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Biological Services Program

FWS/OBS-79/23
JULY 1979

MENT

ECOLOGICAL AND PHYSIOLOGICAL/
TOXICOLOGICAL EFFECTS OF PETROLEUM
ON AQUATIC BIRDS

A Summary of Research Activities FY76 through FY78

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interagency Energy-Environment Research and Development Program

OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY

AND

Fish and Wildlife Service

?<// 33 U.S. Department of the Interior

The Biological Services Program was established within the U.S. Fish and Wildlife Service
to supply scientific information and methodologies on key environmental issues that
impact fish and wildlife resources and their supporting ecosystems. The mission of the
program is as follows:

• To strengthen the Fish and Wildlife Service in its role as a primary source of

information on national fish and wildlfe resources, particularly in respect to
environmental impact assessment.

• To gather, analyze, and present information that will aid decisionmakers in
the identification and resolution of problems associated with major changes in
land and water use.

• To provide better ecological information and evaluation for Department of
the Interior development programs, such as those relating to energy deve-
lopment.

Information developed by the Biological Services Program is intended for use
in the planning and decisionmaking process to prevent or minimize the impact of
development on fish and wildlife. Research activities and technical assistance services are
based on anaysis of the issues, a determination of the decisionmakers involved and their
information needs, and an evaluation of the state of the art to identify information gaps
and determine priorities. This is a strategy that will ensure that the products produced
and disseminated are timely and useful.

Projects have been initiated in the following areas: coal extraction and conver-
sion; power plants; geothermal, mineral, and oil-shale development; water resource
analysis, including stream alterations and western water allocation; coastal ecosys-
tems and Outer Continental Shelf development; and systems inventory, including
National Wetland Inventory, habitat classification and analysis, and information transfer.

The Biological Services Program consists of the Office of Biological Services in
Washington, D.C., which is responsible for overall planning and management; National
Teams, which provide the Program's central scientific and technical expertise and arrange
for contracting biological services studies with states, universities, consulting firms, and
others; Regional Staff, who provide a link to problems at the operating level; and staff at
certain Fish and Wildlife Service research facilities, who conduct in house research
studies.

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FWS/OBS-79/23
JULY 1979

ECOLOGICAL AND PHYSIOLOGICAL/TOXICOLOGICAL EFFECTS
OF PETROLEUM ON AQUATIC BIRDS

A Summary of Research Activities FY76 through FY78

by
Lucille F. Stickel

and

Michael P. Dieter

Patuxent Wildlife Research Center

Fish and Wildlife Service

U.S. Department of the Interior

Laurel, Maryland 20810

Howard D. Tait, FWS Project Officer
National Coastal Ecosystems Team

U.S. Fish and Wildlife Service

NASA-Slidell Computer Complex

1010 Gause Boulevard

Slidell, LA 70458

Clinton Hall, EPA Project Officer

Environmental Protection Agency

Room 635 -West Tower (RD68T)

401 M Street S.W.

Washington, D.C. 20460

This study was conducted

in cooperation with the

Environmental Protection Agency

Office of Research and Development

Performed for

Coastal Ecosystems Project

Biological Services Program

Fish and Wildlife Service

U.S. Department of the Interior

PREFACE

This report summarizes the research conducted from 1 July 1975 to 30 September 1978
by the U.S. Fish and Wildlife Service (FWS) about the effects of petroleum on aquatic birds.
The following assessments were made:

1 . Effects of oiling on hatchability of eggs.

2. Effects of oil ingestion on physiological condition and survival of birds.

3. Effects of oil ingestion on reproduction in birds.

4. Accumulation and loss of oil by birds.

5. Development of analytical methods for identification and quantification of oil break-
down products in tissues and eggs of ducks.

Research was funded by the Environmental Protection Agency (EPA) Federal Inter-
agency Energy-Environment Research and Development Program and the FWS Environmental
Contaminants Evaluation Program, with Dr. Allan Hirsch, Chief, Office of Biological Services,
serving as FWS coordinator. Coastal Ecosystems Project contacts were Drs. William Palmisano
and Howard Tait. Mr. Clinton Hall served as EPA's coordinator. The research was conducted at
the Patuxent Wildlife Research Center, under the direction of Dr. Lucille F. Stickel and Dr.
Michael P. Dieter.

Questions about this research and requests for this publication should be directed to:

Information Transfer Specialist
National Coastal Ecosystems Team
U.S. Fish and Wildlife Service
NASA-Slidell Computer Complex
1010 Gause Blvd.
Slidell, LA. 70458

This report should be cited as follows:

Stickel, L.F., and M. P. Dieter. 1979. Ecological and physiological/toxicological effects
of petroleum on aquatic birds. U.S. Fish and Wildlife Service, Biological Services Program.
FWS/OBS-79/23. 14 pp.

