Co-occurrence of marine and freshwater phycotoxins in oysters, and analysis of possible predictors for management.

Oysters (Crassostrea virginica) were screened for 12 phycotoxins over two years in nearshore waters to collect baseline phycotoxin data and to determine prevalence of phycotoxin co-occurrence in the commercially and ecologically-relevant species. Trace to low concentrations of azaspiracid-1 and -2 (AZA1, AZA2), domoic acid (DA), okadaic acid (OA), and dinophysistoxin-1 (DTX1) were detected, orders of magnitude below seafood safety action levels. Microcystins (MCs), MC-RR and MC-YR, were also found in oysters (maximum: 7.12 µg MC-RR/kg shellfish meat wet weight), warranting consideration of developing action levels for freshwater phycotoxins in marine shellfish. Oysters contained phycotoxins that impair shellfish health: karlotoxin1-1 and 1-3 (KmTx1-1, KmTx1-3), goniodomin A (GDA), and pectenotoxin-2 (PTX2). Co-occurrence of phycotoxins in oysters was common (54%, n = 81). AZAs and DA co-occurred most frequently of the phycotoxins investigated that are a concern for human health (n = 13) and PTX2 and KmTxs co-occurred most frequently amongst the phycotoxins of concern for shellfish health (n = 9). Various harmful algal bloom (HAB) monitoring methods and tools were assessed for their effectiveness at indicating levels of phycotoxins in oysters. These included co-deployed solid phase adsorption toxin tracking (SPATT) devices, toxin levels in particulate organic matter (POM, >1.5 µm) and whole water samples and cell concentrations from water samples as determined by microscopy and quantitative real-time PCR (qPCR). The dominant phycotoxin varied between SPATTs and all other phycotoxin sample types, and out of the 11 phycotoxins detected in oysters, only four and seven were detected in POM and whole water respectively, indicating phycotoxin profile mismatch between ecosystem compartments. Nevertheless, there were correlations between DA in oysters and whole water (simple linear regression [LR]: R2 = 0.6, p < 0.0001, n = 40), and PTX2 in oysters and SPATTs (LR: R2 = 0.3, p = 0.001, n = 36), providing additional monitoring tools for these phycotoxins, but oyster samples remain the best overall indicators of seafood safety.

Effects of Two Toxin-Producing Harmful Algae, Alexandrium catenella and Dinophysis acuminata (Dinophyceae), on Activity and Mortality of Larval Shellfish.

Harmful algal bloom (HAB) species Alexandrium catenella and Dinophysis acuminata are associated with paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) in humans, respectively. While PSP and DSP have been studied extensively, less is known about the effects of these HAB species or their associated toxins on shellfish. This study investigated A. catenella and D. acuminata toxicity in a larval oyster (Crassostrea virginica) bioassay. Larval activity and mortality were examined through 96-h laboratory exposures to live HAB cells (10−1000 cells/mL), cell lysates (1000 cells/mL equivalents), and purified toxins (10,000 cells/mL equivalents). Exposure to 1000 cells/mL live or lysed D. acuminata caused larval mortality (21.9 ± 7.0%, 10.2 ± 4.0%, respectively) while exposure to any tested cell concentration of live A. catenella, but not lysate, caused swimming arrest and/or mortality in >50% of larvae. Exposure to high concentrations of saxitoxin (STX) or okadaic acid (OA), toxins traditionally associated with PSP and DSP, respectively, had no effect on larval activity or mortality. In contrast, pectenotoxin-2 (PTX2) caused rapid larval mortality (49.6 ± 5.8% by 48 h) and completely immobilized larval oysters. The results indicate that the toxic effects of A. catenella and D. acuminata on shellfish are not linked to the primary toxins associated with PSP and DSP in humans, and that PTX2 is acutely toxic to larval oysters.

Oyster hatchery breakthrough of two HABs and potential effects on larval eastern oysters (Crassostrea virginica).

