OCS Study 

MMS 2002-009 

Coastal Marine Institute 

Effects of Simultaneous Exposure 

to Petroleum Hydrocarbons, Hypoxia, 

and Prior Exposure on the Tolerance and 

Sublethal Responses of Marine Animals : 

Blue Crabs and Killifish 

Final Report 

U.S. Department of the Interior CooperativeAgreement 

Minerals Management Service Coastal Marine Institute 

Gulf of Mexico OCS Region Louisiana State University 
OCS Study 

MMS 2002-009 

Coastal Marine Institute 

Effects of Simultaneous Exposure 

to Petroleum Hydrocarbons, Hypoxia, 

and Prior Exposure on the Tolerance and 

Sublethal Responses of Marine Animals : 

Blue Crabs and Killifish 

Final Report 

Author 

William B . Stickle, Jr. 

March 2002 

Prepared under MMS Contract 

14-35-0001-30660-19914 

by 

Coastal Studies Institute 

Louisiana State University 

Baton Rouge, Louisiana 70801 

Published by 

US. Department of the Interior cooperative Agreement 

Minerals management Service Coastal Marine Institute 

Gulf of Mexico OCS Region Louisiana State University 
DISCLAIMER 

This report was prepared under contract between the Minerals Management Service (MMS) and 

Louisiana State University. This report has been technically reviewed by the MMS and approved for 

publication . Approval does not signify that the contents necessarily reflect the views and policies of the 

Service, nor does mention of trade names or commercial products constitute endorsement or 

recommendation for use. It is, however, exempt from review and compliance with MMS editorial 

standards. 

REPORT AVAILABILITY 

Extra copies of the report may be obtained from the Public Information Office (Mail Stop 5034) at the 

following address: 

U.S. Department of the Interior 

Minerals Management Service 

Gulf of Mexico OCS Region 

Public Information Office (MS >034) 

1201 Elmwood Park Boulevard 

New Orleans, LA 70123-2394 

Telephone Number: (504)7-')6-2519 or 

1-800-200-GULF 

CITATION 

Suggested citation : 

Stickle, W.B . 2002 . The Effects of Simultaneous Exposure to Petroleum Hydrocarbons, Hypoxia and 

Prior Exposure on the Tolerance and Sublethal Responses of Marine Animals, Blue Crabs and Killifish: 

Final Report . U.S. Dept. of the Interior, Minerals Management Service, Gulf of Mexico OCS Region, 

New Orleans, Louisiana . OCS Study . MMS 2002-009 . 18 pp . 

ABOUT THE COVER 

The Minerals Management Service gratefully acknowledges the generous permission from Dr. William 

Lang to use his copyrighted O photograph on the front cover. 
ABSTRACT 

Coastal Louisiana has been impacted by oil field activities for the last half century and the waters 

over the continental shelf west of the Mississippi River delta are also subject to extensive periods of 

hypoxia during the summer months. The determination of 28 day tolerance and RNA :DNA ratios, EROD 

activity, growth condition (K) and the liver-somatic index have been used as sublethal biomarkers have 

been used to assess the effects of gradients of oxygen tension and the WSF of South Louisiana crude oil 

alone and in concert were determined for juvenile blue crabs and lesser blue crabs (tolerance and 

RNA:DNA ratios only) and the Gulf killifish . 

Chronic exposure to sublethal concentrations of the WSF of crude oil leads to increased 

tolerance and condition of the Gulf killifish when exposed to the WSF of South Louisiana crude oil . 

Exposure to the WSF under hypoxia produces slight increases in sensitivity to crude oil in comparison 

with exposure under normoxia. Blue crabs and lesser blue crabs are not as sensitive to the additive 

effects of hypoxia and the WSF of South Louisiana crude oil nor to prior exposure history . RNA:DNA 

ratios are not a sensitive indicator of sublethal WSF stress in the Gulf killifish , the blue crab or the 

lesser blue crab. EROD activity, as an indicator of Cytochrome p-450 induction increases in a dose 

related manner in the liver of Fundulus grandis from both locations on both a specific activity and total 

liver activity basis. Fundulus grandis exhibits enhanced adaptations to chronic petroleum hydrocarbon 

exposure but neither juvenile Callinectes sapidus nor Callinectes similis exhibit enhanced adaptation to 

petroleum hydrocarbons with chronic exposure. 

iii 
TABLE OF CONTENTS 

Page 

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v 

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi 

1 .0 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 

2.0 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 

3 .0 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 

3 .1 28-day Tolerance Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 

3 .2 Sublethal Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 

3.2 .1 Blue Crab and Lesser Blue Crab RNA:DNA Ratios . . . . . . . . . . . . . . . 4 

3.2 .2 Sublethal Responses of Gulf Killifish . . . . . . . . . . . . . . . . . . . . . . . . . . 4 

3.2.2 .1 Growth, Condition Factor and Liver-somatic Indices . . . . . . . 5 

3.2.2 .2 Liver RNA:DNA Ratios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 

3.2.2 .3 Liver EROD Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 

3.2 .2 .4 White Muscle RNA :DNA Ratios . . . . . . . . . . . . . . . . . . . . . . 15 

4.0 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 

Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 

iv 
LIST OF FIGURES 

Page 

Figure 

3.1 Mean total body weights (x + SE) of Fundulus grandis from Pass Fourchon 

and from Florida in hypoxia (73 mm Hg OZ) control, hypoxia + low, and 

hypoxia + high water soluble fraction treatments of South Louisiana crude oil . . . . . 6 

