Evidence for a Novel Marine Harmful Algal Bloom:

Cyanotoxin (Microcystin) Transfer from Land to Sea

Otters

Melissa A. Miller1,2*, Raphael M. Kudela2, Abdu Mekebri3, Dave Crane3, Stori C. Oates1, M. Timothy

Tinker4

, Michelle Staedler5, Woutrina A. Miller6, Sharon Toy-Choutka1, Clare Dominik7, Dane Hardin7,

Gregg Langlois8

, Michael Murray5, Kim Ward9, David A. Jessup1

1 Marine Wildlife Veterinary Care and Research Center, California Department of Fish and Game, Office of Spill Prevention and Response, Santa Cruz, California, United

States of America, 2Ocean Sciences Department, University of California Santa Cruz, Santa Cruz, California, United States of America, 3 Water Pollution Control Laboratory,

California Department of Fish and Game, Office of Spill Prevention and Response, Rancho Cordova, California, United States of America, 4 Western Ecological Research

Center, United States Geological Survey, Long Marine Laboratory, Santa Cruz, California, United States of America, 5 Monterey Bay Aquarium, Monterey, California, United

States of America, 6 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, California, United States

of America, 7 Applied Marine Sciences, Livermore, California, United States of America, 8 California Department of Public Health, Richmond, California, United States of

America, 9 Division of Water Quality, State Water Resources Control Board, Sacramento, California, United States of America

Abstract

‘‘Super-blooms’’ of cyanobacteria that produce potent and environmentally persistent biotoxins (microcystins) are an

emerging global health issue in freshwater habitats. Monitoring of the marine environment for secondary impacts has been

minimal, although microcystin-contaminated freshwater is known to be entering marine ecosystems. Here we confirm

deaths of marine mammals from microcystin intoxication and provide evidence implicating land-sea flow with trophic

transfer through marine invertebrates as the most likely route of exposure. This hypothesis was evaluated through

environmental detection of potential freshwater and marine microcystin sources, sea otter necropsy with biochemical

analysis of tissues and evaluation of bioaccumulation of freshwater microcystins by marine invertebrates. Ocean discharge

of freshwater microcystins was confirmed for three nutrient-impaired rivers flowing into the Monterey Bay National Marine

Sanctuary, and microcystin concentrations up to 2,900 ppm (2.9 million ppb) were detected in a freshwater lake and

downstream tributaries to within 1 km of the ocean. Deaths of 21 southern sea otters, a federally listed threatened species,

were linked to microcystin intoxication. Finally, farmed and free-living marine clams, mussels and oysters of species that are

often consumed by sea otters and humans exhibited significant biomagnification (to 107 times ambient water levels) and

slow depuration of freshwater cyanotoxins, suggesting a potentially serious environmental and public health threat that

extends from the lowest trophic levels of nutrient-impaired freshwater habitat to apex marine predators. Microcystinpoisoned sea otters were commonly recovered near river mouths and harbors and contaminated marine bivalves were

implicated as the most likely source of this potent hepatotoxin for wild otters. This is the first report of deaths of marine

mammals due to cyanotoxins and confirms the existence of a novel class of marine ‘‘harmful algal bloom’’ in the Pacific

coastal environment; that of hepatotoxic shellfish poisoning (HSP), suggesting that animals and humans are at risk from

microcystin poisoning when consuming shellfish harvested at the land-sea interface.

Citation: Miller MA, Kudela RM, Mekebri A, Crane D, Oates SC, et al. (2010) Evidence for a Novel Marine Harmful Algal Bloom: Cyanotoxin (Microcystin) Transfer

from Land to Sea Otters. PLoS ONE 5(9): e12576. doi:10.1371/journal.pone.0012576

Editor: Ross Thompson, Monash University, Australia

Received April 26, 2010; Accepted August 2, 2010; Published September 10, 2010

Copyright: 2010 Miller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Financial assistance was provided by the California Division of Water Quality, State Water Resources Control Board (SWRCB), the California Department

of Fish and Game, Division of Spill Prevention and Response and the CDFG Water Pollution Control Laboratory. Co-Author Kim Ward of the SWRCB assisted with

basic concepts of study design and data analysis in relation to ongoing statewide research on microcystins, but the various funding agencies had no directive or

editorial role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: mmiller@ospr.dfg.ca.gov

Introduction

During 2007, 11 dead and dying southern sea otters were

recovered along the shore of Monterey Bay in central California

with lesions suggestive of acute liver failure. Some animals were

diffusely icteric and their livers were enlarged, bloody and friable.

Expected causes for this condition, such as systemic bacterial

infection were excluded via microscopic examination and

diagnostic testing. Livers from affected animals tested positive for

cyanotoxins (microcystins) via liquid chromatography-tandem

mass spectrophotometry (LC-MS/MS) and hepatic lesions consistent with microcystin intoxication were observed microscopically.

Environmental surveillance revealed that some local freshwater

lakes and rivers supported Microcystis blooms during late summer

and autumn, triggering the investigation reported here.

Cyanobacteria (formerly called ‘‘blue-green algae’’) have a

worldwide distribution and can form extensive blooms in freshwater

and estuarine habitat. Toxin production by the cyanobacterium

Microcystis aeruginosa was first reported in 1946 [1] and additional

toxic species have been described. Exposure to environmentally

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stable microcystins in food, drinking water, nutritional supplements

and during medical dialysis can cause significant and sometimes

fatal hepatoxicity and possible tumor induction in humans and

animals [2-6]. Microcystins are fast becoming a global health

concern and recurrent blooms with toxin elaboration have been

reported throughout Europe [7,8], Asia [9,10], Africa [11,12],

Australia [13,14] and North and South America [15–17]. Factors

that contribute to bloom formation and toxin production include

warm water [18,19], nutrient enrichment [19,20] and seasonal

increases in light intensity [2,21]. Rising global temperatures and

eutrophication may contribute to more frequent events and

cyanobacterial ‘‘super-blooms’’, with enhanced risks to human

health [23].

