A Preliminary Risk Assessment of Human Exposure to Phycotoxins in Shellfish: A Review

Over the past few decades, phycotoxins, secondary metabolites produced by toxic phytoplankton, have seen an increase in their frequency, concentrations, and geographic distribution. As shellfish accumulate phycotoxins making them unfit for human consumption, they are considered as an important food safety issue. Thus, a consumer exposure assessment on phycotoxins is necessary. Exposure assessment requires two types of information: contamination and consumption data. Shellfish contamination data on major toxins encountered by at-risk populations (Domoic Acid group, Okadaic Acid group, and Saxitoxin group) have been reviewed. Consumption data have been reviewed for both general and potential high-consumer populations. Then, we undertook acute and chronic exposure assessments, combining available French contamination data and our own consumption data. Studies including exposure assessment were then reviewed. Lastly, risk characterization was undertaken. It can be concluded that both acute and chronic exposure to phycotoxins via shellfish consumption is a matter of concern, mainly for high consumers identified in this review (specific populations and shellfish harvesters). However, the results for risk characterization must be improved. There is a need for (i) toxicological data to establish a Tolerable Daily Intake; (ii) an assessment of consumption and contamination data, undertaken at the same time, so as to assess exposure.     

Are Seafood PCB Data Sufficient to Assess Health Risk for High Seafood Consumption Groups?

Some populations in Washington's Puget Sound area consume much more seafood than the general population. Mean consumption rates are 61?g/person/day for the Tulalip and Squaxin Island Tribes and 117.2?g/person/day for Asian and Pacific Islanders (API). There is concern about possible health risks from seafood PCB exposure for these groups, but exposure evaluation is difficult due to inadequacies of environmental data. Available seafood PCB data were matched to results from recent seafood consumption surveys. To rate quality of matches and identify data gaps, a ranking system based on species specificity, data quality, and location compatibility was developed. Sensitivity of total PCB and congenerspecific PCB testing (for use in TCDD toxic equivalency approaches) necessary for cancer risk assessment was explored and included in the ranking scheme. For the Squaxin Tribe, appropriate total PCB data for risk assessment were available for 58% of seafood consumed, which is dominated by local salmon. For API, appropriate total PCB data were identified for only 4% of seafood consumed, which is dominated by commercial shellfish. Insufficient sensitivity of commercial seafood PCB analysis and overall lack of sufficiently sensitive PCB congener analysis are major gaps in ability to characterize PCB exposure and risk for these groups.     

Consensus document on the prevention of methylmercury exposure in Spain: Study group for the prevention of Me-Hg exposure in Spain (GEPREM-Hg)

The beneficial effects of fish consumption in both children and adults are well known. However, the intake of methylmercury, mainly from contaminated fish and shellfish, can have adverse health effects. The study group on the prevention of exposure to methylmercury (GEPREM-Hg), made up of representatives from different Spanish scientific societies, has prepared a consensus document in a question and answer format, containing the group’s main conclusions, recommendations and proposals. The objective of the document is to provide broader knowledge of factors associated with methylmercury exposure, its possible effects on health amongst the Spanish population, methods of analysis, interpretation of the results and economic costs, and to then set recommendations for fish and shellfish consumption. The group sees the merit of all initiatives aimed at reducing or prohibiting the use of mercury as well as the need to be aware of the results of contaminant analyses performed on fish and shellfish marketed in Spain. In addition, the group believes that biomonitoring systems should be set up in order to follow the evolution of methylmercury exposure in children and adults and perform studies designed to learn more about the possible health effects of concentrations found in the Spanish population, taking into account the lifestyle, eating patterns and the Mediterranean diet.     

Recent trends in paralytic shellfish toxins in Puget Sound,relationships to climate,and capacity for prediction of toxic events

