.05)

70

Table 36. Hematology results of adult male mallards fed weathered North Slope crude oil in the diet for
22 weeks (mean 1 SE).

 

Diet Concentration (ppm)

 

 

Parameter Week 0 200 2,000 20,000
Red Blood 0 2.9 1 0.09 2.9 1 0.05 2.8 1 0.05 2.9 1 0.09
Count 5 3.1 1 0.21 3.0 1 0.03 2.7‘ 1 0.04 2.7' 1 0.07
(10“ce11/mm") 10 2.9 1 0.04 2.9 1 0.05 2.8 1 0.07 2.8 1 0.13
Term. 2.91 0.11 2.71 0.10 2.71 0.07 2.61 0.12
White Blood 0 10520 1 1519 8940 1 1622 10706 1 1231 9760 1 2109
Count 5 14340 1 4557 10100 1 1621 7280 1 702 13700 1 2729
(oells/mma) 10 11020 1 1332 11400 1 3000 7940 1 1399 10520 1 2166
Term. 16340 1 3225 19020 1 4835 19700 1 2710 10720 1 2042
Heterophils 0 47.8 1 5.3 52.2 _+_ 6.2 53.0 1 5.7 44.8 1 5.3
+ Eosinophils 5 55.2 1 7.3 70.6 1 1.5 52.8 1 7.5 52.6 :11.7
(%) 10 70.8 1 4.5 62.8 1 3.6 67.2 1 7.6 53.4 i 3.7
Term. 62.2 i 5.3 58.2 _+_ 8.3 58.6 1 4.2 68.0 1 6.5
Lymphocytes 0 35.8 1 8.2 29.2 _+_ 5.2 30.4 1; 5.5 38.8 _+_ 6.8
(%) 5 34.6 1. 7.7 19.0 _+_ 1.6 31.0 .1 5.9 31.8 110.4
10 15.8 1 4.5 22.4 1 4.1 18.2 1 8.1 34.6 1. 2.2
Term. 29.8 _+_ 4.6 33.8 1 7.3 35.4 1 4.7 23.8 i 7.2
Monocytes 0 15.0 .1 3.4 13.8 i. 4.0 8.8 _+_; 2.0 13.2 1 3.1
(%) 5 7.2 .1 1.6 6.6 1 1.6 12.8 1 2.7 11.8 1 2.7
10 11.4 1 2.0 11.0 1. 0.7 9.2 1 2.3 8.8 1 2.7
Term. 5.4 i 1.6 4.8 1 1.2 4.2 _4; 2.0 3.8 i 1.8
Basophils 0 1.4 .1 0.7 4.8 i 2.1 7.6 i 3.4 3.2 :1. 0.5
(%) 5 3.0 i 0.7 3.8 _+_ 1.0 3.4 .1 0.7 3.8 .2“. 1.2
10 2.0 .1 1.3 3.8 .1 1.0 5.4 .i'. 1.6 3.2 1 1.2
Term. 2.6 _+_ 0.9 3.2 1 1.4 1.8 .t 0.9 4.4 i 0.9

Table 36 (cont’d).

71

 

 

Parameter Week 0 200 2,000 20,000
HGB 0 15.7 1 0.2 15.4 1 0.4 14.7 1 0.3 15.3 1 0.3
(g/dl) 5 15.0 1 0.5 14.9 1 0.1 14.2 1 0.2 14.0 1 0.2
10 15.0 1 0.6 15.4 1 0.3 15.0 1 0.4 14.6 1 0.6
Term. 15.5 1 0.5 15.2 1 0.8 15.6 1 0.4 14.2 1 0.6
Mean Cell 0 167.8 1 4.3 166.6 1 0.9 160.2 1 3.2 161.2 1 3.2
Volume 5 157.2 1 10.0 158.4 1 2.0 169.4 1 1.9 167.6 1 4.4
(1L) 10 168.6 1 3.0 162.8 1 1.7 165.2 1 1.3 164.2 1 1.5
Term. 170.2 1 3.2 169.4 1 2.9 171.8 1 3.8 172.5 1 3.9
Mean Cell 0 54.5 1 1.2 53.8 1 0.6 53.0 1 0.9 52.0 1 1.0
Hemoglobin 5 49.2 1 3.2 50.1 1 0.4 53.2 1 0.6 52.4 1 0.6
(uug) 10 52.4 1 2.2 53.6 1 1.0 54.4 1 0.5 52.8 1 0.7
Term. 54.0 1 3.1 55.3 1 1.9 58.2 1 2.0 54.7 1 1.8
Mean Cell 0 32.6 1 0.2 32.2 1 0.3 33.2 1 0.3 32.3 1 0.3
Hemoglobin 5 31.3 1 0.2 31.7 1 0.4 31.5 1 0.3 31.3 1 0.6
Concentration 10 31.2 1 1.1 32.9 1 0.3 32.9 1 0.4 32.2 1 0.4
(%) Term. 31.7 1 1.7 32.6 1 0.8 33.9 1 1.3 31.8 1 1.2
Packed Cell 0 48.2 1 0.4 47.6 1 0.9 44.2‘ 1 1.1 47.4 1 1.0
Volume 5 48.0 1 1.9 47.2 1 0.9 45.0 1 0.4 44.6 1 1.0
(%) 10 48.2 1 0.4 46.8 1 0.8 45.6 1 0.9 45.4 1 1.9
Term. 49.0 1 1.9 46.4 1 1.4 46.2 1 1.2 44.6 1 1.6
Reticulocyte 0 1.4 1 0.2 1.2 1 0.1 1.5 1 0.4 1.2 1 0.2
Count 5 2.0 1 0.3 1.7 1 0.3 2.2 1 0.5 1.6 1 0.2
4%) 10 2.3 1 0.5 1.6 1 0.4 1.9 1 0.3 2.3 1 0.5

 

‘Significantly different from control (P g 0.05)

72

Table 37 . Reproductive data from mallard hens fed weathered North Slope crude oil
, in the diet for 22 weeks.

