Effects of cell-bound microcystins on survival and feeding of Daphnia spp.

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.

Fate and effects of esfenvalerate in agricultural ponds.

The fate of esfenvalerate was investigated by sampling and chemical analysis after spraying of an artificial pond (25 g a.i./ha) and in the laboratory with [14C]esfenvalerate by trapping of 14CO2 and fractionation of the sediment. The effects were investigated on pelagic communities in enclosures in a natural lake and in the laboratory on surface (Cymatia coleoptrata) and sediment (Chironomus riparius) insects. The latter were used in sediment-plus-water and in water-only tests, measuring effects on emergence and mortality. The measurements in the artificial pond indicated exposure concentrations in the surface microlayer, water column, and sediment of 0.4 microgram/L, 0.05 microgram/L, and 9 micrograms/kg dry weight, respectively, two weeks after application. The degradation studies showed a limited mineralization (26.5%) of [chorophenyl-14C]esfenvalerate during 112 d. Part of the substance was transformed to water-soluble compounds (18.1%) or compounds attached to fulvic acids (26.2%), humic acids (14.2%), or nonextractable sediment constituents (8.8%). The formulated product Sumi-Alpha 5 FW caused 100% mortality to Cymatia coleoptrata after surface application of 0.13 g a.i/ha. Effects on zooplankton were recorded at 0.005 microgram/L of esfenvalerate. The 96-h median lethal concentration for first-instar larvae of Chironomus riparius was 0.13 microgram/L, whereas the delayed emergence lowest-observed-effect concentration was 0.8 microgram/L.

A spontaneous mutant of microcystin biosynthesis: genetic characterization and effect on Daphnia.

Microcystis aeruginosa strain MRC is unique in its' possession of the mcyA-J gene cluster, which encodes microcystin synthetase, but its' inability to produce microcystins. M. aeruginosa strain MRD is genetically identical to MRC at numerous genomic loci examined, but produces a variety of microcystins, mainly with the amino acid tyrosine in the molecule. Zooplankton studies with Daphnia galeata and D. pulicaria, using the mutant (MRC) and its' wild type (MRD), showed for the first time that microcystins other than microcystin-LR can be responsible for the poisoning of Daphnia by Microcystis. Regardless of microcystin content, both Daphnia exhibited significantly reduced ingestion rates when fed with either strain of M. aeruginosa compared with the green alga Scenedesmus acutus. A disruption of the molting process in both Daphnia spp. was noted when these species were fed with MRC cells. Such symptoms on Daphnia have not been previously reported for cyanobacteria and may point to a bioactive compound, other than microcystin, which inhibits the hardening of protein-chitin complexes in Daphnia.

Towards viable, useful and usable human factors design guidance.

This paper investigates the factors relevant to producing effective human factors design guidance, using the Engineering Data Compendium (EDC) as a research vehicle. A series of three exploratory experiments focusing on the factors that affect the usability, usefulness and viability of human factors handbooks was conducted. The results of these studies were interpreted in the context of the process by which the EDC was developed, leading to the following recommendations: (a) human factors guidance should be organized in a manner that is stepped in context; (b) human factors guidance should be based on an explicit requirements analysis; (c) the calibration of designers' perceptions of the cost of obtaining human factors information must be improved; (d) organizational policies must be changed to induce more effective information search behaviour.

Effect of microcystin on growth of single species and on mixed natural populations of heterotrophic nanoflagellates.

Natural populations of heterotrophic nanoflagellates were reduced in numbers and in situ growth rates during a toxic Microcystis bloom in a eutrophic lake. Microcystin was found in the particulate phase (algae) and in the dissolved phase (water) with maximum concentrations of 414 micrograms g freeze-dried algal material-1 and 141 micrograms l-1 lake water, respectively. An average reduction in growth rates of 49% was found when comparing the growth estimates before and after the toxic bloom had peaked. Similar reductions were found in laboratory experiments when growing mixed flagellate populations from two different systems with microcystin, but without predators. Concentrations of 10 micrograms toxin 1-1 reduced the growth rates by 36-41% (significantly different from the controls) and 1 microgram toxin 1-1 reduced the growth rates by 24-28% (not significant). Thus, natural populations of heterotrophic nanoflagellates seem very sensitive to microcystin. The growth characteristic of two cultured species was also tested in the presence of microcystin. Both the cultured species, Heteromita globosa and Spumella sp., grew well at 1-10 micrograms microcystin 1-1 and Spumella sp. was able to grow at 50 micrograms microcystin 1-1. However, the growth curves indicate that the decline in numbers during the stationary phase occurred faster in the presence of microcystin. The ecological consequences of a highly sensitive protozoan community may be that larger zooplankton species (copepods and cladocerans) are affected by reduced availability of food.

Grazing of nonindigenous bacteria by nano-sized protozoa in a natural coastal system.