3.2 Mean liver weights + SE of Fundulus grandis from Florida and Pass Fourchon 

over 28 days in hypoxia (73 mm Hg Oz) control, hypoxia + low, and hypoxia + high 

water soluble treatments of South Louisiana crude oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 

3 .3 Mean EROD activity (picomol resorufin " min "' " mg protein -' ) + SE of 

Fundulus grandis from Florida and Pass Fourchon over 28 days in hypoxia 

(73 mm Hg OZ) control, hypoxia + low, and hypoxia + high water soluble 

treatments of South Louisiana crude oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 

3 .4 Total liver EROD activity (picomol resorufin " min ` - total hepatic protein -') 

+ SE of Fundulus grandis from Florida and Pass Fourchon over 28 days in hypoxia 

(73 mm Hg OZ) control, hypoxia + low, and hypoxia + high water soluble treatments 

of South Louisiana crude oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 

v 
LIST OF TABLES 

Table 

Page 

3 .1 LCso values after 28 days exposure of Callinectes sapidus, C. similis, and 

Fundulus grandis to hypoxia, and the water soluble fraction (WSF) of South 

Louisiana crude oil under normoxia (156 mm Hg OZ) and hypoxia (75 mm Hg 

OZ for F. grandis and 113 mm Hg OZ for C. sapidus and C. similes) . . . . . . . . . . . . . . . . . . . 4 

3 .2 Condition Factor (body weight (mg) / length (mm)' x 100) + SE in Fundulus 

grandis from Florida and Pass Fourchon over 28 days in normoxia (156 mm Hg Oz) 

control, hypoxia (73 mm Hg 02) control, hypoxia + low and hypoxia + high water 

soluble treatments of South Louisiana crude oil (n=16). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 

3.3 Liver somatic Index (LSI, liver wt (mg) / body wt (mg) x 100) + SE in 

Fundulus grandis from Florida and Pass Fourchon over 28 days in normoxia 

(156 mm Hg OZ) control, hypoxia (73 mm Hg Oz) control, hypoxia + low, and 

hypoxia + high water soluble treatments of South Louisiana crude oil (n=16). . . . . . . . . . . . . 9 

3.4 Liver RNA :DNA ratios + SE of Fundulus grandis from Florida and Pass 

Fourchon over 28 days in normoxia (156 mm Hg OZ), hypoxia (73 mm Hg OZ), 

hypoxia + low, and hypoxia + high WSF treatments of South Louisiana 

crude oil (n=16). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 

3.5 RNA:DNA ratios from the white muscle of Fundulus grandis exposed to the WSF 

of South Louisiana crude oil for 28 days. WSF concentrations are given as ng.l"1 total 

aromatics and aliphatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 

vi 
1 .0 INTRODUCTION 

Coastal Louisiana has been impacted by oil field activities for the last half century and the waters 

over the continental shelf west of the Mississippi River delta are also subject to extensive periods of 

hypoxia during the summer months. These hypoxic events are known locally as dead water zones 

because of a paucity of fauna in the water mass during their development . 

Marine and estuarine fish are sensitive to exposure to the water soluble fraction (WSF) of crude 

oil but their tolerance doesn't change much after 12-24h exposure because of the presence of an 

inducible cytochrome P-450 enzyme system for metabolizing aromatic hydrocarbons as illustrated by the 

response of coho salmon, Oncorhynchus kisutch (Stickle et al., 1982). All mortalities occurred during the 

first two days of exposure to toluene and naphthalene even though the experiment ran for 42 days. In 

contrast invertebrates tolerate high concentrations of the WSF for short time periods but their tolerance 

declines during 28 days of exposure (Stickle et al ., 1987; Wang and Stickle, 1988). 

The determination of long term tolerance and two sublethal biomarkers have been used to assess 

the effects of gradients of oxygen tension and the WSF of crude oil on marine and estuarine fauna (Das 

and Stickle, 1993; Kapper and Stickle, 1987; Stickle et. al ., 1989; and Wang and Stickle, 1988). 

This project was designed to test the following null hypotheses: 

Null hypothesis 1 : The WSF of crude oil and hypoxia gradients acting alone and in concert do not alter 

the 28 day LCso of the Gulf killifish, Fundulus grandis, or two conspecific species of portunid crabs, 

Callinectes sapidus and C. similis . 

Null hypothesis 2: Prior exposure to sublethal concentrations of the WSF of crude oil and hypoxia acting 

alone and in concert do not alter the tolerance, RNA:DNA ratio or in all three species and EROD activity 

in the Gulf killifish . In addition, an attempt was made to determine polyamine profiles in the two species 

of crabs but this biomarker could not be reliably determined. 
2.0 MATERIALS AND METHODS 

Juvenile Callinectes sapidus and Callinectes similis and adult Fundulus grandis were collected 

from two sites by dip nets and fish traps and returned to LSU for experimental use. . The site at Port 

Fourchon, LA is impacted by produced water discharge (Rabalais et al., 1991) and the non polluted site 

was the beach, sea grass and local streams in the vicinity of the Florida State Marine Laboratory at 

Turkey Point, FL. Callinectes similis was not found at the Turkey Point site . GCMS aromatic 

hydrocarbon analysis of Callinectes sapidus documented that the body burden was higher at the Port 

Fourchon produced water site (30.6 ng.g wet weight") than in those collected at Turkey Point, FL (11 .9 

ng.g wet weight -') . Likewise the aromatic hydrocarbon body burden of Callinectes similis from the Port 

Fourchon produced water site was (57.1 ng.g wet weight-') higher than that of C. sapidus from Turkey 

Point, FL. 

