In vitro alternatives to whole animal testing. Comparative cytotoxicity studies of divalent metals in established cell lines derived from tropical and temperate water fish species in a neutral red assay

A simple vital dye (neutral red) uptake method for investigation of the acute toxicity of seven divalent metal cations to cell lines derived from the turbot (TF) and bluegill (BF-2) was evaluated. The method was extremely reproducible between replicate experiments on a week to week basis as well as being reproducible between different laboratories. Whilst there were some species-dependent differences in the relative toxicities of the metals there were good correlations between toxicity and the chemical softness (Σp) of the metal ions and also between in vivo and in vitro toxicities. These studies demonstrate that this simple and relatively inexpensive technique carried out in 96-well microplates may be extremely useful for evaluation of structure-toxicity relationships between related groups of compounds.     

Inter-annual variability in larval supply to populations of three invertebrate taxa in the northern California Current

We investigated sources of inter-annual variability in larval supply to crab and sea urchin populations at Bodega Head and Point Reyes in northern California. During the spring and summer upwelling seasons of the years 1992 through 1997 we monitored the weekly settlement rates of nine species of crabs and two species of sea urchins. As observed in previous studies, daily values of alongshore windstress, temperature and salinity provided evidence for the poleward flow of relatively warm, low salinity water from south of Point Reyes, an apparent retention zone, during upwelling relaxation events. In years dominated by these events (1992, 1993, 1995 and 1996) we observed that alongshore windstress, temperature and salinity were coherent and temperature was significantly correlated with cancrid crab settlement. During these years the magnitude of cancrid crab settlement and the fraction of cancrid crabs relative to other crab species settling were high. Over four years of concurrent sampling there was consistently greater cancrid crab settlement at the Point Reyes site, within the retention zone, than at Bodega Head. Settlement of non-cancrid crabs (porcellanids, grapsids, pagurids and majids) was not as closely linked to intra-annual patterns of upwelling and relaxation, possibly due to the shorter seasonal availability of larvae allowing for the influence of fewer relaxation events. Settlement of this group among years was positively correlated with environmental indicators of strong seasonal upwelling; high salinity, Bakun upwelling index and low temperature. Sea urchin settlement events were observed in June and July of 1992, 1994 and 1997 during warming periods when salinity and temperature were increasing and alongshore windstress was low. Across the six years of the study, we found that cancrid crab larvae had a more even seasonal availability than larvae of non-cancrid species, which settled in greatest numbers during the early portion of the upwelling season. Sea urchins settled in greatest numbers during the later part of the upwelling season. Together these patterns demonstrate the taxon-specific way that inter-annual variability in larval supply is forced by the coincidence of larval availability with favorable physical transport mechanisms.     

Dissolved carbon and 33-133-133-1anomalies in the water column caused by hydrocarbon seeps on the northwestern Gulf of Mexico slope

Alkalinity, dissolved inorganic carbon (DIC), and ¹³C profiles from seep sites on the northwestern Gulf of Mexico upper slope show anomalously negative ¹³C values of up to –4.5 PDB, increased levels of DIC of up to 2.45 mmole/liter, and slight alkalinity rises of up to 2.54 meq/liter, relative to water column profiles from a seep-free site (0.63, 2.04 mmole/liter, and 2.39 meq/liter). The observed DIC enrichments coupled with the¹³C-depletions are attributed to the release of CO₂ by microbial oxidation of crude oil in the seep environment, and its migration into the water column. The ¹³C composition of the migrating CO₂ is estimated to be –26.0 on the basis of dissolved carbon inventory. Manifestation of DIC and ¹³C anomalies in the water column caused by hydrocarbon seepage holds promise to be useful for hydrocarbon reconnaissance surveys over large offshore tracts on account of the simplicity of sampling acquisition, and rapidity of analytical techniques in the laboratory.     

Voracious planktonic hydroids: unexpected predatory impact on a coastal marine ecosystem

Hydroids are typically attached, benthic cnidarians that feed on a variety of small prey. During sampling on Georges Bank in spring 1994, we found huge numbers of hydroids suspended in the plankton. They fed on young stages of copepods that are an important prey for fish, as well as on young fish themselves. Two independent methods were used to estimate feeding rates of the hydroids; both indicate that the hydroids are capable of consuming from 50% to over 100% of the daily production of young copepods. These results suggest that hydroids can have a profound effect on the population dynamics of zooplankton and young fish on Georges Bank.     

