Clonal in vitro propagation of peat mosses (Sphagnum L.) as novel green resources for basic and applied research.

As builders and major components of peatlands, Sphagnopsida (peat mosses) are very important organisms for ecosystems and world's climate. Nowadays many Sphagnum species as well as their habitats are largely protected, while their scientific and economic relevance remains considerable. Advanced methods of in vitro cultivation provide the potential to work in a sustainable way with peat mosses and address aspects of basic research as well as biotechnological and economical topics like biomonitoring or the production of renewable substrates for horticulture (Sphagnum farming). Here, we describe the establishment of axenic in vitro cultures of the five peat moss species Sphagnum fimbriatum Wils. and Hook., Sphagnum magellanicum Brid., Sphagnum palustre L., Sphagnum rubellum Wils. and Sphagnum subnitens Russ. and Warnst. with specific focus on large-scale cultivation of S. palustre in bioreactors. Axenic, clonal cultures were established to produce high quantities of biomass under standardized laboratory conditions. For advanced production of S.palustre we tested different cultivation techniques, growth media and inocula, and analyzed the effects of tissue disruption. While cultivation on solid medium is suitable for long term storage, submerse cultivation in liquid medium yielded highest amounts of biomass. By addition of sucrose and ammonium nitrate we were able to increase the biomass by around 10- to 30-fold within 4 weeks. The morphology of in vitro-cultivated gametophores showed similar phenotypic characteristics compared to material from the field. Thus the tested culture techniques are suitable to produce S. palustre material for basic and applied research.

Application of neutral red retention assay to caged clams (Ruditapes decussatus) and crabs (Carcinus maenas) in the assessment of dredged material.

Dredged material management is a key issue for the protection of aquatic environments. The in situ approach using caged bioindicator species has been chosen lately as a new methodology for the assessment of dredged material. In a tier testing approach, neutral red retention (NRR) assay has been applied as a screening tool to detect adverse changes in health status associated with contamination. Nevertheless, to authors' knowledge, little is known about the application and validation of this technique in sediment bioindicator species and under field conditions. Caged Ruditapes decussatus and Carcinus maenas were exposed during 28 days to potentially contaminated sediments at three sites in Algeciras Bay (SW Spain) and one site in Cádiz Bay (SW Spain). Lysosomal membrane stability was measured over time in haemolymph samples of exposed clams and crabs using the NRR assay. Sediment characterization of the study sites was performed in parallel. NRR time did not vary significantly (p > 0.05) over time in organisms from Cádiz Bay. Conversely, significant differences (p < 0.05) in NRR time were found in clams and crabs exposed to sediments from Algeciras Bay, which exhibited a 30-70% decrease in haemocyte lysosome membrane stability compared to day 0. Statistical analysis showed a strong correlation between the drop of haemocyte lysosome membrane stability, in both crabs and clams, and the presence of metals (p < 0.05) and PAHs (p < 0.01) in the studied sediments. The results obtained confirmed the use of NRR assay as a suitable and sensitive method to be used in the assessment of sediment quality using as bioindicator species the clam R. philippinarum and the crab C. maenas.

Validation of Arenicola marina in field toxicity bioassays using benthic cages: biomarkers as tools for assessing sediment quality.

Sediment toxicity assessments using caged organisms present advantages over using laboratory and native community studies. The use of caged Arenicola marina in sediment toxicity assessments was evaluated. Lugworms were exposed in situ to sediments from coastal and port areas in Spain for seven days, and the activities of the biotransformation enzymes ethoxyresorufin O-deethylase, dibenzylfluorescein dealkylase and glutathione S-transferase, the activities of the antioxidant enzymes glutathione reductase and glutathione peroxidase and lipid peroxidation were then analyzed as biomarkers. Biomarker results and sediment physicochemical data were integrated. Cádiz Bay (SW Spain) sediments presented metal contamination that was not linked to a biochemical response. In LPGC Port (SW Spain), Pb contamination exhibited a moderate toxic potential, while PAHs, and presumably pharmaceuticals, provoked biochemical responses that efficiently prevented lipid peroxidation. In Santander Bay (N Spain), exposure to PAHs and, presumably, pharmaceuticals induced biomarker responses, but lipid peroxidation occurred nevertheless. These results indicated that caged A. marina were effective for the assessment of sediment quality and that the selected biomarkers were sufficiently sensitive to identify chemical exposure and toxicity.