Until recently, microcystin intoxication was considered a public

health issue mainly of freshwater habitat, reflected by the vast

body of published literature on potential human health risks due to

microcystin exposure in rivers, lakes, reservoirs and freshwater

aquaculture [4,13,16,24,25,26]. In contrast, monitoring of marine

water and seafood for similar risks has been limited, despite

confirmation of outflows of microcystin-contaminated freshwater

to the ocean [14,17,27,28], detection of impacts by microcystins

on copepods, corals and fish [29–31] and identification of proteins

with protein phosphatase inhibitory activity in seawater, suggesting the existence of an additional class of marine ‘‘Harmful Algal

Blooms’’ (HAB); hepatotoxic shellfish poisoning (HSP) [15].

The ability of potent and environmentally stable cyanotoxins to

magnify trophically poses additional risks: Microcystin accumulation has been demonstrated in fresh and saltwater mussels [7,32],

farmed crustaceans [33,34], fish [30] and possibly humans [26]. In

addition, exposure of estuarine and marine biota to microcystins

may trigger behavioral adaptations, such as decreased feeding on

co-occurring nutritious species, that facilitate trophic transfer

[9,35–38]. Despite these concerns, worldwide shellfish sanitation

and water safety programs do not typically include microcystin

testing.

Along the Pacific coast of the United States, large-scale

Microcystis blooms with toxin production occur each year in lakes

and rivers throughout Washington [39], Oregon [4] and

California [40,41]. In California, Microcystis-contaminated runoff

has been documented at the marine interfaces of the Klamath

River [42] and San Francisco Bay [17]. Here we extend areas of

concern to include the central California coast and document

numerous marine mammal deaths due to microcystin intoxication.

The potential for cyanotoxins to flow to the ocean, resulting in

deaths of marine species is a newly recognized problem.

Demonstration of bioaccumulation in marine invertebrates and

deaths of threatened southern sea otters due to microcystin

intoxication provides strong evidence for significant and recurrent

marine pollution by freshwater-derived microcystins within North

America’s largest national marine sanctuary. Because sea otters

and humans utilize the same coastal habitat and share the same

marine foods, our findings in sea otters are also likely to have

important human health implications.

Materials and Methods

Performance of this research was approved by the California

Department of Fish and Game, Office of Spill Prevention and

Response and the University of California.

1.) Environmental testing

Chemical confirmation of microcystin exposure in tissues from

southern sea otters stranding during 2007 prompted investigation

of local freshwater sources flowing into Monterey Bay for any prior

history of cyanobacterial blooms. This investigation revealed that

Pinto Lake, located approximately 8.5 km inland from Monterey

Bay had a history of severe and recurrent Microcystis blooms with

microcystin production. Visual examination of Pinto Lake, light

microscopy and liquid chromatography-mass spectrophotometry/

mass spectrophotometry (LC-MS/MS) testing in fall, 2007,

confirmed the occurrence of an extensive Microcystis bloom with

high toxin production, leading authorities to post warning signs at

this location for several weeks. Stepwise sampling of water and

surface bloom from Pinto Lake, its drainage into Corralitos Creek,

and the Pajaro River that carries water from this region to

Monterey Bay was performed during the bloom event.

Because bloom events are often ephemeral and patchy, sensitive

methods are required to facilitate source tracking efforts. We

investigated use of resin-based, Solid Phase Adsorption Toxin

Tracking (SPATT) samplers to passively monitor fresh and salt

water for microcystin contamination. SPATT was first proposed

for HAB monitoring in 2004 to circumvent disadvantages

associated with invertebrate bioassays [43]. To evaluate their

performance under laboratory conditions, SPATT bags were

placed into subsamples of concentrated water/Microcystis mixtures

from Pinto Lake that were used for laboratory-based invertebrate

exposures (section 3). Replicate SPATT samplers were also placed

in each exposure tank during the invertebrate studies to assess

consistency and repeatability of microcystin adsorption, and

additional SPATT bags were deployed in the local marine

environment and at the freshwater outflows of selected local rivers.

SPATT bags were constructed from 100 micron Nitex bolting

cloth filled with 3 g (dry weight) HP20 (Diaon) resin. For

activation, bags were soaked in 100% HPLC-grade methanol

(MeOH) for 48 hours, rinsed thoroughly, transferred into a fresh

volume of Milli-Q for MeOH residue removal by sonication and

stored in Milli-Q at 4–6uC prior to use. Plastic embroidery hoops

were used to fasten the bags in place during field deployment.

After exposure, SPATT bags were evaluated using LC-MS for

adsorption of domoic acid and microcystins as described below. In

the laboratory, adsorption of environmentally-relevant concentrations of microcystins (hundreds of ppb) was observed in ,24 h,

following an exponential decay (adsorption) curve in a closed

volume of filtered Pinto Lake water. In prior studies, 100%

recovery of microcystin was achieved with simple extraction

procedures (sequential 50% MeOH column extractions) (data not

shown).

2.) Sea otter necropsy and microcystin testing

Sea otter carcasses were recovered by stranding network

members, chilled with ice and transported to CDFG for necropsy

as previously described [44]. Detailed postmortem examinations

were performed by a veterinary pathologist and all major tissues

were fixed in 10% neutral buffered formalin, trimmed, paraffinembedded and 5 mm-thick, hematoxylin and eosin (H&E)-stained

sections prepared and examined on a light microscope. Supplementary diagnostic testing included bacterial and fungal culture,

immunofluorescence and PCR for common serovars of Leptospira

interrogans and LC-MS/MS analysis of urine, gastrointestinal

content, feces and urine for the presence of microcystin, domoic

acid, okadaic acid, nodularin, yessotoxin and anatoxin-A. The

primary and contributing cause(s) of death were determined based

on gross lesions, histopathology and diagnostic results. Tissues,

urine, serum and gastrointestinal contents were also cryoarchived

at 280 C.

