Detection of ecotoxicological effects at environmentally relevant concentrations of some antifouling and personal care products Hans Blanck Plant & Environmental Sciences Biology University of Gothenburg Sweden

Natural & experimental ecosystems METALS ANTIFOULANTS METALOIDS HERBICIDES Ecotoxicology PERSONAL CARE PRODUCTS NANOPARTICLES AND MATERIALS ANTIFOULANTS PHARMACEUTICALS Ecotoxicogenomics & bioinformatics Community ecotoxicology Mixture ecotoxicology Natural & experimental ecosystems

Mind the gap!!! Regulatory Ecotoxicology mostly describes the immediate physiological consequences of toxicity in simplified systems Ecological status of the environment is the consequence of the a long-term selection pressures (also from mixtures of toxicants) on complex and variable systems

PICT will respond when the most sensitive of these species are replaced by more tolerant ones

Pollution-Induced Community Tolerance PICT PICT works with natural communities sampled in a gradient of toxicant exposure Community tolerance increases when sensitive species are eliminated by toxicants Community tolerance is quantified in short-term tests for suspected toxicants PICT can detect small community effects and ”identify” the cause of the damage

PERIPHYTON - attached microbial communities on submerged surfaces

Periphyton communities Field studies Establishment during environmental exposure Microcosm studies Establishment during controlled exposure Flow-through system

Area for antifouling PICT studies and metasequencing of marine periphyton

Irgarol / Cynbutryn Photosystem II inhibitor used for fouling protection S-triazine

PHOTOSYSTEM II IN PLANTS AND ALGAE IRGAROL INHIBITS PHOTOSYSTEM II IN PLANTS AND ALGAE Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J., Iwata, S., 2004. Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831-1838.

Field study of irgarol tolerance Irgarol levels stable over the years Irgarol and Cu dominated the pleasure craft market in Sweden from 1994 -2004 Blanck et al 2009 Marine Pollution Bulletin

Irgarol-induced succesion DIFFERENTIAL SENSITIVITY IS A REQUIREMENT 1994 2001 No PICT detected in 1994 PICT readily detected 2001 DIFFERENTIAL SENSITIVITY IS A REQUIREMENT

encoding the irgarol target D1 Community-wide sequencing of the psbA gene encoding the irgarol target D1 The diversity in psbA was much greater than expected. 72 haplotypes were found these translated into 40 different D1-proteins haplotypes came from various taxa The distributions of psbA were very dissimilar between control and irgarol-exposed communities Eriksson, K.M., Clarke, A.K., Franzen, L.G., Kuylenstierna, M., Martinez, K., Blanck, H., 2009. Community-Level Analysis of psbA Gene Sequences and Irgarol Tolerance in Marine Periphyton. Applied and Environmental Microbiology 75, 897-906.

Herbicide binding niche D1 protein Herbicide binding niche PEST region Ser264→Gly Picture from from Erickson et al (1989) The Plant Cell (1) 361-371

What about mixture toxicity with other PSII inhibitors ??? Irgarol Atrazine Diuron Isoproturon Terbacil etc. TRIAZINES, PHENYL UREAS, URACILS ETC

Congeneric mixtures of PSII inhibitors Periphyton photosynthesis Observed April 1998 CA model provides good prediction Arrhenius, A., Gronvall, F., Scholze, M., Backhaus, T., Blanck, H., 2004. Predictability of the mixture toxicity of 12 similarly acting congeneric inhibitors of photosystem II in marine periphyton and epipsammon communities. Aquatic Toxicology 68, 351-367.

Congeneric and non-congeneric mixtures of PSII inhibitors Periphyton community structure Diuron Isoproturon Fluometuron Diuron Isoproturon Fluometuron Irgarol Diuron Terbacil Porsbring, T., Backhaus, T., Johansson, P., Kuylenstierna, M., Blanck, H., 2010. Mixture toxicity from photosystem II inhibitors on microalgal community succession is predictable by Concentration Addition. Environmental Toxicology and Chemistry 29, 2806-2813.

Clotrimazole Very toxic to aquatic organisms OSPAR Commission 2005: ”currently no clear environ-mental risk from clotrimazole” RQ > 2 for low tide marine scenarios OSPAR Commission 2006: listed clotrimazole for priority action

Clotrimazole inhibits 14 α-demethylase in plant-sterol metabolism

Precursors increase and products decrease

Sterol metablism in periphyton affected at environmental concentrations (50 pM) MEC: 100 pM PNEC: 300 pM PEC: 600 pM Porsbring, T., Blanck, H., Tjellstrom, H., Backhaus, T., 2009. Toxicity of the pharmaceutical clotrimazole to marine microalgal communities. Aquatic Toxicology 91, 203-211.

