OCS Study 

•

MMS 2001-048 

Effects of Biologically Degraded Oil 

on Marine Invertebrate and Vertebrate 

Embryos and Larvae 

Final Technical Summary 

Final Study Report 

U.S. Department of the Interior 

Minerals Management Service

Pacific OCS Region 

Effects of Biologically Degraded Oil 

on Marine Invertebrate and Vertebrate 

Embryos and Larvae 

Final Technical Summary 

Final Technical Report 

Authors 

Gary N. Cherr 

Frederick J. Griffin 

Principal Investigators 

Prepared under MMS Cooperative 

Agreement No. 14-35-0001-30761 

by 

Southern California Educational Initiative 

Marine Science Institute 

University of California 

Santa Barbara, CA 93106 

U.S. Department of the Interior 

Minerals Management Service Camarillo

Pacific OCS Region April 2001
Disclaimer 

This report has been reviewed by the Pacific Outer Continental Shelf Region, Minerals Management 

Service, U.S. Department of the Interior and approved for publication. The opinions, findings, 

conclusions, or recommendations in this report are those of the authors, and do not necessarily reflect 

the views and policies of the Minerals Management Service. Mention of trade names or commercial 

products does not constitute an endorsement or recommendation for use. This report has not been 

edited for conformity with Minerals Management Service editorial standards. 

Availability of Report

Extra copies of the report may be obtained from: 

U.S. Dept. of the Interior 

Minerals Management Service 

Pacific OCS Region 

770 Paseo Camarillo 

Camarillo, CA 93010 

phone: 805-389-7621 

A PDF file of this report is available at: 

http://www.coastalresearchcenter.ucsb.edu/SCEI/ 

Suggested Citation 

The suggested citation for this report is: 

Gary N. Cherr and Frederick J. Griffin. Effects of Biologically Degraded Oil on Marine Invertebrate 

and Vertebrate Embryos and Larvae. MMS OCS Study 2001-048. Coastal Research Center, Marine 

Science Institute, University of California, Santa Barbara, California. MMS Cooperative Agreement 

Number 14-35-0001-30761. 14 pages. 
Table of Contents 

FINAL TECHNICAL SUMMARY ....................................................................... 1 

FINAL TECHNICAL REPORT............................................................................ 3 

 Background.......................................................................................................... 3 

 Objectives............................................................................................................ 6 

 Description .......................................................................................................... 6 

 Degradation of crude oil................................................................................ 6 

 Quantification of BWSF................................................................................ 6 

 Multixenobiotic Resistance Protein (MXR) Measurements.......................... 7 

 BWSF Exposures .......................................................................................... 7 

 Conclusions ......................................................................................................... 7 

 Study Results....................................................................................................... 8 

References .......................................................................................................... 14 

Final Study Report – Cherr & Griffin 

1 

FINAL TECHNICAL SUMMARY 

STUDY TITLE: Effects of Biologically Degraded Oil on Marine Invertebrate and Vertebrate 

Embryos and Larvae

REPORT TITLE: Effects of Biologically Degraded Oil on Marine Invertebrate and 

Vertebrate Embryos and Larvae

CONTRACT NUMBER: 14-35-0001-30761 

SPONSORING OCS REGION: Pacific

APPLICABLE PLANNING AREA: Southern California

FISCAL YEARS OF PROJECT FUNDING: FY96, FY97 

COMPLETION DATE OF THE REPORT: March 2001 

COSTS: FY 96 - $78,853, FY 97 - $81,547, FY 98 - $85,770, FY 1999-2000 – no cost 

CUMULATIVE PROJECT COST: $246,170 

PROJECT MANAGER: Russell J. Schmitt

AFFILIATION: University of California, Santa Barbara

ADDRESS: Coastal Research Center, Marine Science Institute, University of California, 

Santa Barbara, CA 93106

PRINCIPAL INVESTIGATORS: Gary N. Cherr and Frederick J. Griffin, Bodega Marine 

Laboratory, Univ. Calif. Davis, PO Box 247, Bodega Bay, CA 94923 

KEYWORDS: multixenobiotic resistance protein transporter, biodegraded crude oil, 

invertebrate embryo, fluorescence excitation

BACKGROUND: A by-product of microbial degradation of crude oil is neutral water 

soluble hydrocarbons (PAHs). Due to the hydrophobic nature of many PAHs and other oil 

constituents, they are able to freely cross the plasma membranes of cells and perturb cell 

function. This process can have profound implications to biota that are repeatedly subjected 

to PAH exposure, such as in the Santa Barbara channel near sites of natural oil seeps and noncatastrophic releases associated with oil production or transport. To defend against exposure 

to such compounds, some marine organisms possess mechanisms that bind to PAH 

compounds and transport them out of cells. This study investigates one such mechanism 

present in marine invertebrates, the multixenobiotic resistance transport protein (MXR), 

which is analogous to the mammalian multidrug resistances transporter MDR. Organisms that 

express less MXR transporter will likely be more susceptible to oil-derived hydrocarbons. 

