Domoic Acid Toxicity in California Sea 

Lions (Zalophus californianus) 

Stranded Along the Central California 

Coast, May-October 1998 

Report to the National Marine Fisheries Service Working 

Group on Unusual Marine Mammal Mortality Events 

Frances Gulland 

U.S. Department of Commerce 

National Oceanic and Atmospheric Administration 

National Marine Fisheries Service 

NOAA Technical Memorandum NMFS-OPR- 17 

December 2000 
'This technical memorandum series is used for documentation and timely communication of 

preliminary results, interim reports, or similar special-purpose information. Although the 

memoranda are not subject to complete formal review, editorial control, or detailed editing, 

they are expected to reflect sound professional work. The views and conclusions expressed 

by the authors are not necessarily those of the National Marine Fisheries Service. In addition, the mention of trade names or commercial firms is for information only and does not 

imply endorsement by the National Marine Fisheries Service. 

In response to large numbers of California sea lions washing ashore dead and in obvious 

physical distress, individuals from the Marine Mammal Center, other stranding network participants, National Marine Fisheries Service, as well as numerous other State and Federal 

partners, were called into action to give aid to the animals and determine the cause of the 

event. In the course of this mortality event, many individuals with expertise in various 

disciplines contributed their efforts and insight into the initial response, sample collection, and 

final analyses. The results of their investigations are in this report. This report was prepared 

by the Marine Mammal Center under contract 40AAND801390. 

Suggested citation: 

Gulland, F. 2000. Domoic acid toxicity in California sea lions (Znlophus cnliforninnus) 

stranded along the central California coast, May-October 1998. Report to the National 

Marine Fisheries Service Working Group on Unusual Marine Mammal Mortality Events. 

U.S. Dep. Cornmer., NOAA Tech. Memo. NMFS-OPR-17, 45 p. 
Domoic Acid Toxicity in California 

Sea Lions (Zalophus californianus) 

Stranded Along the Central 

California Coast, May-October 1998 

Report to the National Marine Fisheries Service Working Group on 

Unusual Marine Mammal Mortality Events 

Frances Gulland 

The Marine Mammal Center 

1065 Ft Cronkhite 

Sausalito, CA 94965 

NOAA Technical Memorandum 

NMFS-OPR-17 

December 2000 

U.S. Department of Commerce 

Norman Y. Mineta, Secretary 

National Oceanic and Atmospheric Administration 

D. James Baker, Under Secretary for Oceans and Atmosphere 

National Marine Fisheries Service 

Penelope D. Dalton, Assistant Administrator for Fisheries 
TABLE OF CONTENTS 

Expert Team of Investigators 

Abstract 

Introduction 

Epidemiology 

Clinical Signs 

Clinical Pathology 

Treatment 

Gross Post Mortem Findings 

Microbiology 

Histopathology 

Toxicology 

Plankton Bloom 

Diagnosis 

Post Release Monitoring 

Discussion 

Acknowledgments 

References 

List of terms 

APPENDICES 

Appendix 1. Accession numbers, names, stranding site and date, age 

and disposition of animals stranding during the unusual 

mortality event. 

la Animals that died during the event 3 7 

lb Animals that survived and were released 39 

Appendix 2. Clinical hematology and chemistries of stranded animals 

2a. Hematology 40 

2b Hematology 4 1 

2c Blood chemistries 42 

Appendix 3. Hematology and chemistries, pre-release 43 

Appendix 4. Neuroclinical examination sheet 4 5 
EXPERT TEAM OF INVESTIGATORS 

A multi-disciplinary team of experts participated in response, data collection, and analyses of this 

mortality event. They are listed by their area of expertise. 

Veterinary Medicine and Pathology Toxicology 

Frances Gulland Mark Busman 

The Marine Mammal Center Marine Biotoxins Program 

Marin Headlands NOAADJational Oceanic Service 

Sausalito, CA 94965 Center for Coastal Environmental Health and 

Biomolecular Research 

2 19 Fort Johnson Road 

Charleston, SC 294 12 

Marty Haulena 

The Marine Mammal Center 

Marin Headlands 

Sausalito, CA 94965 

Greg Doucette 

Marine Biotoxins Program 

NOAMational Oceanic Service 

Center for Coastal Environmental Health and 

Biomolecular Research 

2 19 Ft Johnson Road 

Charleston, SC 294 12 

Tom Lipscomb Greg Langlois 

Armed Forces Institute of Pathology California Department of Health Services 

Veterinary Pathology 2 15 1 Berkeley Way 

Washington DC 20306-6000 Berkeley, CA 94704- 10 1 1 

Linda J. Lowenstine Christine Powell 

Department of Veterinary Pathology, Marine Biotoxins Program1 

Microbiology and Immunology NOAMational Oceanic Service 

University of California at Davis Center for Coastal Environmental Health and 

Davis, CA 95616 Biomolecular Research 

2 19 Fort Johnson Road 

Charleston, SC 294 12 

Teri Rowles Chris Scholin 

National Marine Fisheries Service Monterey Bay Aquarium Research Institute 

13 15 East West Highway P.O. Box 628 

Silver Spring, MD 20910 Moss Landing 

Monterey, CA 95039 

Terry Spraker 

Colorado State University 

College of Veterinary Medicine 

Fort Collins, CO 80523 

Vera Trainer 

Marine Biotoxin Group 

NOAADJMFSECD 

2725 Montlake Blvd. 

Seattle, WA 98 1 12 
Fran Van Dolah 

Marine Biotoxins Program 

NOAA/National Ocenaic Service 

Center for Coastal Environmental Health and 

Biomolecular Research 

2 19 Fort Johnson Road 

Charleston, SC 294 12 

Kathy Lefebvre 

Institute of Marine Science 

University of Santa Cruz 

Santa Cruz, CA 95064 

Pinniped Biology and Management 

Joe Cordaro 

National Marine Fisheries Service 

Southwest Region 

501 West Ocean Boulevard, Suite 4200 

Long Beach, CA 90802-42 13 

Robert DeLong 

National Marine Mammal Laboratory 

7600 Sand Point Way 

Seattle, WA 98 1 15-0070 

Jim Harvey 

Moss Landing Marine Laboratories 

P.O. Box 450 

Moss Landing, CA 95039 

William McLellan 

Biological Sciences 

University of North Caroline, Wilmington 

Wilmington, NC 28403 

Teri Rowles 

National Marine Fisheries Service 

13 15 East West Highway 

Silver Spring, MD 20910 

Epidemiology 

Andrew DeVogelaere 

Monterey Bay National Marine Sanctuary 

299 Foam St. 

Monterey, CA 93940 

L. Grella 

Gulf of the Farallones National Marine Sanctuary 

San Francisco, CA 94 123 

Jan Mortenson 

Farallones Marine Sanctuary Association 

P.O. Box 29386 

San Francisco, CA 94129 

Jan Roletto 

Farallon Islands National Marine Sanctuary 

Ft Mason Bldg. 201 

San Francisco, CA 94 123 

Scott Benson 

Moss Landing Marine Laboratories 

P. 0. Box 450 

Moss Landing, CA 95039 
ABSTRACT 

Between May 15 and June 19, 1998, 70 

California sea lions (Zalophus calijornianus) 

and one northern fur seal (Callorhinus ursinus) 

stranded along the central California coast from 

San Luis Obispo to San Mateo County. Of these 

70 animals, 54 were adult females, with 27 (50 

%) pregnant; three were subadult females; two 

were juvenile females; one was a yearling 

female; six were subadult males and four were 

juvenile males (Appendix 1). All animals were 

in good nutritional condition and displayed 

similar clinical signs that were predominantly 

neurological. The animals had'ievere seizures 

that either became increasingly frequent, 

resulting in opisthotonus, then death, or became 

less frequent with the animals showing ataxia 

and decreased responsiveness to stimuli between 

seizures and eventually becoming clinically 

normal. Forty-eight of the 70 animals (69 %) 

died despite treatment. 

Treatment consisted of supportive care 

with oral and subcutaneous fluids, antibiotic 

cover with penicillin, control of seizures using 

diazepam, lorazepam, and phenobarbitone, and 

reduction of cerebral edema using 

dexamethasone. Induction of parturition using 

dexamethasone and prostaglandin F2a was 

attempted in adult females with open cervices 

and dead intrauterine fetuses. 

Hematological parameters in these 

stranded sea lions were within normal limits, as 

were serum biochemical profiles other than 

creatine kinase levels. The creatine kinase levels 

were elevated in most animals, presumably as a 

consequence of muscular damage during 

seizures. Levels of blood lead were normal in 

the eight affected animals tested, and brain 

cholinesterase levels were normal in the five 

affected animals tested. 

Virus neutralization (VN) tests for 

phocine distemper virus (PDV) detected low 

levels of antibody in 10 of 34 animals (29 %). 

After two weeks, four of these 10 animals 

showed low positive, but rising, titers on VN. 

No signs of respiratory disease or novel 

neurologic signs were detected. After a further 

month, these four animals were seronegative to 

PDV. Three animals that had been in contact 

with seropositive animals remained 

seronegative. A retrospective survey of banked 

sera from 100 adult California sea lions that had 

stranded previously revealed that 20 % had low 

titers to PDV. 

A variety of non-specific lesions were 

observed on gross post mortem examination. 

These included gastric ulceration and erosion 

with associated lymphadenopathy, bile stasis, 

pulmonary congestion and occasional 

subcutaneous hemorrhages. Sea lions that died 

within the first two days of stranding had 

diffusely pale myocardium with occasional focal 

areas of severe pallor. All pregnant females had 

necrotic placentae and dead fetuses. 

Bacteria isolated from tissues at post 

mortern were typical of those isolated from 

stranded California sea lions in recent years. A 

novel calicivirus was isolated from myocardium 

of one animal (#3709). 

The predominant histologic lesion in 

affected animals was neuronal necrosis, that was 

most severe in zones CA3 and CA4 of the 

hippocampi and the dentate gyri. There were 

also intramyelinic and neuropil edema and 

occasional foci of gliosis. In some animals that 

died within 48 hours of stranding, there was 

multifocal myocardial necrosis and 

inflammation. 