 

Diet Concentration (ppm)

 

 

Parameters 0 200 2,000 20,000
Eggs Laid 730 711 778 616
Eggs Laid/Max Laid'(%) 69.5 72.6 74.1 62.9
Eggs Cracked/Eggs Laid(%) 4.1 1.8 4.0 3.4
Fertile Eggs/Eggs Set(%) 97.4 99.4 94.4 98.4
Viable 21-day Embryos/

Fertile Eggs(%) 94.4 94.2 92.4 93.5
Ducklings/Fertile Eggs(%) 84.5 82.2 84.6 84.0
14-Day Survivors/Hatch(%) 98.7 97.9 98.0 98.6

 

’ Max Laid = the maximum # of egg laying days (70)

73

Table 38. Reproductive data from mallard hens fed weathered North Slope crude oil
in the diet for 22 weeks - eggs set.

 

Diet Concentration (ppm)

 

 

Parameters 0 200 2,000 20,000
Eggs Laid 730 711 778 616
Shell-less Eggs Laid 5 0 0 56
Eggs Removed for

Thickness/Strength - 51 46 52 37
Eggs Cracked 30 13 31 19
Eggs Cracked During

Incubation 9 1 3 4

Eggs Set 644 654 695 504

74

Table 39. Reproductive data from female mallards fed weathered North Slope crude
oil for 22 weeks - number hatched.

 

Diet Concentration (ppm)

 

 

Parameters 0 200 2,000 20,000
Eggs Laid 730 711 778 616
Eggs Cracked 30 13 31 19
Eggs Set 644 654 695 504
Fertile Eggs 627 650 656 496
Viable 21-day Embryos 592 612 608 464
Ducklings 530 534 555 417

14-day Old Survivors 523 523 544 411

 

75

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77

33.3 .v. .3 383338 88.3 333233333 33333833333333 .

 

 

 

 

 

 

 

 

3.3 3 3.333 3.3 3 3.33 3.3 3 3.333 3.3 3 3.33 3.3 3 3.333 3.3 3 3.33 3.3 3 3.3: 3.3 3 3.33 33
3.3 3 3.333 3.3 3 3.3.3 3.3 3 3.333 3.3 3 3.33 3.3 3 3.33.3 3.3 3 3.33 3.3 3 3.33.3 3.3 3 3.33 3
3.3 3 .3333 3.3 3 3.33 3.3 3 3.333 3.3 3 3.33 3.3 3 .3333 3.3 3 3.33 3.3 3 3.333 3.3 3 3.3.3 3
3.3 3 3.333 3.3 3 .333 3.3 3 3.333 3.3 3 .333 3.3 3 3.333 3.3 3 3.33 3.3 3 3.333 3.3 3 3.33 3
3.3 3 3.33.3 3.3 3 3.33 3.3 3 .3333 3.3 3 .333 3.3 3 .3333 3.3 3 3.33 3.3 3 3.33.3 3.3 3 3.33 3
3.3 3 3.333 3.3 3 3.33 3.3 3 3.333 3.3 3 3.33 3.3 3 3.333 3.3 3 .333 3.3 3 3.333 3.3 3 3.33 3
3.3 3 3.333 3.3 3 .333 3.3 3 .3333 3.3 3 .333 3.3 3 3.333 3.3 3 .333 3.3 3 3.333 3.3 3 3.33 3
3.3 3 .3333 3.3 3 3.33 3.3 3 .3333 3.3 3 3.33 3.3 3 .3333 3.3 3 3.33 3.3 3 3.333 3.3 3 3.33 3
3.3 3 .33: 3.3 3 3.33 3.3 3 .3333 3.3 3 3.3.3 3.3 3 3.333 3.3 3 3.3.3 3.3 3 3.333 3.3 3 3.33 3
3.33 33.333 3.3 3 3.33 3.3 3 3.333 3.3 3 3.33 3.3 3 3.3.33 3.3 3 3.33 3.3 3 3.333 3.3 3 3.33 3
35-33 3333333 33.33 33333 3333.33 33333 35-33 3333 3383
33333 3333 333
335 338838.39
.333 3 333383

338.. 33 33.3 33.33 233 33 35 33.5 23333 33332 323338: 333 3323 33 33.333323 3333333 33 33 333333: .3333 3235. 333-33 333 3.3333 3833 .33 233.3.

78

Table 43. Mortalities observed in hatchlings of mallard hens fed weathered North
Slope crude oil in the diet for 22 weeks.

 

Diet Concentration (ppm)

 

#Dead/#Exposed/#Exhibiting Toxic Sign
O 6/ 529/0
200 10/ 546/0
2,000 9 / 557 / 0
20,000

7/416/0

 

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