OBJECTIVES: 

The objectives of this study were to: 1) determine the susceptibility of embryonic and 

larval stages of selected marine invertebrates and vertebrates to the water-soluble fraction of 

biodegraded crude oil (BWSF) from the Santa Barbara Channel; 2) determine the basis for 

species sensitivity to BWSF; 3) identify biomarkers in these animals for predicting both 

exposure and effect of Santa Barbara Channel BWSF. 
Effects of Biologically Degraded Oil on Marine Invertebrate and Vertebrate Embryos and Larvae 

2 

DESCRIPTION: 

(1) Crude oil was obtained from a Santa Barbara channel oil platform and artificially 

weathered and biodegraded in the lab using microbes enriched for their oil degrading ability; 

(2) Concentration of biodegraded water soluble fraction (BWSF) of crude oil was quantified 

using fluorescence excitation; 

(3) Multixenobiotic resistance protein (MXR) levels were quantified in invertebrate tissues; 

and 

(4) Embryos were exposed to increasing concentrations of BWSF. 

SIGNIFICANT CONCLUSIONS: 

The tolerance to degraded oil hydrocarbons in select phyla can be attributed to the 

presence of a multi-xenobiotic membrane transporter (MXR) which is homologous to the 

mammalian multi-drug resistance transporter (MDR1) common in cancer cells treated with 

chemotherapeutics. The degraded Santa Barbara Channel oil is a substrate for the MXR 

transporter in echiuroid and mollusk embryos and they are able to effect an efflux of these 

compounds. This was demonstrated using dye efflux assays based on mammalian cell 

technology, as well as the use of mammalian cell lines which over and under express the 

MDR/MXR transporter and show differential sensitivity to degraded crude oil. 

STUDY RESULTS: 

This study has shown that organisms which do not express the MXR transporter are at 

high risk when exposed acutely or chronically to degraded crude oil. Sites which contain high 

proportions of species which do not express the MXR transporter (e.g. selected echinoderms, 

some fish) should be considered “high risk” sites with respect to oil production activities.

STUDY PRODUCTS: 

This study will result in two peer-reviewed publications that are presently in the final 

stages of preparation for submission. The first will include the methods of preparation of 

biologically degraded water soluble crude oil (BWSF), the responses of early life stages of 

different phyla to BWSF exposure, and the linking of the presence of the multi-xenobiotic 

resistance transporter (MXR) in species which are more resistant to BWSF constituents. The 

second paper will characterize the MXR transporter in the Echiurid worm Urechis caupo, and 

will show substrate specificity and how BWSF can serve as a substrate for this membrane 

transport protein. These publications will provide direct evidence that the MXR transporter in 

organisms in the Santa Barbara Channel provides a direct mechanism for adaptation to 

hydrocarbon-enriched environments. This is turn indicates that organisms lacking transport 

activity will be more susceptible to oil-derived PAH exposure due to oil exploration activities.
Final Study Report – Cherr & Griffin 

3 

FINAL STUDY REPORT 

Background

 The removal of volatile compounds through weathering of crude oil results in the 

release of low boiling point aromatic and saturated hydrocarbons. It has been thought that 

those components hold the greatest toxicity to marine life (Capuzzo, 1987; Galt et al., 1991; 

Payne et al., 1991; Venkateswaran et al., 1995). Although biodegradation of crude oil can be 

considered a component of the weathering process, the process continues well after initial 

weathering and the elimination of volatile compounds has occurred. Known results of this 

continued microbial degradation include a measurable decrease in sediment crude oil along 

with a measurable organic enrichment in those sediments (Spies, 1987). 