The constant hypoxia exposure system was described in detail in Kapper and Stickle (1987) . 

Oxygen tensions were determined daily by injecting water samples taken anaerobically into a syringe 

and injected into a water jacketed Strathkelvin flow through oxygen electrode-chamber system that was 

connected to a Strathkelvin oxygen meter. 

The flow through system used to generate the WSF of South Louisiana crude oil was designed 

and is in use in the Laboratory of Dr. Stanley Rice at the NMFS laboratory in Auke Bay, Alaska. 

Different amounts of South Louisiana crude oil and pentane were poured into a commercial cement 

mixer that contained a known amount of three-eighths inch pea gravel and mixed for five minutes. After 

five minutes of mixing the oil coated pea gravel was poured onto plywood, raked regularly and allowed 

to dry. Known weights of different doses of oiled gravel were put into WSF generators constructed of 

12" PVC pipe that was 1 meter high. A perforated plate was placed in the bottom of each generator so 

sea water flowed into the bottom. Seawater (30 °/.S) flowed through each generator by gravitational 

flow by entering via an inlet at the bottom and exiting at the top at a rate controlled to -50 ml.miri' . Oil 

doses were monitored weekly by extraction into methylene chloride using standard methods established 

at the Auke Bay, Laboratory NMFS Laboratory (Larsen et al ., 1992) and analyzed by HPLC . 

The duration of all bioassays and experimental exposures was 28 days. Animals were collected 

at 0, 1, 7, 14 and 28 days in dosing experiments. Crabs were frozen whole in liquid nitrogen and killifish 

were dissected into white muscle and liver then stored into an ultracold freezer until analyzed for RNA, 

EROD, or polyamine profile analysis. 

Two morphological indexes of fitness were utilized to determine the fitness of Fundulus grandis 

following exposure to hypoxia, the WSF of South Louisiana crude oil and a combination of the two 

stressors . Killifish condition factor ((K), body weight (mg) / length (mm)' x 100) was utilized to 

determine bioenergetic constraints on the entire fish. The liver somatic index (liver wt (mg) / body wt 

(mg) x 100) was determined in order to determine treatment effects on the organ in which most of the 

induction of aromatic hydrocarbon metabolism occurs . 

RNA: DNA analysis followed procedures outlined in Das and Stickle (1993). EROD activity was 

determined in the liver of Fundulus grandis as the microsomal O-dealkylation of ethyoxyresorufin 

according to modification of the method of Burke et al . (1985) . Protein concentration was determined by 

the method of Lowry et al . (1951). Activity was calculated as picomols resorufin/mg protein/min . 

2 
Statistical analyses were performed with the Statistical Analysis System (SAS Institute; 1996). 

Data on the LCso of all species (Table 1) were analyzed using probit analysis (Hamilton et al ., 1977). 

Non-overlap of 95% fiducial limits were used as a criterion to determine significant differences between 

individual LCso values. 

3 
3.0 RESULTS 

3.1 28-day Tolerance Studies 

No significant difference existed in the 28 day LCso values of either species of crab from the less 

contaminated site from Turkey Point, FL and the produced water site at Port Fourchon, LA when 

exposed to the WSF of South Louisiana crude oil under either normoxia or hypoxia . In contrast, 

Fundulus grandis from the produced water site in Louisiana tolerated the WSF of South Louisiana crude 

oil better than those from Turkey Point, FL under both normoxia and hypoxia but the difference in 

tolerance was of small magnitude (Table 3 .1) . 

Table 3 .1 . LCso values after 28 days exposure of Callinectes sapidus, C. similis, and Fundulus grandis to 

hypoxia, and the water soluble fraction (WSF) of South Louisiana crude oil under normoxia (156 mm Hg 

OZ) and hypoxia (75 mm Hg OZ for F. grandis and 113 mm Hg OZ for C. sapidus and C. similis) . W.F. 

data are given in n.l .-' of total aromatic hydrocarbons. 

Stressor Species Louisiana Florida 

Hypoxia Callinectes sapidus 111 113 

Callinectes similis 45 xxx 

Fundulus grandis 28 33 

W.F. + Normoxia Callinectes sapidus 27 26 

Callinecles similis 26 xxx 

Fundulus grandis 22 17 

W.F. + Hypoxia Callinectes sapidus 28 29 

Callinectes similis 26 xxx 

Fundulus randis 17 13 

3.2 Sublethal Responses 

3.2 .1 Blue Crab and Lesser Blue Crab RNA :DNA Ratios 

There was no significant difference in the RNA:DNA ratio of either Callinectes sapidus or C. 

similis exposed to 0, 9, and 35ng oil . 1`over 28 days. In contrast, RNA:DNA ratios of C. sapidus 

exposed to the WSF of South Louisiana crude oil declined with WSF concentration and duration of 

exposure (Wang and Stickle, 1988). 

32 .2 Sublethal Responses of Gulf Killifish 

Several physiological indices indicated differences in the sublethal responses of 

Fundulus grandis from the polluted and chronically polluted sites (Szeto, 1998). The condition factor 

(K), liver somatic index (LSI), RNA:DNA ratios and ethoxyresorufin-O-deethylase (EROD) activity 

4 
indicated that the Gulf killifish from the Port Fourchon was better adapted to the WSF of South 

Louisiana crude oil than the Turkey Point Florida population. 