Temporal and spatial trends in paralytic shellfish toxins (PSTs) in Puget Sound shellfish and their relationships with climate are investigated using long-term monitoring data since 1957. Data are selected for trend analyses based on the sensitivity of shellfish species to PSTs and their depuration rates, and the frequency of sample collection at individual sites. These criteria limit the analyses to the shellfish species Mytilus edulis at 20 sites from 1993 to 2007. Blue mussel toxicity is highly variable, but typically exceeds the regulatory limit for human consumption from July to November annually, with most closures occurring early in fall. Using blue mussel data only, we find no robust evidence to suggest that the frequency, magnitude, duration, or geographic scope of PST events in Puget Sound increased between 1993 and 2007. However, there is a significant basin-wide trend for closures to occur earlier in the year. There are no significant correlations between annual indices of mussel toxicity and aspects of the local and large-scale climate. Case studies of daily variations in local environmental factors leading up to exceptionally toxic events identify a combination of conditions that generally precedes most closures from 1993 to 2007. These results suggest that periods of warm air and water temperatures and low streamflow on sub-seasonal timescales may facilitate toxin accumulation in mussels. No relationships were found between water residence times in the surface layer and either streamflow or mussel toxicity. Recommendations are made for future monitoring to improve forecasting of PST risks in Puget Sound, an important region for recreational, commercial, and tribal subsistence shellfish harvesting.     

Calculating Background Levels for Ecological Risk Parameters in Toxic Harbor Sediment

Establishing background levels for biological parameters is necessary in assessing the ecological risks from harbor sediment contaminated with toxic chemicals. For chemicals in sediment, the term contaminated is defined as having concentrations above background and significant human health or ecological risk levels. For biological parameters, a site could be considered contaminated if levels of the parameter are either more or less than the background level, depending on the specific parameter. Biological parameters can include tissue chemical concentrations in ecological receptors, bioassay responses, bioaccumulation levels, and benthic community metrics. Chemical parameters can include sediment concentrations of a variety of potentially toxic chemicals. Indirectly, contaminated harbor sediment can impact shellfish, fish, birds, and marine mammals, and human populations. This paper summarizes the methods used to define background levels for chemical and biological parameters from a survey of ecological risk investigations of marine harbor sediment at California Navy bases. Background levels for regional biological indices used to quantify ecological risks for benthic communities are also described. Generally, background stations are positioned in relatively clean areas exhibiting the same physical and general chemical characteristics as nearby areas with contaminated harbor sediment. The number of background stations and the number of sample replicates per background station depend on the statistical design of the sediment ecological risk investigation, developed through the data quality objective (DQO) process. Biological data from the background stations can be compared to data from a contaminated site by using minimum or maximum background levels or comparative statistics. In Navy ecological risk assessments (ERA's), calculated background levels and appropriate ecological risk screening criteria are used to identify sampling stations and sites with contaminated sediments.     

Maximizing recovery and detection of Cryptosporidium parvum oocysts from spiked eastern oyster (Crassostrea virginica) tissue samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Linking fish consumption patterns and health risk assessment of mercury exposure in a coastal community of NW Mexico

We aimed at characterizing the methylmercury (MeHg) exposure through fish consumption in two populations (common general and fishing-related population (FRP)) using a probabilistic health risk assessment in children, women of childbearing age, and adults in Mazatlán. The hazard quotients (HQs) were obtained from fish consumption, defined through a survey, and the levels of mercury in fishery products, obtained from published information. The average fish ingestion rate (IR_food) was higher in the FRP (167.85 g d^?1) than in the general population (GP) (140.9 g d^?1). However, HQs were significantly (p < 0.05) higher in the GP (ranging from 0.18 to 10.91) compared to the FRP (0.20 to 2.48); significant differences were also found among groups of both populations. Remarkably, children in both populations exhibited the highest proportions of risk, reaching up to 97% in GP. For all populations, fish consumption was the most important variable influencing MeHg exposure. Overall, for MeHg exposure, there is no safe level of fish consumption without risk, and actions should be taken to mitigate possible risk; further research with current data is needed to assess potential health risks associated with MeHg exposure, particularly in children.     

TRPV1 as a key determinant in ciguatera and neurotoxic shellfish poisoning

Ciguatera fish poisoning and neurotoxic shellfish poisoning are distinct clinical entities characterized by gastrointestinal and neurological disturbances, following the consumption of certain reef fish and shellfish containing toxic polyether compounds sporadically present in certain toxic marine dinoflagellates. The biotransformation and bioaccumulation of gambierol and brevetoxin, and their congeners, are believed to be involved in the pathogenesis of these "food-chain diseases", for which no effective treatments are available. Here, we describe for the first time the potent effect of gambierol and brevetoxin on TRPV1 channels, a key player in thermal and pain sensation. Our findings may lead to promising new therapeutic interventions.     