 As petroleum-degrading microbes are well suited to metabolizing hydrocarbons of 

weathered oil, enhancing such microbial biodegradation has successfully been used as an 

effective means of cleansing oiled sites (Mueller et al., 1992; Bragg et al., 1994). Fertilizers 

were employed after the Exxon Valdez oil spill in 1989 to augment the growth of microbial 

beach populations. Not only was there an enhanced reduction in deposited oil from the 

fertilized plots, there was also an enhanced loss of extractable organic matter from the 

remaining weathered oil (Claxton et al., 1991). 

 Studies (including our laboratory) have demonstrated that a by-product(s) of microbial 

degradation of artificially weathered Alaska North Slope crude oil is a ten fold increase in 

neutral water soluble hydrocarbons that exhibits significantly high toxicity to developing 

atherinid and clupeoid fish embryos (Middaugh et al., 1996, 1998). Biodegradation of crude 

oil occurs in regions of natural seepage (e.g. Coal Oil Point) as well as in regions of oil 

production and transport where elevated populations of crude oil-degrading microbes are 

purported to exist (Spies, 1987). It can be assumed that the process of oil biodegradation at 

these sites is an ongoing process and that the products of that biodegradation are chronically 

present. The fact that the biodegradation of crude oil is now known to produce water soluble 

fractions that may be toxic to marine life, means there are profound implications to the biota 

in the Santa Barbara Channel near sites of natural oil seeps and non-catastrophic release 

(associated with oil production or transport). In particular, we have been interested in how 

select species of marine organisms tolerate chronic exposure to microbially degraded oil 

constituents. 

 Due to the hydrophobic nature of many PAHs and other moderately hydrophobic oil 

constituents, they are able to freely cross the plasma membranes of cells and perturb cell 

function. To defend against exposure to such compounds, some marine organisms possess 

mechanisms to bind these compounds at the plasma membrane and transport them out of cells 

(Epel, 1998). One such mechanism involves the binding of these compounds at the plasma 

membrane and subsequent transportation out of cells due to the presence of a multixenobiotic 

resistance transport protein (MXR), a member of the ABC (ATP Binding Cassette) transporter 

protein superfamily homologous to the mammalian multidrug resistance transporter (aka. 

MDR, p-glycoprotein). Organisms that express less MXR transporter will likely be more 

susceptible to oil-derived hydrocarbons. MXR transporters in marine organisms function as a 

“first line of defense” against exposure to organic compounds in the marine environment 

(Epel, 1998) Organically rich sediments, such as mudflats of estuaries and bays, contain an 

array of natural and anthropogenic cytotoxic compounds. Although neither their chemical 
Effects of Biologically Degraded Oil on Marine Invertebrate and Vertebrate Embryos and Larvae 

4 

identities nor biological effects are well known, low to moderately hydrophobic compounds 

present in these environments are often toxic. Furthermore, natural products from degradation 

or metabolism of marine organisms (e.g. dinoflagellates and algal toxins) may also be toxic. 

Due to the hydrophobic nature of some of these toxicants, they are able to freely cross the 

plasma membranes of cells and perturb cell function. 

 Mammalian MDR proteins have been well studied and are involved in resistance of 

humans to chemotherapeutic drugs (reviewed by Ambudkar et al., 1999). The P-glycoprotein 

in mammalian systems, as well as the MXR transporter in invertebrate systems, is up 

regulated upon exposure to chemotherapeutic drugs and possibly xenobiotics. In 

invertebrates, a MXR-like transporter has been described for sponges, bivalves, arthropods 

and echiuroid worms (Epel, 1998). Limited studies suggest the MXR transporter may be 

absent in echinoderms (embryo and larval stages) (Holland-Toomey and Epel, 1993). In 

clams, individuals from polluted sites show increased transporter activity (Kurelec et al., 

1996). In oysters and mussels, increased expression of MXR transporter mRNA was 

observed in animals from polluted sites (Minier et al., 1993), although it was not known if 

actual transporter activity was increased. MXR protein levels can be induced in mussel 

hemocytes in the laboratory by exposure to the alkaloid vincristine (Minier and Moore, 1996). 

An indicator of expression of MXR transport glycoproteins can often be the appearance of the 

non-glycosylated nascent MXR polypeptide chain which appears on Western blots as a lower 

molecular weight polypeptide (Chin et al., 1990). We were able to study MXR transport 

activity using two different fluorescent dyes as well as a commercially available antibody 

directed against the highly conserved ATPase region on the mammalian MDR1 protein. 