ECOLOGICAL AND PHYSIOLOGICAL/TOXICOLOGICAL EFFECTS
OF PETROLEUM ON AQUATIC BIRDS

INTRODUCTION

Oil and gas development and exploration in
marine coastal areas and the Great Lakes will
result in unavoidable spills of polluting oil.
Although large oil spills may kill thousands of
birds and stimulate much public concern, the
bulk of oil that reaches aquatic environments is
released in the course of normal operations,
with a total input into the world's oceans esti-
mated at 6 million metric tons per year. The
effects of sublethal low-level oil pollution may
be more deleterious to bird populations over
the long term than the spectacular bird kills
resulting from oil spills.

The physiological and ecological effects of
oil on waterbirds were examined in a series of
laboratory and field experiments, including
studies of the effects of oiling on hatchability
of eggs; the effects of an oil-contaminated diet
on physiological condition, reproduction, and
survival; and the accumulation of oil in body
tissues. Chemical methodology was developed
in support of these studies.

EFFECTS OF OILING ON HATCHABILITY
OF EGGS

Very small quantities of oil applied to
aquatic bird eggs in the laboratory caused
embryo mortality (fig. 1). As little as 5 jul
(about one drop) of South Louisiana crude oil,
No. 2 fuel oil, Kuwait crude oil, Bunker C fuel
oil, or Prudhoe Bay crude oil applied to the
shell surface on the eighth day of incubation
reduced hatching of mallard eggs by 90%, 70%,
68%, 62%, and 26%, respectively. No embryos
survived application of 50 nl of any of the
tested oils. Fifty microliters of oil represented
approximately 1 ppm of the egg.

When 50/il of an alkane mixture of com-
pounds naturally occurring in crude oils
(paraffins) or propylene glycol was applied to
the shell surface, embryo mortality did not
occur even though the shell surface was coated.
This indicated that some other component of
the oil was affecting the embryos, and that
they were not dying because of oxygen depriv-
ation caused by clogged shell pores.

The toxicity of various oils to embryos is
not confined to mallards. Common eider, great
black-backed gull, laughing gull, Louisiana
heron, and Sandwich tern eggs were oiled arti-
ficially both in the field and the laboratory.
Variations in toxicity from that demonstrated
for mallards can be attributed to differences in
embryo size, which change the dose-to-weight
relationship, and differences in ages of embryos
at treatment (younger embryos are more
sensitive). As an example, mortality was 26% in
16 clutches of naturally incubated common
eiders treated in the nest with 20 jul of No. 2
fuel oil and opened 7 days after treatment
(table 1). Mortality was 60% among 25 clutches
of great black-backed gulls (table 2). Embryos
of both species are approximately twice the
size of mallard embryos and those tested were
of mixed ages at the time of treatment. Morta-
lity of 8-day-old mallard embryos treated with
20 /il of No. 2 fuel oil and artificially incubated
was 100%.

Thirty clutches of great black-backed gulls
treated with 20 *d of fuel oil were incubated to
term by the parents on the Isles of Shoals,
Maine. Hatching success was again one-half that
of controls and production of fledglings was
one-third that of controls. External environ-
mental factors did not appreciably modify the
toxic effects of the oil in this or other field
studies.

No. 2 fuel oil and Prudhoe Bay crude oil
were weathered over water in outdoor troughs
at the Patuxent Center. After 2 weeks of

Figure 1. Very small amounts (5 to 50 n\) of oil were applied to mallard eggs with

chemist's syringe, as shown.

Table 1. Effects of No. 2 fuel oil on embyos of the
common eider (Albers and Szaro 1978).

% of clutch alive

No. Mean Mean of

. of % clutch transformed Mean
Treatment Nests eggs alive size percentages2 % '

Control 13 60 98 4.6 75.42 93.7

No. 2 fuel

oil (5m0 19 84 94 4.4 72.54 91.0

No. 2 fuel

oil (20 Ail) 16 72 74 4.5 59.40 74.1

Three nests were found; 9 nests were abandoned or des-
troyed by predation.

2
Arcsine transformation for binomial proportions; angle

equals arcsine \J percentage. Significant one-way analysis
of variance, P <0.05; 20 jul group significantly dif-
ferent from control group, f-test, /><0.025.

^Mean of transformed percentages converted back to
percent.

Table 2. Embryo survival in naturally incubated great

black-backed gull eggs 8 days after treatment with

No. 2 fuel oil (Coon et al. 1979).

Condition of
embryo

No. of Survival No. of %

Treatment clutches Index eggs Alive Dead alive

Control 28 95.8 81 72 9 88.9

No. 2 fuel

oil (5 Ml) 26 90.3 72 58 14 80.6

No. 2 fuel

oil (20 mD 25 32.7' 72 29 43 40.3

Most clutches contained three eggs; however, some
contained only two at the time of treatment. For each
clutch, a percentage of the total embryos alive 8 days
after treatment was computed. Clutch survival data
were evaluated statistically after angular transforma-
tion, arcsin\/X. This transformation is applicable to
binomial data expressed as percentages and covering a
wide range of values (Steel and Torrie 1960). The Sur-
vival Index reported can be described by the following
expression: [sin (l/n£arcsin\/%)] 2and is a transforma-
tion back to the original scale. Statistical comparisons
were made on the transformed scale, rather than on the
reported values.