Evidence from heterologous expression of glutathione S-transferases A and A1 of the plaice (Pleuronectes platessa) that their endogenous role is in detoxification of lipid peroxidation products

cDNA clones for glutathione S-transferases A (GST-A) and A1 (GST-A1) from plaice (Pleuronectes platessa) were expressed as N-terminally 6XHis tagged proteins in Escherichia coli and purified to homogeneity from Ni-NTA silica. GST-A was an efficient catalyst for conjugation of unsaturated alkenals derived from peroxidation of polyunsaturated fatty acids with the highest activity observed with trans-non-2-enal (8 micromol min(-1) mg(-1)). GST-A1 was a very efficient Se-independent glutathione peroxidase with an activity towards cumene hydroperoxide of 25 micromol min(-1) mg(-1). Although the enzymes exhibited moderately high activities towards the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) they exhibited little or no activity towards other common prototypical xenobiotic substrates. Together with data for ontogeny, tissue distribution and inducibility of these enzymes, we contend that a primary function of these enzymes is protection from the harmful effects of lipid peroxidation products generated naturally or exacerbated by xenobiotic exposure.     

Short-term and interannual variability of redox-sensitive chemical parameters in hypoxic/anoxic bottom waters of the Chesapeake Bay

A combination of CTD casts, discrete bottle sampling and in situ voltammetric microelectrode profiling was used to examine changing redox conditions in the water column at a single station south of the Bay Bridge in the upper Chesapeake Bay in late July/early August, 2002–2005. Short-term (2–4 h) fluctuations in the oxic/suboxic/anoxic interface were documented using in situ voltammetric solid-state electrodes. Profiles of dissolved oxygen and sulfide revealed tidally-driven vertical fluctuations of several meters in the depth and thickness of the suboxic zone. Bottom water concentrations of sulfide, Mn²⁺ and Fe²⁺ also varied over the tidal cycle by approximately an order of magnitude. These data indicate that redox species concentrations at this site varied more due to physical processes than biogeochemical processes. Based on analysis of ADCP data, tidal currents at this station were strongly polarized, with the principal axis of tidal currents aligned with the mainstem channel. Together with the chemical data, the ADCP analysis suggests tidal flushing of anoxic bottom waters with suboxic water from north of the site. The present study is thus unique because while most previous studies have focused on processes across relatively stable redox interfaces, our data clearly demonstrate the influence of rapidly changing physical mixing processes on water column redox chemistry.Also noted during the study were interannual differences in maximum bottom water concentrations of sulfide, Mn²⁺ and Fe²⁺. In 2003, for example, heavy spring rains resulted in severe hypoxia/anoxia in June and early July. While reported storm-induced mixing in late July/early August 2003 partially alleviated the low-oxygen conditions, bottom water concentrations of sulfide, Mn²⁺ and Fe²⁺ were still much higher than in the previous year. The latter implies that the response time of the microbial community inhabiting the suboxic/anoxic bottom waters to changing redox conditions is slow compared to the time scale of episodic mixing events. Bottom water concentrations of the redox-sensitive chemical species should thus be useful as a tracer to infer prior hypoxic/anoxic conditions not apparent from ambient oxygen levels at the time of sampling.     

Characterization and molecular analysis of hepatic microsomal UDP-glucuronosyltransferases in the marine fish, Pleuronectes platessa

Here we report the results of the first molecular enzymological study of fish UDP-glucuronosyltransferase (UDPGT). UDPGT activities of plaice liver microsomes were greatest for planar phenols and there was low but measurable conjugation of bilirubin, testosterone and androsterone. A highly purified preparation was isolated which possessed activities towards 1-naphthol, bilirubin and testosterone, containing several molecular weight species of M_r 52–56 kDa. On immunoblot analysis these proteins cross-reacted with a polyspecific anti-rat UDPGT antibody, suggesting that a number of plaice UDPGT isoforms with epitopes in common with the corresponding rat enzymes had been purified.