Figure 1: How MXR or the

MDR transporter keeps 

hydrophobic contaminants 

from entering the cell. Dye,

cytostatic drug, or PAH-like

compounds enter the 

membrane and are bound by

the transporter which then 

transports the compound out 

of the cell in an ATPdependent manner. If a 

competitive inhibitor (in 

higher concentration) is 

present (e.g. verapamil), the

dye or toxicant enters the cell 

as the transporter is “busy” 

transporting verapamil. In 

the case of Calcein –AM dye 

used for transporter activity 

analyses, esterases cleave the 

methoxy ester and thus the 

Calcein which enters the 

cytoplasm cannot be 

transported out of the cell. 
Final Study Report – Cherr & Griffin 

5 

Objectives 

 The objectives of this study were to: 1) determine the susceptibility of embryonic and 

larval stages of selected marine invertebrates and vertebrates to the water-soluble fraction of 

biodegraded crude oil (BWSF) from the Santa Barbara Channel; 2) determine the basis for 

species sensitivity to BWSF; 3) identify biomarkers in these animals for predicting both 

exposure and effect of Santa Barbara Channel BWSF. 

Description 

Degradation of crude oil 

 Crude oil that was obtained from a Santa Barbara Channel oil platform, shipped to 

BML at 4oC, and artificially weathered and biodegraded using microbes enriched for their oil 

degrading ability. The crude oil was artificially weathered by autoclaving at 374o

C, distilled 

at 1 atm, and stored in aliquots at -70oC (ISO/DIS 8708, 1994). The enriched microbe 

population was obtained from a seep sediment starter that was collected from Coal Oil Point 

in collaboration with UCSB researchers (Keith Seydel). Agar plates containing BushnellHaas nutrients, sea water, and 0.2% weathered oil were inoculated with the seep starter and 

allowed to incubate for 3 days. Further purification of oil degrading microbe colonies was 

accomplished by serial inoculations (2x) of growing colonies to new culture plates. Selective 

enrichment steps (using crude oil agar plates) were successful since they resulted in dramatic 

increases in oil degrading capabilities and yielded a more uniform (primarily gram negative) 

bacterial population. 

 Biodegraded oil was obtained utilizing the artificially weathered oil and our enriched 

microbe population. Solutions of seawater containing 0.2% weathered crude oil (w/v) were 

incubated for 14 days at 15oC with or without enriched cultures of microbes from Coal Oil 

Point. While complete emulsification of the crude oil/microbe mixture was achieved in ~10 

days, the weathered crude oil/sea water mixture without microbes showed virtually no 

emulsification. Following incubation, both microbially degraded and non-degraded oil-water 

mixtures were serially filtered through 0.7 μm and 0.45 μm filters to remove particulate oil 

materials and microbes, aliquoted, and stored at -70o

C. Aliquots were lyophilized for 

biological experiments as well as additional chemical fractionations. All fractions were 

filtered to 0.22 μm at the time experiments were initiated. Flasks containing only artificial sea 

water, Bushnell-Haas nutrients, and weathered oil were aerated and used as non-biodegraded 

controls. Aeration was utilized to insure that any volatile by-products of degradation were 

removed from the water. The resultant seawater from the flasks containing microbes, the 

BWSF, possessed a distinctive yellow coloration and a much reduced pH (pH =5-5.7). The 

nondegraded flasks (minus the microbes) contained what was denoted as the nondegraded 

water soluble fraction (NWSF), a clear liquid with a pH of approximately 7.8. Based on the 

appearance of the different flasks, the crude oil-microbe treatment was almost completely 

emulsified as compared to the treatment without microbes. 

Quantification of BWSF 

The concentration of BWSF in seawater following degradation was quantified using 

fluorescence excitation at 290 nm and emission at 353 nm. A known concentration (W/V) of 

crude oil was dissolved in methylene chloride and diluted into dimethyl sulfoxide (DMSO) at 

increasing dilution. A standard curve was established (R2

=0.96) for dilutions of the crude oil 

and established as the standard in which BWSF concentrations were extrapolated from. 
Effects of Biologically Degraded Oil on Marine Invertebrate and Vertebrate Embryos and Larvae 

6 

Multixenobiotic Resistance Protein (MXR) Measurements 

MXR protein levels were quantified in invertebrate tissues on Western blots following 