Complicated response pattern: some algae stimulated some inhibited Chlorophyll a Pigment profiles Xanthophyll cycle

Clotrimazole summary

MIXTURES of pharmaceuticals & personal care products

MIXTURES of pharmaceuticals and personal care products Good predictions by both IA and CA Strong hormesis at low concentrations of mixture Effects based on total pigment content Periphyton EC50 for triclosan is 1-2 nM depending on endpoint

Something from ”Nothing”.... Periphyton communities 5-component mixture at the NOECs of the individual components yield a 28.3% inhibition, predicted to be 30% according to IA. Prediction based on previously generated data gave 23 % (IA) and 30% (CA). Backhaus, T., Porsbring, T., Arrhenius, A., Brosche, S., Johansson, P., Blanck, H., 2011. SINGLE-SUBSTANCE AND MIXTURE TOXICITY OF FIVE PHARMACEUTICALS AND PERSONAL CARE PRODUCTS TO MARINE PERIPHYTON COMMUNITIES. Environmental Toxicology and Chemistry 30, 2030-2040.

Antibiotics and periphyton - tetracycline Environmentally relevant tetracyline concentrations (< 1 – 100 microgram/l) in US 0.5 ug tetracycline per litre affected: Antibiotic resistance Abundance and productivity of bacteria Algal and cyanobacterial biomass Nematode abundance At higher doses bacterial productivity recovered, although the food web did not US EPA´s Stream Mesocosm Facility Quinlan EL et al. 2011.Temporal dynamics of periphyton exposed to tetracycline in stream mesocosms. Env Sci Technol. In press

Ecological considerations Ecological Energy Flow: 100 % through primary producer and decomposer compartments Huge diversity and many phyla Huge diversity implies broad set of potential target molecules and species Potential for cascading effects in food webs (e.g. deficiency in nitrogen, sterols, energy, or food preferencies for species ,sizes, shapes, nutrient content etc ) Selected Test Organisms are Examples not Models...

SCENIHR Scientific Committee on Emerging and Newly Identified Health Risks Assessment of theAntibiotic Resistance Effects of Biocides

Triclosan favours antibiotic resistance in periphyton biofilms

Cu Exposure under Field Conditions Coselects for Antibiotic Resistance as Determined by a Novel Cultivation-Independent Bacterial Community Tolerance Assay Berg et al 2010. Env Sci Technol 44: 8724-28

Chemical Hazard and Risk Assessement Present procedures tend to ignore the role of biodiversity and genetic diversity Present procedures tend to ignore the role of chemodiversity and mixture complexity

Emergent comments... Emerging pollutants suggest that present control of chemicals fails Why are we surprised that pharmaceuticals and preservatives are causing effects in the environment WFD is ambitious but its present approach to chemical status is disappointing. We tend to monitor those that we can handle. Monitor ALL biocides, pharmaceuticals, PBT, vPvB, metals, HVCs at selected sites. WFD requires good understanding of environmental mixtures

The NICE project Novel instruments for effect-based assessment of chemical pollution in coastal ecosystems Use present chemical monitoring data to provide predictions of mixture toxicity in Swedish coastal waters Select sites and extend chemical analysis Use biological monitoring tools (e.g. PICT, fish biomarkers) to confirm suspected ecotoxicological effects or detect new ones Explore the sensitivity and specificity of ecotoxicogenomics in selected biota (periphyton,fish, invertebrates)

Thank YOU !!!!!!

A EUROPEAN BIOFILM NETWORK IS BEING ESTABLISHED PICT vision PICT detects ecological effects from toxicants in the environment PICT indicates the toxicant (group) that caused the effect. PICT is used systematically in Europe e.g. under the Water Framework Directive PICT is used together with community ecotoxico-genomics for mechanistic understanding, for higher specificity – and for mutual validation... A EUROPEAN BIOFILM NETWORK IS BEING ESTABLISHED

Irgarol, PICT and the psbA gene Irgarol induced community tolerance in periphyton, but only slowly. No differential sensitivity in 1994 Mutations in PEST region favoured by irgarol selection pressure in diatoms (not cyanobacteria) A tolerance mechanisms for PS-II inhibitors proposed in the 1970´s, was discovered in nature. A mechanistic understanding of irgarol PICT

Antibiotic contaminated sediment increase gene frequencies of resistance genes and genes involved in horizontal gene transfer Kristiansson, E., J. Fick, et al. (2011). "Pyrosequencing of Antibiotic-Contaminated River Sediments Reveals High Levels of Resistance and Gene Transfer Elements." Plos One 6(2).  

Antibiotic resistance # biocides # overlapping resistance mechanims # mobile genetic elements (plasmids) # biocides may help maintaining plasmids for cross-resistance # dissemination of plasmids of cross-resistance in biofilms creates a potential risk of AR development