Seppo I. Niemelä

Toxic cyanobacteria (blue-green algae) in Finnish fresh and coastal waters

A survey of the occurrence of toxic blooms of cyanobacteria in Finnish fresh and coastal waters was made during 1985 and 1986. Toxicity of the freeze-dried water bloom samples was tested by mouse-bioassay (i.p.). Forty-four per cent (83/188) of the bloom samples were found to be lethally toxic. Hepatotoxic blooms (54) were almost twice as common as neurotoxic ones (29). Anabaena was the most frequently found genus in toxic and non-toxic blooms and it was present in all neurotoxic samples. Statistical associations were found between hepatotoxicity and incidence of Microcystis aeruginosa, M. viridis, M. wesenbergii, Anabaena flos-aquae and Anabaena spiroides. Neurotoxicity was statistically associated with Anabaena lemmermannii, Anabaena flos-aquae and Gomphosphaeria naegeliana. Isolation of strains of cyanobacteria confirmed the occurrence of hepatotoxic and neurotoxic strains of Anabaena, as well as hepatotoxic strains of Microcystis and Oscillatoria species.Toxic blooms caused cattle poisonings at three different lakes during the study period. Toxic blooms also occurred in drinking water sources. Our study shows that toxic cyanobacteria are more common in Finnish lakes than would be expected on the basis of animal poisonings. The results of this study show the existence of toxic cyanobacteria in Finnish water supplies and the need for their continued study as agents of water based disease.

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N2 fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.

Biodegradability and adsorption on lake sediments of cyanobacterial hepatotoxins and anatoxin‐a

Cyanobacterial hepatotoxins and anatoxin‐a, a neurotoxin, were shown to be degraded when crude extracts of lysed toxic laboratory strains of cyanobacteria were exposed to natural populations of micro‐organisms from lakes. While anatoxin‐a decayed equally fast with all the inocula from lake sediment and water, the degradation rate of hepatotoxins was higher with inocula from places at which cyanobacterial water blooms had occurred than with inocula from places with no known mass occurrences of cyanobacteria. Degradation was slowest when an inoculum from a humic lake was used. A part of the loss of the toxins was shown to be due to adsorption on lake sediments.

Endotoxins associated with cyanobacteria and their removal during drinking water treatment

The aim of this study was to investigate endotoxin concentrations in cyanobacterial water blooms and strains, and to assess the removal of endotoxins during drinking water treatment. Endotoxin concentrations were measured from 151 hepatotoxic, neurotoxic and non-toxic cyanobacterial water blooms by using Limulus amebocyte lysate (LAL) assay, and the results were compared to bacterial data. Endotoxin activities ranged from 20 to 3.8 x 10(4) endotoxin units (EU) per ml. Endotoxicity of the samples correlated with phycobiliprotein concentration that was used to assess cyanobacterial abundance, heterotrophic plate count, and Aeromonas spp. but it did not correlate with the number of coliforms or streptococci. The high endotoxin concentrations occasionally detected in the water bloom samples were probably due to Gram negative bacteria that existed together with cyanobacteria since the 26 axenic cyanobacterial strains from different genera that were studied showed very low endotoxin activity. No differences in endotoxin activity were detected between hepatotoxic, neurotoxic and non-toxic strains. Removal of endotoxins during drinking water treatment was studied at nine waterworks that previously had been associated with high numbers of cyanobacteria and that used different processes for water purification. Endotoxin concentration in raw waters ranged from 18 to 356 EU ml(-1). The treatment processes reduced 59-97% of the endotoxin activity; in the treated water the concentration ranged from 3 to 15 EU ml(-1). The most significant reduction occurred at the early stages of water treatment, during coagulation, settling and sand filtration. Activated carbon filtration either increased or had no effect on endotoxin concentration. Ozonation and chlorination had little effect on the endotoxin concentrations.

Toxicity and isolation of the cyanobacterium Nodularia spumigena from the southern Baltic Sea in 1986

Three water bloom samples were collected in August 1986 from the southern Baltic Sea. Acute toxicity of the samples was determined by mouse bioassay and the toxins were further studied by HPLC. The bloom samples contained equal amounts of cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae and were hepatotoxic. Two hepatotoxic Nodularia spumigena strains were isolated from the samples. The isolates produce a toxic peak indistinguishable from the bloom material in the HPLC analysis. The toxicity of the fractions was verified by mouse bioassay. Thus the toxicity of the bloom samples was in all likelihood caused by Nodularia spumigena.