Domoic acid, a biotoxin produced by a 

diatom, was detected in serum of 3 of 7 animals, 

urine 7 of 14 animals, and feces of 3 of 9 

animals. Two of the positive fecal samples were 

examined by electron microscopy, and frustules 

of Pseudo-nitzschia australis were observed. No 

domoic acid was found in kidney, stomach 

washings, cerebrospinal fluid, or brain samples 

from affected animals. Analyses were carried 

out using a receptor binding assay (RBA) and 

High Performance Liquid Chromatography with 

Ultraviolet detection (HPLCIUV). HPLC 1 mass 

spectrometry (HPLCMS and HPLCMSMS) 

carried out on a subset of samples from each 

tissue type provided independent confirmation 

of the chemical identity of the biotoxin. 

A bloom of Pseudo-nitzschia australis 
(P.australis) occurred in Monterey Bay during 

the latter half of May 1998, reaching its peak on 

or about May 22. The greatest concentration of 

P. australis recorded was -200,000 cells per 

liter. Cells were concentrated near shore, 

possibly in response to nutrients of terrestrial 

origin brought to coastal waters by enhanced 

river outflow. Plankton samples were also 

analyzed for domoic acid using a receptor 

binding assay. The rise and fall of domoic acid 

in these samples corresponded to the rise and 

fall of P. australis observed in the plankton. 

Anchovies collected from the bay on May 22, 

1998 had levels of domoic acid of 105.6 pg 

domoic acidtg tissue, whereas fish collected on 

June 10, 1998 had no detectable levels of 

domoic acid. Anchovies collected during the 

peak of the P. australis bloom that contained 

high amounts of domoic acid had P. australis 

frustules within their stomachs. In Monterey 

Bay, the bloom of P. australis was followed by 

a bloom of P. pseudodelicatissima. Anchovies 

collected from Monterey Bay reflected this 

change in species abundance in their stomach 

contents. No P. australis frustules or domoic 

acid were detected in stomach contents of these 

fish, and no domoic acid was detected in 

plankton during the P. pseudodelicatissima 

bloom. 

Three of the 23 sea lions (#38 15, #3822 

and #3815) that survived treatment during the 

event in May and June 1998 were equipped with 

satellite and radio-transmitters prior to their 

release on November 15, 1998. Battery life of 

the satellite transmitters was three months. 

Satellite and re-sight data indicated that sea lions 

survived for at least 48, 64 and 94 days, 

respectively. Two sea lions traveled as far south 

as the Channel Islands, whereas the third sea 

lion remained in the vicinity of Aiio Nuevo 

Island. All three sea lions that were 

sightedduring the three-month telemetry period 

appeared healthy and displayed normal 

behavior. 

During routine surveys around the 

Monterey Bay during the months of May and 

June 1998, a 3.5 fold increase in numbers of 

dead beach-cast birds and pi~ipeds was 

detected compare to the same months of the 

previous year. The majority of the dead birds 

were Common Murres, Surf Scoters and Sooty 

Shearwaters. Large numbers of stranded sea 

lions were also detected along other central 

California beaches at the time. The extent to 

which these mortalities was due to domoic acid 

toxicity rather than as a consequence of 

starvation due to El Niiio conditions is unclear, 

since most animals were not examined. 

From July 2 to October 17, 1998, an 

additional 11 California sea lions stranded 

displaying similar clinical signs to those that 

stranded during late May and early June. Nine of 

these animals stranded between October 3 and 

October 17. Four animals survived and seven 

died. The dead animals had histologic lesions 

similar to those in the earlier cases. Domoic acid 

was detected in urine of two of these animals by 

the microplate assay. At the same time (early 

October), cells of P. australis were observed in 

Monterey Bay around the Santa Cruz and 

Capitola piers, but were at relatively low 

concentrations (<10,000 cells per liter). 

However, further offshore (outside of Monterey 

Bay), concentrations of P. australis were in 

excess of 100,000 cells per liter. It is likely that 

the source of domoic acid affecting sea lions 

during October was a bloom outside of 

Monterey Bay. 

In summary, the combination of clinical 

signs, histopathological, toxicological, 

epidemiological and oceanographic changes led 

to the conclusion that domoic acid toxicity was 

the cause of this Marine Mammal Unusual 

Mortality Event. Domoic acid was first reported 

as a cause of toxicity in humans in 1987, when 

four people died and approximately one hundred 

were clinically ill following ingestion 

ofcontaminated mussels on Prince Edward 

Island, Canada. This event is the first 

documented occurrence of domoic acid toxicity 

in marine mammals. - 
INTRODUCTION 

Within the California Marine Mammal 

Stranding Network, response to stranded marine 

mammals is shared between a number of 

organizations under the direction of the NMFS 

Southwest Regional Stranding Coordinator, Joe 

Cordaro. The Marine Mammal Center (TMMC) 

responds to calls on live animals from San Luis 

Obispo County in the south to Mendocino 

County in the north. Calls on dead marine 

mammals in the same range are responded to by 

a number of organizations, usually a different 

one in each county. The type of response and 

extent of sampling beyond the collection of level 

A data varies between agencies within the 

California Marine Mammal Stranding Network, 

due to different interests of volunteer members. 

In El Niiio years, there is typically an increase in 

the number of pinnipeds, especially California 

sea lion (Zalophus californianus) pups and 

yearlings, stranding along the central California 

coast (Cordaro, 1997). In late 1997 and early 

1998, strandings of emaciated animals increased 

as predicted in association with changes in sea 

surface temperature and decreasing food 

availability. The majority of these animals were 

emaciated, heavily parasitized California sea 

lions. In May and June 1998, 334 yearling sea 

lions were admitted to TMMC, compared to 92 

in the same months of 1997. Strandings of adults 

are rare, and usually a consequence of human 

interactions or neoplasia (Gulland el al., 1996). 

Thus, although TMMC admitted 334 yearling 

California sea lions in May and June 1998, the 

stranding of 70 adults and subadults in good 

nutritional condition showing severe neurologic 

signs was an unusual occurrence. 

EPIDEMIOLOGY 

Live stranded sea lions 

From May 18 to June 19, 1998, 70 

California sea lions (Zalophus californianus) 

and one northern fur seal (Callorhinus ursinus) 

stranded live along the central California coast 

between Oceano Dunes, San Luis Obispo 

County in the south and Half Moon Bay, San 

5 

Mateo County, in the north. This unusual 

mortality event has been defined by these dates. 

A second event of 11 sea lions showing similar 

signs stranded between July 12 and October 17, 

with nine of them stranding in the two weeks 

from October 3, 1998. This second mortality 

event will be described in the discussion section 

of this document. 

Criteria for inclusion in this unusual 

mortality event were as follows. All live 

animals included in this study displayed severe 

neurologic signs, were in good nutritional 

condition, and showed no other clinical signs of 

primary traumatic diseases or chronic infectious 

diseases. Dead animals were included if they 

showed good nutritional condition and severe 

neurologic signs prior to death or consistent 

pathologic findings of neurologic lesions like 

those described in the following pathology 

section. Evidence of tumors or "normal" 

parasitic diseases did not exclude animals in this 

study if the above criteria were met. Dead 

animals for which there was minimal diagnostic 

information were not included, therefore the 70 

animals identified are likely to be an 

underestimate of the actual numbers of animals 

involved. 

Figure 1 shows the spatial and temporal 

assessment of the strandings of the animals 

included in this event. The majority of the 

animals that stranded in the first week of the 

mortality event stranded along Oceano Dunes. In 

the second week of the mortality event, animals 

stranded in both Monterey Bay and Oceano 

Dunes, and on June 14 one animal stranded 

north of Monterey Bay, in San Mateo County. 

All live animals admitted were transported to 

TMMC by volunteers following calls from 

thepublic about animals observed in distress. No 

surveys for affected animals were undertaken. 

The distribution of stranding sites was therefore 

affected by the intensity of public visitation to 

beaches along the coast. The area between the 

south end of Monterey Bay and Oceano Dunes, 

Big Sur, is inaccessible to the public due to lack 

of roads along the beach, so it is unclear whether 

or not clinically affected sea lions stranded along 

this stretch of coastline. 
The majority of clinically affected animals were 

adult females, 50% of which were pregnant 

(Table 1). Although juvenile animalswere of 

both sexes, there were no adult male sea lions 

affected. This may be due to sex differences in 

dispersal along the California coast. The 

majority of the other 334 California sea lions 

that were admitted to TMMC in May and June 

1998 were emaciated yearlings of both sexes. 

Sea lions (other than yearlings) were aged by 

counting the number of dentine annuli in 

longitudinal sections of the upper left canine by 

Dr. Sue Chivers at the South West Fisheries 

Science Center, La Jolla California and Dr. R. 

DeLong, at the National Marine Mammal 

Laboratory (NMML) in Seattle, Washington. 

Ages of tooth-aged animals are listed in 

Appendix 1, and ranged from 3 to 13 years. 

Table 1. Age stnicture of total affected and number of California sea lions which stranded or survived from May 16 

to June 14, 1998. 

Figure 1. Stranding sites for sea lions stranding during the Unusual Mortality event in 

successive time periods in 1998. Numbers of animals stranding in each time period at each 

site are given within the circles. 

Males, survived 

4 

2 

Age Class 

Adult 

Subadult 

Juvenile 

Weanling 

Females, affected 

54 

1 

Females, survived 

12 

1 

2 

Males, 

affected 

6 

4 
Dead stranded marine mammals and mortality 

of other species 

Data on stranded marine birds and dead marine 

mammals in the Monterey Bay were collected 

by the Monterey Bay National Marine Sanctuary 

BeachCOMBER program. The monitoring plan 

examines the sandy beaches within Monterey 

Bay and Carmel Bay, divided into 10 roughly 

equal-length segments for sampling. Data on 

beach-cast birds and mammals along the sandy 

beaches north of Aiio Nuevo Island to Bodega 

Head, Sonoma County, were collected by the 

Gulf of the Farallones National Marine 

Sanctuary (GFNMS) Beach Watch program. 

Pairs of volunteers survey these pre-defined 

segments on a monthly basis. Surveys are 

conducted during the first week of each month at 

low tide; therefore, encountered beach-cast 

animals represent those deposited during the 

previous month. For each encountered carcass, 

the following information is recorded: species, 

stage of decomposition, age, sex, evidence of 

scavenging, evidence for the cause of death, the 

presence of oil, and whether or not a photograph 

was obtained. A comments section is provided 

for documentation of any tags present on the 

carcass, length measurements, photograph roll 

and frame numbers, or any notes that would aid 

in post-identification of the encountered carcass. 

