Gonyaulax monilata: Population Growth and Development of Toxicity in Cultures

SYNOPSIS. The development of three 8-liter and four 12-liter cultures of the photosynthetic dinoflagellate Gonyaulax monilata was followed for 4 months. Weekly estimates were made of population levels of this chain-forming flagellate, along with incidence of cells in chains and toxicity to fish. Guppies ( Lebistes reticulatus ) were used to assay toxicity. Populations reached a peak when cultures were 3–5 weeks old, declined during weeks 6–10, and tended to stabilize thereafter thru the 17th (final week). The percentage of cells in chains was related to the slope of the population curve; rapidly increasing populations had the highest proportion of long chains, suggesting that incidence of chains is an index of the growth phase in G. monilata. Peak toxicity was not reached until culture populations had been steadily declining for a month, indicating that most toxin is released by autolysis. The reproducibility of culture population and toxicity levels recommend the methods used for future studies.     

The influence of Gomphosphaeria aponina on the growth of Gymnodinium breve and the effect of aponin on the ichthyotoxicity of Gymnodinium breve.

Cells of the marine blue-green alga Gomphosphaeria aponina survive in mixed culture with the marine dinoflagellate, Gymnodinium breve (the Florida red tide organism), but G. breve cells lysed within 4-7 days. It has been established that the cytolytic effect of G. aponina, not nutrient competition, is responsible for the decrease in number of cells. The material elaborated by G. aponina has been termed aponin and has been extracted from the cells. The effect of aponin on the ichthyotoxicity of G. breve cultures was measured using Poecilia sphenops, adapted to sea water, as the assay organism. Aponin is not ichthyotoxic toward P. sphenops, though this material, when incubated with G. breve cultures does destroy the cells and increases the ichthyotoxicity of the cultures. At certain concentrations of aponin, the ichthyotoxicity of G. breve cultures appeared to be mitigate d.     

Vectorial transport of toxins from the dinoflagellate Gymnodinium breve through copepods to fish

Toxins from the dinoflagellate Gymnodinium breve were tracedthrough experimental food chains from dinoflagellates, throughcopepod grazers, to juvenile fish. The generality of this foodweb transfer was demonstrated using three different combinationsof copepods and juvenile fish during different seasons. Fishwere not exposed directly to the toxic dinoflagellates but werefed toxin-laden copepods in order to examine sublethal vectorialintoxication. Toxins were shown to move from fish viscera tomuscle tissue within periods of 2–6 h to 25 h. A new toxindetection method was used in this first stepwise demonstrationof multi-trophic-level intoxication of a planktonic food chainby G.breve. Micellar electrokinetic capillary electrophoresiswith laser-induced fluorescence allowed measurements of toxinsat trace levels and nanoliter-sized volumes critical for planktonicfood web transfer studies.     

Critical micelle concentrations of allelopathic substances produced by Nannochloris oculata which affect a red tide organism, Gymnodinium breve

Laboratory cultures of the green algae Nannochloris oculata and Nannochloris eucaryotum are known to cause lysis of Gymnodinium breve, which is Florida's red tide organism. Two cytolytic agents were previously identified as methyl palmitate and methyl stearate. In this study, the critical micelle concentrations of these substances were determined by ultraviolet light and turbidimetric methods to be 3.5 +/- 0.3 ppm (methyl stearate) and 4.3 +/- 0.6 (methyl palmitate). There were no significant differences in results obtained using the two methods.     

Size-Dependent Toxicity of Silver Nanoparticles to Bacteria,Yeast, Algae,Crustaceans and Mammalian Cells In Vitro

The concept of nanotechnologies is based on size-dependent properties of particles in the 1–100 nm range. However, the relation between the particle size and biological effects is still unclear. The aim of the current paper was to generate and analyse a homogenous set of experimental toxicity data on Ag nanoparticles (Ag NPs) of similar coating (citrate) but of 5 different primary sizes (10, 20, 40, 60 and 80 nm) to different types of organisms/cells commonly used in toxicity assays: bacterial, yeast and algal cells, crustaceans and mammalian cells in vitro. When possible, the assays were conducted in ultrapure water to minimise the effect of medium components on silver speciation. The toxic effects of NPs to different organisms varied about two orders of magnitude, being the lowest (∼0.1 mg Ag/L) for crustaceans and algae and the highest (∼26 mg Ag/L) for mammalian cells. To quantify the role of Ag ions in the toxicity of Ag NPs, we normalized the EC₅₀ values to Ag ions that dissolved from the NPs. The analysis showed that the toxicity of 20–80 nm Ag NPs could fully be explained by released Ag ions whereas 10 nm Ag NPs proved more toxic than predicted. Using E. coli Ag-biosensor, we demonstrated that 10 nm Ag NPs were more bioavailable to E. coli than silver salt (AgNO₃). Thus, one may infer that 10 nm Ag NPs had more efficient cell-particle contact resulting in higher intracellular bioavailability of silver than in case of bigger NPs. Although the latter conclusion is initially based on one test organism, it may lead to an explanation for “size-dependent“ biological effects of silver NPs. This study, for the first time, investigated the size-dependent toxic effects of a well-characterized library of Ag NPs to several microbial species, protozoans, algae, crustaceans and mammalian cells in vitro.     