Mesocosms (∼4.5 m(3)) situated in a closed bay area were used to investigate the effect of protozoan predation on nonindigenous bacteria. Pseudomonas fluorescens strain Agl was released into mesocosms as a single inoculum of 1 × 10(5) cells ml(-1) (final concentration) or as four inocula (same concentration each) at intervals of 3 days. Mesocosms that had received growth media corresponding to the inoculum served as controls. Numbers of P. fluorescens Ag1 decreased rapidly whether released as single or multiple inocula. Direct estimation of protozoan predation using fluorescently labeled P. fluorescens from log phase and starved cultures, respectively, revealed that natural populations of heterotrophic nanoflagellates consumed substantial amounts of the nonindigenous bacterial strain. The volume of fluorescently labeled cells prepared from starved cells was 68% of log phase cell volume, but the individual clearance of the small cells was five to seven times higher than that of the log phase bacteria. The natural populations of nanoflagellates consumed 34-62% of P. fluorescens Ag1 daily if starved bacteria were offered as food, and 3-13% if the cells were in the logarithmic growth phase. This suggests that the effect of protozoan predation on nonindigenous bacterial strains is substantial because cultured bacteria are likely to starve in natural environments. The addition of P. fluorescens Ag1 and the growth medium enhanced the abundance of natural bacteria, chlorophyll a, heterotrophic nanoflagellates, and ciliates, but it did not improve the growth conditions for the released strain. The effects on the indigenous populations were more pronounced after addition of fresh medium than following inoculation with cells, which possibly was due to the lower nutrient content of spent medium. However, these results, based on direct estimation of protozoan predation on log phase and starved nonindigenous bacteria, point to the conclusion that mortality induced by bacterivorous predators is the key factor determining removal of nonindigenous bacteria introduced in natural aquatic systems.

Survival of Bacillus licheniformis in Seawater Model Ecosystems.

The fate of Bacillus licheniformis DSM 13 was monitored after introduction into laboratory microcosms and mesocosms established in the Knebel Vig estuary, Denmark. The model organism was detected by a combination of immunofluorescence microscopy and nonselective plating followed by colony blotting. This allowed simultaneous quantification of intact cells and culturable cells. B. licheniformis DSM 13 adapted poorly to the conditions in filtered (0.2-mum-pore-size filter) seawater. Results from additional microcosm studies using natural seawater demonstrated that protozoan grazing also was important in regulating the population of the introduced model organism. In experiments using mesocosms, B. licheniformis DSM 13 also showed a rapid die-off. The introduction of the organism led to increased nutrient levels and to increased growth of both autotrophic and heterotrophic components of the plankton community compared with those of control enclosures. Thereby, a more intensive predation impact on the bacterioplankton community was induced. The combination of microcosm and mesocosm experiments provides a scenario in which the influence of single biotic and abiotic factors on survival of introduced organisms can be tested and in which the effect of the introduction on ecosystem structure and function can be evaluated. This test concept might prove useful in risk assessment of genetically modified microorganisms.

Qualitative importance of the microbial loop and plankton community structure in a eutrophic lake during a bloom of cyanobacteria.

Plankton community structure and major pools and fluxes of carbon were observed before and after culmination of a bloom of cyanobacteria in eutrophic Frederiksborg Slotssø, Denmark. Biomass changes of heterotrophic nanoflagellates, ciliates, microzooplankton (50 to 140 µm), and macrozooplankton (larger than 140 µm) were compared to phytoplankton and bacterial production as well as micro- and macrozooplankton ingestion rates of phytoplankton and bacteria. The carbon budget was used as a means to examine causal relationships in the plankton community. Phytoplankton biomass decreased and algae smaller than 20 µm replacedAphanizomenon after the culmination of cyanobacteria. Bacterial net production peaked shortly after the culmination of the bloom (510 µg C liter(-1) d(-1) and decreased thereafter to a level of approximately 124 µg C liter(-1) d(-1). Phytoplankton extracellular release of organic carbon accounted for only 4-9% of bacterial carbon demand. Cyclopoid copepods and small-sized cladocerans started to grow after the culmination, but food limitation probably controlled the biomass after the collapse of the bloom. Grazing of micro- and macrozooplankton were estimated from in situ experiments using labeled bacteria and algae. Macrozooplankton grazed 22% of bacterial net production during the bloom and 86% after the bloom, while microzooplankton (nauplii, rotifers and ciliates larger than 50 µm) ingested low amounts of bacteria and removed 10-16% of bacterial carbon. Both macro-and microzooplankton grazed algae smaller than 20 µm, although they did not control algal biomass. From calculated clearance rates it was found that heterotrophic nanoflagellates (40-440 ml(-1)) grazed 3-4% of the bacterial production, while ciliates smaller than 50 µm removed 19-39% of bacterial production, supporting the idea that ciliates are an important link between bacteria and higher trophic levels. During and after the bloom ofAphanizomenon, major fluxes of carbon between bacteria, ciliates and crustaceans were observed, and heterotrophic nanoflagellates played a minor role in the pelagic food web.

Simultaneous quantification by double rocket immunoelectrophoresis of apolipoproteins A-I and B in blood spotted on filter paper.