'Significantly different from control, P<0.05 (Stu-
dent's r test).

weathering, No. 2 fuel oil was significantly less
toxic than fresh oil when applied to mallard
eggs. Prudhoe Bay crude oil was less toxic than

its fresh counterpart after 3 weeks of weath-
ering. Even after the oils weathered 4 weeks,
however, 20 ix\ of each still caused 50% morta-
lity when applied to mallard eggs (fig. 2).

The minute quantities of oil that produce
marked reductions in embryonic survival sug-
gest that oil pollution could seriously affect
marine and estuarine bird populations by trans-
fer of oil from the plumage of incubating birds
to their eggs. Laughing gulls were captured at
their nest sites in Texas to examine this possi-
bility. Forty-two were treated with 2.5 ml of
No. 2 fuel oil applied with a syringe to the
feathers over and surrounding their brood
patches. Twenty additional birds were treated
with water. All were released immediately after
treatment. After 5 days, their eggs were col-
lected and examined. Embryo mortality was
41% in the eggs incubated by the oiled gulls ,
but only 2% in those incubated by the water-
treated gulls (table 3).

Paired mallards were kept in pens con-
taining water troughs in which they could swim
during the breeding season. Prudhoe Bay crude
oil was added to the water during the first
week of incubation. One-third of the troughs
were treated with 100 ml of oil per square
meter of surface area, another one-third were
treated with 5 ml of oil per square meter, and
the last group were left untreated. Hatching
success was 45%, 85%, and 95% respectively.

Other indications of toxicity include tera-
togenic effects (production of malformed indi-
viduals) when oil is applied to eggs during the
first few days of embryo development. Treat-
ment of mallard eggs at 24 hr of development
with 5 /il of crude oil produced a significant
number of abnormal survivors (table 4). The
most common abnormalities included de-
formed bills, incomplete ossification of the
wing or foot bones, reduction in size of the
liver lobes, and stunting. Teratogenicity was
increased when vanadium, nickel and mercury,
metals normally found in petroleum, were
added.

Mortality was greater when artificially
formulated mixtures of aromatic compounds
were applied to the egg surface than in controls

1/1
on

UJ

o

un

o

OIL WEATHERING TIME (WEEKS)

ioo r

£ 60 -

<

>

>
en

on 40 -

20 -

50 Mld

i/->

un
o

U1

O

2 3 4 0

C OIL WEATHERING TIME (WEEKS)

Figure 2. Survival 6 days after treatment and hatching success of mallard embryos treated (on
the 8th day of incubation) with petroleum hydrocarbons (A and B, No. 2 fuel oil; C and D,
Prudhoe Bay crude). Note: Treatments or weeks that do not have a letter in common were sig-
nificantly different at a<0.05, using Tukey's multiple comparison procedure. Differences be-
tween controls and 5 nl of fresh oil (weathered 0 weeks) were significant at a<0.05, using a
binomial test. Sample size is 47 for all groups except the controls, where «=94 (Szaro et al.
1979).

or those treated with the alkanes or paraffin
components. Mortality was particularly en-
hanced by the presence of the tetracyclic aro-
matic chrysene even at half its normal concen-
tration in oil (0.2% in Kuwait crude oil and
0.5% in South Louisiana crude oil).

Table3. Embryonic mortality in eggs of laughing gulls

with breast feathers treated with 2.5 ml No. 2 fuel

oil (King and Lefever unpubl).

Na

Dead

Infertile

Group

No. %

No. %

Control
Oil-treated

53
105

1 2

43 41b

2 4

2 2

N=number of eggs recovered after 5 days of incuba-
tion.

Significantly different from controls, P<0.01, x2test.

EFFECTS OF OIL INGESTION ON PHYSI-
OLOGICAL CONDITION AND SURVIVAL
OF BIRDS

In two 6-month studies, adult mallards
were fed South Louisiana crude oil (SLC) and
an artificial reconstituted aromatic mixture
(RAM) composed of 10 aromatic and 9 ali-
phatic compounds typical of those found in
South Louisiana crude oil. SLC was fed at
0.25% and 2.5% of the total diet and the RAM
was fed at 0.04% and 0.4% of the diet (levels
that represent the aromatic content of diets
containing 0.25% and 2.5% oil). Adult mallards
could realistically ingest these concentrations
of oil at spill sites. All birds maintained their
body weights and ingested contaminated food
at rates comparable to ingestion of clean food
by control animals. There was no mortality in
either study. Indocyanine green dye, a com-
pound that is metabolized by the liver, was used

Table 4. Effects of South Louisiana crude oil on mallard duck embryos (Hoffman 1978).

