Alessio Gomiero 1,2, Lara Boatti 2, Alessandro Dagnino 2, Cristina Nasci 1, Fabrizio Perin 1, Aldo Viarengo 2. Thetis S.p.A, 2

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1 Development of new bioassays to assess the quality of coastal and estuarine environments: the potential of the cosmopolitan single cell marine protozoa Euplotes crassus Alessio Gomiero 1,2, Lara Boatti 2, Alessandro Dagnino 2, Cristina Nasci 1, Fabrizio Perin 1, Aldo Viarengo 2 1 Thetis S.p.A, 2 Department of Environmental and Life Sciences (DiSAV) - University of Eastern Piedmont A. Avogadro Venice, 6-8 April 1 La Jolla, 15 th November 1 Thetis, II 6 Panel - 8 April meeting 11

2 Background The contamination of the aquatic environments by pollutants has become a serious problem in the recent years since thousands of organic and inorganic chemicals are continuously discharged in lakes and rivers by atmospheric deposition, runoff and as final effluents of domestic and industrial wastewaters The interest in the toxicity of complex mixtures of pollutants and their biological effects, has increased during the last decades as they enter waterways and may be transported to estuaries and coastal sites potentially causing harm to a large variety of non-target wildlife organisms Pollutant concentration in sediments and interstitial sediment water can be more than 1 - times higher than that present in the overlying water column It is of increasing concern to identify a panel of organisms displaying sensitive responses to environmental perturbations An approach based on the estimation of sub-lethal stress indexes (biomarkers) can provide useful information for identifying those situations requiring a close investigation at an early stage

3 Objectives and approaches The development of valuable bioassays to be exploit in standardized laboratory procedures for evaluating the toxicity of chemical compounds on polluted environmental matrices Identification of sensitive and reliable biological markers of stress for the evaluation of organisms health status Validate bioassay application by performing a pilot biomonitoring program Target species: the protozoa Euplotes crassus, Dujardin 1841 (Ciliophora, Hypotrica) A single cell, free-crawling, interstitial marine ciliate Why protozoa?...

4 Protozoa as bioassay Strenghts Ecological relevance: These microorganisms mediate the flow both of biological substances and energy from one trophic level to the next making them suitable models for predicting the effects of chemicals on aquatic communities. Good biological model: Due to their nature as a eukaryotic cell/organism, protists exhibit a relatively simple organization and a high degree of specialization. Significance of responses: these marine protozoa can be simply cultured in the laboratory under natural-like conditions, so that their biological responses are more reliable.

5 Biomarker approach A biomarker is a xenobiotically, sub-lethal induced variation in cellular or biochemical components, processes or functions, that is measurable in a biological system Being measurable at different levels of biological organization, they provide a correct snapshot of organisms health status anticipating changes at higher levels of biological structure Biomarkers = early warning tools in environmental quality assessment The responses of two typical ecotoxicological high-level endpoint tests like mortality and replication rate were combined with sensitive, sublethal, early-warning biomarkers of stress like: The lysosomal membrane stability: was estimated by the retention time of lysosomal dye Neutral Red The endocytotic rate: was assessed by quantifying cellular intake of fluorescent bio particles Escherichia coli K-12 Strain to provide a clear estimation of organism s health status

6 Experimental design Three environmentally relevant chemicals tested: Cu (essential trace element) Hg (not essential trace element) Benzo(a)pyrene (organic pollutant model) Study of effects of toxicant-exposure: Aliquots of logarithmically growing cells in artifical seawater. on mortality on replication rate on endocytosis rate Estimation of LC5, LC, LC1, NOEC Mortality < 1% Evaluation of biological effects induced by sub-lethal concentrations of toxicants and mixtures of them on lysosomal membrane stability To provide basic knowledge about the linkage Pollutants toxicity Estimation of EC5, EC25, EC1, LOEC, NOEC Ecological relevance