We describe double rocket immunoelectrophoresis for simultaneous quantification of apolipoprotein A-I (apo A-I) and B (apo B) in blood on filter paper. The apolipoproteins from blood spots on filter paper were eluted with detergents (sodium dodecyl sulfate and Triton X-100). The eluates were subjected to electrophoresis on agarose gel containing antisera against both apolipoproteins. Within- and between-assay CVs for apo B/A-I ratios were less than 5.5% and 7.2%, respectively. The apo B/A-I ratio was influenced by length and temperature of storage. In results for 121 venous blood samples, the apo B/A-I ratios in dried blood spots correlated well with those in serum (r = 0.92) and correlated somewhat with the ratios for low-density lipoprotein/high-density lipoprotein cholesterol in serum (r = 0.87). Of these specimens, 68 were from patients with known familial hypercholesterolemia, all of whom had an apo B/A-I ratio greater than 0.90. We think this method will be of value for detecting familial hypercholesterolemia and possibly familial hyperapobeta- and hypoalphalipoproteinemia.

Carcinoma of the urinary bladder after treatment with cyclophosphamide for non-Hodgkin's lymphoma.

We observed nine cases of transitional-cell carcinoma of the urinary bladder among patients who had had long-term treatment of other cancers with cyclophosphamide. Seven of the bladder carcinomas occurred within a cohort of 471 patients treated for non-Hodgkin's lymphomas. In this cohort the relative risk of bladder cancer was 6.8 (95 percent confidence interval, 3.2 to 14.2). The cumulative risk (mean +/- SE) was 3.5 +/- 1.8 percent 8 years after the start of treatment with cyclophosphamide and 10.7 +/- 4.9 percent after 12 years. Three of the nine patients were 50 years of age or younger; seven died with progressive bladder cancer. Subsequently, an additional patient had acute nonlymphocytic leukemia. Hemorrhagic cystitis was observed in 33 patients (cumulative risk, 11.8 +/- 2.1 percent after five years). Development of carcinoma of the urinary bladder was not related to previous hemorrhagic cystitis. The results caution against long-term treatment with cyclophosphamide for diseases with a favorable prognosis.

Prognosis and thallium-201 scintigraphy in patients admitted with chest pain without confirmed acute myocardial infarction.

Exercise and rest thallium scintigraphy and exercise electrocardiography were performed after discharge in 158 patients aged less than 76 years admitted with chest pain in whom a suspected diagnosis of acute myocardial infarction had not been confirmed. During a follow up of 12-24 months (median 14 months) there were 10 cardiac events--that is, non-fatal acute myocardial infarction or cardiac deaths. Transient thallium defects and abnormal ST response (that is ST segment deviation or uninterpretable ST segment) during exercise were correlated significantly with an unfavourable prognosis. One hundred and four patients with neither of these characteristics were at lower risk of a cardiac event than the 19 patients with both of these characteristics. The percentages of patients in these two groups without a cardiac event after one year were 98.1 and 78.8 respectively. Thallium scintigraphy, alone or in combination with exercise electrocardiography, can be used to identify groups at high and low risk of future cardiac events, in patients with chest pain in whom acute myocardial infarction is suspected but not found.

Unemployment and mortality in Denmark, 1970-80.

Relative mortality in the period 1970-80 was studied among Danish men and women who were unemployed and employed on the day of the 1970 census. The study population consisted of the total labour force in the age range 20-64 on 9 November 1970--that is, about 2 million employed and 22,000 unemployed people. Relative mortality was analysed by a multiplicative hazard regression model (as a natural extension of the standardised mortality ratio) and a multiplicative regression model with extra-Poisson variation. A significantly increased death rate (40-50%) was found among the unemployed after adjusting for occupation, housing category, geographical region, and marital state. Analysis of five main causes of death showed increased mortality from all causes, but especially from suicide or accidents. In areas where the local unemployment rate was comparatively high the relative mortality among the unemployed was lower. The increased mortality among the unemployed was interpreted as a consequence of health related selection as well as increased susceptibility associated with the psychosocial stress of unemployment.

Ventricular premature beats on Holter monitoring in patients admitted with chest pain, in whom acute myocardial infarction is not confirmed. The prognostic value and relationship to scars or ischemia on thallium-201 scintigraphy.

Ambulatory 24-h Holter monitoring was carried out in 198 patients who had been admitted because of suspected acute myocardial infarction (AMI) due to chest pain, but in whom AMI was not confirmed. During a follow-up period of 12-24 months (median 14 months) 16 cardiac events (i.e., nonfatal AMI or cardiac death) occurred. Ventricular premature beats (VPBs) were found in 65.2% of the patients, complex VPBs in 28.8%. Pairs of VPBs which were seen in 10.0% of the patients were the only important type of VPBs significantly related to an impaired prognosis. Thallium-201 scintigraphy was performed in 144 of the patients. VPBs were significantly related to scar formation (i.e., to permanent defects, but not to ischemia, specifically, to transient defects). It is concluded that ventricular arrhythmias in this patient category indicate presence of chronic ischemic heart disease, and that pairs of VPBs seem to identify patients at risk for cardiac events.