Development and use of wild game consumption rates in human health risk assessments

Clearer guidance is recommended for evaluating site-specific chemical risks due to the consumption of wild game in human health risk assessment (HHRA), particularly in estimating consumption rate values for site-specific exposure modeling. This study reviewed site-specific HHRAs, survey data, regulatory documents, and scientific literature to evaluate the state of the practice for evaluating site-specific chemical risks via wild game consumption. Consumption rates of distinct wildlife species groups varied over three orders of magnitude (increasing from lowest to highest: waterfowl, terrestrial birds, small mammals, and cervids), and rates for all groups except cervids were lower than wild fish consumption rates. Wild game consumption rate values should be explicitly derived for site-specific HHRAs using one or more of the following approaches (in general order of lowest to highest uncertainty): (1) Empirical site-specific surveys; (2) Empirical surveys of comparable communities; (3) Modeling of local game harvest data; (4) Use of a nationally applicable default value; (5) Modeling of legally harvestable game limits; and (6) Modeling based on estimates of total meat consumption. Data are readily available such that approximate nationally applicable default values could be developed. More precise guidance and data for different wildlife species groups, regions, and consumers would improve HHRAs including this exposure pathway.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium minutum (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium minutum (Dinophyceae) are a worldwide concern from both the economic and human health points of view. For population genetic studies of A. minutum distribution and dispersal, highly polymorphic genetic markers are of great value. We isolated 12 polymorphic microsatellites from this cosmopolitan, toxic dinoflagellate species. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the estimate of gene diversity was from 0.560 to 0.862 across the 12 microsatellites; these loci have the potential to reveal genetic structure and gene flow among A. minutum populations.     

Arsenic in Seafood: Speciation Issues for Human Health Risk Assessment

Major sources of arsenic exposure for humans are foods, particularly aquatic organisms, which are called seafood in this report. Although seafood contains a variety of arsenicals, including inorganic arsenic, which is toxic and carcinogenic, and arsenobetaine, which is considered nontoxic, the arsenic content of seafood commonly is reported only as total arsenic. A goal of this literature survey is to determine if generalizable values can be derived for the percentage of total arsenic in seafood that is inorganic arsenic. Generalizable values for percent inorganic arsenic are needed for use as default values in U.S. human health risk assessments of seafood from arsenic-contaminated sites. Data from the worldwide literature indicate the percent of inorganic arsenic in marine/estuarine finfish does not exceed 7.3% and in shellfish can reach 25% in organisms from presumably uncontaminated areas, with few data available for freshwater organisms. However, percentages can be much higher in organisms from contaminated areas and in seaweed. U.S. site-specific data for marine/estuarine finfish and shellfish are similar to the worldwide data, and for freshwater finfish indicate that the average percent inorganic arsenic is generally < 10%, but ranges up to nearly 30%. Derivation of nationwide defaults for percent inorganic arsenic in fish, shellfish, and seaweed collected from arsenic-contaminated areas in the United States is not supported by the surveyed literature.     

Probabilistic Estimation of Regional Dietary Exposure to Dioxins in Fish in Japan on the Basis of Market and Fish Distribution Network Data

Dietary consumption of fish is the greatest contributor to dioxin exposure of the general population in Japan. Unlike with other routes of exposure to dioxins, exposure via foodstuffs does not necessarily occur from local sources. Clarifying the distribution of fish from the catch area to local markets helps to control dioxin exposure from the head of the distribution chain down. We analyzed the data from 30 major central wholesale fish markets to determine both the market share of fish by catch area and the market share of major fish types in fish consumption markets. Probabilistic estimation of dietary exposure of the general population to dioxins in fish was conducted in seven regions in Japan. Probabilistic density functions (PDFs) were assigned to express the variability of data from monitoring of dioxin levels in fish by catch area or by fish type. From histograms of dioxin levels in fish by catch area, the mean and 5th to 95th percentile range of dietary exposure to dioxins in the region with the largest 95th percentile value were estimated, respectively, as 55.5 and 12.9–172.9 pg-TEQ/day. No statistically significant differences in dietary exposure to dioxins in fish were observed among the seven regions. Some specific coastal catch areas and some types of fish that had a greater impact than others on exposure were pinpointed for each region.     