SDS-PAGE using a commercial antibody raised against mammalian MDR-1 (C219, 

Centocor). MXR transporter efflux activity of embryos, larvae, and cells, before and after 

exposure to BWSF, were performed using the rhodamine dye exclusion assay (HollandToomey and Epel, 1993), or calcein-AM dye accumulation (Hollo et al, 1994). For 

rhodamine dye efflux, Rhodamine B (1 μM final concentration) was added and embryos 

incubated in the dark at 14oC for 30 min in the presence or absence of the dye efflux inhibitor 

verapamil (20 μM) or BWSF. Fluorescence measurements in response to various treatments 

were made using a temperature controlled, stirred cell Photon Technology International (PTI) 

spectrofluorometer (546 nm excitation and 590 nm emission). PTI quantitative fluorescence 

software was used to determine the fluorescence of up to 500 embryos/larvae at one time. 

This software enabled determination of the emission intensity of samples over time as a direct 

measure of the inhibition of MXR activity. In other experiments, the methoxy ester form of 

Calcein, Calcein-AM, was used. In this case, Calcein-AM crosses cell membranes and is a 

substrate for MDR1-like transporters (Hollo et al 1994). The methoxy ester form of Calcein 

is not fluorescent. Once inside the cell, endogenous esterases cleave the methox ester to yield 

fluorescent Calein salt that cannot cross the cell membrane and is thus trapped inside of cells. 

In this way, dye accumulation in cells and embryos can be determined without concern over 

the dye coming out of the cells (as can occur with Rhodamine B) during the experiment. 

Quantification of dye efflux with Calcein-AM treated embryos/larvae (0.5 μM) was 

conducted using the cuvette-based fluorometric method as described above, or using video 

imaging on an Olympus BX50-WI fixed stage upright microscope using xenon-illumination 

and a cooled stage. Images of 3-5 individual larvae/field (5-10 fields/treatment) were 

collected using Universal Imaging MetaMorph software. Verapamil (3 μM) and the highly 

specific MDR1 inhibitor Reversin (Sharom et al, 1999) were used as positive controls along 

with BWSF and control seawater. 

BWSF Exposures 

All experiments were conducted in triplicate and included a seawater control. Embryo 

exposures were conducted in 20 ml volumes in acid-washed borosilicate glass vials containing 

50-100 embryos/ml. Eggs were fertilized, washed, and introduced into increasing 

concentrations of BWSF or the NWSF. Embryos were cultured to the hatching stage (7-10 

days at 12oC) for Pacific herring (Clupea pallasi), to trochophore stage for Urechis caupo, 

pluteus stage for Lytechinus anamesus, and veliger stage for Crassostrea gigas. Normal 

morphology and survival were scored for all treatments, with stage-specific morphology based 

on the seawater controls for each experiment. Reference exposures to NWSF were also 

conducted in sea urchin. 

Study Results 

Particularly striking are the dramatic differences in susceptibility observed in larvae 

from two species native to the California coast, Lytechinus anamesus (urchin) and Urechis 

caupo (innkeeper worm). BWSF contains a >10 fold increase in PAH-like compounds based 

on fluorescence emission (Fig. 2). Our research shows that BWSF exerts dramatic effects 

even at low concentrations on sea urchin embryos. Continuously exposed urchin embryos 

exhibit dose-dependent delay in development in concentrations as low as 0.2 ppm BWSF 
Final Study Report – Cherr & Griffin 

7 

(corresponding to a 1:400000 dilution of crude oil) in filtered seawater (Fig. 3). NWSF, the 

non-degraded water soluble fraction of weathered oil, was less toxic than BWSF in sea 

urchins (data not shown). 

0

5

10

15

20

25

30

35

40

45

50

BWSF NWSF

ppm oil hydrocarbons 0

0.2

0.4

0.6

0.8

1

0 1 2 3

[Degraded Oil Hydrocarbons] ppm

Proportion Normal

Development

Urchin Urechis

*

*** ***

***

* = P<0.05, ***=P<0.005

The effects were evident in BWSF exposed embryos not only at the pluteus stage (Fig. 

3) but were also manifested earlier in a delay to hatching. In contrast, concentrations of BWSF 

up to 3 ppm had no effect on Urechis trochophore development, even at 96 hrs. of exposure 

(Fig. 4). 

Figure 3. Lytechinus 

anamesus and Urechis 

caupo were exposed to 

different concentrations

of BWSF in seawater 

post-fertilization and 

followed through to the

pluteus and trochophores 

stages. BWSF has 

almost no effect on 

Urechis as compared to

Lytechinus. 