Analysis of water, tissue and digesta for microcystins was

performed at the California Department of Fish and Game

(CDFG) Water Pollution Control Laboratory or at the University

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of California, Santa Cruz. The preferred method of analysis postextraction was high performance liquid chromatography tandem

mass spectrometry (LC-MS/MS), following the protocols of

Mekebri et al. [45]. Prior to testing, tissue samples were first

homogenized using a Bucchi B-400 mixer equipped with a

titanium knife assembly. Pre-weighed samples were mixed with

methanol: water (90:10) using a PolytronH homogenizer for four

minutes, followed by sonication for one hour. The target analytes

were microcystin (MCY)-RR, -Desmethyl RR, -LR, -Desmethyl

LR, -LA, -LF, -LW, -LY and -YR, domoic acid, nodularin and

okadaic acid. Certified calibration solution standards purchased

from Sigma Aldrich and NRC-CNRC (Certified Reference

Materials Program, Institute for Marine Biosciences, National

Research Council of Canada) were used for method development,

analyte identification and quantitation. HPLC-grade solvents

(acetonitrile, methanol, water), glass fiber filters (Type A/E,

90 mm, 1 mm), Gelman AcrodiscH CR PTFE syringe filters

(13 mm, 0.45 mm), and mobile phase additives, ACS grade formic

acid (98%) and trifluoroacetic acid (99%) were also used. A

combined intermediate MCY standard working solution was made

in methanol and used to prepare a matrix spiking solution

(20 ppb), which was serially diluted to develop a seven level

calibration curve ranging from 0.2 to 200 ppb.

To determine total microcystin concentration and congener

type(s) in water, the cyanobacterial cell walls were ruptured by

repeated freeze-thawing and sonication and a 100 ml aliquot was

filtered under vacuum through a glass fiber filter. Water and filters

were extracted separately and filters containing planktonic material

were extracted twice with 15 mL of methanol-acidified water

(90:10, v/v) by homogenizing for 1–2 minutes using a PolytronH,

followed by 10 minutes of sonication in an ultrasonic bath.

For SPATT detection systems, only dissolved toxins were

measured, so cell disruption was not required. Samples were

analyzed using an Agilent 1200 liquid chromatograph (LC)

connected to a 6130 quadrupole MS, using Selected Ion Monitoring

(SIM). For all other samples, an Agilent 6410 triple quadrupole

(QqQ) LC-MS was used for LC-MS/MS analysis. The following

microcystin ions (m/z) were monitored: 519.8 -RR and 512.8 -

desmethyl RR are both [M+2H]2+; 105.6 -YR, 995.7 -LR, 981.7 -

desmethyl LR, 910.6 -LA, 1026.6 -LW, 987.6 -LF and 825.5 NODR were monitored using [M+H]+ using multiple reaction

monitoring (MRM) mode. Full scan was also collected over the

range 100–1100 amu. The MRM windows were established for

microcystins using the MSMS product ions, which are the Adda

fragments of m/z 135.2 and m/z 213 produced by the transition of

the protonated parent ions. Agilent Mass Hunter software was used

to collect and process data. The estimated method detection limits

(MDL) and reporting limits (RL) for water samples were 0.02 mg/L

(ppb) and 0.05 mg/L (ppb) for MCY and DA respectively, and

0.01 mg/L (ppb) and 0.02 mg/L (ppb) respectively for OA. The

estimated method detection limit and reporting limit for tissues were

0.500 ng/g and 1.00 ng/g wet weight, respectively, for all toxins.

3.) Laboratory exposure of marine invertebrates

To assess microcystin uptake and retention by marine invertebrates consumed by humans and sea otters, freshwater/cyanobacterial mixtures were collected during a summer, 2009 Microcystis

bloom at Pinto Lake. Dominance of Microcystis was confirmed

microscopically and total microcystin concentrations were determined via LC-MS/MS. Live marine invertebrates were collected

from Monterey Bay or purchased from commercial vendors,

including species that are commonly farmed or harvested, such as

Pacific oysters (Crassostrea gigas), manila clams (Tapes semidecussatus),

mussels (Mytilus edulis), snails (Tegula spp.), red rock crabs (Cancer

productus) and dungeness crabs (Cancer magister).

Three 1,022 L, temperature-controlled seawater tanks were used

to complete the invertebrate exposure studies. The tanks were

designed to permit water sampling at the top, middle and bottom of

each tank, so that microcystin distributions could be followed

through time and compared with results from invertebrate testing.

Invertebrates were divided randomly between control (Tank 1), low

exposure (Tank 2) and high exposure (Tank 3) tanks and allowed to

acclimatize for 3 to 7 d. Bivalves and snails were placed in wire

cages or plastic mesh bags and suspended at least 20 cm below the

water surface. Large crabs were placed in plastic mesh enclosures

that allowed them to range from just below the tank surface to just

above the bottom. Snails were fed fresh Macrocystis kelp fronds and

crabs were provided with chopped capelin (Mallotus villosus) every

other day. Filter-feeders were exposed to plankton in continually

flowing seawater from Monterey Bay until initiation of the

microcystin exposure, and then from day 4 to day 21 of the

experiment. Starting 4 days post-exposure, all tanks were

continually flushed with clean seawater and water and invertebrate

sampling continued for 21 days to determine post-exposure

depuration characteristics for freshwater microcystins.

