Algal Toxins in Arizona, Common Misconceptions and

Future Research

• Analysis of algal toxins in watersheds surrounding the Phoenix Metro Area since 2000 (comprehensive since 2002).

• Analyses include quarterly sampling of microcystin, anatoxin-a, cylindrospermopsin, and saxitoxin (analyses performed by either Greg Boyer at SUNY-CESF or Paul Zimba at USDA)

• Algal counts and ID’s in addition to suites of physico-chemical and chemical variables (partnered with ADEQ and AzG&F).

• Development of a large, comprehensive database.

Rodeo-Chedeski Fire

• The largest wildfire in Arizona recorded history began on June 18th, 2002.

• This was a “re-setting” event in the Salt River and downstream reservoirs.

Primary Production in Roosevelt

Primary Production in Apache, Canyon, and Saguaro

Components:

Chl_a_mgPerm3

DOC_ppm

TOC_ppm

Ammonia_N_mgPerL_asN

NitrateNitrite_N_ppm

Total_P_ppm

Total_Kjeldahl_Nitrogen_mgPerl_

Prin Comp 1

Prin Comp 2

Prin Comp 3

Prin Comp 4

Prin Comp 5

Prin Comp 6

Prin Comp 7

Chl_a_m

DOC_ppm

TOC_ppm

Ammonia

Nitrate

Total_P

Total_K

x

y

z

Spinning Plot

Potentially Toxic Cyanobacteria/Algae Found in Salt

River Reservoirs• Aphanizomenon flos-aquae• Anabaenopsis circularis• Anabaena laxa• Anabaena schremetievi• Anabaena torulosa• Anabaena variabilis• Cylindrospermopsis raciborskii• Merismopedia elegans• Microcystis sp.• Pseudanabaena sp.• Oscillatoria aghardii• Oscillatoria limnetica• Prymnesium parvum• Gymnodinoids• and several more

Research Highlights• During 08/2000, over 140 µg/L of

anatoxin-a found in Saguaro.• Both anatoxin-a (33% of all fish)

and microcystin (67% of all fish) found at toxic levels in bluegill and threadfin shad stomachs taken from Apache Reservoir during 2004.

• No anatoxin-a found in aqueous samples.

• Numbers of potentially toxic species were relatively low during the 08/2000 event.

• All cylindrospermopsin results (with the exception of a concentrated plankton tow) were non-detects.

• Numbers of potentially toxic species increase after toxic events.

• Anatoxin-a is readily degraded by sunlight and alkalinity; conditions not lacking in surface waters in Arizona during the summer.

• Half-life may only be a few hours under environmental conditions in these reservoirs during spring and summer.

Prymnesium parvum

• Found in Salt River Reservoirs by AzG&F, Spring 2005.

• Working with AzG&F on counts and ID, sampling methodology, etc.

• Caused massive fish kills in Texas since the 1980’s.

• Highly heterotrophic and toxin production may be used to slow prey (bacteria, other algae) prior to ingestion.

• Causes massive hemorrhaging of exposed gill tissue in fish, bivalves, and zooplankton eventually spreading to internal organs.

• Initially believed to be an estuarine species requiring high salinities and relatively low water temperatures.– Seems to be adapting to a wide variety of

habitats.

• Environmental conditions for toxin production are not well known but nitrogen limitation may be a trigger.

• Has a dormant, encysted stage in sediments.

Image From Texas Parks and Wildlife Dept.

Image From Texas Parks and Wildlife Department

P. Parvum Background in Salt River Reservoirs

• Large fish kills occurred in early June of 2004 and 2005 and the riverine zone of Saguaro most affected.

• Small kills noticed beginning in March of both 2004 and 2005.

• Riverine zone of Saguaro is, essentially, hypolimnetic water from Canyon.

• Canyon has pump-back storage.

• Riverine sections of reservoirs usually very low in nitrogenous compounds early in the year.

• Thermal stratification and subsequent hypolimnetic accumulation of ammonia/ammonium released into downstream reservoirs may trigger a decrease in toxin production.

• Again, numbers alone do not directly correspond with toxicity.

• Arizona Game and Fish and Applied Biosciences recently sponsored a meeting where several colleagues from Texas shared their expertise.

• We continue to collaborate with colleagues in Texas and elsewhere on P. parvum issues.

Common Misconceptions re. HAB’s in Arizona and Elsewhere

• Correlation between numbers of toxic species present and the amount of toxin being produced. – The only way to determine amount of

toxin in the water is through direct quantification of toxin(s).

Misconceptions (cont.)

• Species produce constant or “set” amounts of toxins.

• Environmental conditions for toxin production are well-known.

• Algal toxins exert their influence in isolation.

• Genes coding for toxin production are known.

• No new or novel toxins exist.

Misconceptions (cont.)

• Genomic fingerprinting, without first determining toxicity of all strains, will provide an early-warning system.

• Genomic fingerprinting provides information of environmental variables responsible for toxin production.

• Toxicity is consistent with genetic analysis.

Misconceptions (cont.)

• All genomic analysis is created equal. – Much exists “downstream” of 16S

rRNA– No correlation between 16S rRNA

genetic analysis and toxicity of Microcystis strains (Neilan et al 1997a)

New Research

• In collaboration with ADEQ and AzG&F.

• Co-PI’s: Paul Zimba (USDA) and JoAnn Burkholder (NCSU).

A De-Constructionist Approach

• Simplicity and sound research design prioritized.

• Changes in sampling methodology to reflect current knowledge.

• Composite samples taken from all reservoirs.

• Split samples between UA and Dr. Burkholder.

• Identification to species.• Isolation of all suspect species into

axenic cultures.• Bioassays to screen toxin producers.

– Expose organisms to•Individual algae•Algal supernatant•Mixed population

• Quantification of toxins by Dr. Zimba– Anatoxin-a, microcystin,

cylindrospermopsin, saxitoxin, and prymnesin.

• Examine the role of bioactive peptides (“chemotype” differentiation of species producing, aeruginosins, cyanopeptolins, microginins, microviridins etc.).

• Examination of “novel” toxins– Euglenoids– Gymnodinoids

Genetic Analysis of Known Toxin Producers

• Only after toxin-producing species have been isolated will genetic analysis occur.

• Who cares what strains are present if only a given few produce toxin?

Dendrogram of cyanobacterial 16 S genes (generated by RFLP). Scale bar = similarity coefficients. Numbers 1–4 = clades. Symbols: neurotoxic; non-toxic; hepatotoxic; toxicity not determined; type of toxicity unknown.Lyra et al. (2001)

Dendrogram of cyanobacterial genomic fingerprints (generated by REP- and ERIC-PCR). Scale = similarity coefficients. Numbers 1–4 = groups. Symbols: neurotoxic; non-toxic; hepatotoxic; toxicity not determined; type of toxicity unknown. Lyra et al., 2001

Avoiding Hysteria• Tempe Town Lake and “Killer

Microcystis”• Fish kills in Saguaro likely not due to

cyan-toxins, or any toxin that can harm humans.– This does not mean there are no human-

health implications from other toxins.

• No matter how carefully-worded, several put all their research eggs in the C. raciborskii basket

• Responses given to the media or public following an event should be based on sound research, not speculation.– It’s OK to say “we don’t know but

we’re working on it”

• Research not driven by alarmism.

Summary

• Successful management of HAB’s can only occur through the enhanced understanding that research provides. – Which strains/toxins– Environmental factors (eutrophication,

allelopathy, grazing pressure, water movement, etc.)

Questions?