3 .2 .2 .1 Growth, Condition Factor and Liver-somatic Indices 

Significant differences in condition factor ((K), body weight (mg) / length (mm)3 X 100) were 

observed among the two populations . Condition factor did not change over 28 days in the normoxia 

controls . Florida fish had a lower (K) than Pass Fourchon (Table 32). Condition factor means of 

hypoxia controls and hypoxia and the water soluble fraction of crude oil concentrations decreased in fish 

from Florida over time, whereas Pass Fourchon losses reached an asymptote by day 7. Fish from Florida 

gradually lost weight over time (Figure 3.1) . Weight of fish exposed to hypoxia + WSF concentrations 

for both populations were not significantly different from their respective hypoxia-control treatment by 

day. Over the course of the experiment a significant decline in (K) compared to day 0 and day 1 

hypoxia-control treatments were observed on days 7, 14, and 28 in both populations . Percentage 

differences of (K) were greatest in the Florida population at -22.6% in the day 28 hypoxia high + WSF 

concentration compared to the day zero fish. The decline in the day 28 high treatment in Pass Fourchon 

fish was -15.6%. 

Table 3 .2 . Condition Factor (body weight (mg) / length (mm)' x 100) + SE in Fundulus grandis from 

Florida and Pass Fourchon over 28 days in normoxia (156 mm Hg OZ) control, hypoxia (73 mm Hg Oz) 

control, hypoxia + low and hypoxia + high water soluble treatments of South Louisiana crude oil 

(n = 16) . 

Florida 

WSF Added Day 0 Day 1 Day 7 Day 14 Day 28 

hypoxia-control 1 .22+0.02 1 .21+0.02 1 .12+0.02 1 .02 + 0.03 * 0.97 + 0.02 

hypoxia + low - 1 .12+0.03 1 .08 +0.03 * 0.99+ 0.01 * 0.99 + 0.03 *a 

hypoxia + high - 1 .1810.02 1 .09 +0.03 * 1 .01 + 0.04 * 0.94 + 0.02 *b 

normoxia-control 1 .22+0.02 - - - 1 .36+0.14 

Pass Fourchon 

WSF Added Day 0 Day 1 Day 7 Day 14 Day 28 

hypoxia-control 1 .33+0.03 1 .34+0.03 1 .17 + 0.04 * 1 .21+0.03 1 .16 + 0.03 

hypoxia + low - 1 .35+0.02 1 .07 + 0.04 * 1 .16 ±0.02 * 1 .12 + 0.03 *a 

hypoxia + high - 1 .29+0.03 1 .18 + 0.02 * 1 .17 +0.02 * 1.12 + 0.02 *b 

normoxia-control 1 .33+0.03 - - - 1 .28+0.05 

* Significantly different from day 0 and 1 hypoxia-control and day 0 and day 28 normoxia-control 

(a) n = 8; (b) n = 5 
16 

14 

.. 

°v° 12 

r 

b0 

3 io 

8 

Total Body Weight Florida Population 

6 -

0 

WSF 

--EY- Hypoxia Control 

- Hypoxia + Low 

--p- H yp oxia + Hi g h 

1 

1 

r 

5 10 15 20 25 30 

Day 

Total Body Weight Louisiana Population 

16 -

14-

.. 

°~° 12--

r+ 

bD 

3 10 -

8 -

6 -- 

0 5 10 15 20 25 30 

Day 

Figure 3.1 . Mean total body weights (x + SE) of Fundulus grandis from Pass Fourchon and from 

Florida in hypoxia (73 mm Hg OZ) control, hypoxia + low, and hypoxia + high water soluble fraction 

treatments of South Louisiana crude oil . n = 16; n = 8 in Florida population day 28 hypoxia + low WSF 

concentration ; n = 5 in Florida population day 28 hypoxia + high WSF concentration . * Significantly 

different from day 1 hypoxia-control . 

6 
Mean liver weights analyzed by ANCOVA with fish length as the covariate showed a trend in 

which weights increased with increasing WSF concentration exposure, and with days (Figure 3 .2) . 

Initial mean liver weights of both populations were similar on day 1 but final weights of the Florida 

population was greater . Liver weights of fish in the Florida population exposed to hypoxia + high WSF 

concentration increased rapidly from days 1 to 14 and reached an asymptote on days 14 to 28 . The fish 

exposed to hypoxia + low WSF concentration increased liver weight at a slower rate, but by day 28, liver 

weights of fish exposed to hypoxia + low and high WSF concentration converged (Figure 3.2) . Increases 

in liver weight of Pass Fourchon fish were not as great as in fish from Florida. Significant increases in 

liver weights occurred in Florida fish only on day 28 in the low WSF concentration (Figure 3 .2) . The 

high WSF concentration liver weights had high variabilty (n = 5) and were not significant . 

The increase in mean liver weights in the Florida population on day 28 in the hypoxia + WSF 

treatments corresponded with increases in liver somatic indices (LSI) and hepatic RNA:DNA ratios 

(Tables 3 .3, 3 .5) . Although RNA:DNA ratios increased in the Pass Fourchon population increases in the 

corresponding liver weights were not significant . Pass Fourchon RNA:DNA ratios were less than the 

ratios in the Florida population, and LSI and mean liver weights were not as high as those in the Florida 

population . 

The liver somatic index (liver wt (mg) / body wt (mg) x 100) of both populations of Fundulus 

grandis responded in a similar manner to the WSF treatments . The LSI tended to increase with WSF 

concentration and with time. Increases were significant only in the Florida population (Table 3 .3) on day 

28 in the hypoxia and WSF concentrations which differed from the day 0 and day 1 hypoxia control and 

the hypoxia control on day 28. There were no difference in LSI in the Pass Fourchon treatments. 