At the start of the exposures, a less concentrated Microcystis

mixture collected from Pinto Lake during a bloom event (2.2 ppm

[2,195 ppb] aqueous microcystin–LR mixed with suspended

Microcystis) was added to the low exposure tank (Tank 2). A more

concentrated mixture (10.6 ppm [10,600 ppb] aqueous microcystin–LR mixed with suspended Microcystis) was added to the high

exposure tank (Tank 3) at the same time, while Tank 1 (the

negative control) contained only seawater. Microcystin LR

concentrations were measured in invertebrates and seawater

sampled from the top, middle and bottom of each tank at regular

intervals (24 H, 48 H, 72 H, 7 D, 14 D, and 21 D postexposure).

During the first 96 H postexposure, invertebrates in tanks 2 and 3

were continually exposed to the microcystin-contaminated inoculum, while Tank 1 contained only recirculating, clean seawater

and served as a negative control. Positive controls consisted of nonexposed water and invertebrate tissues spiked with known

concentrations of a commercial preparation of microcystin-LR

prior to LC-MS/MS testing.

Following 96 H of continuous microcystin exposure, all 3 tanks

were flushed with clean seawater and sampling continued through

21d post-exposure. Prior to seawater flushing, water was collected

from tanks 2 and 3, refrigerated and sub-sampled at the same

intervals as invertebrates to determine persistence of microcystin

toxin in seawater. Invertebrates were washed in tapwater prior to

dissection to remove any surface contamination by Microcystis or

microcystin. Invertebrate sub-sampling techniques reflected patterns of consumption by humans or otters: For snails, the entire

body and shell was homogenized and tested whole, while the soft

parts of bivalves and crabs were removed and the shells and

carapace were discarded. The gastrointestinal tract and/or

hepatopancreas was collected and screened for the presence of

microcystin-LR in addition to archiving whole bodies and muscle

tissue for evaluation as funds permit. All samples were refrigerated

or frozen at 280 C prior to LC-MS/MS testing.

Results

1.) Environmental testing

Analysis (LC-MS/MS) of water from Pinto Lake in fall, 2007

confirmed the occurrence of an extensive Microcystis bloom with

high toxin production (Fig. 1). During this period, microcystin

concentrations in scum from the surface of Pinto Lake exceeded

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2,100 ppm (2.1 million ppb) MCY-LA, or approximately

2,900 ppm (2.9 million ppb) total microcystins, which is one of

the highest microcystin concentrations ever reported from an

environmental sample. During this same period, stepwise sampling

of water and surface bloom from Pinto Lake, its drainage through

Corralitos Creek and the Pajaro River confirmed the presence of

Microcystis and microcystins from Pinto Lake to the river channel

within 1 km of the ocean (Fig. 1). Recurrent Microcystis blooms

with toxin production were also confirmed microscopically and via

chemical analysis in samples from Pinto Lake and surrounding

waters in 2008 and 2009 (data not shown). The most common

microcystin congener that was detected in samples from Pinto

Lake and the adjoining watershed during the 2007 event was

MYC-LA, but MCY-RR, MCY-LR, MCY-Desmethyl-LR,

MCY-LF and MCY-YR were also detected, and MCY-LR was

repeatedly detected in Pinto Lake using SPATT between 2009 and

2010. No other biotoxins were detected in freshwater samples.

Deployment of SPATT into microcystin/water mixtures collected

from Pinto Lake demonstrated 100% adsorption of free MCY

in ,24 h (Fig. 2). In addition, higher sensitivity of SPATT for

microcystin detection in water, when compared to intermittent

‘‘grab’’ samples, was also demonstrated (Fig. 3). During laboratory

invertebrate studies, good agreement between duplicate SPATT

bags suspended in each tank was noted for Tanks 2 and 3 (low and

high microcystin exposure, respectively), while SPATT bags hung

in Tank 1 (negative control) tested negative for microcystin (data

not shown).

Using field-deployed SPATT, ocean water and the marine

interfaces of selected coastal rivers flowing into Monterey Bay

tested negative for microcystins during the dry season in summer

and early fall of 2009, when freshwater runoff was minimal.

However, at the onset of the fall rainy season (October-November,

2009), SPATT deployed at the marine outfalls of the Pajaro and

Salinas Rivers tested positive for microcystins via LC-MS (data not

shown). SPATT samplers deployed weekly in the ocean at the

Santa Cruz Municipal Pier (Figure 4) during 2008 and 2009

routinely tested negative for microcystins, suggesting that the main

source of these toxins for sea otters was not marine in origin.

However, low levels of MCY-LR were detected at the pier (located

near the mouth of the San Lorenzo River), after the first major

storm event of Fall, 2009. Limited testing of fresh- and seawater

and invertebrates from other regions of Monterey Bay did not

reveal additional sources of microcystin-contamination (data not

shown). Sea otter stranding patterns suggest that similar pollution

events occurred in other coastal areas, but were not detected.

