Effect of irrigation with lake water containing microcystins on microcystin content and growth of ryegrass, clover, rape, and lettuce.

The effect of irrigation with lake water containing a variety of microcystins on accumulation of toxins, or toxin metabolites, and plant growth in ryegrass, clover, rape, and lettuce, was investigated in a glasshouse experiment. The plants were grown in sand culture and received either three or six applications of lake water, which was applied either directly to the sand surface or to the plant shoots. As determined by LC-MS, each plant received 170 mug of a mixture of 10 different microcystins per application. Microcystins in plant samples were extracted with 70% methanol and analyzed by Adda-specific ELISA. For the shoot application treatment, microcystins were not present at measurable levels in shoots of ryegrass or rape, but were present in lettuce [0.79 mg/kg dry weight (DW)] and clover (0.20 mg/kg DW). Total microcystin concentration in roots did not vary greatly depending on whether treatment water was applied directly to the sand, or reached the roots via run-off from the shoots. Microcystins in roots were highest in clover (1.45 mg/kg DW), intermediate in lettuce (0.68 mg/kg DW) and low in ryegrass (0.20 mg/kg DW), and rape (0.12 mg/kg DW). There was no evidence for root-to-shoot translocation of microcystins. Three applications of microcystins reduced shoot DW of ryegrass, rape and lettuce, and increased root DW of ryegrass and lettuce. Clover DW was not changed by treatment with microcystins. The results show that irrigation with water containing microcystins has the potential to move microcystins into farm animal and human food chains at concentrations that can exceed recommended tolerable limits.

Changes in concentrations of microcystins in rainbow trout, freshwater mussels, and cyanobacteria in Lakes Rotoiti and Rotoehu.

Microcystin concentrations in cyanobacteria and their accumulation in rainbow trout (Oncorhynchus mykiss) and freshwater mussels (Hyridella menziesi) in Lakes Rotoiti and Rotoehu (New Zealand) were investigated. Hatchery rainbow trout were added to an enclosure in Lake Rotoiti where concentrations of microcystins in the phytoplankton and cyanobacterial cell concentrations could be closely monitored. Rainbow trout that were free to roam in the entire area of each lake were also included in the study. Freshwater mussels were suspended subsurface in cages in the enclosure. Phytoplankton samples, rainbow trout liver and muscle tissue, and the tissues of mussels were analyzed for microcystins using the ADDA-ELISA method, and selected samples were analyzed using LC-MS. A maximum concentration of microcystins in the phytoplankton samples of 760 microg L(-1) was recorded in Te Weta Bay, Lake Rotoiti, in March 2004. ELISA results confirmed microcystin immunoreactivity in rainbow trout liver and muscle tissues and in freshwater mussels. The microcystin congeners LR, YR, RR, AR, FR, LA, and WR were detected by LC-MS in caged freshwater mussels in Lake Rotoiti but were not detected in either muscle or liver tissue of rainbow trout. The daily tolerable intake limit of microcystins for human consumption recommended by the World Health Organisation is 0.04 microg kg(-1) day(-1). Modeling was carried out for the human intake of microcystin compounds from rainbow trout muscle tissue, and the potential health risks were estimated, assuming the ADDA-ELISA was determining compounds of toxicity equivalent to microcystin-LR.

Use of ELISA to identify Protoceratium reticulatum as a source of yessotoxin in Norway.

Picked cells of Protoceratium reticulatum collected from five locations in Norway were shown by ELISA analysis to contain yessotoxins (YTXs). The production of yessotoxin (YTX) was verified by culturing followed by LC-MS analysis of one of the Norwegian isolates. This is the first report of the biogenic origin of YTXs in Norway. The sensitivity of the ELISA method made it possible to quantitate YTXs in algal cultures, net-hauls, and in single cells of P. reticulatum. The cells picked from cultures and net-hauls contained 18-79 pg YTXs per cell. Dilution series and analyses of cells from non-YTX-producing algal species demonstrated the presence of only minimal matrix effects on the ELISA, probably attributable to the presence of salts. The sensitivity of this method makes it possible to search for other possible producers of YTXs, and might also make it possible to follow the YTXs through the food chain. This method allows, for the first time, measurement of the variability in toxin content within a population of dinoflagellate cells--rather than just the average amount of toxin per cell.

Integrated enzyme-linked immunosorbent assay screening system for amnesic, neurotoxic, diarrhetic, and paralytic shellfish poisoning toxins found in New Zealand.

Enzyme-linked immunosorbent assays (ELISAs) were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.

Urinary excretion of immunoreactive sporidesmin metabolites in sheep in relation to factors influencing susceptibility to sporidesmin intoxication.

AIM: To study the urinary disposition of orally administered sporidesmins A and D in sheep and identify factors influencing their kinetics, particularly the influence of breeding for resistance and susceptibility to sporidesmin, the mycotoxin responsible for the hepatogenous photosensitisation, facial eczema. METHODS: A competitive ELISA was used to monitor urinary output of immunoreactive metabolites after the intraruminal administration, to female Romney sheep, of either sporidesmin A or sporidesmin D, the nontoxic analogue. Preliminary characterisation of metabolites was carried out using HPLC with fractions monitored by ELISA. RESULTS: Maximum urinary excretion rates of immunoreactive metabolites occurred 2-8 h after dosing with sporidesmin D and 15-30 h after dosing with sporidesmin A. Sporidesmin D caused no liver injury, as detected by changes in serum enzyme activity, while the liver injury caused by sporidesmin A was greatest for the sheep with the highest cumulative output of metabolite. When sporidesmin D was administered in two separate doses to sheep bred for either resistance or susceptibility to facial eczema, the variability of metabolic output between sheep within groups was much less after the second dose. The mean urinary metabolite excretion was greater for the susceptible than the resistant sheep but the difference was not significant. Potentiation (caused by pre-administration of small doses of sporidesmin A) resulted in a more severe reaction to the dosed sporidesmin A. Urinary output of metabolite was less in the potentiated than in the unpotentiated sheep. When resistant and susceptible sheep were dosed with sporidesmin A after potentiation there was no difference between them in their cumulative totals or excretion rates of immunoreactive metabolites. However, the volume of urine produced by the susceptible sheep was lower and less variable than the resistant sheep and consequently the concentration of their urinary metabolites was higher. Preliminary ELISA examination of HPLC-fractionated urine from a sheep dosed with sporidesmin A indicated the presence of several metabolites of sporidesmin. CONCLUSION: Sporidesmin A and metabolites are rapidly excreted in urine but not as rapidly as sporidesmin D and its metabolites. Only minor differences between sheep bred for resistance and susceptibility were seen. Potentiation caused a more severe reaction to sporidesmin A and less urinary excretion of the sporidesmin and its metabolites. CLINICAL RELEVANCE: This work is part of a programme with the aim of identifying FE-resistant animals without the need for sporidesmin dosing.