7 
200 

180 

.. 

160 

s 

3 iao 

L 

7 

Mean Liver Weight Florida Population 

a 120 

100 

200 

180 

bA 

160 

bD 

3 140 

~. 

a 120 

100 

80 

Liver Weight Louisiana Population 

WSF 

~ Hypoxia Control 

-~ Hypoxia + Low 

-~ Hypoxia + High 

1 

1 

0 5 10 15 20 25 30 

Day 

Figure 3 .2 . Mean liver weights + SE of Fundulus grandis from Florida and Pass Fourchon over 28 days 

in hypoxia (73 mm Hg OZ) control, hypoxia + low, and hypoxia + high water soluble treatments of South 

Louisiana crude oil . n = 16; n = 8 in Florida population day 28 hypoxia + how WSF concentration ; n = 5 

in Florida population day 28 hypoxia + high WSF concentration . * Significantly different than day 1 

hypoxia-control by analysis of covariance with length as covariate. 

8 

80 

0 5 10 15 20 25 30 

Day 
Table 3.3 . Liver somatic Index (LSI, liver wt (mg) / body wt (mg) x 100) + SE in Fundulus grandis 

from Florida and Pass Fourchon over 28 days in normoxia (156 mm Hg Oz) control, hypoxia (73 mm Hg 

OZ) control, hypoxia + low, and hypoxia + high water soluble treatments of South Louisiana crude oil (n 

=16) . 

Florida 

WSF Added Day 0 Day 1 Day 7 Day 14 Day 28 

hypoxia-control 0.84+0.07 0.84+0.07 1 .04+0.09 0.91+0.07 1 .21+0.10 

hypoxia + low - 1 .0+0.08 1 .09+0.14 1 .22+0.14 1 .61 + 0.14 * a 

hypoxia + high - 0.82+0.04 1 .13+0.08 1 .25+0.13 2.38 + 0.41 *#b 

normoxia-control 0.84+0.07 - - - 0.95+0.25 

Pass Fourchon 

WSF Added Day 0 Day 1 Day 7 Day 14 Day 28 

hypoxia-control 0.87+0.06 0.88+0.06 0.86+0.08 1 .06+0.08 0.88+0.15 

Hypoxia + low - 1 .17+0.07 0.97+0.08 1 .01+0.06 1 .25 +0.13 a 

hypoxia + high - 1 .20+0.11 1 .13+0.07 1 .04+0.08 1 .17 +0.13 b 

normoxia-control 0.87+0.06 - - - 1 .32+0.36 

* Significantly different from day 0 and day 1 hypoxia-control, and day 0 and day 28 normoxia-control . 

# Significantly different from its respective control by day (a) n = 8 ; (b) n = 5 . 

There were no significant differences in LSI in the hypoxia-controls of either population over 28 

days as a result of exposure to hypoxia. There was no change over 28 days in LSI in the normoxic 

controls. The differences seen in the hypoxia + WSF treatments were a result of exposure to the WSF of 

crude oil . 

The Florida population LSI values differed from day zero in the day 28 hypoxia + high WSF 

concentration (2 .38) and hypoxia + low WSF concentration (1 .61). The high concentration LSI value 

was greater than all but the day 28 hypoxia low concentration by at least 2 fold and mean liver weights 

of the Florida population were significantly greater in the low WSF treatment (Figure 3 .3) . The day 28 

high concentration had high variability (n = 5) and was not significantly increased. Increases in liver 

weight in the hypoxia + high WSF concentration were 58% in the Florida population and 26% in the 

Pass Fourchon population . Both decreases in body weight and increases in liver weight contributed to 

the change in LSI . Liver weights on day 28 corresponded with the significant increase in LSI of the 

Florida population . Liver weight and LSI did not differ in Pass Fourchon fish. 

9 
3 .2 .2.2 Liver RNA:DNA Ratios 

Liver RNA:DNA ratios differed between populations over 28 days; ratios were higher in the 

Florida population due to experimental exposure. RNA:DNA ratios increased in both populations with 

time and with increasing WSF concentration ; this was most pronounced on days 14 and 28 in the 

Louisiana population (Table 3 .4) . RNA:DNA ratios in the low and high treatments of day 28 of the 

Florida population were increased compared to the day 1 hypoxia-control (Table 3 .4) . The normoxic 

controls did not change over the 28 days of the experiment. 

The highest increases in liver RNA:DNA ratios were observed on day 28 in the Florida 

population; the hypoxia + low WSF treatment ratio (2.61) and high treatment ratio (3 .40) were 1 .6 fold 

and 2.0 fold greater than the day 1 hypoxia-control ratio, respectively (Table 3 .4) . In the Pass Fourchon 

population, an increase in RNA :DNA ratios of fish exposed to the high WSF concentration compared to 

the day 1 hypoxia-control occurred earlier, on day 14 and remained so to day 28. Increases in Pass 

Fourchon were 1 .6 fold greater than the day 1 hypoxia-control . RNA:DNA increases in fish from Florida 

occurred later (day 28), in the hypoxia and WSF treatments . 

3 .2 .23 Liver EROD Activity 

EROD activity differed between the populations and was greater in the Pass Fourchon 

population .(Figure 32). In both populations, peak EROD activity occurred on day 7 in the hypoxia + 

high WSF concentration and did not differ in the hypoxia-controls of the populations. 