Control

Paraffin

Crude oil

1/il

5 Hi

Number treated

65

65

65

65

Percentage survival (days 3-18)

97

94

65a

9a

Sex ratio, M: F

45:55

44:56

53:57

50:50

Embryonic weight (g)

15.72±1.78*

Male

16.34H.49

16.2911.89

15.8310.49

Female

15.75±2.03

15.98+1.58

15.3311.77

14.90+3. 15c

Combined

15.74+1.90

16.1411.54

15.7411.82

15.7311.84

Crown-rump length (mm)

Male

84.1±3.7

84.914.0

84.913.8

86.7+5.1

Female

82.7±3.7

83.413.0

80.114.4

70.213.8C

Combined

82.8±3.7

84.U3.5

82.214.1

77.314.5C

Bill length (mm)

Male

13.2±0.9

13.310.5

13.010.6

13.010.6

Female

13.1+0.6

12.9+0.6

12.710. 7C

10.810.9f

Combined

13.1+0.7

13.H0.6

12.810. 7C

ll^+O^

Percentage that were abnormal

survivors

4.8

3.3

4.8

66.7"

"Significantly different from control and paraffin-treated groups by yi, p-<0.01.

*Mean +SD.

^Significantly different by one-way analysis of variance (p<0.01) and the Duncan multiple range test (p<0.05).

These included abnormal conjoined (1.6%) and stunted (3.2%) in the control group: hydrocephaly with micro-
phthalmia and beak defect (1.6%) and incomplete ossification of ribs and vertebrae (1.6%) in the paraffin group;
cervical vertebrae missing (2.4%) and incomplete ossification of sacral vertebrae (2.4%) in the 1-^1 of crude oil
group; incomplete ossification of the phalanges (16.7%); incomplete ossification of the ischium (16.7%), severe
edema with blisters (16.7%). and abnormal feather formation (16.7%) in the 5-pil of crude oil group.

to measure liver function in the mallard drakes
fed the RAM. Plasma clearance rates were
enhanced in the drakes fed the RAM at 0.4% of
the diet, suggesting that adult waterfowl may
be able to increase liver function to eliminate
high concentrations of petroleum hydro-
carbons. Although several plasma enzymes
(enzymes that appear in the blood because of
damage to specific organs) and electrolytes were
monitored monthly in both studies, none were
elevated above control levels. Adult waterfowl
are apparently able to adapt to and tolerate
high concentrations of petroleum hydro-
carbons in their diet when not otherwise
stressed. However, other adult mallards, given
seawater as drinking water and subjected to
mild cold stress (3° C) while being fed food
contaminated with 3% Kuwait or South Loui-
siana crude oil, died (table 5).

Mallard ducklings survived from hatching
to 8 weeks of age on diets containing 0.025%
to 5% South Louisiana crude oil or No. 2 fuel
oil. Those fed 2.5% and 5% oil, however, were
stunted and failed to develop flight feathers
(fig. 3). Subtle biochemical and behavioral
changes were detected in ducklings fed as little
as 0.25% oil. It is not surprising that young
birds would be more seriously affected than
adults because they are in the critical rapid
growth phase.

I able 5. The effects of petroleum-contaminated diets

on the mortalities of scawatcr-adaptcd ducks main-

tained for 50 days at 27° C followed bj a 50-day period

of continuous mild cold stress at 3° C (adapted from

Holmes et al. 1978).

EFFECTS OF OIL INGESTION ON
REPRODUCTION IN BIRDS

Mallard hens fed diets containing 2.5%
South Louisiana crude oil (25,000 ppm) for a
6-month period produced 50% as manv eggs as
controls. Over a 90-day period, the oil-dosed
birds laid an average of 35 eggs per hen, com-
pared with 69 for controls (table 6). Although
fewer eggs were laid by the oil-dosed birds,
those that were laid hatched as well as control
eggs when artificially incubated, and the hatch-
lings weighed as much as control hatchlings. In
contrast, hens on diets containing 0.25% (2,500
ppm) of oil performed nearly as well as controls.

Dr. W. Holmes (under contract to the Patux-
ent Wildlife Research Center) reported similar
results in studies with South Louisiana and Ku-
wait crude oils. Egg laying was not affected In
1% of either oil in the diet. Egg laying decreased
by 75% on a diet containing 3% South Louisiana
crude oils and completely ceased on a diet con-
taining 3% Kuwait crude oils.

Paired mallards were fed vanadium, a metal
contaminant of crude oils. The vanadium accu-
mulated to higher concentrations in the bone
and liver than in other tissues. Concentrations in
the bones of hens were five times those in the
bones of drakes, suggesting an interaction be-
tween vanadium and calcium mobilization in
laying hens. Lipid metabolism was altered within
3 weeks in laying hens fed 100 ppm vanadium
and within 12 weeks in hens fed 10 ppm vana-
dium. Vanadium concentrations in crude oil can
range up to 1,400 ppm.

