Process and mechanistic concepts in toxicity testing. Ronny Blust Department of Biology University of Antwerp

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1 Process and mechanistic concepts in toxicity testing Ronny Blust Department of Biology University of Antwerp INFLAME Workshop, Birmingham January 2012

2 The law of the minimum (Liebig, 1840) The functioning of humans and ecosystems depends on a multitude of processes The structure and organisation determine the potential of a system The law of the minimum determines the capacity of the system

3 The law of tolerance (Shelford, 1913) The success of an organism, population or community depends on a complex of conditions Both too much or too little of one or the other can be limiting Every condition approaching or exceeding the boarder of tolerance is limiting Nutrient-Toxicant

4 Toxic substances in the environment Tens of thousands of substances introduced in the environment by human activities Many of these substances potentially dangerous for plants, animals and man In many cases neither the exposure concentrations or effects are well documented

5 Toxic substances in the environment Examples of important groups of substances - Heavy metals - Polyaromatic hydrocarbons - Halogenated hydrocarbons - Solvents and surface active agents - Insecticides, herbicides, fungicides - Pharmaca- and cosmetics - Radioactive materials

6 The European REACH programme REACH stands for Registration, Evaluation and Authorisation of Chemicals For chemical substances with a production of more than 1 ton per year manufacturers have to submit a registration file This file has to provide detailed information concerning exposure risk and effects for man and environment Substances with a high risk have to be phased out and replaced by safer alternatives.

7 The European Water Framework Directive Regulatory framework to improve the quality of surface, ground and coastal waters within the European Union Integrated approach taking into account the emission and imission site of the problem Compilation of lists of priority substances and scientific underpinning of environmental and human health quality criteria Rigorous characterisation and monitoring of waste streams and environmental compartments to achieve pre-set goals

8 The process of environmental risk assessment

9 In the human environment there are many different receptors Large differences in structural and functional organization Large differences in sensitivity to toxicants related to: - Exposure and elimination pathways - Instrinsic sensitivity of targets - Internal processing and repair

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11 Status The physiological condition-impairment curve Fatal effects Health Disturbance Disease Death Permanent impairment Limit of permanent repair Limit of compensatory capacity Limit of normal adjustment Homeostasis Compensation Breakdown Failure Physiological condition

12 Endocrine disturbance as an example of too little too late Imposex and the disappearance of the purple snail Nucella lapillus Imposex is the partial development of male reproductive organs in female individuals (or vice-versa)

13 Endocrine disruption in relation to male fertility Sperm concentration in semen of Danish males Sperm characteristics in prenatally PCB exposed Taiwanese boys

14 Statement 1 Establishing empirical or preferably functional linkages between exposure and effects across time scales remains a challenge. Within this context determining toxicity thresholds or other relevant respones in an as early stage as possible is crucial. If the toxicity thresholds are not properly defined than the estimation of risk quotients becomes meaningless and misleading.

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16 Water-Air Bioaccumulation Food-Soil Compartmentalisation Toxicity Exposure Concentration

17 Bio-Active Pool Bio-Inactive Pool k 01 BAM k 21 BIM k 10 k k 20 Toxicity 0 BIM-BAM Microcontaminant Accumulation Toxicity Model

18 Effect level Exposure toxicity relationship EC50 NOEC 0 LOEC Internal or external concentration

19 Response Microcontaminant Bioaccumulation & Toxicity Processes Water Food-Sediment Bio-Active Pool Bio-Inactive Pool Elimination Time Toxicity Threshold Bio-Active Pool

20 Microcontaminantl Accumulation Toxicity 2 Pool Model k 21 k 10 BAM BIM k 01 k 12 k 20 Exposure=1 µg.l -1 Total Body BioActive BioInActive Toxicity Threshold k(0,1)=0.1 - k(1,0)=0.1 - k(2,1)=0.1 - k(1,2)=0.1

21 Microcontaminantl Accumulation Toxicity 2 Pool Model k 21 k 10 BAM BIM k 01 k 12 k 20 Metal Exposure=1 µg.l -1 Total Body BioActive BioInActive Toxicity Threshold k(0,1)=0.1 - k(1,0)=0.1 - k(2,1)=0.1 - k(1,2)=0.01

22 Microcontaminantl Accumulation Toxicity 2 Pool Model k 21 k 10 BAM BIM k 20 k 01 k 12 Exposure=1 µg.l -1 Total Body BioActive BioInActive 75 days Toxicity Threshold k(0,1)=0.1 - k(1,0)=0.1 - k(2,1)=0.1 - k(1,2)=1

23 Macro and microscopic aspects of metal uptake

24 Fluorescence Fluorescence Intracellular zinc response of FluoZin-3 loaded fish hepatocytes Time (sec) Time (sec)

25 Number of species Toxicant species sensitivity distributions P=0.50 P=0.05 No effect exposure concentration No effect tissue concentration

26 Chronic Toxicity Relating acute to chronic toxicity Slope gives acute to chronic ratio Acute Toxicity

27 In vivo toxicity Relating in vitro to in vivo toxicity Slope gives in vitro to in vivo ratio In vitro toxicity

28 Statement 2 The relationship between exposure, uptake and toxicity is highly dynamic. Large differences in management strategies and intrinsic sensitivities among species and tissues within a given species exist. This results in uncertainty concerning the most essential and sensitive targets and extrapolation factors from in vitro to in vivo and from short term to long term.

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31 Non-mechanistic versus mechanistic approaches Microcontaminant Cell or organism Mechanistic responses need to be expressed on a physiologically relevant scale in terms of dose and effects

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34 Tools to interrogate cells, tissues & organisms Treated Cells RNA RNA nucleotide Template strand RNA polymerase Direction of transcription Toxicogenomics

35 Toxicoproteomics

36 ToxicoMetabolomics

37 ToxicoCellomics

38 Biomarker based In vitro assays and biosensors Endogenous Gene Promoter Structural Gene inducing agent transcription

39 A case study with TBBPA and zebrafish

40 Liver gene expression profiles of TBBPA exposed zebrafish

41 Functional classification of gene expression TBBPA exposed zebrafish

42 Major differentially expressed proteins in liver of TBBPA exposed zebrafish Comparison of gene and protein level responses is partially confirmatory but also complementary.

43 Statement 3 The so called omic approaches provide in depth information of the mode of action of microcontaminants. At the same time they have also revealed the molecular complexity and the multiple targets and pathways involved. Nonetheless this allows to classify compounds on a biological mode of action basis. However, establishing the modes that are actually expressed in real life situations and can form the basis for risk assessment criteria remains a challenge.