Fig. 2

Concentration of

soluble crude oil

hydrocarbons in 

BWSF vs. NWSF. 

This was based on 

a crude oil 

standard curve. 
Effects of Biologically Degraded Oil on Marine Invertebrate and Vertebrate Embryos and Larvae 

8 

Figure 5. Effects of BWSF exposure

on development of plutei at 96 hrs 

post-fertilization. A. Control embryos 

incubated in seawater. B. Embryos in

0.44 ppm BWSF in seawater. C. 1.1 

ppm BWSF in seawater. D. 2.2 ppm 

BWSF in seawater. Concentrations of 

BWSF were based on a fluorescence 

emission of a crude oil standard curve 

(see Methodology). 

0

0.2

0.4

0.6

0.8

1

0 0.5 1 1.5 2 2.5 3

[Degraded Oil Hydrocarbons] ppm

 Previous research on stress responses of urchins has suggested that urchins do not 

express the full complement of stress responsive genes until after gastrulation. To investigate 

the hypothesis that BWSF exerts all of its effects on early life stages (prior to hatching) we 

assessed normal development in urchin larvae exposed to BWSF only at or after hatching 

(Fig. 6). Similar effects were observed to those seen in continuously exposed embryos. We 

have also investigated the possibility that the difference in susceptibility to BWSF observed 

between the two species is caused by the different times taken to develop to larval stage at 

similar temperatures. We exposed Urechis to BWSF for 96 hours (corresponding to the time 

taken for normal urchins to reach the pluteus stage) and found no significant effect of BWSF 

(Fig. 4). 

Figure 4. Even when 

Urechis trochophores 

were incubated with 

BWSF for up to 96 hrs., 

the longest period for sea 

urchin embryo/larval 

exposures), BWSF (up to 

3 ppm) still has no effect 

on morphology or 

swimming. 

A B

C D
Final Study Report – Cherr & Griffin 

9 

Figure 6. BWSF exposure results in 

delayed hatching of blastula stage sea 

urchin embryos ~18 hrs postfertilization. A. Control embryos 

incubated in seawater. B. Embryos in

0.44 ppm BWSF in seawater. C. 1.1 

ppm BWSF in seawater. D. 2.2 ppm 

BWSF in seawater. Concentrations of 

BWSF were based on a fluorescence 

emission of a crude oil standard curve 

(see Methodology). 

 Overall, clear phylogenetic differences were found with respect to embryo responses to 

BWSF. Pacific herring and sea urchins were approximately ten times more sensitive to 

BWSF than Urechis and oyster embryos (Figs. 7 and 8). 

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

0

20

40

60

80

100

Herring 

Oyster

Sea Urchin

Urechis

Effect of BWSF on Development of Marine Embryos

% BWSF

% Normal development  (normalized)

A B

C D

Figure 7: Responses of 

herring, sea urchin, oyster, 

and Urechis embryos to 

BWSF fraction. Note that 

Urechis and oysters are an 

order of magnitude less 

sensitive than herring and

sea urchin embryos. 

Concentrations here are 

expressed as percentage 

BWSF (1% = 0.44 ppm). 

Figure 8: Sea Urchin (L. anamesus), Urechis, and 

oyster (C. gigas) larvae which were exposed to 2.2 ppm

BWSF or control medium immediately after fertilization

and cultured to the larval stage. Note the severe 

abnormalities in the sea urchin pluteus larva, while the 

Urechis trocophore and the oyster veliger larvae are 

relatively normal at this concentration of BWSF.
Effects of Biologically Degraded Oil on Marine Invertebrate and Vertebrate Embryos and Larvae 

10 

 

Rhodamine Rhod. + BWSF Rhod. + Ver.

0

10000

20000

30000

40000

50000

60000

Oyster

Sea Urchin

Urechis

Competitive Fluorescent Dye Transport Assay

Treatment

Fluorescence intensity (counts/sec.)

Our results indicate that BWSF acts as a competitive inhibitor of ATPase mediated 

dye efflux. Quantitative rhodamine dye efflux experiments show that BWSF exposure causes 

an increase in dye accumulation in Urechis and oyster embryos, but not in sea urchin embryos 

(Fig. 9). Our data suggest that this efflux is mediated by a homologue of the mammalian 

multi-drug resistance protein (MXR). Figure 10 shows the expression of multi-xenobiotic 

resistance protein (MXR) throughout development in both sea urchins and Urechis. 