2.) Sea otter necropsy and microcystin testing

Between 1999 and 2008, livers from 21 southern sea otters with

gross and/or microscopic evidence of liver disease (Figs. 5 and 6)

tested positive for microcystins via LC-MS/MS (Table 1). On the

microscope, livers of microcystin-positive sea otters exhibited

hepatocellular vacuolation, apoptosis, necrosis and hemorrhage

(Fig. 6) consistent with previous descriptions of microcystin

intoxication in humans and animals [1,12,13,46,13,47]. In

contrast, livers from 2 captive sea otters (Table 1) and 19 wild

otters without evidence of primary liver disease (data not shown)

tested negative for microcystin. Carcasses of otters dying due to

microcystin intoxication appeared to cluster near river mouths,

Figure 1. Tracing freshwater contamination by microcystins

from land-to-sea. Inset: Sample of surface water collected during a

‘‘super-bloom’’ of Microcystis in Pinto Lake in fall, 2007 (Caution: Nitrile

gloves and other appropriate personal protective equipment should be

used to prevent dermal contact when collecting environmental samples

of Microcystis and microcystins). Main figure: Time-matched microcystin-LA concentrations (ppb) in samples from Pinto Lake, just

downstream in Corralitos Creek and the receiving waters of the Pajaro

River within 1 km of Monterey Bay. Asterisks (*) indicate sampling

locations where Microcystis was detected microscopically.

doi:10.1371/journal.pone.0012576.g001

Figure 2. Evaluation of Solid Phase Adsorption Toxin Tracking

(SPATT) sampler adsorption characteristics for freshwater

microcystins. SPATT adsorption characteristics for microcystin were

tested in the laboratory using Pinto Lake water amended with a known

quantity of microcystin-LR. A control sample (open symbols) showed no

change in microcystin concentration over time; In contrast, SPATT HP20

resin-based samplers (solid symbols: error bars represent standard

deviation of 3 replicates) show rapid microcystin adsorption, with neartotal depletion of microcystins from a controlled volume of water

within ,24 hours.

doi:10.1371/journal.pone.0012576.g002

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coastal ponds, embayments and harbors, all areas with significant

potential to receive and retain plumes of contaminated fresh water

(Fig. 4).

The earliest confirmed case was in 1999 (Table 1). The greatest

number of cases detected/year was in 2007, with 11 LC-MS/MSconfirmed sea otter deaths due to microcystin intoxication. Based

on preliminary test results, 71% of known, microcystin-associated

sea otter deaths have occurred since 2005 and 81% of affected

animals stranded within Monterey Bay, California. However,

additional cases were detected along the Big Sur and South-central

California coastline, suggesting that multiple point-sources for

microcystin exposure exist along the central California coast. One

additional case was suspected based on lesions observed on

histopathology, but the liver tested microcystin-negative, perhaps

due to near-total loss of hepatocyte mass in the liver of this animal.

Pathology associated with microcystin intoxication in sea otters

will be described in greater detail in a subsequent manuscript.

Hepatic microcystin concentrations varied from 1.36 to

348 ppb wet weight (ww; Table 1). Various forms of microcystin

(MCY-RR, -LR and -desmethyl LR) were detected in sea otter

livers, with the majority of otters (19/21) testing positive for MCYRR, compared to 2/21 and 1/21 for MCY-LR and MCYdesmethyl-LR, respectively. One liver tested positive for both

MCY-RR and -LR. Feces of one otter recovered during necropsy

also tested positive for microcystin, but it is unclear whether this

represents ingestion of contaminated prey or enterohepatic toxin

circulation. The microcystin congener that was detected at the

highest concentration in a sea otter liver was MCY-LR (348 ppb

ww). All water and tissue samples tested negative for okadaic acid,

nodularin, yessotoxin and anatoxin-A. Urine from some otters

with microcystin-positive livers also tested low-positive for domoic

acid on LC-MS/MS; this biotoxin is widely distributed in

sediments within Monterey Bay [48] and low levels of domoic

acid are commonly detected in sea otter urine at necropsy (M.

Miller, pers. commun.).

Four microcystin-positive otters had been previously captured

and fitted with intraperitoneal VHF transmitters as part of longterm studies on habitat use, prey selection, and environmental

exposure to biological pollutants. The home ranges of all four

otters overlapped at the same point in south Monterey Bay (Fig. 7),

suggesting a common source of microcystin exposure. Based on

the spatial distribution of home ranges for radio-tagged southern

sea otters [49], the likelihood of any four animals having

overlapping home ranges by chance is ,5.8%. Moreover, bivalve

mollusks were the first or second most frequently consumed prey

type for three of the four otters (for the fourth otter, no foraging

data were available). This trait was shared with just 27% of all

tagged animals; the majority preferentially fed on non-bivalve prey

such as crabs, abalone or urchins [50]. Similar stranding clusters

were identified for other microcystin-poisoned otters, leading to

identification of several high-risk sites for microcystin intoxication

along the shores of Monterey Bay (Fig. 4). Collectively these

findings raised suspicion of bivalve prey as a possible vehicle for

microcystin poisoning of sea otters.

Figure 3. Variation in microcystin detection between conventional ‘‘grab’’ samples and Solid Phase Adsorption Toxin Tracking

(SPATT). Comparison of microcystin (MCY-LR) detection in fresh water using intermittent ‘‘grab’’ sampling (sample periods indicated by black

circles) and SPATT (solid line indicating weekly averaged toxin values) in Pinto Lake, demonstrating the higher sensitivity of SPATT for microcystin

detection. Grab samples were collected at the beginning of each weekly SPATT deployment, and from the same sample location, so each 7-day

integrated SPATT deployment is bracketed by two grab samples.

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3.) Laboratory exposure of marine invertebrates

Intact Microcystis cyanobacteria initially accumulated at the top

of the seawater tanks, but were almost completely lysed after

48 hours. Microcystin–LR was detectable in seawater for the

duration of the study (21 days) and microcystin concentrations in

refrigerated seawater had declined only 44–71% from mean one

hour post-exposure levels after 3 weeks (Table 2).

Significant bioconcentration of microcystin by marine bivalves

(clams, mussels and oysters) and snails, but not large marine crabs,

was documented (Table 3), with tissue concentrations of microcystin–

LR up to 107 times higher in invertebrate tissues than in adjacent

seawater. Microcystin concentrations in gastrointestinal tissues

ranged from negative to 1,324 ppb wet weight (ww) in invertebrates,

with the highest concentrations observed in clams, mussels and

oysters sampled between 24 and 48 hours post-exposure.

