Catalá, M.1, García-Cambero, J.P.2, García-Cortés,H.1, Valcárcel Rivera, Y3.
1Department of Biology ande Geology, Physics and Inorganic Chemistry. Higher School of Science and Technology. Rey Juan Carlos University. Campus de Móstoles c/Tulipán s/n E-28933 Móstoles
2National Centre for Environmental Health. Institute of Health Carlos III. Ctra Majadahonda-Pozuelo km 2, 28220 Majadahonda, Madrid, Spain
3Yolanda Valcárcel Rivera. Grupo de Investigación en Salud Ambiental y Ecotoxicología. Area de Medicina Preventiva y Salud Pública. Facultad de Ciencias de la Salud. Universidad Rey Juan Carlos.
E-mail contact: email@example.com
Several studies have found cocaine and its main active metabolite benzoylecgonine (BE) in the aquatic environment and natural waters  derived from their high consumption by humans and the inability of water treatment processes to eliminate it . As an emerging substance, research on BE has primarily focused on improving methodologies for its determination, while hardly any study has tackled the impact that such waste can have on wild biota. Only a couple of works have addressed BE ecotoxicology on animals [3-4] and no data have been published of the ecotoxicity of BE on plants or microalgae.
Concerning fish, there is growing interest in the use of fish embryos as an alternative tool to examine the presence and potency of aquatic toxicants. On one hand, it is generally considered that initial phases of development are more susceptible to environmental pollution than adults, what is determinant for the viability of natural populations . On the other hand, new endpoints, beyond simple lethality, have emerged to assist toxicity identification in fish embryos, including the study of developmental effects and sublethal effects relative to cardiotoxicity, hepatotoxicity or neurotoxicity among others.
A microbioassay based on the development of fern spores/gametophytes has been developed some years ago, turning into a robust and sensitive method for the assessment of the toxicity of industrial pollutants , pharmaceuticals  or environmental samples .
So, the aim of this publication is to provide information on the toxicity of environmental concentrations of BE during animal and vascular plant development. To evaluate fish toxicity, we have used a combined model (embryo-larvae) to identify sensitive biomarkers, e.g. those related to neurodevelopment . For plant toxicity, we have used the fern spores model. Data from this study will contribute to a better understanding of the impact of such emerging substances in different organisms of the freshwater ecosystems, necessary for environmental risk assessment.
2. Materials and methods
A stock solution of 1 mg mL-1 of BE in methanol was prepared. The concentrations (ranged from 10-4 to 10 µg L-1) were selected to cover the environmental concentrations of BE detected in fluvial systems. In order to assess the toxicity of BE on zebrafish embryos, several toxicological endpoints were monitored: viability, alterations in development (morphology, biometry and hatching), cardiotoxicity (presence of edema, impaired circulation and heartbeat frequency), and finally larvae locomotion. Two different endpoints were used to evaluate acute phytotoxicity (48 hours): mitochondrial activity for cell energetic status and DNA quantification for lethality.To evaluate subchronic phytotoxicity (168 hours), chlorophyll autofluorescence was also measured. The mitochondrial activity was measured according to with minor modifications . For DNA quantification, samples were sent to the Genomic Service of Pompeu Fabra University for DNA quantification with the PicoGreen® method (QuantiFluor ST Fluorometer and Paradigm Detection Platform, Beckman). Chlorophyll autofluorescence was measured (λexc: 485nm, λem: 635 nm) in a SPECTRAFluor Plus microplate reader (Tecan Group Ltd. Männedorf, Switzerland).
3. Results and discussion
3.1. Toxicity during fish embryo development
In spite of the potential cardiotoxic and neurotoxic effects of cocaine, the main metabolite BE (0.01 to 1000 µg L-1) did not seem to affect the heartbeat rate -indicative of cardiotoxicity- nor the locomotion at day 6pf (behavior) in this zebrafish embryo-larval bioassay. Embryos exposed to BE did not show malformations, and their growth and hatching were comparable to the control group. The low toxicity of BE to zebrafish embryos is in line with the poor toxic activity of BE found in other experimental vertebrates, such as rats .
