Genetic identification of three CITES-listed sharks using a paper-based Lab-on-a-Chip (LOC).

Threatened shark species are caught in large numbers by artisanal and commercial fisheries and traded globally. Monitoring both which shark species are caught and sold in fisheries, and the export of CITES-restricted products, are essential in reducing illegal fishing. Current methods for species identification rely on visual examination by experts or DNA barcoding techniques requiring specialist laboratory facilities and trained personnel. The need for specialist equipment and/or input from experts means many markets are currently not monitored. We have developed a paper-based Lab-on-a-Chip (LOC) to facilitate identification of three threatened and CITES-listed sharks, bigeye thresher (Alopias superciliosus), pelagic thresher (A. pelagicus) and shortfin mako shark (Isurus oxyrinchus) at market source. DNA was successfully extracted from shark meat and fin samples and combined with DNA amplification and visualisation using Loop Mediated Isothermal Amplification (LAMP) on the LOC. This resulted in the successful identification of the target species of sharks in under an hour, with a working positive and negative control. The LOC provided a simple "yes" or "no" result via a colour change from pink to yellow when one of the target species was present. The LOC serves as proof-of-concept (PoC) for field-based species identification as it does not require specialist facilities. It can be used by non-scientifically trained personnel, especially in areas where there are suspected high frequencies of mislabelling or for the identification of dried shark fins in seizures.

Ranking sediment samples from three Spanish estuaries in relation to its toxicity for two benthic species: the microalga Cylindrotheca closterium and the copepod Tisbe battagliai.

The present study assesses the sediment toxicity levels of three Spanish estuaries, as well as the suitability of two microorganisms, the benthic microalga Cylindrotheca closterium and the harpacticoid copepod Tisbe battagliai, as test organisms in whole-sediment toxicity assays. The sensitivity of both species to potentially polluted sediments was compared. Three sites at the southwest of the Iberian Peninsula were chosen: the Ría of Huelva, the Guadalquivir Estuary, and the Bay of Algeciras. Inhibition data were based on growth for C. closterium and fecundity for T. battagliai. No toxicity was recorded for the microalga in the Guadalquivir Estuary and the Bay of Algeciras. However, for T. battagliai, inhibition of fecundity was approximately 50% in those zones, indicating higher sensitivity. Samples from stations in the Ría of Huelva were the most toxic of all those assayed; inhibition values higher than 90% were obtained for both organisms. The highest values for total metal concentrations such as arsenic (As), cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), tin (Sn), and zinc (Zn) were found in the Ría of Huelva, which can be classified as severely impacted. The Guadalquivir Estuary and the Bay of Algeciras can be considered moderately impacted. In general, both methodologies are suitable for application in ecotoxicological studies.

Sensitivity of Cylindrotheca closterium to copper: influence of three test endpoints and two test methods.

The present study checked the suitability of Cylindrotheca closterium (Bacillariophyceae) as a test species for ecotoxicology studies. To date, only limited use has been made of microphytobenthos in ecotoxicology, and C. closterium has been employed as a target organism in this study because the biological group is considered to be very relevant ecologically. The main objective was to assess the response of C. closterium to a contaminant-type (copper) using three different test endpoints (esterase activity, chlorophyll fluorescence and population growth) and two different test methods (Erlenmeyer flasks and microplates), to evaluate which combination of test conditions would provide the most sensitive approach for assessment of effects. Regardless of the endpoints, the response of C. closterium to copper was very similar; however lower sensitivity (EC50 of 27.8 +/- 0.7 microg Cu L(-1) was observed when tests were carried out in microplates. Chlorophyll fluorescence measured by flow cytometry as total FL3 was slightly more sensitive (EC50 of 4.7 +/- 0.1 microg Cu L(-1)) than the other parameters measured, probably because it takes into account the effect on chlorophyll fluorescence and cell density simultaneously. The test method (Erlenmeyer flask or microplate) was the determining factor for the observed differences in sensitivity. These differences found for the two methods are explained by the higher metal adsorption capacity of microplate vessel walls (more than 40%), which decreases the available copper. C. closterium was demonstrated to be a suitable organism for adoption in ecotoxicological studies, given the reliability of the three endpoints and also of the two test methods evaluated here.