The highest EROD activity in the Pass Fourchon population was on day 7 in the hypoxia + high 

WSF concentration (301 .5 + 48 .9 picomol resorufin " min "' " mg protein "') and this was the highest 

activity observed in either population. A secondary peak EROD activity occurred at day 14 in the 

hypoxia + high WSF concentration (135 .8 + 57.7 picomol resorufin " min -' " mg protein -') (Figure 

3 .3) . EROD activity in the Pass Fourchon population decreased by day 28 . EROD activity of fish 

exposed in the day 7 and 14 hypoxia + high WSF concentrations were significantly different from their 

respective hypoxia-control by day, and the day 0 hypoxia-control (8 .5 + 3.5 picomol resorufin - min-' 

mg protein "') . In the Florida population, EROD activity increased on day 7 (135.7+ 22.0) and day 28 in 

the hypoxia + high WSF concentration (128 .1 + 28.9 picomol resorufin " min -' " mg protein -') (Figure 

3 .3) compared to the day 0 control (8 .0 + 3.1 picomols resorufin " min -' " mg protein -') . 

10 
Table 3.4 . Liver RNA:DNA ratios + SE of Fundulus grandis from Florida and Pass Fourchon over 28 

days in normoxia (156 mm Hg OZ), hypoxia (73 mm Hg OZ), hypoxia + low, and hypoxia + high WSF 

treatments of South Louisiana crude oil (n = 16). 

Florida 

WSF Added Day 1 7 14 28 

hypoxia-control 5 .43+0.33 4.54+0.39 6.43+0.32 4.70+0.60 

hypoxia + low 5 .98+0.27 5 .32+0.41 4.17+0.24 4.64 + 0.21 a 

hypoxia + high 5 .49+0.30 4.89+0.40 5.55+0.30 3 .28 + 0.61 6 

normoxia-control - - - 5 .62+0.33 

Pass Fourchon 

WSF Added Day 1 7 14 28 

hypoxia-control 6 .03+0.40 6.16+0.42 6.47+0.75 5 .26+0.65 

hypoxia + low 5 .62+0.48 5 .08+0.48 4.71+0.21 4.04 + 0.53 a 

hypoxia + high 5.71 +0.43 5 .28+0.31 4.41+0.51 3 .42 + 035 *b 

normoxia-control - - - 6.32+0.23 

* Significantly different from day 1 hypoxia-control, and day 28 normoxia-control ; (a) n = 8 ; 

(b)n=5 . 

EROD activity of Florida fish in the day 7 hypoxia + high WSF concentration was equal to the 

secondary activity peak of the Pass Fourchon fish (day 14 hypoxia + high WSF concentration) . EROD 

activity of the Pass Fourchon population on days 7 and 14 were at least 1 .5 fold greater than the 

corresponding Florida EROD activity . Induction magnitudes for the high WSF concentrations were 9.1 

X, 11 .0 x, 4.2 X, 6.6 x for fish from Pass Fourchon, and -1 .72 x, 4.8 x, 2 .6 x, 4.7 x, for fish from Florida 

on days 1, 7, 14, and 28 respectively compared to the hypoxia-control activities by day. 

The Pass Fourchon activity in day 7 in the hypoxia + high WSF concentration was 2.2 x higher 

than the corresponding activity in the Florida population (p < 0.05). 

11 
400 

v 

O 

L 

a 350 

an 

8 

300 

a 

250 

c 

i 200 

0 h 

,yd 

1 50 

0 

0 100 

u 

a 

50 

0 

EROD Activity Florida Population 

400 

w+ 

O 

O. 350 

en 

8 

300 

c 

250 

0 

°, 200 

0 

d 

a 150 

0 1 7 14 28 

Day 

EROD Activity Louisiana Population 

0 

0 100 

u 

fir 

50 

0 1 7 14 28 

Day 

Figure 3 .3 . Mean EROD activity (picomol resorufin " min "' " mg protein "' ) + SE of Fundulus grandis 

from Florida and Pass Fourchon over 28 days in hypoxia (73 mm Hg OZ) control, hypoxia + low, and 

hypoxia + high water soluble treatments of South Louisiana crude oil . n = 16; n = 8 in Florida population 

day 28 hypoxia + low WSF concentration ; n = 5 in Florida population day 28 hypoxia + high WSF 

concentration ; * Significantly different from Day 0; t Significantly different from its respective hypoxiacontrol 

Total liver EROD activity was calculated by multiplying EROD activity per mg of hepatic 

protein by the total amount of hepatic protein of each liver. Increases in total hepatic EROD activity was 

observed in both populations (Figure 3.4) . In the Florida population, increases occurred on day 7 

(8891 .4 f 3353.5 picomol resorufin " min -' " total hepatic protein -') and 28 (5453 .2 t 1766.7 picomol 

resorufin " min -' " total hepatic protein ') in the hypoxia + high WSF concentrations . The day 7 

12 
activity was significantly increased compared to the day 0 control and to its hypoxia control by day. The 

day 28 activity was greater than the day 0 hypoxia-control only. The Pass Fourchon population had 

increased activity on day 7 (11719.6 f 2688.8 picomols resorufin " min total hepatic protein -') in the 

hypoxia + high WSF concentration . 

Total EROD activity in the Florida population in the hypoxia + high WSF concentration on day 7 

and 28 was 4.7 x, and 3 .9 X greater than the hypoxia-control activities by day, respectively. The Day 7 

hypoxia + high WSF concentration of the Pass Fourchon population was 7.6 x higher than its hypoxiacontrol activity by day, and 1 .3 x higher than the corresponding activity in the Florida population. The 

day 7 hypoxia + high WSF concentration activities of the two populations were significantly different . 