7 Significance of an integrated approach POTENTIAL EFFECT: BIODIVERSITY DECLINE LC - High level Endpoint Risk for biodiversity LC5 - Biological vulnerability LC - LC1 - LC3 - EC5 - EC - EC1 - < 3% survival capability Sub-lethal endpoint Stress Level Enhanced compound-specific comprehension of dose / effects relationship knowledge of changes at higher ecological levels

8 Results chronic tests Mortality rate (%) Mortality rate 1 1,1 µm Hg 5 1 1,1,1 mm Cu ,1 C (5% µm B(a)p Dmso) Replication rate Replication rate (%) nm Hg µm Cu 3 5 nm B(a)p Good linear dose / effect relationship in both mortality and replication rates tests

9 Results acute tests Endocytosis rate OD (%) nm Hg µm Cu nm B(a)p X X X X X X X Control Cu 5µM Cu 1 µm Cu 25 µm Cu 5 µm Cu 75 µm Cu µm Transmitted light Cu 75 µm Study of binary mixtures.. Mix 1 EC 25 Cu + EC 25 Hg Exp EC 5 (?) Mix 2 EC 25 Hg+EC 25 B(a)p Exp EC 5 (?) Mix 3 EC 25 Cu+EC 25 B(a)p Exp EC 5 (?) Additive, synergic or antagonistic effects?... EC x

10 Lysosomal membrane stability NRRT (%) nm Hg µm Cu 3 5 nm B(a)p X X LMS Estimated as time to observe the release of NR in the 5% of scored protozoa... Control t= Control t=3 Good linear responses among lysosomes permeability and toxicants concentrations X X Same binary mixtures tested.. Mix 1 EC 25 Cu + EC 25 Hg Exp EC 5 (?) Mix 2 EC 25 Hg+EC 25 B(a)p Exp EC 5 (?) Mix 3 EC 25 Cu+EC 25 B(a)p Exp EC 5 (?) Cu 25 µm t= Cu 25 µm t=3 7 5 EC x (%) 3 1

11 Conclusions Experimental activity Both endocytosis rates and lysosomal membrane stability were able to detect biological effects of n/µm concentrations of Hg, Cu and B(a)p Results of binary mixtures reported additive and/or synergic toxic effects on protozoa Developed biomarkers were easy to perform and provided rapid and objective results All available evidence indicates that the here presented new bioassay is fast, sensitive and easy to handle Suitability for field biomonitoring programs?...

12 Case study: the Venice Lagoon Represents coastal area with a peculiar ecosystem characterized by an high ecological and economic value Sediments from 4 sites selected according to well known degrees of anthropic pressure were examined S1 Located close to the industrial district of Porto Marghera High levels of environmental contamination S1 S3 FC S2 Located in the southern part of Venice lagoon Site under moderate agriculture run-off S2 S3 Located close to the historic centre of Venice Site affected by household wastewater FC Located close to Lido inlet Low levels of environmental contamination Sediment samples were collected and processed (elutriated) according to standardized US-EPA methods

13 Case study: Results Moderate increments of mortality were recorded only in site S1 ( 7-1 %) Significant effects on chronic high-level endpoint tests Reduction of replication rate Average higher alterations were recorded by sub lethal biomarkers of stress Replication rate (%) Replication rate ** Anova * = p<,5 ** = p<,1 OD (%) Endocytosis rate * ** Anova * = p<,5 ** = p<,1 NRRT (%) Lysosomal membrane stability * ** Anova * = p<,5 ** = p<, Lab Control FC S1 S2 S3 Lab Control FC S1 S2 S3 Lab Control FC S1 S2 S3

14 Main conclusions The approach based on a battery of biomarkers in conjunction with high-level endpoint tests may help follow the pollutant-induced stress syndrome in the organisms, from early sub lethal effects to more deleterious effects (i.e. death) From biological vulnerability To risk for biodiversity The combination of all tests were able to discriminate a gradient of stress within the sampling sites The presented bioassay based on protozoa appears suitable for environmental biomonitoring purposes This leads to consider the future application of E. Crassus assay for the screening of sediments toxicity of coastal sites

15 La Jolla, 15 th November 1 Thetis, II 6 Panel - 8 April meeting 11