For seabirds, a toe is clipped each month in 

which the carcass is encountered, allowing 

determination of residence times and of the 

number of newly deposited birds. Prior to 

clipping a toe, the volunteer documents the 

number of toes previously removed. Beach-cast 

marine mammals are not marked. 

The results of surveys in May and June 

1997 and 1998 are shown in Table 2. They refer 

only to newly deposited birds, defined as birds 

with no previously clipped toes. Total beachcast deposition of marine birds during May and 

June 1998 was greater at all beaches throughout 

the study area compared to the same period in 

1997. In GFNMS, numbers of encountered 

carcasses were higher than the mean for these 

months over the previous three years (1994- 

1997). The causes of this elevated deposition are 

not clear. Many foraging guilds seem to have 

been affected, and overall species diversity was 

greater in 1998. In particular, overwintering 

seabirds were more frequently found beach-cast 

during the May and June 1998 surveys 

compared to the same period in 1997 (even 

though May 1997 was the first survey and 

therefore would be expected to have higher 

counts because it includes deposition for >1 

month). Although no comparable data exist for 

April 1997, deposition rates of overwintering 

birds for April 1998 were also high. This 

indicates that resource limitations (perhaps 

caused by El Niiio) during the winter months 

may have been a factor contributing to elevated 

seabird mortality from March through early 

June. 

Table 2. Number of Dead Marine Birds and Mammals Reported by BeachCOMBER in Monterey Bay 

1 Marine Birds + Common Murre 

I Pacific Loon 

May 1997 June 1997 April 1998 May 1998 June 1998 

Total 

83 

12 

10 

9 

9 

% 

51.2 

7.4 

6.2 

5.6 

5.6 

Total 

46 

4 1 

10 

11 

5 

% 

32.2 

28.7 

7.0 

7.7 

3.5 

Total 

83 

15 

2 

46 

45 

% 

13.3 

2.4 

0.3 

7.4 

7.2 

Total 

149 

69 

5 

12 

46 

% 

26.2 

12.1 

0.9 

2.1 

8.1 

Total 

239 

110 

7 

1 

29 

% 

44.2 

20.3 

1.3 

0.2 

5.4 

9 

Clark'sIWestern 

Ashy Storm-Petrel 

Laysan Albatross 

1 0.2 

1 0.2 
There was a notable increase in beachcast Common Murre (Uria aalge), Sooty 

Shearwater (PufJinus griseus), Surf Scoter 

(Melanitta perspicillata), Pacific Loon (Gavia 

pacifica), Common Loon (Gavia immer) and 

Cassin's Auklet (Ptychoramphus aleuticus) in 

May and June 1998 compared to the previous 

year. The causes for these differences are not 

known; however, several necropsied Surf 

Scotersexhibited large parasite loads 

(acanthocephalans). Cassin's Auklet, a small 

planktivore, may have been more susceptible 

than other species to the combination of resource 

limitation and severe winter storms caused by El 

Niiio conditions. Increases in beach-cast marine 

birds have occurred in this area in previous El 

Niiio events (Bodkin and Jameson 1991). 

However, it is possible that piscivorous 

Common Murre, Sooty Shearwater, Pacific 

Loon and Common Loon were impacted by 

domoic acid in sardines. By comparing live bird 

counts in Monterey Bay to beach-cast 

birdcounts, it is clear that the increased 

deposition was not simply an artifact of a greater 

number of birds in the Bay (Figure 2). 

at each survey, therefore, the results outlined in 

Table 2 and 3 include all carcasses found, 

regardless of residence time. Marine mammal 

deposition was relatively constant from March 

through early May of 1998, and the May 1998 

count was nearly identical to the number 

encountered during May 1997. This pattern 

contrasts sharply with the results described 

above for seabirds. A large (4-fold) increase in 

the deposition of marine mammals occurred 

between the May 1998 and June 1998 samples, 

with the June 1998 sample being nearly three 

times higher than the June 1997 sample. The 

most frequently encountered species was the 

California sea lion(Za1ophus calijornianus), 

comprising about 60% of all marine mammals in 

both years. The increase in marine mammal 

deposition between the months of June 1997 and 

June 1998 may have been caused by the unusual 

presence of female California sea lions in 

Monterey Bay during late spring/early summer. 

Previous studies have found a general migration 

northward from southern California breeding 

grounds during periods of resource limitation 

associated with El Niiio (Trillmich and Ono 

1991). 

In contrast with seabirds, marine 

mammal carcasses could not reliably be marked 

Total 

12 

3 

2 

5 

2 

1 

% 

36.4 

9.1 

6.1 

15.2. 

6.1 

3.0 

Marine Mammals 

California sea lion 

Unidentified 

otariid 

Northern elephant 

seal 

Harbor seal 

Unidentified 

phocid 

Unidentified 

Total 

83 

23 

9 

12 

0 

9 

% 

59.7 

15.8 

6.5 

8.6 

0 

6.5 

Total 

31 

3 

0 

11 

1 

4 

Total 

3 

3 

2 

3 

Total 

16 

3 

0 

2 

0 

2 

% 

59.6 

5.8 

0 

21.2 

1.9 

7.7 

% 

55.9 

8.8 

2.9 

8.8 

5.9 

8.8 

% 

59.3 

11.1 

0 

7.4 

0 

7.4 
t \ 

Sooty Shearwater 

!"_ 

1000 

500 S 0 

z 

Ppril May June July 

Month 

\ / 

f \ 

Common Murre 

5 300 

250 

200 

O 150 

t loo n 50 

0 z 

April May June July 

Month 

\ / 

Figure 2. Counts of live and beachcast Common Murres and Sooty Shearwaters in Monterey Bay during 

1998. 
Table 3. Number of dead California sea lions reported to the California Marine Mammal Stranding Network in May 

and June 1998. 

MAY 1998 1 

County 

San Luis Obispo 

Monterey 

Santa Cruz 

I JUNE 1998 1 

Condition 

Dead 

Alive 

San Mateo 

Dead 

Alive 

Dead 

Alive 

Pup 

0 

11 

27 8 8 3 9 6 1 10 214 

Dead 

Alive 

County Condition Pup 

0 

I 

9 

0 

0 

Monterey 

Santa Cruz 

CLINICAL SIGNS 

Yearling 

0 

25 

3 

3 

Yearling 

0 

San Mateo 

Total 

The characteristic clinical signs in 

affected sea lions were ataxia, head weaving, 

muscle tremors, tetanic convulsions, rubbing 

and lethargy. Forty-eight (69 %) of the initial 70 

affected California sea lions died or were 

euthanized. In animals that died, seizures 

became increasingly frequent, often progressing 

to status epilepticus before the animal died. If 

animals were repeatedly in status epilepticus for 

over an hour, and showed no improvement in 

clinical signs over subsequent days, or were 

comatose, they were euthanized (n=22). The 

seizures varied from a few minutes in duration, 

to over 30 minutes. The seizure duration was 

often affected by treatment, as seizuring sea 

lions were treated with diazepam, lorazepam and 

phenobarbitone symptomatically to alleviate 

tetany. Frequency of seizures varied from one to 

3 

3 1 

0 

13 

Alive 

Dead 

Alive 

Dead 

30 in 24 hours. In animals that survived, the 

frequency of seizures gradually decreased over a 

one month period. Seizures were usually 

bilateral, but two animals repeatedly had 

unilateral seizures. Between seizures, sea lions 

showed varylng degrees of lethargy and 

occasionally a characteristic rubbing behavior, 

in which the animals would rub the back of their 

necks and dorsum by rolling repeatedly on the 

pen floor. As assessment of the lethargy was 

subjective, a neurologic examination sheet 

(Appendix 4) was developed that could be used 

to give animals a numeric score. This could be 

evaluated daily by different personnel, and 

scores compared between days to determine an 

animal's clinical progress. A higher Lscore 

would equate with a worse neurologic condition. 

In the first days after stranding, most animals 

had scores above 20. Apparently healthy 

animals prior to release had scores of between 7 

Subadult 

0 

24 

6 

10 

Subadult 

----- 0 

Alive 

Dead 

Alive 

2 

7 

0 

4 

4 

12 

6 

Adult 

2 

3 0 

1 

1 

Adult 

2 

0 

3 

3 

2 8 

1 

17 

0 

8 

Unknown Total 

13 

20 

8 

3 6 

Unknown 

6 

0 

3 

0 

3 1 

24 

7 

126 

Total 

8 

79 

2 

0 

0 

0 

8 

12 

14 

9 

64 

0 

25 

2 

0 

4 

5 

3 

46 

15 

14 

1 

11 

2 

4 

0 

20 

0 

3 

0 

3 3 

47 

56 

0 

4 

0 

20 

37 

40 

13 

24 1 
and 10. 

Other clinical signs observed included 

retching blood-tinged mucus, diarrhea, moist 

cough, and blepharospasm. Although animals 

retched mucus, no vomitus was observed, and 

on post mortem examination of animals that 

died, stomachs were empty. 

Electrocardiograms were performed on 

three clinically affected animals both during 

seizures and between seizures. No abnormalities 

were detected. 

Pathologic examination of animals that 

died or were euthanized during the event 

suggested that fetuses had died in utero 

sometimes days prior to the female's death. 

Ultrasonography was used to examine five 

animals that had distended abdomens. Of the 

five animals examined, all were diagnosed as 

pregnant, with no detectable fetal heart beat. 

Magnetic resonance imagery (MRI) was 

used to examine the brain of one affected animal 

under anesthesia (# 3768) and one control 

animal. Although examination of histologic 

sections of the brain(#3768) after death revealed 

characteristic lesions of neuronal necrosis in the 

hippocampus, no lesions were detected by MRI 

when compared to the control animal. 

CLINICAL PATHOLOGY 

Blood analyses 

Hematological results from affected sea 

lions at admission are shown in Appendix 2a. 

Hematocrits were elevated in many individuals, 

presumably as a consequence of dehydration. 

Three individuals (#4209, #4024 and #4220) had 

elevated leukocyte counts due to neutrophilia. 

These three animals stranded after the initial 

event, and all three had chronic abrasions. 

