Effects of active pharmaceutical ingredients in mussel mytilus galloprovincialis

More than 3000 types of active pharmaceutical ingredients (APIs) are applied in Human and veterinary medicine practice. These compounds are considered an emergent class of environmental contaminants with the ability to cause damage and unexpected effects to aquatic organisms, namely in species of high commercial value. APIs are ubiquitous in the environment being frequently detected in influents and effluents of waste water treatment plants (WWTPs), surface waters and more distressingly in the public tap water in concentrations ranging from ng to μg.L-1. Considering these premises, the present thesis focused on APIs detection in the Arade river water, the impact of summer period in APIs’ concentration alterations applying the passive sampler device, POCIS (polar organic compound integrative sampler), as well as, the assessment of the effects caused by non-steroidal anti-inflammatory drugs (NSAID) ibuprofen (IBU) and diclofenac (DCF) and antidepressant selective serotonin reuptake inhibitor (SSRI) fluoxetine as single and mixture exposures along with a classical contaminant copper (Cu) on a non-target species, mussel Mytilus galloprovincialis. For this purpose, a multibiomarker approach was applied namely including biomarkers of oxidative stress (antioxidant enzymes activities of superoxide dismutase – SOD, catalase – CAT, glutathione reductase – GR and Phase II glutathione-S-transferase), damage - lipid peroxidation (LPO), neurotoxic effects (through the activity of acetylcholinesterase enzyme - AChE) and endocrine disruption (through vitellogenin-like proteins measurement applying the indirect method of alkali-labile phosphate - ALP) after exposure of mussel species’ to selected APIs at environmental relevant concentrations. The main results highlighted the occurrence of 19 APIs in the river Arade from several distinct therapeutic classes. Stimulant caffeine, antiasthmatic theophylline, NSAID ibuprofen and analgesic paracetamol presented the highest concentrations. Summer impact was inconclusive due to each API transient concentration in each month. The multibiomarker results revealed distinct responses towards each selected API (as single exposure or as mixtures) that were tissue and time dependent. Several multistressor interactions were proposed for each biomarker. The results also revealed APIs potential to induce oxidative stress, LPO, neurotoxicity and endocrine disruption even at extremely low concentrations on a species extremely vulnerable to APIs presence highlighting the urgency on the development of methodologies able to prevent its entrance in the aquatic environment.

Assessment of the mineralogenic toxicity of aquatic pollutants

In ecotoxicology a major focus is in the aquatic environment, not only because it presents a great economic value to man but it is an ecosystem widely affected by the growing anthropogenic pollution. Most of the studies performed relate to adverse effects in development, reproductive or endocrine disruption but little is known about the possible effects in bone formation and skeletal development. In this study, we set out to evaluate the effects of 8 aquatic pollutants on the skeletal development using an in vivo system, the zebrafish larvae aged 20 days post-fertilization, through chronic exposure. Several endpoints were considered such as the cumulative mortality, total length, occurrence of skeletal deformities and marker gene expression. We were able to establish LD50 values for some pollutants, like 3-methylcholanthrene, lindane, diclofenac, cobalt and vanadate and found that the total length was not affected by any of the pollutants tested. Cobalt was the most harmful chemical to affect hatching time, severely affecting the ability of the zebrafish embryos to hatch and overall the number of deformities increased upon exposure to tested chemicals but no patterns of deformities were identified. We also propose that 3-methylcholanthrene has an osteogenic effect, affecting osteoblast and osteoclast function and that op levels can act as a mediator of 3-methylcholanthrene toxic stress to the osteoblast. In turn we found naphthalene to probably have a chondrogenic effect. Our results provided new insights into the potential osteotoxicity of environmental pollutants. Future studies should aim at confirming these preliminary data and at determining mechanisms of osteotoxicity.

Endocrine regulation of extracellular matrix proteins in calcified tissue in teleost fish

Tese dout., Biologia, Universidade do Algarve, 2005