COMPARATIVE POPULATION DYNAMICS OF FUR SEALS

The population sizes, trends, exploitation, and life history parameters for the ten fur seal species and subspecies are summarized. The largest population is that of Arctocephalus pusillus pusillus with approximately two million seals, and the smallest is A. townsendi with approximately 7,000 individuals. Most populations are legally protected, although controlled harvesting may occur. None of the fur seal populations is currently known to be decreasing. Data are presented for parameters related to the survival of pups, juveniles, adults, and territorial males, and to reproduction, including the age of attainment of territorial status, aggregation sizes, age of first parturition, pregnancy rates, sex ratios of young animals, and information on the birth seasons of the different species. Since pinnipeds are often of concern in fisheries management, their daily consumption rates are of importance, and consequently data on body masses are summarized and the paucity of data on consumption rates as a function of body mass noted. A simplified age-structured model is developed, and the results of this model are compared with results from more detailed models based on two published life tables for Callorhinus ursinus. This comparison shows that the use of the simplified age-structured model is justified to explore changes in population growth rate. However, the simplified model does show exaggerated age structure effects compared to the more detailed models. This model is used to compare the population dynamics of those species for which sufficient data are available. Areas in which limited, or no, data are available for the different fur seal species are highlighted.     

GENE FLOW AND INEFFECTIVE ANTIPREDATOR BEHAVIOR IN A STREAM-BREEDING SALAMANDER

Predators often feed on prey that show ineffective antipredator behavior. Gene flow among populations may constrain evolution of effective antipredator ability in larvae of the streamside salamander, Ambystoma barbouri, a species that occupies distinctly different habitats with conflicting selection pressures. Some streams are ephemeral, where larvae should be active to feed and reach metamorphosis before stream drying. In contrast, other streams are more permanent and contain pools with predatory fish, where larvae should remain inactive to avoid fish predation. Feeding rates and predator escape behavior were assayed for laboratory-reared larvae from 15 populations. Larval survival was also compared among populations in artificial streams with natural predators. Five populations represented streams subjected to fish predation along a gradient of genetic and geographic isolation from populations without fish; the remaining 10 populations were ephemeral and without fish. Individuals from populations with fish had significantly stronger behavioral responses to fish (i.e., decreased feeding rate associated with the presence of fish and increased escape response) than individuals from fishless populations. Larvae from populations containing fish that were more isolated from fishless populations showed stronger antipredator responses than less isolated populations. Further, larvae from more isolated populations survived longer in the predation experiment, indicating that the behaviors measured were related with survival. These results suggest that gene flow between populations with conflicting selection pressures limits local adaptation in some salamander populations with fish. While previous studies have typically focused on the role of gene flow in pairs of populations, the results of this study suggest that gene flow is acting to swamp local adaptation across several populations.     

Purification of five azaspiracids from mussel samples contaminated with DSP toxins and azaspiracids

Human intoxications during toxic episodes in shellfish are a very important concern for public health, as well as for economic interests of producer regions. Although initially each toxin appeared in a determined geographical zone, nowadays many of them are found in multiple places worldwide. In addition, more toxic compounds (new toxins or new analogs of known toxins) are being isolated and identified, which bring about new risks for public health. An example of this situation is the group of azaspiracids (AZAs). Initially these toxins were concentrated in Irish coasts but today appear in many different geographic locations; in the first toxic episode only three analogs were isolated, but now it is known that the group is comprised of at least eleven identified compounds. A substantial problem associated with all these new toxins is the extreme difficulty associated with the study of their toxic effects and mechanisms of action due to the very small quantities of purified toxin available. Therefore, the study of procedures to isolate them from contaminated shellfish or to synthesize them is of tremendous importance. In this paper we design a complete procedure to obtain AZAs analogs from mussels contaminated with DSP toxins and azaspiracids by means of three consecutive steps: an extraction procedure to remove toxins from shellfish, a solid phase extraction (SPE) to clean the samples and separate DSP toxins and AZAs, and a preparative HPLC to isolate each analog. In all the steps LC/MS is used to detect and quantify the toxins. Large amounts of AZA1, AZA2, AZA3, AZA4 and AZA5 were obtained by use of this procedure, which can be utilized in future studies relating to the toxins such as the production of certified materials and standards.     