Isolation and properties of isolated nuclei from the Florida red tide dinoflagellate Gymnodinium breve (Davis)

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA : RNA : acid-soluble protein : acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.     

Isolation and Properties of Isolated Nuclei from the Florida Red Tide Dinoflagellate Gymnodinium breve (Davis)1

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA: RNA: acidsoluble protein: acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.     

Diverse Responses of Planktonic Crustaceans to Fish Predation by Shifts in Depth Selection and Size at Maturity

We investigated the behaviour and life histories of large zooplankton in the Esch-sur-Sûre reservoir (Luxembourg). We found that the decrease in size at maturity, as well as diurnal refuge in deep waters, were the adaptive responses (concurrently or alternatively) adopted by large zooplankters to cope with the increasing predation risk throughout the summer. Daphnia galeata initiated a more severe trade-off to cope with high summer predation relative to other cladoceran species. Diaphanosoma brachyurum and Daphnia cucullata seem less susceptible to fish predation as indicated by the low alteration of their size structure and their vertical distribution. The copepod Eudiaptomus gracilis took refuge in deep waters and achieved important modifications of its mature size.     

Jezierska,B. and Witeska,M. (2001) Metal Toxicity to Fish

Reviews in Fish Biology and Fisheries -     

Comparative studies of skeletal soluble matrices from some Scleractinian corals and Molluscs

The soluble organic matrices extracted from aragonitic skeletons of five Scleractinia and two Molluscs were analyzed using FTIR spectrometry, HPLC and 2-D gel electrophoresis. All these methods show that scleractinian and molluscan matrices are different. Both matrices are acidic with poorly separated molecular weights. The scleractinian matrices are highly glycosylated, whereas the molluscan matrices are not as shown by stains in 2D-electrophoresis. These results were in accordance with earlier data: high S contents in Scleractinia skeletons and low S contents in Mollusca shells. These results confirm the control of the morphology and the chemical composition of aragonite biocrystals in various taxa.     

Properties of Respiratory Pigments in Bimodal Breathing Animals: Air and Water Breathing by Fish and Crustaceans

SYNOPSIS. Oxygen is nearly 30 times more available in air comparedwith water, whereas the carbon dioxide capacity of water is{small tilde}28.5-fold greater than for oxygen, presenting bimodalbreathing species with two very different respiratory milieus.The respiratory pigment plays a variable role in animals switchingbetween the two media. In vertebrates the transition to airbreathing involves two main strategies: a decrease in oxygenaffinity and changes in other haematological parameters suchas haematocrit. When appropriately analyzed, data reveal a decreasein blood oxygen affinity during transition to air. This mayarise via differences in the intrinsic affinity as occurs insome amphibians, or be due to increasesin the organic phosphate:haemoglobin ratio when acclimating to air breathing. Adoptingair breathing often promotes increased haematocrit. It is difficultto discern trends in haemocyanin functioning. Many but not allbimodal and air breathingspecies of crab contain haemocyaninwith high affinity for oxygen. As with haemoglobin there issome tendency for blood haemocyanin concentration to increasewith air breathing but bimodal species are quite variable inthis regard. Different strategies for breathing air are employedby various bimodal crustaceans, some of which involve modulationof haemocyanin oxygenaffinity. The exact mechanisms are oftenspecies dependent and in all bimodal breathing organisms therole of the pigment is best appreciated when the demands ofthe local environment and the behaviour of the species are considered.     

Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts

In October of 1996, a Gymnodinium breve bloom occurred in shellfish harvesting waters of Alabama, Mississippi and Louisiana, Gulf of Mexico, USA. Bloom densities reached 5.6x10(5) cells liter(-1) and bloom residence at shellfish sampling stations ranged from 3 to 28 days. Brevetoxin-2 dominated G. breve toxin profiles in bloom seawater extracts. Shellfish toxicity, assessed by mouse bioassay, exceeded the guidance level for up to 75 days after the bloom had dissipated. Cytotoxicity assays and mouse bioassays showed similar temporal patterns of shellfish toxicity, but the two methods differed in estimations of brevetoxin-3 equivalent toxicity by a factor of 93 to 1. LC-ESI-MS showed the temporal patterns in shellfish toxicity reflected metabolism of G. breve toxins. The molecular ions m/z 1004, 1017 and 1033 dominated LC-ESI-MS spectra of toxic chromatographic fractions from the extracts and were identified as brevetoxin metabolites on the basis of LC-APCI-MS-MS. The discrepancy between cytotoxicity and mouse bioassay estimates of brevetoxin-3 equivalent toxicity resulted from the difference in extraction efficiency of solvents used in the respective methods and the relative sensitivity of the assays to toxin metabolite mixtures present in the extracts. The normalized cytotoxicity assay showed 75% agreement with mouse bioassay positive test samples and 64% agreement with mouse bioassay negative test samples. Published in 1999 by John Wiley & Sons, Ltd.     