Consistent with observations in the purple urchin (Holland-Toomey and Epel, 1993), 

Lytechinus do not express MXR at any stage of development. In contrast, Urechis express 

relatively uniform amounts of this protein throughout development, regardless of BWSF 

exposure. Thus the expression of MXR is consistent with the observed absence of stage 

specificity in susceptibility of urchins or Urechis. Oyster embryos also possess the MXR 

transporter based on Western blot analysis (data not shown). 

To confirm that the dye efflux results observed in sea urchins and Urechis were not 

due to species-specific differences in membrane permeability to dye we developed a dye 

efflux assay using calcein-AM (acetoxymethyl ester). The AM form of calcein is membrane 

permeable, while the free form is not. Moreover only the free form is fluorescent. Because the 

Figure 9: Quantitative fluorescence 

measurements of rhodamine dye in 

seawater (“Rhodamine”), seawater + 

0.44 ppm BWSF (“Rhod + BWSF”),

and a positive control (“Rhod + Ver.”), 

verapamil (20 μM). Note that for 

Urechis and oyster embryos, there is a 

significant increase in fluorescence in

the presence of BWSF and verapamil. 

There is no change in sea urchins, 

indicating an absence of MXR 

transporter. 

Fig. 10. Western blot analyses of Urechis and sea urchin embryos using a commercial 

monoclonal (C219) antibody raised against mammalian MDR1. All stages of Urechis embryos 

contain the transporter, but it is absent from sea urchin embryos. All lanes contain equal 

amounts of protein (30 μg) and blots were developed using a chemiluminescent method which

is the most sensitive method of detection available. 
Final Study Report – Cherr & Griffin 

11 

AM group is rapidly cleaved off by intracellular esterases the assay measures only the 

fluorescence of calcein trapped within the cells. 

The results are identical to those previously reported using the dye rhodamine. In 

addition, we have included a highly specific inhibitor of MDR1 in mammalian cells, 

“Reversin” (Sharom et al., 1999). This peptide is even more effective than equivalent 

concentrations of verapamil (Fig. 11, 12). 

Calcein Accumulation in Urechis Trocophore Embryos 

0

50

100

150

200

250

300

350

400

BWSF 5.0% (n=37) Reversin 3.0uM (n=40) Verapamil 3.0uM (n=29)

Inhibitor

As previously described, the ability of the MXR transporter to rid cells of hydrophobic 

toxic compounds, is functionally similar to multidrug resistance (MDR) in humans. The 

biochemical similarity is evidenced by the fact that antibodies to a human MDR-glycoprotein, 

MDR1, recognize the MXR transporter in both Urechis and C. gigas. The MDR transport 

proteins have been implicated in transport of metabolites, ions and both endogenous and 

exogenous toxins (Gottesman and Pastan, 1988; Horio et al, 1988; Sarkadi et al, 1992). To 

unequivocally demonstrate that the MXR transporter is responsible for BWSF tolerance, we 

examined two variations of a human fibroblast cell line, NIH-3T3 cells. One was a nontransfected 3T3 cells that did not possess the MDR gene and thus was unable to express MDR 

transporters, and the second was an MDR-transfected sub-line of 3T3 cells that does express 

Fig. 11. Urechis trochophore larvae incubated with 

Calcein-AM (O.5 μM) in seawater (control), Reversin 205, 

BWSF, and verapamil. In seawater, the Calcein dye is 

efficiently removed via efflux. Reversin, at a similar 

concentration as BWSF is a potent inhibitor of dye efflux

as has been reported in mammalian cells. BWSF at the 

lowest concentration of biological effects is also a 

competitive inhibitor of dye efflux. Verapamil in excess is 

a potent inhibitor as been observed previously. 

Fig. 12. Calcein dye 

accumulation in Urechis

trochophores as compared 

with seawater controls. 

BWSF (2.2 ppm) induces a 

57% increase in dye 

accumulation, whereas 

Reversin (at 2.4 ppm) results 

in a >200% increase. 