The most consistent serum biochemical 

abnormality was an increase in creatine kinase 

ranging from 169 to 6,288 KT/L in animals 

tested (Appendix 2b). This elevation was 

believed to be a consequence of muscle damage 

during seizures and transport. Thirteen of 41 

animals tested (32%) on admission had gammaglutamyl transferase levels over 100 mg/dl. 

This elevation may have been due to bile stasis, 

as many animals on necropsy had distended gall 

bladders. Both hypo- (#3734) andhypernatremia 

(# 390 1, #42 17, #37 19, #38 15) were observed. 

Hyperphosphaternia was also observed in three 

animals (# 3734, #3806, and #3812). Two of the 

sea lions with hyperphosphatemia, # 3734 and # 

3806, also had increased blood urea nitrogen, 

suggesting some of these biochemical changes 

could have resulted fiom impaired renal 

function. Both these animals were female and 

#3806 was euthanized; whereas #3734 died. 

Histologic examination of kidney tissue fiom # 

3806 revealed interstitial nephritis, indicating 

that this clinical pathologic change was indeed a 

consequence of renal damage. However, no 

abnormalities were reported in kidney tissue 

from #3734. 

Neurologic analyses 

To rule out chemical pollutant exposure 

as a cause of the seizures, brain cholinesterase 

levels were assayed in five affected animals and 

control animals at California Veterinary 

Diagnostic Laboratory Systems (CVDLS) and 

were found to be normal (> 1.5 M/g/min) as 

compared to normal mammalian ranges. In 

addition, blood lead levels were examined in 

eight affected and control animals and were 

found to be below detectable limits. 

Cerebrospinal fluid samples from four 

animals (#3837, #3794, # 3746, #3796) were 

spun and smears were examined after staining 

with Wright's stain. No inflammatory cells were 

observed. 

Fecal analyses 

Fecal samples from 18 animals were 

examined by Dr. R. DeLong at the National 

Marine Mammal Laboratory ( NMML) for 

presence of prey species, but fish bones were 

only detected in samples from three sea lions. 

Vertebrae from anchovies (Engraulis spp.) were 

identified in 2 samples (#3801 and #3796) and 

probable sardine (Sardinops sagax) otoliths in 

one sample (#3760). These are common prey 

species of California sea lions in spring and 

summer (Antonelis et al., 1984). 
TREATMENT 

Seizures were controlled symptomatically with 

diazepam (0.1 - 0.2 mgkg intramuscularly (IM)' 

or intravknously (IV) up .to four times daily), or 

lorazepam (0.03 - 0.04 mgkg LM up to twice a 

day). The latter was found to be more effective 

at controlling seizures. Long term control was 

achieved with phenobarbitone at 2 mgkg IM 

initially, then orally once the sea lions- recovered 

sufficiently to eat. Dexamethasone at 0.5 mgkg 

IM was also given to reduce cerebral edema 

following seizures. 

Supportive care was achieved with subcutaneous 

fluids (either Ringer's or 0.9% NaCl at 60 ml/kg 

subcutaneously) until the animals were eating 

consistently. 

Parturition was induced in the five' pregnant 

animals in which no fetal heart beat could be 

detected.' Induction was performed in four 

animals by intramuscular injection of 500 g 

prostaglandin F2 (Estrumate), and in one animal 

by intravaginal administration of prostaglandin 

E (Misoprostol). The four given prostaglandin 

F2 gave birth to dead fetuses after 36 hours, 

while the one that received the drug 

intravaginally died within 12 hours. Post 

mortem examination revealed that this animal 

had a uterine rupture; an intra-abdominal fetus, 

and peritonitis of several days duration. 

Two of the seizuring animals gave birth 

to live pups. One of the pups died within 48 

hours of birth, the other did not nurse and the 

female showed no interest in it, so it was 

removed for hand-rearing. This pup' did not 

survive. 

GROSS POST MORTEM FINDINGS 

were considered features of the toxicity 

eventwere pallor of the myocardium (1 6/48), 

fibrinous plaques on the epicardium (5/48), and, 

more rarely, fibrinous fluid in the pericardial sac 

(2148) (Table 4). These lesions were not 

observed in all cases, but were more frequent in 

sea lions that died within 48 hrs of stranding. 

Four animals with pallor of the myocardium had 

friable livers with a nutmeg pattern on the 

surface. Congestion of the meningeal blood 

vessels was observed in two cases, cerebral 

edema in one. Twenty three of the forty five 

females that died or were euthanized (51%) 

were peri-parturient. Placental necrosis was 

observed in five peri-parturient female sea lions 

that had not delivered a fetus prior. to death. 

Uterine pathologes were observed in five sea. 

lions. Two animals had uterine ruptures with 

intra-abdominal delivery of the fetuses, two had 

uterine torsions and one had a uterine prolapse 

that had become necrotic. Other gross lesions 

were those associated with parasitism - gastric 

ulceration and erosion and enlargement of the 

gastric lymph nodes (associated with Anisakid 

nematodes), cholecystitis (associated with 

Zalophatrema hepaticum) and pulmonary 

congestion (associated with Parafilaroides spp.) 

(Table 4). These lesions are common in animals 

normally stranded in this region. Enlarged 

tonsils were observed in 10 cases, and pus 

within the laryngeal pouches in two cases. 

Abscesses within the skeletal muscle and fascia 

were observed in three cases, one of which was 

gun-shot. 

Five female sea lions had neoplasms. 

Two had leiomyomas of the uterus, and three 

had rough plaques of the vaginal mucosa that 

were found to be early epithelial carcinoma 

when examined histologically. 

All animals that died or were euthanized 

were in good nutritional condition with blubber 

layers over the sternum between 20 and 50 mm. 

The most common gross lesions observed that 
Table 4. Frequency of gross post mortem findings in 48 California sea lions. 

Lesion 

Pallor of the myocardium 

Fibrinous fluid in the pericardial sac 

Epicardial fibrinous plaques 

Left atrial endocardia1 fibrosis 

Friable liver, nutmeg pattern 

Thickening of gall bladder wall 

Atresia of common bile duct 

Swelling of the kidneys 

Placental necrosis 

Uterine rupture 

Uterine torsion 

Prolapsed uterus 

Uterinelvaginal neoplasia 

Congestion of meningeal blood vessels 

Cerebral edema 

Keratitisluveitis 

Oral ulcers 

Gastric erosions and ulcers 

Enlarged gastric lymph nodes 

Swollen gastric rugae 

Blood tinged mucus in oropharynx 

Pulmonary congestion 

Pulmonary interstitial edema 

Blood tinged froth in trachea and bronchi 

Pus in laryngeal pouches 

Abscess within muscle fascia 

# animals 

16 

2 

5 

1 

4 

6 

1 

13 

5 

2 

2 

1 

5 

2 

1 

5 

6 

22 

5 

9 

7 

14 

6 

3 

2 

3 (gunshot 1) 

MICROBIOLOGY 

Bacteriology 

A variety of bacteria were isolated from 

tissues of the sea lions after routine culture of 

swabs taken at post mortem examination 

(summarized in Table 5.). These bacteria species 

are not atypical from bacteria found in sea lions 

that strand along the California coast at other 

times (Thornton et al., 1998). Uterine mucosa 

samples from 25 animals were submitted to 

National Veterinary Services Laboratory in 

Ames, Iowa, for Brucella isolation. All cultures 

were negative. 

Virology 

Swabs from pericardium, lung, liver and 

whole blood were submitted to Dr. Carol House, 

USDA, Plum Island, for viral isolation. A new 

calicivirus, designated San Miguel Sea Lion 

virus 18, was isolated from a pericardial swab 

from sea lion "Dean", #3709. This virus was 

identified using electron microscopy. 

Virus neutralization (VN) tests for 

phocine distemper virus (PDV) detected low 

levels of antibody in sera from 10 of 34 animals 

(29 %). After two weeks, four of these 10 

animals showed low positive, but rising, titers on 

VN. No signs of respiratory disease or novel 

neurologic signs were detected. After a further 

month, these four animals were seronegative to 

PDV. Three animals that they had been in 

contact with were also seronegative. A 

retrospective survey of banked sera from 100 

adult California sea lions that had stranded 

previously revealed a seroprevalence of 20 % of 

low titers to PDV. The significance of these 

findings is under investigation by Drs. Carol 

House, USDA, Plum Island and Don King, 

University of California at Davis. 
HISTOPATHOLOGY 

Tissues from 48 California sea lions, 4 

California sea lion fetuses and one northern fur 

seal were examined microscopically with special 

emphasis on the central nervous system. 

Histopathology was performed at the University 

of California, Davis, Veterinary Medical 

Teaching Hospital (Dr. Linda Lowenstine and 

Dr. Paul Silvagni, pathology resident) (n =20 + 

3 fetuses ), the Armed Forces Institute of 

Pathology (under the direction of Dr. Tom 

Lipscomb) (n = 9 + one fetus ), and at Colorado 

State University (Dr. Terry Spraker) (n = 19). 

Brain 

The most significant lesions were in the 

brain, and these varied with duration post 

stranding. Acute: All animals that died within 

the first 2 days (acute cases, n = 27) had 

vacuolation (presumed edema) in both the gray 

and white matter in many morphologc areas of 

the brain. There was always a strilung laminar 

pattern of micro-vesicular vacuolation in the 

stratum radiatum of the anterior ventral 

hippocampus along with marked edema in the 

pyramidal cell layer of the dentate gyms and the 
hippocampus. In addition, 15 of these animals 

had severe acute neuronal necrosis involving 

most of the neurons of the dentate gyms and 

many pyramidal cells in sectors CA3, CA4, CA 1 

and CA2 (in descending order of frequency). In 

another six animals there was minimal to mild 

necrosis in these same areas. All the 

hippocampal changes were segmental, involving 

almost exclusively the most ventral and anterior 

portions of "Ammond's horn". Sometimes the 

necrosis extended into the laminar cortex of 

theadjacent pynform lobe and amygdala, or 

rostrally to involve the ventral rhinencephalon. 

In seven animals, no acute neuronal necrosis 

was observed in sections examined. In addition 

to the hippocampal and rhinencephalic lesions, 

which appeared to be contiguous, there was also 

necrosis of thalamic nuclei in two animals, and 

in the cuneate nucleus of the medulla in a further 

two animals. 