Sportsfish Consumption Surveys: A Risk Assessment Practitioner's Wish List

In the United States, risk assessments that include sportsfish consumption often rely on default values derived by the USEPA. However, local, regional, and cultural differences can result in substantial differences in consumption rates, species of fish caught, parts of fish consumed, and fish preparation methods—all of which contribute to the extent of contaminant exposure. Because of these differences, site-specific data are important for the reliable prediction of exposure. This article reviews the types of information important to a risk assessor to enhance the applicability of the exposure assessment to the population being evaluated. It provides a method for setting priorities based on the sensitivity of the exposure assessment to each type of information. Consumption rate and parts of fish consumed are the most important information to obtain. This information should be obtained for each fish species consumed, and should be obtained for both children and adults. Body weight and exposure duration have the next level of priority, and cooking method has the lowest level of priority. Results from surveys should be presented in a manner that allows the reader to understand the variability observed in the data and the survey questions that were asked. Correlations between variables should be evaluated and presented.     

The association between parasite infection and growth rates in Arctic charr: do fast growing fish have more parasites?

Trophically transmitted parasites are known to impair fish growth in experimental studies, but this is not well documented in natural populations. For Arctic charr [Salvelinus alpinus (L.)], individual growth is positively correlated with food consumption. However, increased food consumption will increase the exposure to trophically transmitted parasites. Using a correlative approach, we explore the association between parasite abundance and the individual growth of Arctic charr from five lakes within the same watercourse. The studied parasite species differ in their life cycles and cost to the host. We predicted a positive association between parasite abundance and fish growth for parasites of low pathogenicity reflecting high consumption rates, and a negative association at higher parasite abundances for more costly parasites. We found no direct negative associations between parasite abundance and fish growth. The relationship between parasite abundance and growth was linearly positive for the low costly Crepidostomum sp. and concave for the more costly Eubothrium salvelini. In natural fish populations, the negative effects of parasites on fish growth might be outweighed by the energy assimilated from feeding on the intermediate host. However, experimental studies with varying food consumption regimes are needed to determine the mechanisms underlying our observations.

     

Population-Based Fish Consumption Survey and Probabilistic Methylmercury Risk Assessment

A fish consumption survey was developed and administered by telephone to 820 Wyoming fishing license holders. Survey respondents provided the frequency, species, and quantity of Wyoming-caught and store-bought fish consumed for license holder and household members. Deterministic and probabilistic methylmercury exposure distributions were estimated by multiplying fish consumption by species-specific mercury concentrations for each household member. Risk assessments were conducted for children, women of childbearing age, and the rest of the population by comparing methylmercury exposure distributions to levels of concern. The results indicate that probabilistic risk assessment likely provides a more realistic view of the risk to the study population. The results of this study clearly indicate that: (1) there is no level of fish consumption that is without risk of methylmercury exposure, (2) fish advisories may be warranted for children and women of childbearing age, and (3) that store-bought fish generally contribute more to methylmercury exposure than do Wyoming-caught fish.     

A novel antibody-based biomarker for chronic algal toxin exposure and sub-acute neurotoxicity

The neurotoxic amino acid, domoic acid (DA), is naturally produced by marine phytoplankton and presents a significant threat to the health of marine mammals, seabirds and humans via transfer of the toxin through the foodweb. In humans, acute exposure causes a neurotoxic illness known as amnesic shellfish poisoning characterized by seizures, memory loss, coma and death. Regular monitoring for high DA levels in edible shellfish tissues has been effective in protecting human consumers from acute DA exposure. However, chronic low-level DA exposure remains a concern, particularly in coastal and tribal communities that subsistence harvest shellfish known to contain low levels of the toxin. Domoic acid exposure via consumption of planktivorous fish also has a profound health impact on California sea lions (Zalophus californianus) affecting hundreds of animals yearly. Due to increasing algal toxin exposure threats globally, there is a critical need for reliable diagnostic tests for assessing chronic DA exposure in humans and wildlife. Here we report the discovery of a novel DA-specific antibody response that is a signature of chronic low-level exposure identified initially in a zebrafish exposure model and confirmed in naturally exposed wild sea lions. Additionally, we found that chronic exposure in zebrafish caused increased neurologic sensitivity to DA, revealing that repetitive exposure to DA well below the threshold for acute behavioral toxicity has underlying neurotoxic consequences. The discovery that chronic exposure to low levels of a small, water-soluble single amino acid triggers a detectable antibody response is surprising and has profound implications for the development of diagnostic tests for exposure to other pervasive environmental toxins.