Marine bivalves were also slow to depurate ingested microcystin;

despite continuous seawater flushing beginning at 96 hours postexposure, gastrointestinal microcystin concentrations at 14 days

Figure 4. Map of Monterey Bay showing distribution of sea otters dying due to microcystin intoxication (yellow circles). Note spatial

association of sea otter strandings with coastal locations of river mouths, harbors, coastal ponds and embayments. Habitat utilization distributions for 4

radio-tagged, microcystin-poisoned otters are plotted as kernel density distributions fit to daily re-sighting locations (red shading, with regions of most

intense shading corresponding to the habitats most frequently utilized by affected animals). Locations of freshwater samples collected during a ‘‘Superbloom’’ of Microcystis in 2007 are indicated by green circles, with numbers that correspond with the microcystin concentrations listed in Figure 1.

doi:10.1371/journal.pone.0012576.g004

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PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e12576
Figure 5. Microcystin detection in sea otter tissues was linked to bivalve consumption, liver damage and icterus. A.) Wild southern sea

otter (Enhydra lutris nereis) consuming a clam in Elkhorn Slough, Monterey Bay. B.) Diffuse icterus of oral mucous membranes of an otter poisoned by

microcystin, due to severe hepatic damage and elevated plasma bilirubin. C.) Severe icterus of cartilage at the costochondral junction in a sea otter

that died due to microcystin intoxication.

doi:10.1371/journal.pone.0012576.g005

Figure 6. Gross and microscopic hepatic lesions of microcystin intoxication in sea otters, compared to control livers. A.) Gross

appearance of normal sea otter liver. B.) Swollen, hemorrhagic liver from a sea otter that died due to microcystin intoxication. C.) Microscopic view of

normal sea otter liver, D.) Microscopic appearance of liver from an otter that died due to microcystin intoxication, demonstrating hepatocyte swelling,

cytoplasmic vacuolation, necrosis or apoptosis and parenchymal hemorrhage. Small greenish-gold accumulations of bile are apparent at the upper

left and center-right portions of the photomicrograph.

doi:10.1371/journal.pone.0012576.g006

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post-exposure were at 120%, 14% and 6.5% of 24 hour postexposure concentrations for oysters, clams and mussels, respectively

(Table 3). Mussel digestive tract remained microcystin-positive

(30.5 ppb ww) 21 days after the initial exposure period and

following 17 days of continuous exposure to clean seawater, which

was the longest post-exposure timepoint evaluated during this study

Some variation in microcystin concentration between sample

periods was attributed to differences in toxin uptake by individual

bivalves, similar to the approximately 4-fold individual variability

noted for bioaccumulation of domoic acid and saxitoxin in

individual mussels from California coastal waters (R. Kudela, pers.

commun.). All non-exposed controls (ambient seawater and

invertebrates) tested negative for microcystin throughout the study

(data not shown), which is consistent with a proposed freshwater

(not marine) source of cyanotoxin exposure for sea otters.

Discussion

Here we provide the first documentation of microcystin

intoxication in a marine mammal. Our research confirms deaths

of threatened southern sea otters from microcystin intoxication

and incorporates 3 distinct, but interconnected lines of scientific

inquiry to address the hypothesis that land-sea flow of microcystins

with trophic transfer through marine invertebrates is the most

likely route of sea otter exposure: 1) Time-integrative passive

samplers were deployed in fresh and marine systems along the

central California coast, confirming the presence of microcystins;

2) Necropsy, histopathology and chemical analysis of tissues from

stranded southern sea otters and 3) Determining dynamics and

persistence of freshwater microcystin uptake from contaminated

seawater by marine invertebrates using controlled laboratory

experiments. Although trophic transfer of biotoxins from plankTable 1. Stranding information and microcystin (MCY) concentrations (ppb wet weight) for wild, microcystin-positive sea otters

and captive controls.

Animal number Stranding date Stranding region Sample tested MCY-RR MCY-LR MCY-Desmethyl LR

1280-04 (Captive control) 6/27/2002 N/A Liver nd1 nd nd

1485-06 (Captive control) 11/14/2001 N/A Liver nd nd nd

3216-99 7/28/1999 Monterey Bay Liver 1.36 nd nd

3377-00 6/26/2000 Monterey Bay Liver 2.04 nd nd

3858-03 3/17/2003 Estero Bay Liver nd 11.8 nd

3955-03 5/8/2003 Monterey Bay Liver 3.19 nd nd

4240-04 6/5/2004 Monterey Bay Liver nd nd 1.53

4294-04 8/25/2004 Monterey Bay Liver 13.13 nd nd

3110-98 5/14/2006 Monterey Bay Liver 9.52 nd nd

4811-06 8/25/2006 Estero Bay Liver 7.71 nd nd

4844-06 9/24/2006 Monterey Bay Liver 3.62 nd nd

4913-07 1/30/2007 Monterey Bay Liver 61.58 nd nd

5020-07 6/9/2007 Monterey Bay Liver 38.45 348 nd

5023-07 6/9/2007 Monterey Bay Liver 104.46 nd nd

5036-07 6/25/2007 Monterey Bay Liver 2.69 nd nd

5082-07 8/16/2007 Monterey Bay Liver 5.29 nd nd

5108-07 9/23/2007 Monterey Bay Liver 14.39 nd nd

5167-07 11/21/2007 Monterey Bay Liver & feces 18.7 & 16.4 nd nd

5174-07 11/30/2007 Monterey Bay Liver 6.18 nd nd

5179-07 12/1/2007 Monterey Bay Liver 3.76 nd nd

5182-07B 12/2/2007 Monterey Bay Liver 4.8 nd nd

5185-07 12/6/2007 Estero Bay Liver 1.97 nd nd

5416-08 11/8/2008 Big Sur Liver 7.58 nd nd

1

nd = microcystin concentration was below minimum detection limits on liquid chromatography-tandem mass spectrophotometry.