Subacute 

In animals that survived more than four 

days (short-term survivors, n = 21), edema and 

acute neuronal necrosis were much less apparent 

in the hippocampus, but there was obvious loss 

of neurons in dentate gyms and hippocampal 

sectors as evidence of past necrosis. As survival 

time increased, reactive lesions such as capillary 

proliferation, mild diffuse gliosis, and mild 

perivascular lymphocyte accumulation became 

more apparent although ongoing acute necrosis 

of individual neurons could sometimes be 

appreciated. In some animals there was grossly 

apparent post necrotic hippocampal atrophy. 

Overt malacia (necrosis of large areas) with 

phagocytic glial cell ("gtter" cell) response was 

noted in the amygdala and pynform lobes in 

nine animals and in midbrain (inferior 

colliculus/ lateral geniculate region) in one 

animal. The lesions in the amygdala and 

pynform lobes were often grossly evident in 

fixed brain sections. In one animal that survived 

for six days, there was severe sub-acute 

asymmetrical edema causing a mass-like lesion 

and midline shift. 

An additional finding in both groups of 

animals (rapid death and short-term survivors) 

was non-suppurative meningoencephalitis and 

choroiditis that varied from minimal to florid. 

In some of the short-term survivors, this was 

clearlya response to neuronal damage, but in 

others and in the acute death group, the lesion 

may have been preexisting. Non-suppurative 

encephalitis is a common finding in both 

California sea lions and southern sea otters 

stranding on the California coast. Chronic viral 

or protozoal infections are suspected, but not 

proven. One of the animals from this event did 

have protozoal tissue cysts in the brain and 

another one had possible endothelial 

tachyzooites, butinflammation did not appear to 

be directed toward them. Two additional 

animals had neuronal inclusions suggestive of a 

viral etiology, but electron microscopy, 

immunohistochemical stains and Reverse 

Transcriptase- Polymerase Chain Reaction (RTPCR) were negative for morbillivirus (AFIP). 

Cardiac 

The heart was the second most 

frequently affected organ in terms of lesions that 

could not be attributed to known etiologic 

agents. Areas of myocardial pallor thought to 

represent necrosis and edema were grossly 

evident in several animals. Acute myofiber 

necrosis and edema were confirmed 

histologically in 21 of the animals that died early 

in the event. These varied from scattered areas 

of individual fiber necrosis to regonally 

extensive myocardial damage in two animals. In 

the short-term survivors, small areas of 

myocardial fibrosis, nuclear hyperplasia and 

hypertrophy, and mild non-suppurative 

myocarditis were noted. In one animal, small 

tachyzooite-like structures were associated with 

inflammation and in another animal Sarcocystislike tissue cysts were seen. Rhabdomyolysis 

(muscle necrosis) was also seen in skeletal 

muscle of diaphragm and elsewhere in many of 

the animals and was thought to be secondary to 

the abnormal exertion of seizure activity. 
Other 

Other lesions identified histologically 

confirmed the gross diagnoses. These included 

cholecystitis and dochitis secondary to 

Zalophatrema, parasite-induced ulcers and 

stress-type acute gastric erosions and 

hemorrhages, pulmonary nematodiasis and 

verminous pneumonia. Additional common 

histologic lesions included: enteritis associated 

with trematodes or acanthocephlans, parasitic 

granulomas in liver and regional lymph 

nodes,lymphocytic interstitial nephritis, 

microfilaremia, vagnitis and cervicitis, cystitis, 

pharyngitis associated with acariasis, larval 

cestodiasis in colon, superficial colitis and acute 

enteritis. 

Neoplasias 

Interesting incidental findings were 

several neoplasms including: three cases of very 

early genital epithelial carcinoma; two uterine 

leiomyomas (one of which was external and 

pedunculated); one epithelium-predominant 

teratoma associated with the surface of the right 

kidney; one pharyngeal cleft cyst associated 

with the pineal gland; and one islet cell tumor of 

the pancreas. The prevalence of genital epithelial 

neoplasia in California sea lions in this group 

was 6.3% and the over-all prevalence of tumors 

(pharyngeal cyst excluded) was 14.6%. Another 

interesting. case was a very old female with 

systemic amyloidosis. 

Ocular lesions 

The eyes from 12 animals (including 

one fetus) were examined. No lesions were 

noted in three animals and mild' to moderate 

lesions noted in nine animals. The most common 

lesion (n = 7, including the fetus) was 

vacuolation (presumed edema) in the ganglion 

cell layer of the retina. Two animals had retinal 

hemorrhage, one animal had ophthalmitis, 

onehad bilateral corneal ulceration, one had 

degeneration of the outer molecular layer, and 

one had degeneration of the internal molecular 

layer. Four animals had more than one lesion. In 

none of the animals did the pattern of lesions 

mimic those described in rats with experimental 

domoic acid intoxication. 

Reproductive lesions 

Most of the females (51%) were either 

pregnant or recently post partum at the time of 

necropsy. Placentitis was apparent is five 

animals. Tissues from only four fetuses were 

examined histologically, due to the advanced 

autolysis of most of the fetuses recovered at 

gross examination. Of these four fetuses, one 

was severely autolyzed. One had a bacterial 

infection acquired in utero that had 

causedomphalophlebitis and pneumonia. The 

other two had large numbers of amniotic 

squames filling the bronchioles of the lungs. 

This is suggestive of fetal distress. In none of the 

fetuses were severe brain lesions noted 

(although domoic acid has been demonstrated to 

cross the placenta in rats). 

In summary, the principal lesions that were 

unique to this unusual stranding event were in 

the brain and heart. The acute cerebral edema 

and neuronal necrosis were most severe in the 

dentate gyms and pyramidal layers of ' the 

hippocampus in the anterior ventral regon, with 

other areas of the brain less severely or 

consistently affected. Extent of lesions in these 

areas, especially pynform lobe and amygdala, 

rhinencephalon, thallamic nuclei and lateral 

geniculate body and inferior colliculus of the 

midbrain may have been underestimated due to 

sectioning. In about 20% of the animals, only 

edema without necrosis was detected in sections 

of brain examined including hippocampus. Short 

term survivors had evidence of neuronal loss 

and gliosis (scarring) in the same regions as the 

acute cases with more evidence of extension into 

the pynform lobe and amygdala in which overt 

malacia were often seen. The acute lesions in the 

hippocampus were compatible with exposure to 

an excitotoxin such as domoic acid as described 

in human cases and in experimental rats and 

macaques. Myocardial edema and necrosis were 

also features in animals that died rapidly during 

this event, and there was evidence of repair of 
previous lesions in the short-term survivors. 

Myocardial necrosis has been reported in rats 

exposed to excitotoxins (kanic acid and Nmethyl-D-aspartic acid), although it has not been 

previously described in domoic acid toxicosis 

(Rockhold et al., 1989). Cardiac arrhythmia 

have been noted in humans exposed to domoic 

acid (Per1 et al., 1990). 

TOXICOLOGY 

Sea lion serum, urine, kidney, fecal 

samples, and stomach washings were analyzed 

by the Northwest Fisheries Science Center 

(NWFSC) Marine Biotoxin group (Trainer), the 

National Ocean Service (NOS) Marine 

Biotoxins Program (Van Dolah, Powell, 

Doucette and Busman), and the University of 

California at Santa Cruz (UCSC) (Lefebvre) for 

domoic acid. 

Four analytical techniques were used to 

detect the presence of domoic acid in either 

tissues or fluids. These methods were: receptor 

binding assay (Van Dolah et al, 1997), high 

performance liquid chromatography-with 

standard ultraviolet absorptance (HPLC-UV), 

high performance liquid chromatography with 

mass spectroscopy (HPLC-MS), and high 

performance liquid chromatography with 

tandem mass spectroscopy (HPLC-MSMS). 

The limit of detection for domoic acid in serum 

using the receptor binding assay is 

approximately 50 ng/rnL. The limit of detection 

of the HPLC-UV method is approximately 0.5 

ug/ml with a spiked serum recovery of 98.7%. 

All samples were tested in NWFSC and NOS 

labs by receptor binding assay. All samples 

were analyzed by a receptor binding assay (Van 

Dolah et al., 1997) which uses a cloned 

glutamate receptor and dilutions of a domoic 

acid standard of known concentration. A 

competitive displacement curve is generated and 

sample results are compared to controls. 

Glutamate dehydrogenase is incubated with all 

samples prior to analysis toeliminate 

endogenous glutamate activity. Samples with 

high levels of domoic acid on receptor binding 

assay were confirmed using high performance 

liquid chromatography with MS/MS .at the NOS 

laboratory. 

Serum 

Serum from fourteen clinically affected 

animals were analyzed by the NWFSC lab, and 

serum from fourteen clinically affected animals 

(five of which were the same as that tested by 

NWFSC lab) and three negative controls were 

analyzed by the NOS laboratory using the same 

microplate receptor binding assay for domoic 

acid (Van Dolah et al., 1997). At the NWFSC 

lab, domoic acid was detected in the serum of 

one (#3806) of 14 clinically affected sea lions 

(Table 6). At the NOS lab, two serum samples 

(CSL# 3734 and #3724) were positive with low 

levels of domoic acid (Table 6). Because high 

levels of glutamic acidmay cause false positives 

in the receptor binding assay, the presence of 

domoic acid was further investigated at the NOS 

lab using the HPLC-UV method of Quilliam 

(1989), and by HPLCIMS. A total of 23 clinical 

samples and three control sera were analyzed by 

the HPLC-UV method. All were negative for 

domoic acid. The two serum samples positive by 

receptor assay (#3724 and.#3734) also showed 

low levels of domoic acid by HPLCMS. 

Urine 

At the NWFSC lab, urine samples from 

four animals were tested (two affected animals 

and two control animals). Domoic acid was 

detected in the urine of two affected sea lions (# 

3807 and #3794) as compared to urine from two 

control animals using the receptor binding assay. 

One of these urine samples from the affected 

animals (#3807) also had weakly positive levels 

of domoic acid as detected by high performance 

liquid chromatography (HPLC) using standard 

UV absorbance at 242 nm. At the NOS lab, 

urine from eight clinically affected animals and 

three control animals were tested by both 

thereceptor binding assay and HPLC-UV. Four 

of these were positive by both methods (Table 

6), and three additional samples were positive by 

receptor assay at levels below the detection limit 
of the HPLC method. Urine from one animal 

(#3707) was also analyzed by HPLC/MS and 

confirmed positive for domoic acid. 