Harmful algal bloom (HAB) dinoflagellate species Karlodinium veneficum and Prorocentrum cordatum (prev. P. minimum) are commonly found in Chesapeake Bay during the late spring and early summer months, coinciding with the spawning season of the eastern oyster (Crassostrea virginica). Unexplained larval oyster mortalities at regional commercial hatcheries prompted screening of oyster hatchery water samples for these HAB species. Both HAB species were found in treated hatchery water during the oyster spawning season, sometimes exceeding bloom cell concentrations (≥ 1,000 cells/mL). To investigate the potential for these HAB species, independently or in co-exposure, to affect larval oyster mortality and activity, 96-h laboratory single and dual HAB bioassays with seven-day-old oyster larvae were performed. Treatments for the single HAB bioassay included fed and unfed controls, K. veneficum at 1,000; 5,000; 10,000; and 50,000 cells/mL, P. cordatum at 100; 5,000; 10,000; and 50,000 cells/mL. Subsequently, the 1,000 cells/mL K. veneficum and 50,000 cells/mL P. cordatum treatments were combined in a co-exposure treatment for the dual HAB bioassay. At all cell concentrations tested, K. veneficum swarmed oyster larvae and caused significant larval oyster mortality by 96 h (Karlo1,000: 21 ± 5%; Karlo5,000: 93 ± 2%; Karlo10,000: 85 ± 3%; Karlo50,000: 83 ± 5%, SE). In contrast, there was no significant difference in larval oyster mortality between the control treatments and any of the P. cordatum treatments by 96 h. By 24 h, larval oysters were significantly less active (immotile) in the presence of either HAB species as compared to control treatments (e.g., Karlo1,000: 37.8 ± 4.1%; Proro100: 47.3 ± 7.4%; Fed: 10.8 ± 3.2%; Unfed: 10.1 ± 4.9%, SE). In the dual HAB bioassay, larval oyster mortality associated with 1,000 cells/mL K. veneficum (44 ± 9%, SE) was not changed by the addition of 50,000 cells/mL P. cordatum (55 ± 7%, SE), demonstrating that K. veneficum was primarily responsible for the observed mortality. This study demonstrated that even low cell concentrations of K. veneficum and P. cordatum are harmful to larval oysters, and could contribute to reductions in oyster hatchery production through impacts on this critical life stage.

Spatiotemporal distribution of phycotoxins and their co-occurrence within nearshore waters.

Harmful algal blooms (HABs), varying in intensity and causative species, have historically occurred throughout the Chesapeake Bay, U.S.; however, phycotoxin data are sparse. The spatiotemporal distribution of phycotoxins was investigated using solid-phase adsorption toxin tracking (SPATT) across 12 shallow, nearshore sites within the lower Chesapeake Bay and Virginia's coastal bays over one year (2017-2018). Eight toxins, azaspiracid-1 (AZA1), azaspiracid-2 (AZA2), microcystin-LR (MC-LR), domoic acid (DA), okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and goniodomin A (GDA) were detected in SPATT extracts. Temporally, phycotoxins were always present in the region, with at least one phycotoxin group (i.e., consisting of OA and DTX1) detected at every time point. Co-occurrence of phycotoxins was also common; two or more toxin groups were observed in 76% of the samples analyzed. Toxin maximums: 0.03 ng AZA2/g resin/day, 0.25 ng DA/g resin/day, 15 ng DTX1/g resin/day, 61 ng OA/g resin/day, 72 ng PTX2/g resin/day, and 102,050 ng GDA/g resin/day were seasonal, with peaks occurring in summer and fall. Spatially, the southern tributary and coastal bay regions harbored the highest amount of total phycotoxins on SPATT over the year, and the former contained the greatest diversity of phycotoxins. The novel detection of AZAs in the region, before a causative species has been identified, supports the use of SPATT as an explorative tool in respect to emerging threats. The lack of karlotoxin in SPATT extracts, but detection of Karlodinium veneficum by microscopy, however, emphasizes that this tool should be considered complementary to, but not a replacement for, more traditional HAB management and monitoring methods.