doi:10.1371/journal.pone.0012576.t001

Figure 7. Overlapping home ranges of 4 tagged southern sea

otters that died due to microcystin intoxication. Note the spatial

overlap of all 4 home ranges on the north central Monterey Peninsula

near Monterey Harbor (bracket): This harbor appears to be one of several

high-risk locations for microcystin poisoning of sea otters, possibly due to

prolonged retention of microcystin-contaminated water.

doi:10.1371/journal.pone.0012576.g007

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tonic species to higher vertebrates has been demonstrated for

marine biotoxins like brevetoxin and domoic acid within estuarine

and marine systems [51,52], here we provide the first documentation of putative biotoxin transfer from the lowest trophic levels of

nutrient-impaired freshwater habitat to top marine predators at

the land-sea interface. Our findings provide the first hint of a

serious environmental and public health threat that could

negatively impact marine wildlife and humans.

We confirmed that Pinto Lake, a recreational water body located

just inland from Monterey Bay exhibits substantial and recurrent

Microcystis blooms. Biochemical testing of samples from Pinto lake

during fall, 2007 revealed total microcystin concentrations of almost

2,900 ppm (2.9 million ppb), one of the highest microcystin

concentrations ever reported from an environmental sample; the

World Health Organization limit for microcystin contamination of

finished drinking water is 1 ppb (0.001 ppm) [53]. Stepwise sampling

of downstream tributaries during the late dry season confirmed the

presence of Microcystis and microcystins throughout the lower Pajaro

watershed to within 1 km of the ocean. Factors that facilitate

development of cyanobacterial ‘‘super-blooms’’ in fresh water include

elevated nutrient concentrations and salinity, warm temperatures,

enhanced vertical stratification of lakes, summer droughts and

increased light intensity; all factors that are exacerbated by global

climate change [19,22,23,54]. Cyanobacteria can exploit these

conditions by developing intracellular gas vesicles and accumulating

in dense surface blooms that ‘‘shade out’’ nontoxic phytoplankton like

diatoms and green algae. They can also increase local water

temperatures through light absorption, creating a positive feedback

loop that helps ensure local dominance [23]. Once formed, these

biotoxins can exert their effects in areas that are remote from sites of

toxin production and can bioaccumulate in invertebrates and fish,

suggesting a biologically plausible route for marine mammal (and

human) exposure to freshwater toxins at the land-sea interface

[32,51,52].

We demonstrated the excellent adsorption characteristics of

SPATT resin-based systems for microcystin detection in both fresh

and salt water. These passive samplers were more sensitive than

periodic grab samples for field detection of microcystins and the

ability to evaluate samples for the presence of multiple biotoxins

simultaneously is an additional bonus. Preliminary environmental

Table 2. Microcystin-LR concentrations (ppb) at the top, middle, and bottom of seawater tanks at two exposure concentrations

(Tank 2 and Tank 31

) and varying postexposure intervals.

Inoculum 2,195 ppb Tank 2 (low exposure)1

1 H 12 H 24 H 48 H 72 H 7 Days

Surface 2.34 0.87 5.18 0.75 1.32 Nd2

Middle 0.756 0.71 1.37 0.87 1.03 nd

Bottom 0.500 0.650 1.90 1.32 1.01 nd

Inoculum 10,600 ppb Tank 3 (high exposure)

1 H 12 H 24 H 48 H 72 H 7 Days

Surface 32.8 5.88 22.2 4.02 6.58 nd2

Middle 5.53 5.01 7.46 6.09 4.95 nd

Bottom 5.03 5.03 7.66 3.69 6.75 nd

Mean 5.31 12.44 4.6 6.1 nd

1

All samples from Tank 1 (seawater control) tested negative for microcystin-LR. All 3 tanks were flushed with fresh seawater starting 96 H postexposure). 2

nd = microcystin concentration was below minimum detection limits on liquid chromatography-tandem mass spectrophotometry.

doi:10.1371/journal.pone.0012576.t002

Table 3. Microcystin LR concentrations (ppb wet weight) in marine invertebrate gastrointestinal tissues collected from Tank 3

(high microcystin exposure tank) at various time intervals post-exposure1

.

Invertebrate Spp.2 24 Hour 48 Hour 72 Hour 7 Days 14 Days 21 Days

Manila clam 1,324 110 125 295 183 nd3

Mussel 979 3.13 14.3 45 64.4 30.5

Oyster 102 373 68.3 158 122 4

Dungeness crab nd nd 1.01 2.7 --- ---

Red rock crab nd nd nd nd nd nd

Tegula snail --- --- 170 175 --- ---

Seawater in tank5 12.4 4.6 6.1 nd nd nd

1

All tanks were flushed continually with clean seawater beginning at 96 H post-exposure.

2

n = 1 or 2 pooled invertebrates of each species at each sample point, except snails, where n = 7.

3

nd = microcystin concentration was below minimum detection limits on liquid chromatography-tandem mass spectrophotometry.

4

--- = not tested.

5

Average microcystin-LR concentration across the top, middle and bottom of Tank 3 at each time point.

doi:10.1371/journal.pone.0012576.t003

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PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12576
surveillance using SPATT revealed no detectable microcystin in

nearshore marine waters of Monterey Bay until the fall rainy

season, when the marine outfalls of the 3 most nutrient-impaired

local waterways; the Salinas, the Pajaro and the San Lorenzo

Rivers all tested microcystin-positive.