Feces 

Feces from seven clinically affected 

animals and two control samples were analyzed 

at the NOS lab by receptor assay and HPLC-W. 

Fecal samples were extracted using a mussel 

extraction and SAX clean-up method of 

Quilliam et al., (1 995) before analysis by HPLC 

with spiked extraction recovery of 88.4 + 8.4%, 

n = 3. Three samples were positive for domoic 

acid (Table7) and good quantitative agreement 

was seen between the receptor binding assay and 

HPLC analyses (Table 7). 

Feces from eleven clinically affected sea 

lions were also analyzed at UCSC for the 

presence of domoic acid by HPLC using the 

extraction method of Quilliam et al., (1989). 

Domoic acid was detected in samples from the 

same three animals as at the NOS lab (Table 7). 

In addition, fecal samples from' seven control sea 

lions were analyzed. These animals were three 

captive sea lions from Long Marine Laboratory 

(LML # 1, 2, 3), three captive sea lions from 

Marineworld in Vallejo, CA, and one sea lion 

from Maverick's Beach, CA (Clutch) that was 

euthanized at TMMC due to severe gunshot 

injury two months after the event (August 1998). 

Domoic acid was not detected in any of these 

samples. 

Kidney, Liver, Muscle, Brain 

Five kidney samples were analyzed at 

the NOS lab by HPLC-UV and were negative 

for domoic acid (Table 7). Samples were 

extracted and cleaned using the same procedure 

as described for feces, with a spiked ludney 

extraction recovery of 24.6%, n = 1. Liver, 

muscle, brain, intestinal mucosa and kidney 

samples from two animals (#3765 and # 

3824)analyzed by HPLC at UCSC were also 

negative for domoic acid. 

Stomach washings 

A single stomach washing sample from 

a clinically affected animal (CSL 3708) 

analyzed by receptor ,assay at the NOS lab was 

negative for domoic acid. 

Confirmation of domoic acid in fluids by mass 

spectrometry 

Serum and urine samples with the 

highest concentrations of domoic acid, as 

determined by HPLC-UV, were subjected to 

HPLC/MS and HPLC-MS/MS analysis at the 

NOS lab for confirmation of the identity of the 

HPLC peak in question. For both methods, 

sample extracts were fractionated on a C18 

column eluted with a gradient of 1-95% 

methanol in 0.1% TFA. A PE SCIEX API-111 

triple quadrupole mass spectrometer was used. 

The ionspray source was operated in positive ion 

mode utilizing nitrogen for the nebulization gas. 

The first quadrupole was used to pass only ions 

of nominal 312 m/z. The conditions in the 

second quadrupole were adjusted to allow a 

substantial amount of collisionally-induced 

dissociation. The third quadrupole was operated 

in multiple ion monitoring mode, where 

fragments ions of 161 and 266 m/z, as well as 

the residual parent ions at 3 12 m/z were allowed 

to pass to the mass spectrometer ion detector. 

The presence of domoic acid was confirmed in 

urine from # 3707 and #3758 and feces from # 

3783 (Table 7). 

Pseudo-nizchtia frustules 

Ten of the eleven fecal samples 

analyzed at UCSC were also examined for the 

presence of diatom frustules (glass skeletons) 

using a compound microscope, (there was 

insufficient sample from CSL #3734 after HPLC 

analyses). Diatom frustules were only found in 

fecal samples that had tested positive for domoic 

acid (#3758 and #3783). Using scanning 

electron microscopy, frustules were identified as 

Pseudo-nitzschia australis. 

Anchovies 

Domoic acid was detected in anchovies 

(Engraulis mordax) collected from Monterey 

Bay and Morro Bay by HPLC-W at the NOS 
lab. Two samples of anchovies 'collected fiom 

Monterey Bay on May 22, 1998 had detectable 

levels of domoic acid, while fish collected on 

June 10, 1998 had no detectable levels (Table 8). 

Two samples of anchovies from Morro Bay on 

June 4 also had detectable levels of domoic acid 

(Table 8). Domoic acid was confirmed in two 

anchovy samples by HPLCMS and one by 

HPLCMSMS (Table 8) 

Summary of Toxicology 

These results indicate that there is 

consistency between domoic acid assays 

performed at NOS, NWFSC and UCSC labs. 

The NOS and NWFSC labs run the same 

microplate receptor binding assay as a rapid 

screening method for domoic acid. Although 

highly effective as a screening method, positive 

results should be confirmed by analytical 

methods when testing novel tissue matrices. The 

HPLC-UV method is suitable only when tissue 

concentrations are sufficiently high, because the 

detection limit is poorer than that of the receptor 

assay. HPLCIMS or HPLCMSMS provides the 

most rigorous analytical confirmation, but are 

expensive and time-consuming to run. In the 

current study HPLCMSIMS provided the 

necessary independent confirmation of the 

chemical identity of the toxin to unambiguously 

conclude the involvement of domoic acid in this 

event. The greater detection rate in feces and 

urine than serum (Table 9) suggests feces and 

urine should be routinely collected for detection 

of domoic acid in marine mammal die-offs. The 

low detection rate in serum is probably due to 

the rapid clearance of this water soluble toxin 

fiom blood. Clearance in rodents and primates 

following intravenous inoculation is under four 

hours (Truelove and Iverson 1994). Although 

stomach contents might be valuable for 

analysis,the stomachs of clinically affected sea 

lions in this event were empty, presumably due 

to vomiting and the time elapsed between 

ingestion and post mortem examination. Feces, 

thus, proved to be the most valuable sample 

fortoxicological analyses, as well as for 

determining diet. 

Table 6. Presence of Dornoic Acid in Serum from California Sea Lions 

Animal ID 

CSL 3299 C 

CSL 345 1 C 

CSL 3489 C 

CSL 3510 C 

CSL 3512 C 

CSL 3670 

NWFSC 

Receptor 

assay 

Negative 

Negative 

NOS 

Receptor 

assay 

N/D 

negative 

negative 

negative 

NWFSC 

HPLCIUV 

NOS 

HPLCNV 

negative 

N/D 

negative 

negative 

NOS 

HPLCMS 

N/D 

N/D 

N/D 

negative 

NOS 

HPLCMSMS 

N/D 

N/D 

N/D 
Table 7 Presence of Domoic Acid in Urine, Feces and Tissues from California Sea Lions 

22 

CSL 3724 

CSL 3727 

CSL 373 1 

CSL 3732 

CSL 3733 

CSL 3734 

Animal ID 

URINE 

CSL 345 1 C 

CSL 3707 

CSL 3726 

CSL 3741 

CSL 3742 

CSL 3749 

N/D 

N/D 

N/D 

N/D 

N/D 

Negative 

negative 

negative 

negative 

negative 

negative 

0.17 pg 

eq./mL 

negative 

negative 

negative 

0.2 Pg 

eq ./mL 

positive 

N/D 

N/D 

N/D 

positive 

NWFSC 

Receptor 

assay 

negative 

UC Santa 

Cruz 

HPLC/W 

NOS 

Receptor 

assay 

g eq./mL 

3.72 

0.12 

0.03 

N/D 

0.72 

NOS 

HPLCNV 

g/rnL 

14.05 

2 68 

negative 

negative 

2.38 

NWFS 

C 

HPLCI 

UV 

NOS 

HPLCI 

MS 

posltlve 

N/D 

N/D 

N/D 

N/D 

NOS 

HPLCMSMS 

pos~tive 

N/D 

N/D 

N/D 

pos~tive 
Table 8. Domoic acid levels in Anchovies from the Central California Coast 

Sample site & Date 

5122198a Monterey Bay 

5122198b Monterey Bay 

614198a Morro Bay 

614198b Morro Bay 

6110198a Monterey Bay 

6110198b Monterey Bay 

Receptor Assay 

NOS 

7 1.30 

69.67 

2.53 

0.27 

N/D 

NID 

HPLC/UV 

100.35 

123.04 

4.05 

0.82 

Negative 

Negative 

HPLCMS 

N/D 

Positive 

Positive 

N/D 

N/D 

N/D 

HPLCMSMS 

Positive 

NID 

N/D 

N/D 

N/D 

N/D 
Table 9. Samples positive for Domoid Acid by different laboratory analyses 

PLANKTON BLOOM 

Within Monterey Bay, plankton samples 

are routinely collected several times per week fiom 

the Santa Cruz wharf and Monterey Coast Guard 

pier. Samples are also collected along several sites 

running due west of Moss Landing as part of the 

Monterey Bar Aquarium Research Institute 

(MBARI) upper water column time series project. 

Samples are subjected to DNA probe-based tests as 

a means of identifyng and quantifyng a variety of 

toxic and non-toxic Pseudo-nitzschia species as 

well as the toxigenic dinoflagellate Alexandrium 

tamarense (e.g., Scholin et al. 1996, 1997, 

submitted;). An example of the time series data 

collected from the Santa Cruz wharf is shown in 

Figure 3. During thespring bloom of diatoms, 

toxigenic Pseudo-nitzschia species were largely 

absent and no domoic acid was detected in 

plankton samples, (Fig. 3, arrow #I). As the spring 

bloom progressed, A. tamarense was detected, and 

shellfish samples collected from the same area 

tested positive for paralytic shellfish poisons (Fig. 

3, arrow #2). As the bloom ,of Alexandrium 

declined, the number of Pseudo-nitzschia australis 

increased sharply (arrow #3). The amount of 

domoic acid (= bars) in water samples rose in 

concert with increasing numbers of P. australis 

(arrow #4). As the bloom of P. australis reached its 

maximum, toxigenic cells were concentrated in a 

narrow band near shore. It is possible, although in 

no way proven, that the cells responded to nutrient 

enrichment of near shore waters as a result of 

enhanced river flows following late spring rains. 

