1. 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

2. 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

3. 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

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

5. 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

6. PERIPHYTON
- attached microbial communities on submerged surfaces

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

8. Area for antifouling PICT studies and metasequencing of marine periphyton

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

10. 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., Architecture of the photosynthetic oxygen-evolving center. Science 303,

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

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

13. 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., Community-Level Analysis of psbA Gene Sequences
and Irgarol Tolerance in Marine Periphyton. Applied and Environmental Microbiology 75,

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

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

16. 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., Predictability of the mixture toxicity of 12 similarly acting congeneric inhibitors of photosystem II in marine periphyton and epipsammon communities. Aquatic Toxicology 68,

17. 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., Mixture toxicity from photosystem II inhibitors on microalgal community succession is predictable by Concentration Addition. Environmental Toxicology and Chemistry 29,

18. 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

19. Clotrimazole inhibits 14 α-demethylase in plant-sterol metabolism

20. Precursors increase and products decrease

21. 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., Toxicity of the pharmaceutical clotrimazole to marine microalgal communities. Aquatic Toxicology 91,

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

23. Clotrimazole summary

24. MIXTURES of pharmaceuticals & personal care products

25. 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

26. 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., SINGLE-SUBSTANCE AND MIXTURE TOXICITY OF FIVE PHARMACEUTICALS AND PERSONAL CARE PRODUCTS TO MARINE PERIPHYTON COMMUNITIES. Environmental Toxicology and Chemistry 30,

27. 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 Temporal dynamics of periphyton exposed to tetracycline in stream mesocosms. Env Sci Technol. In press

28. 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...

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

30. Triclosan favours antibiotic resistance in periphyton biofilms

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

32. 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

33. 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

34. 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)

35. Thank YOU !!!!!!

36. 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

37. 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

38. 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).

40. 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