Short-term toxicity tests on the harpacticoid copepod Tisbe battagliai: lethal and reproductive endpoints.

Short-term bioassays based on lethal and reproductive responses of Tisbe battagliai were applied to determine responses of copepods to copper and LAS. Percentage of spawning females, fecundity (F), and total newborn production (N) for 48 and 72 h were calculated for both substances. It was observed percentage of spawning females was not affected by sublethal concentrations of both compounds. Following values were obtained: EC(50)(N)-48 h of 670+/-30 microgLASL(-1) and EC(50)(F)-48 h of 670+/-30 microgLASL(-1); and EC(50)(N)-72 h of 44.5+/-1.8 microgCuL(-1) and EC(50)(F)-72 h of 30.8+/-1.1 microgCuL(-1). Lethal effects of the two substance-types were also assessed, obtaining the LC(50)-24h of 1980+/-160 microgLASL(-1); and LC(50)-48 h of 83.1+/-10.5 microgCuL(-1) for nauplii; and LC(50)-72 h of 157+/-25 microgCuL(-1), and LC(50)-72 h of 2660+/-270 microgLASL(-1) for adults. Fecundity and total newborn production are sensitive endpoints for determining effects of toxicants.

Sediment integrative assessment of the Bay of Cádiz (Spain): an ecotoxicological and chemical approach.

This study consisted of the sediment toxicity assessment of the Bay of Cádiz based on two endpoints: growth inhibition for Cylindrotheca closterium (benthic microalgae) and fecundity inhibition for Tisbe battagliai (harpacticoid copepod). A new methodology to eliminate (but not as storage technique) the autochthonous biota present in the sediment samples by immersing them in liquid nitrogen (-196 degrees C) was also assessed. Sediment toxicity data showed different toxicity levels for both organisms. In general, T. battagliai was more sensitive; however a good correlation (r=0.75; p<0.05) between sediment toxicity results for both species was found. Data in pore water (pH, redox potential, and toxicity for microalgae and copepod) and sediment (pH, redox potential, organic carbon, and metal concentrations) demonstrated that ultra-freezing did not alter sample characteristics; thus, this technique can be adopted as a pre-treatment in whole-sediment toxicity tests in order to avoid misleading results due to presence of autochthonous biota. Multivariate statistical analysis such as cluster and principal component analysis using chemical and ecotoxicological data were employed. Silt and organic matter percentage and lead concentration were found to be the factors that explain about 77% of sediment toxicity in the Bay of Cádiz. Assay methodology determined in this study for both assayed species is considered adequate to be used in sediment toxicity monitoring programs. Results obtained using both species show that the Bay of Cádiz can be considered a moderately polluted zone.

Effects of cold-dark storage on growth of Cylindrotheca closterium and its sensitivity to copper.

Cylindrotheca closterium cells were maintained at low temperature (4+/-1 degrees C) and dark conditions up to 21 weeks to assess the effect on survival and physiological status. From a control culture under standard conditions, three densities were prepared: (A) 2 x 10(4), (B) 10 x 10(4), and (C) 25 x 10(4) cells ml(-1). Weekly, inoculums of each stored density were exposed to continuous light and at 20+/-1 degrees C. Sensitivity to copper for microalgal cultures was evaluated in order to assess possible changes in cells sensitivity due to storage. Concurrently, assays with a control culture were carried out in order to assess the sensitivity of C. closterium to copper and to be able to generate a standard sensitivity control chart with a mean value of EC50-72 h+/-2SD (standard deviation). Density-C presented higher cell yield values, between 40% and 80% relative to control culture. Cell density showed to be important feature that may be taken into account in cell storage experiments. There was an increase in sensitivity of cells submitted to storage; however results always kept in the range established as standard sensitivity with no statistically significant difference with regards to control culture. EC50-72 h mean value for the control culture was 29+/-10 mug Cul(-1), while for densities-A, B and C were 22+/-7; 23+/-9 and 23+/-8 microg Cul(-1), respectively. In spite of drastic changes in the environmental conditions due to storage, it is concluded that C. closterium cells stored during 5 months remained metabolically active and with no significant change in its sensitivity.