Anchovies collected from Monterey Bay 

during the height of the P. australis bloom 

contained high levels of domoic acid and high 

numbers of P. australis in their stomachs 

(Fig. 4). The P. australis bloom was replaced 

by a large P. pseudodelicatissima bloom (not 

shown on Fig. 3). Some P. pungens and P. 

multiseries were also present, but at relatively 

low levels. During this transition in species 

abundance, the concentration of domoic acid 

declined rapidly and precipitously in the 

upper water column (Fig. 3, arrow #5). Gut 

contents of anchovies collected at this time 

reflected this change in species abundance as 

well (Fig. 5), as they did not contain 

detectable levels of domoic acid. Reports of 

sickened sea lions frornblonterey Bay 

decreased in concert with the loss of P. 

australis fiom the upper water column and the 

rise in dominance of P. pseudodelicatissima. 

Notably, cultures of P. australis isolated fiom 

Monterey Bay produce domoic acid whereas 

isolates of P. pseudodelicatissima from the 

same area do not. 

During the P. pseudodelicatissima 

bloom inside Monterey Bay, high 

concentrations of toxigenic P. multiseries 

were found south and especially north of that 

region. Thus, species composition of Pseudonitzschia populations found within Monterey 

Bay and contiguous waters of the central 

California coast can differ substantially. 

Throughout the remainder of the year P. 

australis was not abundant in Monterey Bay. 
a.*po?pp?s,,, 

-...,.:a I... "-I*'E,m-r. 

Figure 3. 1998 time series data collected at the Santa Cruz wharf station showing results of sandwich 

hybridization assays (optical density, O.D.) for Pseudonizchtia australis, P. pungens, P. multiseries and 

North American strains of Alexandrium tamarense/catanella plotted as a function of time (Julian day). 

Increasing O.D. indicates a rise in species abundance; the dashed line indicates lower limit of detection. 

Corresponding amounts of domoic acid associated with a particulate fraction of plankton are given as 

pmol toxin per mL water filtered (top panel, bars). Arrows refer to particular events described in the text. 
Anchovy gut contents containing Pserrdo-~tdc austraiis, Nlag 29,1998. 

Figure 4. 

Scanning electron micrographs of anchovy stomach contents at the height of the 

P. auslralis bloom. Anchovies caught in the Monterey Bay area had stomachs packed with 

P. australis frustules (A = overview, B and C = close-up showing frustules are 

P. australis). 
Anchovy gut contents 6/ 10198 

Figure 5. 

Scanning electron micrographs of anchovy stomach contents after the P. australis bloom. 

Overview micrograph shows that anchovies caught in the Monterey Bay area had stomachs 

packed primarily with P. delicatissima (C), the dominant Pseudonizchtia in the water column 

at the time. Fragments of P. australis were also found (B), as well as frustules of P. multiseries 

(a), but the latter two species were not abundant. 
DIAGNOSIS 

The combination of clinical signs 

(scratching, seizuring, anchovies in fecal 

matter), histopathological (hippocampal 

necrosis), toxicologcal (domoic acid in 

serum, urine, feces, anchovies) and 

epidemiological findings (simultaneous 

mortality in sea lions and the presence of 

toxin-producing blooms) led to the 

diagnosis of domoic acid toxicity in the sea 

lions. This intoxication was due to blooms 

of Pseudonizchtia australis that were 

ingested by anchovies that in turn were 

eaten by California sea lions. Although a 

variety of incidental histopathologic and 

serologic changes were detected in the sea 

lions examined, they were not considered 

to be the cause of the mortality event. 

However, virus neutralization titers to 

phocine distemper virus, presence of 

protozoa in the brain and early neoplastic 

lesions warrant further investigation to 

determine their role in health of the 

California sea lion population. 

POST RELEASE MONITORING 

Three sea lions that survived the 

event were released with telemetry devices 

to monitor movements and survival after 

release. Adult females were selected, as 

animals of this sex and age are currently 

being tagged and monitored off San Miguel 

Island by Dr. Robert De Long and Sharon 

Melin from the National Marine Mammal 

Laboratory, and could be used as normal 

controls. The three animals selected had all 

shown typical clinical signs during the 

"event". One had seizures for only 48 hrs, 

one had repeated seizures for up to a week, 

and the third had seizures for up to one 

month post stranding. They were thus 

classed as mild, moderate and severe cases. 

None of the animals had been pregnant 

when stranded. As the tags are fixed to the 

animals using glue on the hair, the animals 

were not released until after the annual 

molt in September. 

The' three sea lions (CSL #3812, 

#3815 and #3822) were sedated with 

midazolam intramuscularly at 0.02 mgkg 

(mixed with atropine at 0.02 mgkg), then 

masked with isoflurane until relaxed but 

with intact gag reflex. Satellite-linked 

transmitters or platform transmitter 

terminals (ST-10 Argos PTT model; 

Telonics, Mesa, AZ) were glued to the 

dorsum between the scapulae using 5- 

minute epoxy. These tags were 

approximately 14 x 5 x 1 cm and weighed 

0.2 kg. To conserve battery power and 

prolong the life of the PTTs, the duty 

cycle was set to transmit for 24 hrs every 

other day. Location data were collected by 

Service Argos. Radio transmitters 

(Advanced Telemetry Systems, Isanti, 

MN) were attached to the heads of each 

sea lion. These transmitters weighed 0.05 

kg each and had 30 cm long whip 

antennae. 

The sea lions were tagged on 

November 4 and released on November 6 

at Weston Beach, Point Lobos State 

Reserve, Camel, California (Fig. 6). This 

site was chosen as it was within the 

Monterey Bay, the area in which the sea 

lions stranded during the event, and is a 

protected area frequented by this species. 

Two of these three sea lions were 

resighted. One animal was observed on 

December 10 1998 with a satellite 

transmitter and a VHF transmitter on Afio 

Nuevo Island (Morris, pers. comm.), 

although its ID number was not 

determined. #3 8 12 and #3 8 15 were 

sighted on December 23 1998, 48 days 

after release. On this day, #3815 had a 

radio transmitter, but had lost the satellite 

transmitter. Satellite data indicate that the 

transmitter detached from the sea lion 

while on shore. Transmissions were 

received from the transmitter until 

February 7 1999. Sea lion #38 12 was also 

sighted on January 8 1999 at Afio Nuevo 

Island, with both transmitters attached. 
Sea lion #3822 swam as far south 

as Point Benett, San Miguel Island (-325 

km), where it remained for five days, 

before traveling north again to areas off the 

Big Sur Coast. Sea lion #3822 also made a 

second trip down to San Miguel Island, and 

returned north again (Figure 6a). A total of 

170 positions was received for 64 days, 

until transmissions ceased on January 8 

1999 off Point Lobos (36.519N, 

121.954W). It is unclear whether signal 

cessation was a result of mortality, tag 

malfunction or tag loss. A total of 333 

positions was received for sea lion #3812, 

which traveled as far north as the Farallon 

Islands, and as far south as the Channel 

Islands. Sea lion #38 12 remained near the 

Channel lslands for one week, but returned 

to areas around Aiio Nuevo Island (Figure 

6b). The last transmission from #38 12 was 

94 days post release on February 7 1999 off 

Pescadero Point, California (3 7.24 1 N, 

122.488W). Sea lion #3815 primarily 

remained within and around areas adjacent 

to Monterey Bay while the satellite tag 

was attached. Although the date of tag 

detachment from this animal is unclear, 

transmissions ceased on February 7, 1999. 

The failure of transmissions from two tags 

within a day of each other suggests that 

this was due to battery expiration rather 

than death of the sea lions. 

At least two of the three satellitetagged sea lions survived up to three 

months post release and data indicated 

they were using areas where wild sea lions 

could be found. Melin et al. (1993) found 

that female California sea lions from San 

Miguel Island, which were tagged with 

satellite-linked time depth recorders, 

foraged northwest of San Miguel lsland 

during winter and spring months. 

Furthermore, tagged sea lions from San 

Miguel Island traveled as far as 460 km 

north along the mainland coast of 

California (Melin et al. 1993). Thus, 

movements of the three rehabilitated sea 

lions may have been typical. 
DISCUSSION 

This is the first report of domoic 

acid toxicity 'in marine mammals. However, 

as algal blooms producing domoic acid have 

been observed previously along the 

California coast (Walz et aL, 1994), and 

domoic acid toxicity was reported in brown 

pelicans off Monterey in 1991 (Work et al., 

1993), it is likely that this toxicosis has 

occurred previously in marine mammals. 

Clusters of stranded adult sea lions that 

showed similar neurologc signs to those in 

the animals in this 1998 event have been 

observed at other times along the California 

coast, although no cause was determined. In 

July 1978, 40 animals stranded in Ventura 

County displaying neurologic signs 

(Gilmartin et aL, 1979); in 1986, 11 sea 

lions showing opisthotonus and convulsions 

were admitted to TMMC (Vandenbroek et 

al., 1987); in 1988, 38 sea lions and ten 

northern fur seals (Callorhinus ursinus) with 

similar signs were admitted to TMMC 

(Gage et aL, 1989) and in 1992 18 sea lions 

stranded in San Luis Obispo County 

displaying similar signs (Beckrnen et aL, 

1995). Similar histologcal changes in the 

hippocampus to those in sea lions from this 

1998 event were observed by Dr. Linda 

Lowenstine in animals that died in the 1992 

event. Two stomachs from seizuring sea 

lions during the 1992 seizure event were 

submitted for domoic acid analysis with 

negative results. However, the stomachs 

were devoid of contents. No suitable 

samples,i.e. serum, urine or feces, are 

available from earlier animals for 

retrospective analysis. As there was a bloom 

of P. australis in Monterey Bay in 1992 

(Walz et aL, 1994), it is likely that domoic 

acid toxicity affected sea lions off California 

earlier than 1998. In addition, since this 

event there has been another similar 

occurrence in October 1998. 

From July 12 to October 17, 1998 

an additional eleven California sea lions 

stranded with similar clinical signs to the 

animals descnbed in this report. Nine of the 

animals stranded from October 3 to October 

17. Six animals were euthanized or died, 

and four animals recovered and were 

released. Four of six urine samples from 

sea lions in the October event analyzed 

for domoic acid by HPLC-UV were 

positive (Table 6), while one CSF sample 

was negative. Serum, urine, and 

cerebrospinal fluid (CSF) were tested by 

receptor binding assay only. Domoic 

acid was detected by RBA in three out of 

six urine samples from clinically affected 

animals. All serum and CSF samples 

tested were negative for domoic acid. 