Biomarker responsiveness in different tissues of caged Ruditapes philippinarum and its use within an integrated sediment quality assessment.

Biomarkers comprising activities of biotransformation enzymes (ethoxyresorufin-O-deethylase -EROD-, dibenzylfluorescein dealkylase -DBF-, glutathione S-transferase -GST), antioxidant enzymes (glutathione reductase -GR- and glutathione peroxidase -GPX), lipid peroxidation -LPO- and DNA strand breaks were analyzed in the clam Ruditapes philippinarum caged at Cádiz Bay, Santander Bay and Las Palmas de Gran Canaria (LPGC) Port (Spain). Sediments were characterized. Digestive gland was the most sensitive tissue to sediment contamination. In Cádiz Bay, changes in LPO regarding day 0 were related with metals. In LPGC Port, DBF, EROD, and GST activity responses suggested the presence of undetermined contaminants which might have led to DNA damage. In Santander Bay, PAHs were related with EROD activity, organic and metal contamination was found to be associated with GR and GST activities and DNA damage presented significant (p < 0.05) induction. R. philippinarum was sensitive to sediment contamination at biochemical level. Biomarkers allowed chemical exposure and sediment quality assessment.

Sediment-quality assessment using the polychaete Arenicola marina: contamination, bioavailability, and toxicity.

The sediment quality of Cádiz Bay, Las Palmas de Gran Canaria (LPGC) Port, Santander Bay, Algeciras Bay, and Huelva Estuary (Spain) was evaluated by analysing a battery of biochemical biomarkers-activities of biotranformation enzymes ethoxyresorufin O-deethylase [EROD], dibenzylflourescein dealkylase [DBF], and glutathione S-transferase [GST]; activity of antioxidant enzyme glutathione reductase [GR]; and lipid peroxidation [LPO]-in the polychaete Arenicola marina after laboratory sediment exposure. Huelva Estuary polychaetes showed significantly (p < 0.05) enhanced LPO, GST, and EROD activities compared with control lugworms related to metals and presumably polychlorinated biphenyls. EROD activity significant (p < 0.05) induction was associated polycyclic aromatic hydrocarbons after Santander Bay sediment exposure. Nickel appeared to significantly (p < 0.05) induce GR activity and LPO in LPGC Port sediment-exposed organisms. DBF activity significantly (p < 0.05) increased in polychaetes exposed to sediments from sewage-contaminated areas. A. marina was sensitive at the biochemical level. Integration of sediment characterization and biomarker results allowed the identification of polluted sites as well as the cause of possible sediment toxicity.

In situ evaluation of sediment toxicity in Guadalete Estuary (SW Spain) after exposure of caged Arenicola marina.

A 7 day in situ assay to assess sediment toxicity was carried out per replicate in two sites from Guadalete Estuary (Guad1 and Guad2) (South-West, Spain) using the polychaete Arenicola marina (n = 20) in benthic cages. After the exposure period, a battery of biomarkers were determined to asses the sediment toxicity. Five of them were exposure biomarkers [ethoxyresorufin O-deethylase (EROD), dibenzylfluorescein (DBF) dependant CytP450, gluthation reductase (GR), and gluthation-S-transferase (GST) activities] and one of them was an effect biomarker [lipid peroxidation (LPO)]. Toxicity due to metals bound to the sediment was found in Guad2. Metals provoke the inhibition of EROD, DBF, and GR and the induction of GST, which is showed to carry out antioxidative stress defense. Toxicity caused by metals and also PAH was detected in Guad1. The presence of these chemicals leads to GST induction in and presumably in consequence, to LPO inhibition. GST activity resulted to be the main protection mechanism against oxidative stress in Arenicola marina. We suggest further research in Guadalete Estuary focusing on the detection and toxicity assessment of pharmaceutical and personal care products.