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"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. Microcystin-poisoned 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.

In November-December 2007 a widespread seabird mortality event occurred in Monterey Bay, California, USA, coincident with a massive red tide caused by the dinoflagellate Akashiwo sanguinea. Affected birds had a slimy yellow-green material on their feathers, which were saturated with water, and they were severely hypothermic. We determined that foam containing surfactant-like proteins, derived from organic matter of the red tide, coated their feathers and neutralized natural water repellency and insulation. No evidence of exposure to petroleum or other oils or biotoxins were found. This is the first documented case of its kind, but previous similar events may have gone undetected. The frequency and amplitude of red tides have increased in Monterey Bay since 2004, suggesting that impacts on wintering marine birds may continue or increase.

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are useful for monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) methodologies, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a log10 improvement when samples were first processed by IMS. The most sensitive method for the detection of C. parvum in tank-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive.The wild mussel study was conducted to evaluate estuarine and nearshore marine Mytilus spp. as bioindicators of fecal contamination in coastal ecosystems of California. Hemolymph samples from 4680 mussels were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Cryptosporidium genotypes detected in hemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event.The freshwater clam study evaluated Corbicula spp. as bioindicators of fecal protozoan contamination using three approaches: 1) clam tissue spiking experiments to compare several detection techniques, 2) clam tank exposure experiments to evaluate clams that that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions, and 3) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. The spiking and tank experiments showed that DFA, IMS in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. In the tank experiment, oocyst dose and collection time were significant predictors for detection of C. parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS in combination with DFA analysis of clam digestive glands, with significant variation by sampling year and season. These bivalve studies have shown that clams and mussels can be used to monitor water quality in California and suggests that humans and animals ingesting fecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.