During this time period, cells of P. 

australis did reach -10,000 cells per liter 

at the Santa Cruz pier, however, much 

higher cell concentrations were observed 

outside of Monterey Bay, again 

highlighting the difference between water 

masses within and outside of the Bay. In 

this case, it is likely that the bloom of P. 

australis was fueled by upwelling and 

that cells and their associated toxin did 

not penetrate Monterey Bay to any great 

extent. This recent episode illustrates 

the quick response that stranding rates 

and pimipeds may have to toxic blooms 

of P. australis. 

The combination of 

oceanographic data, evidence of domoic 

acid in the prey eaten by the -sea lions, 

clinical signs and histopathology are 

important in making a diagnosis of 

domoic acid toxicity, as detection of 

domoic acid in sea lion tissues is limited. 

The detection of domoic acid in only 

three serum samples by receptor binding 

assay is not surprising, as the plasma 

half-life is short in other species (21.6 

minutes in rats, 114 minutes in monkeys; 

(Truelove and Iverson, 1994). Blood 

samples were not collected from sea lions 

until they arrived at TMMC after initial 

stranding, detection of the animal by the 

public and transport by road up to 200 

miles. It is therefore likely that the 

domoic acid had been cleared from 

plasma by the time blood was collected 

in most cases. As domoic acid is water 

soluble and excreted in urine, it is also 

not surprising that it was detected in 

urine rather than plasma in some animals. 
The even higher detection rate in feces may 

reflect poor absorption from the gastrointestinal tract due to poor lipid solubility. 

After oral administration of - domoic acid to 

rats, recovery from feces was complete 

(Iverson et al., 1989). 

In contrast to the toxicologic results, 

both clinical signs and histopathologic 

changes were characteristic of domoic acid 

toxicity as reported in other species, 

although a variety of incidental signs and 

lesions were detected. The seizures were 

dramatic, and presumably a consequence of 

domoic acid binding in the brain. Domoic 

acid is an excitatory neurotoxin that binds to 

neurones via high-affinity AMPA- and 

kainate-sensitive glutamate receptors to 

produce excitotoxic cell death (Iverson and 

Truelove, 1994; Lam et al., 1997). In 

between seizures, the scratching behavior 

was reminiscent of the hallmark scratching 

syndrome documented in mice that 

distinguishes paralytic shellfish poisoning 

from domoic acid toxicity (Todd, 1993). 

Similar behavior was also described in 

pelicans intoxicated with domoic acid 

(Work et al., 1993). The ability of animals 

to survive exposure, as demonstrated by the 

satellite-tracking data, is also consistent with 

the survival of exposed humans in the 

Canadian outbreak in people who ate 

contaminated mussels (Per1 et al. 1990). 

The significance of the virus neutralization 

titers to phocine distemper virus (PDV) in a 

few animals is unclear. However, the lack of 

accompanying histopathological changes 

consistent with morbillivirus infection, 

negative RT-PCR for morbillivirus on brain 

tissue, and the similar seroprevalence of 

PDV antibodies in banked sea lion sera 

suggest the presence of antibodies in sea 

lions dylng during the event did not play a 

role in this event. In contrast, in the recent 

Mediterranean monk seal die-off, a 

controversy as to the cause of the die-off 

occurred, due to the isolation of a 

morbillivirus from tissue of affected 

animals, in addition to detection of 

saxitoxin (Osterhaus et al., 1997; Costas 

and Lopez-Rodas, 1 998). 

Over the time frame of the 

"seizuring sea lion" event, large numbers 

of dead pinnipeds and sea birds were 

observed on the beaches around 

Monterey Bay. The causes of death of 

these animals were not determined. 

However, many were severely emaciated. 

Based on gross appearance and 

predictions of species likely to be 

affected by changes in food availability 

during El Niiio events (Bodlun and 

Jameson 199 1, Trillmich and Ono 199 l), 

it is likely that many of these mortalities 

were a consequence of starvation due to 

food shortage. It is thus unclear how 

many sea lions in total were affected by 

domoic acid. The overall mortality due to 

domoic acid is likely to be more than 57 

individuals, but is unknown. As the 

overall California sea lion population is 

estimated at 167,000 individuals, an 

event causing mortality in 0.00004 % 

does not constitute a threat to the 

population. However, the rapid and 

dramatic nature of the event suggests the 

potential for similar harmful algal blooms 

to impact endangered species, such as the 

Steller's sea lion or Hawaiian monk seal, 

before control measures can be 

implemented. In addition, the 

susceptibility of humans to domoic acid 

suggests that outbreaks of toxicity in 

marine mammals should be fully 

investigated and measures taken to 

prevent human morbidity and mortality. 

Acknowledgments. 

This investigation would not have been possible without the willing collaboration and 

enthusiasm of each of the organizations involved. Special thanks are due to Diane Paschal1 for 

assistance in putting this report together, the Monterey Bay Aquarium Research Institute for 

funding a workshop in the wake of the event to bring investigators together and facilitate 

exchange of data, Wanda Dietrich for editing and formatting this document, and the staff and 

volunteers of TMMC for their hard work caring for the sea lions during the event. 
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Natural Toxins 2: 271-279. 
List of Terms 

dl 

g or gm 

HPLC 

HPLCIUV 

HPLCMS 

HPLCMSMS 

IM 

IV 

kg 

mg 

ml 

mm 

M 

mlz 

NOS 

NWFSC 

RB A 

SAX 

SSP 

TFA 

TMMC 

u g 

UCSC 

deciliter; one-tenth of a liter 

gram 

High Performance Liquid Chromotography 

High Performance Liquid Chromotography with ultraviolet 

detection 

High Performance Liquid Chromotography with mass 

spectrometry 

High Performance Liquid Chromotography with tandem mass 

spectrometry 

intramuscular 

intravascular 

kilogram; one thousand grams 

milligram; one-thousandth of a gram 

milliliter; one-thousandth of a liter 

millimeter; one-thousandth of a meter 

mass 

masslcharge 

National Ocean Service, National Oceanic and Atmospheric 

Administration, Charleston, South Carolina 

Northwest Fisheries Science Center, National Marine Fisheries 

Service, Seattle, Washington 

Receptor binding assay 

strong anion exchange 

species 

Trichloroacetic acid 

The Marine Mammal Center, Sausalito, California 

microgram; one-millionth of a gram 

University of California, Santa Cruz 
V) 

V) 

- m 

0 

s 

2 uuuuu 

v, LLLLLLLLLLZLLLLLLLLLLLLLZLLLLLLLLZLLLLLLLLLLZLLLLLL 

mmmm m- mm 

- +

3 

u 

2 

=uuuuuuu 

5 ~ u 0 

x a 

m 

w U a 0 

3 

x .- O~~~ob~-~~~~~~~~~~

u mmmmooOa~NNammme~*eeew*emm(DCDCDbaa0)m 

CDCDCDCDbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb 

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm 

AJJJJAJAAJAJJJAAJAAAJAJJAAAAJJJAAJ 

no mmmmmmm/,m/,mwmmm/,mmmmmmmmmmmmmmmmmm 4 0000000000000000000000000000000000 

V) 7 

i3 

c 

0 .- 

Y 

aa 

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Appendix 2C, Admit Blood Chemistries 

BUN 

rngldl 

Acc. # 

CSL 3901 (Palm 

CSL 3950 t~thena 

Creat 

rngidl 

Name 

167 

156 

Prot 

gmldl 

Na 

rneqfl 

6 ( 112 1 90 66 j 3.1 

6.1 1110 1 82 1 61 11.3 

Alb 

gm/dl 

K 

rneqll 

10.2 

8.1 

Glob 

gddl 

CI 

me@ 

Glu 

mgldl 

3.3 16.9 11.2 1 11 1 12 10.5 10.2 1105 34 15613017601 96 

1.9 / 6.2 ( 1.3 19.6 / 11 ] 0.5 10.2 1188 1 34 16518217181 155 

2881 62 1 na 

1381 24 1 na 

Ur. Ac.Ca 

rngldl mgldl 

Phos 

rngldl 

i 

GGT l~lk P 

rngldl lun 

I 

Tot Bil 

mgldl 

ET 

ull 

- 

Dir Bil 

rng/dl 

ALT 

u/l 

LDH 

ull 

Iron 

rncgldl 

Chol 

rngtdl 

Trigl CK 

rngldl 

I 
Appendix 3A Hematolowv, Pre-release 1 I 

Acc # l~ame IWBC IRBC ~HGB~HCT ~MCV IMCH 

CSL 3731 I JU-~i 1 19.2 14.191 16 144.8 1106.9 

CSL 3733 1 pentobarb 1 12.8 1 4.1 9 1 16 1 46.2 1 11 0.3 

. , 

CSL 3776 Professor p-ppp10.7 4.72 18 52.3 11 0.8 38.6 

CSL 3792 Howell . 15.3 4.2 16 43.7 103 37.4 

I I 

MCHC lsegs segs bands bands lymph lymph monos monos eos eos bas0 bas0 plat 

g/~~ 1% A~S % A~S % A~S % A~S % A~S V, A~S x lo3 

APPENDIX 4: Neurologic examination of the California sea lion 

Acc. No: Name: Date: Time: Observer: 

Posture 

1. Sitting up on fore flippers 

2. Recumbency: ventral, sternal, lateral R L, dorsal; 

3. Body twisted to R L 

4. Opisthotonus 

Responsiveness 

To audible or visual approach: 

1. Charges person 

2. Moves away at speed 

3. Moves away hesitantly 

4. Attempts to move but can't 

5. Lifts head 

6. Moves eyes to observe 

7. No response 

8. Seizuring 

To touching lumbar area: 

I. Does not allow due to aggressive behavior 

2. Moves away rapidly 

3. Moves away slowly 

4. Lifts head 

5. Attempts to move but unable 

6. Unresponsive 

Locomotion 

Ground: 

1. Moves rapidly around pen using all 4 flippers 

2. Moves reluctantly around pen with all 4 flippers 

3. Moves around pen but does not use hind-flippers 

4. Ataxic 

5. Scoots on ventrum without using fore-flippers 

6. Attempts to get up on fore flippers but falls over 

7. Unable to use fore and hind-flippers on same side (hemiplegia) 

8. Circling 

9. Immobile 

Pool: 

1. Swims normally, leaps over side of pool easily 

2. Swims normally, gets out down ramp 

3. Floats without using flippers 

4. Cannot enter pool