M

("o of orig

inality

nul population)

Table 6. Mean egg production ot mallards ted diets
containing South Louisiana crude oil. Total numbers ot

No. ill

Before

During

eggs laid in parent

leses (Coon

and Dieter unpubl).

liird>

cold stress

cold stros

Total

Krcshwater-main-

10

0

20

20

Treatment

Sampling period

taincd controls

Days 1-30

Days 1-60

Dav 1-90

Seawater-main-

Control

24.6"'

49.0''

69 o"'

cained controls

10

0

(.11

60

(197)

(392)

(552)

South Louisiana
crude oil

9

22.2

66.7.

88.9

10,000 ppm paraffin

2 3.6''
(189)

47. lab

(377)

68.6"
(549)

Kuwait crude oil
No. 2 fuel oil

9

1 1

0
35.7

66.7
42.7

66.7
78.4

2,500 ppm SLC
25,000 ppm SLC

19.3"''
(154)

n.o''

(88)

37.1"''
(297)

27.3*

(218)

52.1" ''
(417)

34 V
(279)

'Means followed b\ different letters are significant!)
different for a given sampling period, Scheffe's test.
P<0.05.

aba abb c

O)

s

>■

•a
a

14-

13-

12

11-

10-

9

8

7

6

5

4

3_

2-

1

0

a a a b

a a a
* * *

;; ffH* 1

TTT

1 2 3

Mill

12 3 4 5

T I I I I

12 3 4 5

I I I II

12 3 4 5

3

I I I I

12 3 4

4

TT

1 2

TTT

3 4 5

5

TTT

1 2 3

TT

4 5

TTT

1 2 3

1 I I I

2 3 4 5

■14
■13
■12
•11
•10

■ 9

■ 8
7

■ 6

• 5

• 4
3
2

. 1
, 0

TT

4 5

i r

4 5

Figure 3. Growth in mallard ducklings fed (1) control diet; (2) 0.025% South Louisiana

crude oil (SLC); (3) 0.250% SLC; (4) 2.5% SLC; and (5) 5.0% SLC. Treatments within a

given age group that do not have a letter in common were significantly different at P<0.05

by Scheffe's (1959) procedure for pairwise comparisons (Szaro et al. 1978).

ACCUMULATION AND LOSS OF OIL
BY BIRDS

After consideration of several invertebrate
and waterfowl species, the freshwater crayfish
and mallard were selected for food-chain studies.
Both can be successfully maintained in sufficient
numbers in the laboratory and are available from
commercial suppliers. Crayfish are accepted by
mallards as a food item.

Food-chain studies were performed to esti-
mate the uptake of aromatic compounds and
their distribution in mallard tissue after inges-
tion of contaminated crayfish. Crayfish were
exposed to the water-soluble fraction of No. 2
fuel oil containing known quantities of
[14C] naphthalene. (The water-soluble fraction
was prepared by gently stirring a mixture of the
oil and water for 20 hr and siphoning off the
aqueous layer.) After exposure, the live cray-
fish were fed to ducks. After an appropriate
time interval, the birds were sacrificed and their

tissues examined for radioactivity.

Radioactive naphthalene was readily taken
up and underwent biomagnification in the cray-
fish. Accumulation of radioactivity in ducks fed
the crayfish was greatest in the gall bladder,
followed by fat, kidney, liver and blood (table
7).

Table 7. Radioactivity in tissues of male and female
mallard ducks fed 14C-naphthalene-No. 2 fuel oil con-
taminated crayfish (Tarshis unpubl.).

Naphthalene (ppm)

Tissues

Gall bladder

Fat

Kidney

Liver

Blood

Testes

Egg
Oviduct

6

9

1.040

0.496

0.149

0.114

0.045

0.058

0.045

0.032

0.037

0.057

0.011

—

0.020

0.013

DEVELOPMENT OF ANALYTICAL

METHODS FOR IDENTIFICATION AND

QUANTIFICATION OF OIL BREAKDOWN

PRODUCTS IN TISSUES AND EGGS OF

DUCKS

Efforts of chemists involved in this project
have been directed toward several goals:

• The development of methods for the detec-
tion and analysis of hydrocarbons in avian
tissue

• The development of methods of quantifi-
cation of individual compounds and com-
plex mixtures in avian tissues

• Provision of analytical support for biological

studies

A number of problems are encountered in
the analysis of petroleum hydrocarbons in avian
tissue. By their ubiquitous nature, these hydro-
carbons pose continuing contamination prob-
lems for sample analysis. The heterogeneity of
the mixtures necessitates the use of complex
extraction and cleanup procedures. The presence
of hundreds of compounds creates a need for
high-resolution instruments.

During our early attempts to analyze sam-
ples, very high levels of contamination were
detected. To reduce contamination, glass extrac-
tion thimbles cleaned in chromic acid were
substituted for paper thimbles. Later, in simpli-
fying our procedures, we eliminated thimbles
altogether, substituting a Polytron tissue homo-
genizer/extractor for Soxhlet extraction. The
use of nitrogen gas to blow down samples was
abandoned in favor of a flash evaporator. Glass-
ware was at first soaked in chromic acid; this
was an effective but hazardous procedure.
Chromic acid was replaced by an equally effec-
tive but safer technique of soaking the glass in
an ultrasonic bath with 2% 'Micro' solution.