Verapamil at similar 

concentrations results in a 

150% increase in dye 

accumulation. 
Effects of Biologically Degraded Oil on Marine Invertebrate and Vertebrate Embryos and Larvae 

12 

the MDR transporter. NIH-3T3 (ATCC) cells were thawed from liquid nitrogen and cultured 

in standard media (high glucose DMEM (Gibco-BRL) supplemented with 10% fetal bovine 

serum (Hyclone) and sodium 110mg/liter pyruvate) through 2 to 3 passes, and harvested by 

trypsin-EDTA treatment. After a two day recovery, cells were then cultured in 30 mm Falcon 

dishes either in the presence or absence of BWSF at concentrations that ranges from 0.5% to 

5% of the culture media for a period of seven days. Differential growth of cell cultures was 

used as a determinate for whether or not BWSF had an effect on 3T3 cells. 

At the start of each experiment, just under 4 x 105 cells were added to dishes that 

contained 0.22, 0.66, 1.2, or 2.2 ppm BWSF. The differential sensitivity of the MDR and 

non-MDR 3T3 cells to BWSF mirrored that which we observed when comparing echinoderm 

and echiuroid embryos. Two significant responses to BWSF were demonstrated with the 

transfected 3T3 cells. First, transfected 3T3 cells were capable of withstanding concentrations 

that were higher than were the non-transfected cells. Second, the effect of BWSF on cells that 

utilize an MXR transporter is cumulative over time, that is cells or organisms appear to lose 

the ability to tolerate MXR substrates over time. This is demonstrated in Fig. 13. After five 

days of co-incubation, increase in cell number in 0.22, 0.66 or 1.2 ppm BWSF was not 

significantly different from controls (not shown) or from each other. Only cells in the cultures 

containing 2.2 ppm BWSF exhibited a significant difference in the 5 day cell number 

(compared with cultures of controls and the lower BWSF concentrations). Continued 

exposure to BWSF lowers the effective dosage from 2.2 to 1.2 (Fig. 13). After seven days of 

co-incubation, cell growth was inhibited significantly in both 2.2 and 1.2 ppm BWSF. 

Positive cell growth was obtained in the two lower concentrations (0.22 & 0.66 ppm), but not 

in the 1.2 and 2.2 ppm BWSF exposures. Again, these cells tolerated BWSF concentrations 

similar to those that can be withstood by Urechis and C. gigas embryos. 

Conclusions 

This study has demonstrated that Santa Barbara Channel crude oil that is biologically 

degraded with Coal Oil Point sediment microbes, is far more toxic than the water soluble 

fraction of crude oil. This is due to a dramatic increase (>10 fold) in water soluble and 

bioavailable hydrocarbons that result from microbial activity. Concentrations of degraded oil 

as low as 0.2 ppm (equaling a 1:400,000 dilution of crude oil) impacts echinoderm embryo 

development. Surprisingly, mollusk and echiuroid worm embryos are ten times more tolerant 

to the degraded oil than echinoderm and fish embryos/larvae. 

Figure 13. NIH-3T3 G185cells, transfected with the MDR 

gene, were grown on plates containing either 0.5 (0.22 

ppm), 1.5 (0.66 ppm), 2.5 (1.2 ppm), or 5 (2.2 ppm) % 

BWSF. The numbers of cells in each treatment were 

scored at 5 and 7 days of exposure. 

A-A* = ANOVA (P<0.05) 

B-B* = ANOVA (P<0.01). 

C-C* = ANOVA (P<0.05). 

Effect of BWSF on NIH-3T3 Cell Growth

0

1000000

2000000

3000000

4000000

5000000

1 2 3

Days of Exposure

Clells/plate

0.50%

1.50%

2.50%

5.00%

0 5 7

 0 5 7

A*

A A A

B

B* B* C

C*
Final Study Report – Cherr & Griffin 

13 

 The tolerance to degraded oil hydrocarbons in select phyla can be attributed to the 

presence of a multi-xenobiotic membrane transporter (MXR) which is homologous to the 

mammalian multi-drug resistance transporter (MDR1) common in cancer cells treated with 

chemotherapeutics. The degraded Santa Barbara Channel oil is a substrate for the MXR 

transporter in echiuroid and mollusk embryos and they are able to effect an efflux of these 

compounds. This was demonstrated using dye efflux assays based on mammalian cell 

technology, as well as the use of mammalian cell lines which over and under express the 

MDR/MXR transporter and show differential sensitivity to degraded crude oil. 

 This study has shown that organisms which do not express the MXR transporter are at 

high risk when exposed acutely or chronically to degraded crude oil. Sites which contain high 

proportions of species which do not express the MXR transporter (e.g. selected echinoderms, 

some fish) should be considered “high risk” sites with respect to oil production activities. 

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