3.2. Phytotoxicity during development
BE resulted to interfere with the development of gametophytes at so low concentrations as 0.001 µg L-1 affecting both metabolic and genetic processes. BE environmental concentrations have a significant effect on total DNA levels as well as a strong inhibitory effect on mitochondrial activity. The reduction of DNA levels (ca. 20%) could be attributed either to the death of part of the spore population or to a cytostatic effect of BE, causing a delay in spore cell cycle. Alkaloids in general and benzoic acid derivatives in particular, are were studied long ago for their allelopathic effects since all seeds and fruits known for their high alkaloid content are strong germination inhibitors . Evenari classified cocaine as a strong germination inhibitor together with caffeine and codeine among others in dilutions of 1/200. BE has demonstrated the ability to disrupt cell function and probably the cell cycle that may, in the long term, lead to the abortion of spore germination or to abnormalities in the developed gametophyte.
BE could pose an environmental risk for riparian ferns gametophyte recruitment and vascular plant germination and growth. These results agree with the allelophathic role described for alkaloids such as benzoylecgonine and their unspecific interference with plant germination. We conclude that the anthropogenic dispersion of alkaloid allelochemicals may pose a risk for biodiversity and irrigated food production that should be further investigated.
- Mendoza a, López de Alda M, González-Alonso S, Mastroianni N, Barceló D, Valcárcel Y. 2014. Occurrence of drugs of abuse and benzodiazepines in river waters from the Madrid Region (Central Spain). Chemosphere. 95:247–55.
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- Pedriali A, Riva C, Parolini M, Cristoni S, Sheehan D, Binelli A. 2013. A redox proteomic investigation of oxidative stress caused by benzoylecgonine in the freshwater bivalve Dreissena polymorpha. Drug Test. Anal. 5:646–56.
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- Marugán J, Bru D, Pablos C, Catalá M. 2012. Comparative evaluation of acute toxicity by Vibrio fischeri and fern spore based bioassays in the follow-up of toxic chemicals degradation by photocatalysis. J. Hazard. Mater. 213-214:117–122.
- Feito R, Valcárcel Y, Catalá M. 2013. Preliminary data suggest that venlafaxine environmental concentrations could be toxic to plants. Chemosphere. 90:2065–2069.
- Esteban S, Fernandez Rodriguez J, Diaz Lopez G, Nunez M, Valcarcel Y, Catala M. 2013. New microbioassays based on biomarkers are more sensitive to fluvial water micropollution than standard testing methods. Ecotoxicol. Environ. Saf. 93:52–59.
- García-Cambero JP, Catalá M, Valcárcel Y. 2012. River waters induced neurotoxicity in an embryo-larval zebrafish model. Ecotoxicol. Environ. Saf. 84:84–91.
- Morishima HO, Okutomi T, Ishizaki A, Zhang Y, Cooper TB. 2001. The disposition of benzoylecgonine in maternal and fetal rats. Neurotoxicol. Teratol. 23:247–253.
- Evenari M. 1949. Germination inhibitors. Bot. Rev. 15:153–194.
Acknowledgement – The authors thank the program “Projects on Health Research 2011-2012” FIS (PI11/00180)” of Carlos III Health Institute
These results were presented in the 25th European Meeting of SETAC next week (6th May) in a poster (WE293) and a brief speech (poster highlight, 15:55, room 112). / Estos resultados fueron presentados en la reunión Europea de la SETAC en Barcelona (6 de Mayo 2015) mediante un póster y una breve exposición oral (poster highlight 15:55, sala 112).
Download the poster highlight brief presentation
See the communication by clicking in Poster BE SETAC 2015
Download by clicking in Poster BE SETAC 2015
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