Briefly, the method of analysis that is being
used in our laboratory is as follows.

The sample is cut into small pieces and me-
chanically extracted with a homogenizer. Bases
are partitioned into acid from pentane and ana-
lyzed by gas chromatography /mass spectrometry.
After the extraction of bases, pentane extracts
from fat samples are saponified. The saponified
mixture is partitioned into hexane.

The hexane or pentane layer from samples
of fat, liver, and kidney (<0.5 g lipid) are
cleaned up on Morisil; the aliphatic and aroma-
tic hydrocarbons are separated on a silicic acid

column; and the residues are screened and ana-
lyzed by gas chromatography and gas chroma-
tography/mass spectrometry/data system.

Livers, kidneys, and fat from untreated mal-
lard drakes and drakes fed 0.4% of the reconsti-
tuted aromatic mixture for 6 months were ana-
lyzed in our laboratory. Significantly higher resi-
dues were found in the tissues of ducks fed the
aromatic mixture than in tissues from controls.

Researchers at the University of New Orleans
developed a hydrocarbon analysis procedure
similar to the one in use at our laboratory while
under contract to the Patuxent Wildlife Re-
search Center. Livers and kidneys from mallard
ducklings and adult birds fed South Louisiana
crude oil were analyzed. There were no signifi-
cant differences in hydrocarbon accumulation in
tissues between adult males and females. Hydro-
carbon concentrations in both ducklings and
adults increased from the control group through
those fed 2.5% oil in the diet.

The research group at the University of New-
Orleans also analyzed tissues from four birds
killed at the Amoco Cadiz oil spill site off the
coast of France. One, a shag (cormorant), was
heavily contaminated with hydrocarbons; the
other three, a herring gull, a razorbill, and a guil-
lemot, contained smaller amounts.

LITERATURE CITED

Albers, P. H., and R. C. Szaro. 1978. Effects of
No. 2 fuel oil on common eider eggs. Mar.
Poll. Bull. 9:138-139.

Coon, N. C, P. H. Albers, and R. C. Szaro.
1979. No. 2 fuel oil decreased embryonic
survival of great black-backed gulls. Bull.
Environ. Contam. Toxicol. 21:152 -156.

Coon, N. C, and M. P. Dieter. Effects of dietary
ingestion of crude oil on condition and sur-
vival of mallard ducks. Patuxent Wildlife
Research Center Study Plan Progress Report,
October 1, 1976-September 30, 1977
(unpubl).

Hoffman, D. J. 1978. Embryotoxic effects of
crude oil in mallard ducks and chicks.
Toxicol. Appl. Pharmacol. 46:183-190.

Holmes, W. N., J. Cronshaw, and J. Gorsline.
1978. Some effects of ingested petroleum on
seawater-adapted ducks (Ancsplatyrh ynchos ).
Environ. Res. 17:177-190.

King, K. A., and C. A. Lefever. Effects of oil
transferred from incubating gulls to their
eggs (unpubl).

Szaro, R. C, N. C. Coon, and W. Stout. Wea-
thered oil: effects on mallard egg hatcha-
bility. J. Wildl. Manage. In press.

Szaro, R. C, M. P. Dieter, G. H. Heinz, and J. F.
Ferrell. 1978. Effects of chronic ingestion of
South Louisiana crude oil on mallard duck-
lings. Environ. Res. 17:426-436

Tarshis, I. B. Oil toxicity to aquatic birds by the
ingestion of oil-contaminated invertebrates.
Patuxent Wildlife Research Center Study
Plan Progress Report, October 1, 1977-June
30, 1978.

APPENDIX A

PUBLISHED REPORTS RESULTING FROM THE HYDROCARBON TOXICITY

STUDIES AS OF 1 MAY 1979

Albers, P. II. 1977. Effects of external appli-
cations of fuel oil on hatchability of mallard
eggs. Pages 158-163 in Wolfe, D. A., ed. Fate
and effects of petroleum hydrocarbons in
marine ecosystems and organisms. Pergamon
Press, New York.

1978. The effects of petroleum on dif-

ferent stages of incubation in bird eggs. Bull.
Environ. Contam. Toxic. 17:624-630.

, and R.C.Szaro. 1978. Effects of No. 2

fuel oil on common eider eggs. Mar. Poll.
Bull. 9:138-139.

Coon, N. C, P. H. Albers, and R. C. Szaro.
1979. No. 2 fuel oil decreases embryonic
survival of great black-backed gulls. Bull.
Environ. Contam. Toxic. 21 : 152-156.

Dieter, M. P. 1976. The effects of petroleum
hydrocarbons on aquatic birds. Pages 438-
446 in Proceedings of symposium on
sources, effects, and sinks of hydrocarbons
in the aquatic environment. American
Institute for Biological Sciences, Washing-
ton, D. C.