Because of their high metabolic rate, small home ranges and

heavy reliance on nearshore-dwelling marine invertebrates as food,

sea otters provide an upper trophic-level compliment to the SPATT

resin for environmental detection of microcystins. Deaths due to

cyanobacterial intoxication were first recognized in 2007, when 11

microcystin-poisoned sea otters were recovered along the shoreline

of Monterey Bay. Microcystin intoxication appears to be an

emerging health problem for southern sea otters. To date, at least 21

southern sea otters have died due to microcystin intoxication and

the frequency of deaths may be increasing over time. Most

microcystin-positive sea otters were recovered near embayments,

harbors or river mouths. Sea otters generally do not venture into

rivers to feed, so upstream exposure to microcystins is unlikely.

For radio-tagged otters that died due to microcystin intoxication, marine bivalves constituted a major portion of their diet. Sea

otters routinely consume 25 to 30% of their body weight in clams,

mussels, snails, crabs and other marine invertebrates daily [50,55].

Marine bivalves are highly efficient biological filters for polluted

water and can bioaccumulate a wide range of terrestrial-origin

pollutants, including protozoa, enteric bacteria, viruses, biotoxins

and anthropogenic chemicals [32,56–58]. Embayments, harbors

and river mouths are favored foraging sites, placing otters directly

in the path of concentrated plumes of polluted water at the landsea interface. A higher risk of exposure to terrestrial-origin

pathogens and chemicals has been reported for sea otters residing

near impaired habitats and those that feed preferentially on filterfeeding invertebrates [44,49,57,59].

Microcystin accumulation has been demonstrated in fresh- and

saltwater mussels, crustaceans, corals, fish and possibly humans

[7,30,31,32]. Our hypothesis that sea otters were most likely to be

exposed to lethal levels of microcystins through consumption of

contaminated invertebrate prey was evaluated through laboratory

experiments where bioconcentration and depuration of freshwater

microcystins by marine invertebrates could be assessed under

defined conditions. We documented significant bioaccumulation

and slow depuration of freshwater microcystins by marine oysters,

clams, snails and mussels, with gastrointestinal tissue concentrations up to 107 times greater than adjacent seawater. Marine

invertebrates were also slow to depurate ingested toxins, with high

microcystin concentrations detected at 2 weeks post-exposure

(Table 3). Freshwater microcystins were also relatively stable in

seawater, with concentrations remaining at 29 to 56% of 1 hour

postexposure concentrations, even after 21 days.

Collectively these data provide compelling evidence implicating

land-sea flow with trophic transfer through marine invertebrates as

the most likely route of biotoxin exposure. Detection of this

problem initially in southern sea otters is not surprising, given the

high level of scientific scrutiny of this federally-listed threatened

species. Due to several unique aspects of their biology, including a

high metabolic rate, a preference to feed near the shoreline and

strong reliance on filter-feeding invertebrates as prey, southern sea

otters have proven to be highly sensitive indicators of health of

nearshore marine ecosystems [44,49,55,60]. Southern sea otters

are also a keystone species [61]; by foraging on kelp-feeding

invertebrates like urchins, sea otters help maintain the complex 3

dimensional structure of the kelp forest that provides critical

habitat for other marine wildlife [62].

Our data appear to strongly support the following hypotheses

relevant to microcystin pollution and toxicity: H1: Significant

concentrations of freshwater-derived microcystins are intermittently

polluting the land-sea interface of central California. H2: These

microcystins are causing mortality of threatened southern sea otters,

possibly through trophic transfer to marine invertebrates feeding in

contaminated freshwater plumes and H3: Wild and farmed marine

bivalves consumed by sea otters and humans exhibit high

microcystin uptake and slow depuration under conditions that

mimic natural exposure. Monterey Bay, the region where the

majority of microcystin-poisoned sea otters were recovered, forms

the heart of the nation’s largest marine sanctuary and is heavily

utilized by humans for water contact recreation, tourism, fishing

and wildlife viewing. No formal surveillance or regulatory system

exists for microcystin detection in water or shellfish in most

countries, including the United States. Because sea otters and

humans consume many of the same marine foods, our research

findings may reveal unrecognized health risks for humans when

consuming invertebrates harvested at the land-sea interface.

Acknowledgments

Sincere thanks go to Bryant Austin at Studio Cosmos and

Robert Ketley at the City of Watsonville for contributing photos

(Part A, Figure 1 and inset of Figure 3, respectively). We thank

Jack Ames, Hannah Ban-Weiss, Francesca Batac, Gloria Blondina, Wayne Carmichael, Mary Curry, Erin Dodd, Traci Fink,

Corrine Gibble, Dominic Gregorio, Michael Harris, Andy

Johnson, Robert Ketley, Jenny Lane, Kamal Mekebri, Megan

Olea, David Paradies, Meiling Roddam, Ben Weitzman and

Karen Worcester for assistance with sample collection, invertebrate exposure and study design. We acknowledge the Monterey

Bay Aquarium, the Marine Mammal Center, USGS-BRD, the

United States Fish and Wildlife Service and CDFG for their efforts

to recover and care for sick, stranded marine animals. Any use of

trade, product, or firm names in this publication is for descriptive

purposes only and does not imply endorsement by the U.S.

Government or the State of California

Author Contributions

Conceived and designed the experiments: MAM RMK SCO CD DH KW

DAJ. Performed the experiments: MAM RMK AM DBC SCO MTT MS

STC CD. Analyzed the data: MAM RMK AM DBC SCO MTT STC.

Contributed reagents/materials/analysis tools: MAM RMK AM DBC

MTT MS WM DH GL MM KW DAJ. Wrote the paper: MAM RMK

AM DBC SCO MTT MS WM STC CD DH GL MM KW DAJ.

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PLoS ONE | www.plosone.org 11 September 2010 | Volume 5 | Issue 9 | e12576