13 
C 

O 

L 

a 

u 

e 15000 

a 

s 

12500 

0 

.+ 

10000 

E 

7500 

0 

L 

O 

5000 

0 

0 2500 

u 

a 

c 

Total Hepatic EROD Activity 

Florida Population 

w+ 

O 

L 

a 

15000 

a 

12500 

0 

10000 

7500 

0 

L 

O 

a sooo 

0 

o Zsoo 

v 

a" 

0 1 7 14 28 

Day 

Total Hepatic EROD Activity 

Louisiana Population 

-k T 

0 

Figure 3 .4 . Total liver EROD activity (picomol resorufin " min ` " total hepatic protein "') + SE of 

Fundulus grandis from Florida and Pass Fourchon over 28 days in hypoxia (73 mm Hg OZ) control, 

hypoxia + low, and hypoxia + high water soluble treatments of South Louisiana crude oil . n = 16; n = 8 

in Florida population day 28 hypoxia + low WSF concentration ; n = 5 in Florida population day 28 

hypoxia + high WSF concentration . * Significantly different from Day 0; t Significantly different from 

its respective hypoxia-control 

14 
3.2.2 .4 White Muscle RNA :DNA Ratios 

White muscle RNA:DNA ratio of Fundulus grandis from both sites varied significantly as a 

function of the population, day, oil concentration, and day by treatment interaction (ANOVA) . Treatment 

means are given in Table 3.5 . 

Table 3 .5 . RNA:DNA ratios from the white muscle of Fundulus grandis exposed to the WSF of South 

Louisiana crude oil for 28 days. WSF concentrations are given as ng.l~' total aromatics and aliphatics . For 

clarity, only mean values and sample sizes (n) are presented in the table. 

Population Day Control 9n .1-' 35 n g.1-1 

Port Fourchon, LA 1 1 .335(8) 1 .335(8) xxx 

7 0.831(13) 5 .168(14) 1 .223(7) 

14 1 .687(16) 4.357(13) 1 .226(16) 

28 2.584(15) xxx 2.826(16) 

Turkey Point, FL 1 1 .458(14) 1 .487(15) 0.836(16) 

7 xxx 1 .468(16) 1 .031(15) 

14 1 .031(15) 1 .157(12) xxx 

28 1 .117 13 1 .375( 8) 1-219(

5 

15 
4.0 DISCUSSION 

The hypoxia tolerance of the Gulf killifish, Fundulus grandis (28 day LCso = 28 Ton 02) , was 

significantly greater than that of the lesser blue crab, Callinectes similis (45 Ton) and the blue crab, 

Callinectes sapidus (111 Ton). Minimal site differences (Florida and Louisiana) existed in hypoxia 

tolerance . There was no difference in the 28 day tolerance of blue crabs to the WSF of South Louisiana 

crude oil under either normoxia or hypoxia. In contrast, Fundulus grandis from the produced water site in 

Louisiana were slightly more tolerant of the WSF of South Louisiana crude oil than those from Florida 

and fish exposed to the WSF of South Louisiana crude oil under normoxia were more tolerant than those 

exposed under hypoxia . Although RNA:DNA ratios are a good biomarker of bionutritional stress 

(starvation), this ratio is not as effective as a biomarker upon exposure of Gulf killifish or blue crabs to 

the WSF of South Louisiana crude oil . Induction of EROD activity occurred in a dose related manner in 

Fundulus grandis from both locations exposed to the WSF of South Louisiana crude oil under hypoxia 

(75 Ton 02), peaked on the seventh day of exposure and was significantly higher in fish exposed to 

35gg oil/liter from the produced water site in Louisiana than in those exposed to 35gg/liter from Florida . 

Statistically significant but minor differences exist in the tolerance of the Gulf killifish from a 

less contaminated site and a more contaminated produced water site . RNA:DNA ratios are not a 

particularly sensitive indicator of sublethal WSF stress in either Gulf killifish or the blue crab. EROD 

activity, as an indicator of cytochrome p-450 induction increases in a dose related manner in the liver of 

Fundulus grandis from both locations, peaks on the 7th day of exposure and is higher in the Gulf killifish 

from the produced water site . Induction of the cytochrome P-450 enzymes by xenobiotics has been 

documented in a number of fish species including the channel catfish Ictalurus punctatus (Winston et al ., 

1989). WSF exposure induced hepatic tissue growth in both populations; hypertrophy was the dominant 

cause of growth in Pass Fourchon fish and hyperplasia in Florida fish ; growth was greatest in fish from 

Florida. Increases in liver size and RNA:DNA ratios were noted with EROD induction. Stimulation of 

growth in white muscle tissue by WSF exposure was observed in the Pass Fourchon population . The 

condition factor (K) of the Florida population decreased more than Pass Fourchon, and LSI were 

increased in Florida fish . Differences in (K) between hypoxia and hypoxia + WSF treatments were 

minor, suggesting small changes in condition would be sufficient to affect survival in F. grandis. 

Therefore, Fundulus grandis, from the chronically polluted site are slightly more tolerant of exposure to 

the WSF of South Louisiana crude oil and exhibit an increased rate of EROD activity compared with the 

Florida population . However, these biological differences in response patterns appear to be minor. 

16 
LITERATURE CITED 

Burke, M.D., S. Thompson, C.R. Elcombe, J . Halpert, T. Haaparanta, and R.T. Mayer. 1985 . Ethoxy-, 

pentoxy-, benzyloxyphen-oxazomes and homologues: a series of substrates to distinguish 

between different induced cytochromes P-450. Biochem . Pharmacol. 34:3337-3345 . 

