Histological biomarkers in liver and gills of juvenile Solea senegalensis exposed to contaminated estuarine sediments: a weighted indices approach.

Young juvenile Solea senegalensis were exposed to three sediments with distinct contamination profiles collected from a Portuguese estuary subjected to anthropogenic sources of contamination (the Sado estuary, western Portugal). Sediments were surveyed for metals (cadmium, chromium, copper, nickel, lead and zinc), a metalloid (arsenic) and organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and a pesticide, dichloro-diphenyl-trichloroethane plus its metabolites), as well as total organic matter, redox potential and particle fine fraction. The fish were exposed to freshly collected sediments in a 28-day laboratorial assay and collected for histological analyses at days 0 (T(0)), 14 (T(14)) and 28 (T(28)). Individual weighted histopathological indices were obtained, based on presence/absence data of eight and nine liver and gill pathologies, respectively, and on their biological significance. Although livers sustained more severe lesions, the sediments essentially contaminated by organic substances caused more damage to both organs than the sediments contaminated by both metallic and organic contaminants, suggesting a possible synergistic effect. Correlation analyses showed that some alterations are linked, forming distinctive histopathological patterns that are in accordance with the severity of lesions and sediment characteristics. The presence of large eosinophilic bodies in liver and degeneration of mucous cells in gills (a first-time described alteration) were some of the most noticeable alterations observed and were related to sediment organic contaminants. Body size has been found to be negatively correlated with histopathological damage in livers following longer term exposures. It is concluded that histopathological indices provide reliable and discriminatory data even when biomonitoring as complex media as natural sediments. It is also concluded that the effects of contamination may result not only from toxicant concentrations but also from their interactions, relative potency and sediment characteristics that ultimately determine bioavailability.

Ionic interfaces and diphtheria toxoid interactions.

We present here a systematic study on the purification of the diphtheria toxoid (Dtxd) produced at the Instituto Butantan, by adding only one step on the entire process of its production. Aliquots of 1.0 ml of Dtxd were added to an equal amount of Q-Sepharose previously equilibrated with 500 mM Tris, pH 5.0-9.0 (increments of 0.5 pH units). The best condition for the Dtxd monomer adsorption was achieved at pH 9.0. The best condition for desorption was obtained with 300 mM NaCl. After studying the gel binding capacity for Dtxd, a column (C20/20) equilibrated with 500 mM Tris, pH 9.0, was prepared. The purification factor for Dtxd was 1.5. The final recovery of Dtxd was 68.75%, with 90.31% purity. The process methodology presented here is a very realistic sequence of separation steps, which is perfectly compatible with the production requirements. Vaccination with "toxoid highly purified toxin" is known to confer a strong immunity on people in the absence of undesirable reactions, which led experts of European Pharmacopoeia to recommend its use both for children and adult vaccination.

Ionic interfaces and diphtheria toxoid interactions

We present here a systematic study on the purification of the diphtheria toxoid (Dtxd) produced at the Instituto Butantan, byadding only one step on the entire process of its production. Aliquots of 1.0 ml of Dtxd were added to an equal amount of QSepharosepreviously equilibrated with 500mM Tris, pH 5.0–9.0 (increments of 0.5 pH units). The best condition for the Dtxdmonomer adsorption was achieved at pH 9.0. The best condition for desorption was obtained with 300mM NaCl. After studyingthe gel binding capacity for Dtxd, a column (C20/20) equilibrated with 500mM Tris, pH 9.0, was prepared. The purification factorfor Dtxd was 1.5. The final recovery of Dtxd was 68.75%, with 90.31% purity. The process methodology presented here is a veryrealistic sequence of separation steps, which is perfectly compatible with the production requirements. Vaccination with ‘‘toxoidhighly purified toxin’’ is known to confer a strong immunity on people in the absence of undesirable reactions, which led experts ofEuropean Pharmacopoeia to recommend its use both for children and adult vaccination.

Delineation of estuarine management areas using multivariate geostatistics: the case of Sado Estuary.

The Sado Estuary is a coastal zone located in the south of Portugal where conflicts between conservation and development exist because of its location near industrialized urban zones and its designation as a natural reserve. The aim of this paper is to evaluate a set of multivariate geostatistical approaches to delineate spatially contiguous regions of sediment structure for Sado Estuary. These areas will be the supporting infrastructure of an environmental management system for this estuary. The boundaries of each homogeneous area were derived from three sediment characterization attributes through three different approaches: (1) cluster analysis of dissimilarity matrix function of geographical separation followed by indicator kriging of the cluster data, (2) discriminant analysis of kriged values of the three sediment attributes, and (3) a combination of methods 1 and 2. Final maximum likelihood classification was integrated into a geographical information system. All methods generated fairly spatially contiguous management areas that reproduce well the environment of the estuary. Map comparison techniques based on kappa statistics showed thatthe resultant three maps are similar, supporting the choice of any of the methods as appropriate for management of the Sado Estuary. However, the results of method 1 seem to be in better agreement with estuary behavior, assessment of contamination sources, and previous work conducted at this site.

Effects of water-borne copper on metallothionein and lipid peroxidation in the marine amphipod Gammarus locusta.

The aim of the current work was to determine over 10 days the effects of water-borne exposure of the marine amphipod Gammarus locusta to 4 microgCu l(-1) on the metallothionein (MT; measured by differential pulse polarography) protection system and lipid peroxidation (LP: thiobarbituric acid-reactive malondialdehyde equivalents) as a measure of oxidative damage. MT levels in exposed animals increased significantly at day 2 (36% > control; P < 0.001) and remained high at days 6 and 10 (55 and 38%, respectively, P < 0.001). The maximum level of MT at day 6 coincided with the highest Cu body-burden. LP increased within I day of exposure, indicative of Cu as an oxidative stressor. However, in contrast to MT, the highest LP level was seen at day 4 (68% > control, P < 0.001) before returning to control values by day 6, indicating a protective role of MT against the pro-oxidant effects of Cu.

Detection of DNA strand breakage in a marine amphipod by agarose gel electrophoresis: exposure to X-rays and copper.

This article describes the leading steps to develop an assay of DNA damage for the marine amphipod Gammarus locusta, using agarose gel electrophoresis (AGE). To test the sensitivity and feasibility of the AGE technique, X-ray assays were performed with naked DNA and with live amphipods. These positive controls demonstrated the effectiveness of the AGE technique to not only discriminate distinct levels of DNA strand breakage in a dose-dependent manner, but also to identify and quantify the type of strand breakage induced. It was also shown that it is possible to detect DNA damage using whole-body DNA extracts from amphipods. To explore the potential of this technique for use in ecotoxicological studies with amphipods, a 96-h waterborne-copper toxicity test was performed. Copper-induced DNA strand breakage was first observed after 24 h of exposure, and was recorded again at 96 h, at a copper concentration of 20 microg l(-1). The absence of strand breakage after 48 h of exposure is discussed in the light of the underlying mechanisms of copper toxicity and DNA repair. These studies demonstrated the feasibility of including DNA damage as a biomarker in ecotoxicological studies with amphipods. Information gained from the use of this biomarker would help with the interpretation of chronic toxicity tests and would contribute to our understanding of the impact of genotoxic insult in marine invertebrates, particularly crustaceans.