.1977. Acute and chronic studies with

waterfowl exposed to petroleum by hydro-
carbons. Pages 35-42 in Hall, C, and W.
Preston, eds. Program review proceedings
of: environmental effects of energy related
activities on marine estuarine ecosystems.
Interagency Energy-Environment Research
and Development Program Report. EPA-
600/7-77-111. Washington, D. C.

Eastin, W. C, and D. J. Hoffman. 1978. Biologi-
cal effects of petroleum on aquatic birds.
Pages 561-582 in Proceedings of the confer-
ence on assessment of ecological impacts of
oil spills. American Institute for Biological
Sciences, Washington, D.C.

Hoffman, D. J. 1978. Embryotoxic effects of
crude oil in mallard ducks and chicks. Toxi-
col. Appl. Pharmacol. 46: 183-190.

.. 1978. Embryotoxic effects of petro-

leum hydrocarbons in avian embryos. Tera-
tology 17(2).-40A (abstract).

Holmes, W. N., J. Cronshaw, and J. C-orsline.
1978. Some effects of ingested petroleum on
seawater-adapted ducks (Anas platyrbyn-
chos). Environ. Res. 17:177-190.

Eawler, G. C, J. P. Holmes, B. J. Fiorito, and L.
L. Laseter. 1978. Quantification of petro-
leum hydrocarbons in selected tissues of
male mallard ducklings chronically exposed
to south Louisiana crude oil. Pages 583-612
in Proceedings of the conference on assess-
ment of ecological impacts of oil spills.
American Institute for Biological Sciences,
Washington, D.C.

Lawler, G. C, W. A. Loong, B. J. Fiorito, D. R.
Carlisle, and J. L. Laseter. 1978. An auto-
mated glass capillary gas chromatographic
system for routine quantitative analysis. J.
Chromatogr. Sci. 15:532-536.

Lawler, G. C, W. A. Loong, and J. L. Laseter.
1978. Accumulation of saturated hydrocar-
bons in tissues of petroleum exposed mallard
ducks (Anas platyrbyncbos). Environ. Sci.
Technol. 11:47-51.

1978.

Accumulation of aromatic
hydrocarbons in tissues of petroleum ex-
posed mallard ducks (Anas platyrbyncbos).
Envion. Sci. Technol. 11:51-54,

Patton, J. F. 1978. Indocyaninc green: a test of
hepatic function and a measure of plasma
volume in the duck. Comp. Biochem.
Physiol. 60A:21-24.

Szaro, R. C. 1977. Effects of petroleum on
birds. Pages 374-381 in Transactions of the

10

42nd North American Wildlife and Natural
Resources Conference.

., and P. H. Albers. 1977. Effects of ex-

ternal applications of No. 2 fuel oil on com-
mon eider eggs. Pages 164-167 in Wolfe, D.
A., ed. Fate and effects of petroleum hydro-
carbons in marine ecosystems and organisms.
Pergamon Press, New York.

_, and N. C. Coon. 1978. Petroleum:

effects on mallard egg hatchability. J. Wild-
life Manage. 42:404-406.

Szaro, R. C, M. P. Dieter, G. H. Heinz, and
J. F. Ferrell. 1978. Effects of chronic in-
gestion of South Louisiana crude on mallard
ducklings. Environ. Res. 17:426-436.

Tarshis, I. B. 1978. Diets, equipment, and tech-
niques for maintaining crayfish in the lab-
oratory. Bull. Ecol. Soc. Am. 59:59-60.

1978. Diets, equipment, and tech-

niques for maintaining crawfish in the lab-
oratory. Pages 259-269 in Proceedings of the
ninth annual meeting of the world maricul-
ture society. Louisiana State Univ., Baton
Rouge.

White, D. H., K. A. King, and N. C. Coon. 1979.
Effects of No. 2 fuel oil on hatchability of
marine and estuarine bird eggs. Bull. Envi-
ron. Contam. Toxicol. 21 :7-10.

11

APPENDIX B

UNPUBLISHED REPORTS RESULTING FROM THE HYDROCARBON TOXICITY

STUDIES AS OF 1 MAY 1979

Albers, P. H. Oil dispersants and wildlife. Manu-
script submitted to Proceedings of the 1979
U.S. Fish and Wildlife Service Pollution Re-
sponse Workshop, St. Petersburg, Fla., May
8-10, 1979.

.. Transfer of crude oil from contami-

nated water to bird eggs. Study Plan Progress
Report, October 1, 1977-September 30,
1978.

Belisle, A., and M. Gay. Development of analy-
tical methodology for separation, detection,
and measurement of petroleum residues in
tissues and eggs. Work Unit Progress Re-
ports, November 1, 1976-September 30,
1978.

Coon, N. C, and M. P. Dieter. Effects of dietary
ingestion of crude oil on condition and survi-
val of mallard ducks. Study Plan Progress
Reports, October 10, 1967-September 30,
1978.

Custer, T. W., and P. H. Albers. Response of cap-
tive breeding mallards to oiled water. Manu-
script submitted to Bull. Environ. Contam.
Toxicol.