Acute Toxicity,Respiratory Reaction,and Sensitivity of Three Cyprinid Fish Species Caused by Exposure to Four Heavy Metals

Using 3 cyprinid fish species zebra fish, rare minnow, and juvenile grass carp, we conducted assays of lethal reaction and ventilatory response to analyze sensitivity of the fish to 4 heavy metals. Our results showed that the 96 h LC₅₀ of Hg²⁺ to zebra fish, juvenile grass carp, and rare minnow were 0.14 mg L⁻¹, 0.23 mg L⁻¹, and 0.10 mg L⁻¹, respectively; of Cu²⁺0.17 mg L⁻¹, 0.09 mg L⁻¹, and 0.12 mg L⁻¹ respectively; of Cd²⁺6.5 mg L⁻¹, 18.47 mg L⁻¹, 5.36 mg L⁻¹, respectively; and of Zn²⁺44.48 mg L⁻¹, 31.37 mg L⁻¹, and 12.74 mg L⁻¹, respectively. Under a 1-h exposure, the ventilatory response to the different heavy metals varied. Ventilatory frequency (Vf) and amplitude (Va) increased in zebra fish, juvenile grass carp, and rare minnows exposed to Hg²⁺ and Cu²⁺ (P<0.05), and the Vf and Va of the 3 species rose initially and then declined when exposed to Cd²⁺. Zn²⁺ had markedly different toxic effects than the other heavy metals, whose Vf and Va gradually decreased with increasing exposure concentration (P<0.05). The rare minnow was the most highly susceptible of the 3 fish species to the heavy metals, with threshold effect concentrations (TEC) of 0.019 mg L⁻¹, 0.046 mg L⁻¹, 2.142 mg L⁻¹, and 0.633 mg L⁻¹ for Hg²⁺, Cu²⁺, Cd²⁺, and Zn²⁺, respectively. Therefore, it is feasible to use ventilatory parameters as a biomarker for evaluating the pollution toxicity of metals and to recognize early warning signs by using rare minnows as a sensor.     

Morphological, Behavioral, and Physiological Characterization of Bimodal Breathing Crustaceans

SYNOPSIS Bimodal breathing crustaceans, while representing astage in the transition from the aquatic to terrestrial habitat,also constitute a distinct group that can be characterized bymorphological, behavioral, and physiological traits. Morphologically,this group displays reduced gill surface area and enlarged branchialchambers. The lining of the branchial chambers, the branchiostegites,also has increased surface area and is highly vascularized.The branchiostegites can be smooth, cutaneous epithelia, orthey can have more complex evaginations or invaginations tofurther increase surface area. In addition, the thickness ofthe branchiostegal epithelium is greatly reduced, compared tothat in the gills, thus minimizing the diffusion distance betweenair and hemolymph. These animals maintain a store of water inthe branchial chamber that covers the gills and allows for simultaneousgas exchange with two media (air and water). There is also apartitioning of gas exchange, with oxygen uptake occurring preferentiallyfrom air across the branchiostegites, and carbon dioxide excretionoccurring across the gills into the branchial water. One criticalfactor that appears to separate bimodal breathing crustaceansfrom fully terrestrial, exclusively air-breathers is the abilityof the latter group to excrete CO₂directly into air across thegills and branchiostegites. It is suggested that the incorporationof the enzyme carbonic anhydrase into the membrane fractionof the branchiostegites may have been one of the key molecularevents which allowed for pulmonary CO₂ excretion into air.     

A Perspective on the Toxicity of Petrogenic PAHs to Developing Fish Embryos Related to Environmental Chemistry

Numerous studies demonstrate polynuclear aromatic hydrocarbons (PAHs) dissolved from weathered crude oil adversely affect fish embryos at 0.5 to 23 μg/l. This conclusion has been challenged by studies that claim (1) much lower toxicity of weathered aqueous PAHs; (2) direct contact with dispersed oil droplets plays a significant role or is required for toxicity; (3) that uncontrolled factors (oxygen, ammonia, and sulfides) contribute substantively to toxicity; (4) polar compounds produced by microbial metabolism are the major cause of observed toxicity; and (5) that based on equilibrium models and toxic potential, water contaminated with weathered oil cannot be more toxic per unit mass than effluent contaminated with fresh oil. In contrast, several studies demonstrate high toxicity of weathered oil; shifts in PAH composition were consistent with dissolution (not particle ablation), embryos accumulated dissolved PAHs at low concentrations and were damaged, and assumed confounding factors were inconsequential. Consistent with previous empirical observations of mortality and weathering, temporal shifts in PAH composition (oil weathering) indicate that PAHs dissolved in water should (and do) become more toxic per unit mass with weathering because high molecular weight PAHs are more persistent and toxic than the more abundant low molecular weight PAHs in whole oil.