Das, T. and W.B. Stickle . 1993 . Sensitivity of the blue crab, Callinectes sapidus Rathbun, the lesser blue 

crab, Callinectes similis Williams, and the southern oyster drill, Stramonita haemastoma to 

hypoxia and anoxia. Mar. Ecol. Prog. Ser. 98 :263-274 . 

Hamilton, M.A., R.C. Russo, and R.V. Thurston. 1977. Trimmed Spearman-Karber method for 

estimating median lethal concentrations in toxicity bioassay. Environ . Sci . Technol. 11 :714-719. 

Kapper, M.A. and W.B . Stickle . 1987. Metabolic responses of the estuarine gastropod Thais 

haemastoma to hypoxia . Physiol. Zool. 60:159-173 . 

Larsen, M., L. Holland, D. Fremgen, J. Lunasin, M. Wells and J. Short. 1992. Standard operating 

procedures for the analysis of petroleum hydrocarbons in seawater, marine sediments, and 

marine faunal tissue at the Auke Bay laboratory. NMFS Internal Document. 74 pp. 

Lowry, O.H., N.J . Rosebrough, A.L. Farr, and R.J . Randall. 1951 . Protein measurement with the Folin 

phenol reagent. J. Biol. Chem. 193 :265-275 . 

Rabalais, N.N., B .A . McKee, D .J . Reed and J.C. Means. 1991 . Fate and effects of nearshore discharges 

of OCS produced waters, Volumes I-III . OCS Study MMS 91-0004 through 91 - 0006. U.S . 

Department of the Interior, Minerals Management Service, Gulf of Mexico Region, New 

Orleans, LA. 

SAS Institute Inc . 1996. SAS/STAT User's Guide, Version 6.11, Fourth Edition, Cary, N.C. 

Stickle, W.B ., M.A. Kapper, L.L. Liu, E. Gnaiger, and S .Y. Wang. 1989. Metabolic adaptations of 

several species of crustaceans and molluscs to hypoxia : tolerance and microcalorimetric studies . 

Biol. Bull . 177:303-312. 

Stickle, W.B ., T.D. Sabourin, and S.D. Rice. 1982. Sensitivity and osmoregulation of coho salmon, 

Oncorhynchus kisutch, exposed to toluene and napthalene at different salinities . In: Physiological 

Mechanisms of Marine Pollutant Toxicity. New York: Academic Press. Pp. 331-348. 

Stickle, W.B ., M.A. Kapper, T.C . Shirley, M.G. Carls, and S.D. Rice. 1987. Bioenergetics and tolerance 

of the pink shrimp (Pandalus borealis) during long term exposure to the water soluble fraction 

and oiled sediment from Cook Inlet crude oil . In : Vernberg, W.B., A. Calabrese, F .P . 

Thurberg, and F.J . Vernberg, eds. Pollution Physiology of Estuarine Organisms, Columbia: 

Univ. South Carolina Press. Pp. 87-106. 

Szeto, H. 1998. Ethoxyresorufin-O-Deethylase induction and physiological indices in two populations of 

Fundulus grandis exposed to the water soluble fraction of crude oil and hypoxia . M.S . Thesis, 

Louisiana State University, 84 pp. 

17 
Wang, S.Y. and W.B. Stickle . 1988 . Biochemical composition of the blue crab, Callinectes sapidus, 

exposed to the water-soluble fraction of crude oil . Mar. Biol. 98:23-30. 

Winston, G.W., S . Narayan, and C.B. Henry. 1989. Induction pattern of liver microsomal 

alkoxyresorufin-O-dealkylases of channel catfish (Ictalurus punctatus): correlation with PCB 

exposure in situ. J. Environ. Sci. Health. B24:277-289. 

18 
The Department of the Interior Mission 

o`` As the Nation's principal conservation agency, the Department of the Interior has responsibility 

for most of our nationally owned public lands and natural resources. This includes fostering 

sound use of our land and water resources; protecting our fish, wildlife, and biological diversity; 

- preserving the environmental and cultural values of our national parks and historical places; 

p - ` and providing for the enjoyment of life through outdoor recreation . The Department assesses 

our energy and mineral resources and works to ensure that their development is in the best 

interests of all our people by encouraging stewardship and citizen participation in their care. 

The Department also has a major responsibility for American Indian reservation communities 

and for people who live in island territories under U.S . administration . 

The Minerals Management Service Mission 

Moreover, i n working to meet its responsibilities, the Offshore Minerals Management Program 

administers the OCS competitive leasing program and oversees the safe and environmentally 

sound exploration and production of our Nation's offshore natural gas, oil and other mineral 

resources. The MMS Minerals Revenue Management meets its responsibilities by ensuring the 

efficient, timely and accurate collection and disbursement of revenue from mineral leasing and 

production due to Indian tribes and allottees, States and the U.S . Treasury . 

As a bureau of the Department of the Interior, the Minerals Management Service's (MMS) 

primary responsibilities are to manage the mineral resources located on the Nation's Outer 

Continental Shelf (OCS), collect revenue from the Federal OCS and onshore Federal and Indian 

lands, and distribute those revenues. 

The MMS strives to fulfill its responsibilities through the general guiding principles of : (1) being 

responsive to the public's concerns and interests by maintaining a dialogue with all potentially 

affected parties and (2) carrying out its programs with an emphasis on working to enhance the 

quality of life for all Americans by lending MMS assistance and expertise to economic 

development and environmental protection .