Hoffman, D. J. Embryotoxic effects of crude oil
containing nickel and vanadium in mallards.
Bull. Environ. Contam. Toxicol. In press.

Embryotoxic and teratogenic effects of

petroleum hydrocarbons in mallards (Anas
platyrynchos). J. Toxicol. Environ. Health
In press.

Embryotoxic and teratogenic effects of

crude oil on mallard embryos on day one of
development. Bull. Environ. Contam. Toxi-
col. In press.

J. Cronshau, and K. P. Cavanaugh.

j>ome effects of ingested petroleum on ovi-

position and reproduction in experimental
colonies of mallard ducks (Anas platyrhyn-
chos). Part II in Studies on the chronic
effects of ingested petroleum in mallard
ducks (Anas platyrhyncbos). Progress Report
1976-1978. U.S. Fish and Wildlife Service
Contract No. 14-16-0008-2032.

Holmes, W. N., J. Cronshaw, and J. Gorsline. Ef-
fects of mild cold stress on the mortality of
seawater-adapted mallard ducks (Anas platy-
rynchos) maintained on petroleum-contami-
nated food. Part I in Studies on the chronic
effects of ingested petroleum in mallard
ducks (Anas platyrynchos). Progress Report
1976-1978. U.S. Fish and Wildlife Service
Contract No. 14-16-0008-2032.

Effects of ingested petroleum on the

synthesis and metabolism of steroid hor-
mones. Part III in Studies on the chronic ef-
fects of ingested petroleum in mallard ducks
(Anas platyrynchos). Progress Report 1976-
1978. U.S. Fish and Wildlife Service Con-
tract No. 14-16-0008-2032.

King, K. A., and C. A. Lefever. Effects of oil
transferred from incubating gulls to their
eggs. Manuscript submitted to Mar. Poll. Bull.

King, K. A., S. Macko, P. L. Parker, and E.
Payne. Consequence of an oil spill: probable
brown pelican fatality. Manuscript submit-
ted to Mar. Poll. Bull.

Laseter, J. L. Quarterly Progress Reports, Janu-
ary 15, 1976-August 31, 1977. Gas chroma-
tography-mass spectrometry-computer ana-
lytical support program for the study of
petroleum hydrocarbons in avian tissues and
related materials. U.S. Fish and Wildlife Ser-
vice Contract No. 14-16-0008-203 1 .

, and G. C. Lawler. Quarterly Progress

Report, December 1, 1977-February 28,

12

1978. Gas chromatography-mass spectrome-
try-computer analytical support program for
the study of petroleum hydrocarbons in
avian tissues and related materials. U.S. Fish
and Wildlife Service Contract No. 14-16-
0008-2031.

Final Report, February 28, 1977. Gas

chromatography-mass spectrometry-compu-
ter analytical support program for the study
of petroleum hydrocarbons in avian tissues
and related materials. U.S. Fish and Wildlife
Service Contract No. 14-16-0008-2031.

Lawler, G. C. Quarterly Progress Reports, Sep-
tember 1, 1977-November 30, 1977; June 1,
1978-November 30, 1978. Gas chromatogra-
phy-mass spectrometery-computer analytical
support program for the study of petroleum
hydrocarbons in avian tissues and related
materials. U.S. Fish and Wildlife Service
Contract No. 14-16-0008-2031.

Patton, J. F., and M. P. Dieter. Effects of petro-
leum hydrocarbons on hepatic function in
the duck. Manuscript submitted to Comp.
Biochem. Physiol.

Szaro, R. C. Effects of No. 2 fuel oil on growth
rate and kidney and liver function of mallard
ducklings. Study Plan Progress Report, Octo-
ber 1, 1977-June 30, 1978.

.. Bunker C fuel oil reduces mallard egg

hatchabiliry. Bull. Environ. Contam. Toxi-
col. In press.

. N. C. Coon, and W. F. Stout. Weathered

petroleum: effects on mallard egg hatchabi-
lity. Manuscript submitted to J. Wildl.
Manage.

Tarshis, I. B. Oil toxicity to aquatic birds by the
ingestion of oil-contaminated invertebrates.
Study Plan Progress Report, October 1,
1977-September 30, 1978.

13

APPENDIX C
MILESTONE SCHEDULE

Mlelto^T FiscaLyeai^

FY75

FY 76

FY77

FY78

1. Acquired staffing

•

•

•

2. Development of analytical methods for
identification and quantification of oil
breakdown products in tissues and eggs
of ducks.

Preliminary report

Final report

•

•

3. Effects of oil ingestion on reproduction
in birds

Annual report
Final report

•

•

•

4. Effects of oil ingestion on physiological
condition and survival of birds
Annual report

Summary report of work on ducks
with report of pilot comparisons
with seabirds

•

•

•

5. Effects of oiling on hatchability of eggs
Annual report
Final report

•

•

•

6. Accumulation of oiled birds
Annual report
Summary report of work on ducks

•

•
•

7. Assessment of joint action of oil and toxic
chemicals on survival and reproduction
Report on experiments

•

•

14