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

Communityecotoxicology

Mixture ecotoxicologyNatural &

experimental ecosystems

Ecotoxicogenomics &bioinformatics

Ecotoxicology

METALS METALOIDS ANTIFOULANTSHERBICIDES

ANTIFOULANTS PHARMACEUTICALS

PERSONAL CARE PRODUCTS NANOPARTICLES AND MATERIALS

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

Microcosm studiesEstablishment during controlled exposure

Flow-through system

Field studiesEstablishment during

environmental exposure

Periphyton communities

Area for antifouling PICT studies and metasequencing of marine periphyton

Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J., Iwata, S., 2004. Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831-1838.

IRGAROL INHIBITSPHOTOSYSTEM II IN PLANTS AND ALGAE

Field study of irgarol tolerance

Blanck et al 2009 Marine Pollution Bulletin

• Irgarol levels stable over the years• Irgarol and Cu dominated the pleasure craft market in Sweden from 1994 -2004

Irgarol-induced succesionIrgarol-induced succesion

1994 2001

Pre-exposure concentration (nM)

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No PICT detected in 1994 PICT readily detected 2001

DIFFERENTIAL SENSITIVITY IS A REQUIREMENT

The distributions of psbA were very dissimilar between control and irgarol-exposed communities

The diversity in psbA was much greaterthan expected. • 72 haplotypes were found• these translated into 40 different D1-proteins • haplotypes came from various taxa

Community-wide sequencing of the psbA gene Community-wide sequencing of the psbA gene encoding the irgarol target D1encoding the irgarol target D1

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.

PEST region

Herbicide binding niche

Ser264→Gly

Picture from from Erickson et al (1989) The Plant Cell (1) 361-371

D1 protein

What about mixture toxicity with other PSII inhibitors ???

• Irgarol• Atrazine• Diuron• Isoproturon• Terbacil• etc.

TRIAZINES, PHENYL UREAS, URACILS ETC

Congeneric mixtures of PSII inhibitorsPeriphyton photosynthesis

Observed April 1998

CA model provides good prediction

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

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.

IrgarolDiuronTerbacil

DiuronIsoproturonFluometuron

DiuronIsoproturonFluometuron

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.

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

Pigment profiles

Xanthophyll cycle

Chlorophyll a

Clotrimazole summary

MIXTURES of pharmaceuticals & personal care products

MIXTURES ofpharmaceuticals 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...

SCENIHRScientific Committee on Emerging and Newly Identified Health Risks

Assessment of theAntibiotic Resistance Effects of Biocides

Triclosan favours antibiotic resistance in periphyton biofilms

Dosing Period (d)

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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 projectNovel 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 !!!!!!

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

• 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