Isolation and identification of 12 microcystins from four strains and two bloom samples of Microcystis spp.: structure of a new hepatotoxin.

Sixteen microcystins, cyclic heptapeptide hepatotoxins, were isolated and purified by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) from four hepatotoxic strains and two Microcystis spp. bloom samples originating from five different lakes in Finland. The structures of a new [Dha7]MCYST-FR and 11 known microcystins MCYST-LR, [D-Asp3]MCYST-LR, [Dha7]MCYST-LR, [D-Asp3, Dha7] MCYST-LR, MCYST-RR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, [D-Asp3,Dha7]MCYST-RR, [L-Ser7]MCYST-RR, MCYST-YR and [Dha7] MCYST-YR were assigned based on amino acid analysis, fast atom bombardment mass spectrometry (FABMS) and tandem FABMS. Four other new compounds allowed only determination of their molecular formulas and amino acid components because of inadequate amounts obtained. [Dha7]MCYST-RR was found most frequently in these samples as the main toxin.

Biological changes in northern spruce forest soil after clear-cutting

Abstract In northern Finland, reforestation has frequently failed in clear-cut areas of coniferous forests. These failures prompted an investigation of the changes in the invertebrate fauna and in the bacterial populations of such forest soils after clear-cutting. Clear-cut areas in four neighbouring sites of various ages were sampled monthly, and the results were compared with those for samples taken simultaneously from a control site in an untreated spruce stand. After clear-cutting, the total biomass of the invertebrate fauna showed a strong increase, mainly due to an increase of the Enchytraeidae populations. The biomass reached its maximum after 7 yr and returned to the original level by year 13 after clear-cutting. Bacterial counts showed the same pattern as the faunal biomass; the numbers at first increased after clear-cutting, then regressed, and approached the control value after 13 yr. Cellulose degradation and soil respiration behaved in the same way. Bacterial numbers showed a significant seasonal variation at all sites, including the control; counts were maximal after the snow melt in June and decreased during the growing season.

Assessment of rapid bioassays for detecting cyanobacterial toxicity

Simple and easy‐to‐use bioassays with Artemia salina (brine shrimp) larvae, luminescent bacteria and Pseudomonas putida were evaluated for the detection of toxicity due to cyanobacterial hepato‐and neurotoxins. The hepatotoxins and a neurotoxin, anatoxin‐a, were extracted from laboratory‐grown cultures and natural bloom samples by the solid phase fractionation method and dissolved in diluent for different bioassays. The toxin concentration of cyanobacterial extracts was determined with HPLC. The Artemia biotest appeared to be quite sensitive to cyanobacterial hepatotoxins, with LC 50 values of 3–17 mg l‐1. The Artemia test was also shown to be of value for the detection of toxicity caused by anatoxin‐a. The fractionated extract of anatoxin‐a was not lethal to Artemia but it disturbed the ability of the larvae to move forwards. Filtered cyanobacterial cultures with anatoxin‐a, on the other hand, caused mortality of Artemia larvae at concentrations of 2–14 mg l‐1. With the solid phase fractionation of cyanobacterial samples, no non‐specific toxicity due to compounds other than hepato‐ and neurotoxins was observed. In the luminescent bacteria test, the inhibition of luminescence did not correlate with the abundance of hepatotoxins or anatoxin‐a. The growth of Ps. putida was enhanced, rather than inhibited by cyanobacterial toxin fractions.

MPN-PCR—quantification method for staphylococcal enterotoxin c 1 gene from fresh cheese

PCR detection methods have been extensively used in diagnostic microbiology. However, a lack of a simple and reliable method for quantification of the PCR products has partly hindered the use of PCR in routine food laboratories. The quantification of PCR products can be done by combining the principles of MPN statistics and PCR technique. We have developed a simple and sensitive MPN-PCR assay for detection and enumeration of enterotoxin C producing Staphylococcus aureus NCTC 10655 from fresh cheese. By amplifying single copy chromosomal enterotoxin C gene fragment, we could detect as little as 20 cfu/g. By Morans test, most of the DNA dilution series appeared to fulfill the basic mathematical assumptions of ordinary MPN methods. The analysis with MPN-PCR took one day to perform compared with three days analysis time with plate counting. This MPN-PCR method can be readily applied with different primer systems without extensive development work.

Comparison of methods for determining the numbers and species distribution of coliform bacteria in well water samples

R.M. NIEMI, M.P. HEIKKILÄ, K. LAHTI, S. KALSO AND S.I. NIEMELÄ. 2001.