1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1212570_28446780.jpg logo_mu_cerne.gif trojlogo.gif Introduction to Ecotoxicology Ludek Blaha + ecotox colleagues 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Global anthropogenic threats ? A safe operating space for humanity & the nine planetary boundaries Rockstrom et al. 2009 (Ecology and Society 14(2): 32; Nature 461, 472-475) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Environmental pollution Any examples ??? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Contamination of water - chemicals ? Degradable „nontoxic“ organic material + nutrients / fertilizers (N/P) „Other“ chemicals 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif CHEMICAL ENTERS THE ENVIRONMENT Bioavailable fraction “EXPOSURE” acute chronic Toxicokinetics biotransformation bioactivation excretion / sequestration Target site “EFFECT” LEVELS, FATE, PROCESSES CHEMICAL ENTERS THE ORGANISM biomonitoring 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Assessment of chemical hazards …to… Humans Other organisms (TOXICOLOGY) (ECOtoxicology) https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcTE7qm7tLqavKV1VUwQpKDwC6XUksMmHXLKvoBzqHIKaqjv q_mACQ https://encrypted-tbn2.google.com/images?q=tbn:ANd9GcRorg12adBHNbxSdT8AiWMQmO7b7vltAdBZohD_pqfDehNn re7fkg https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcQvHwZACb-TU516T7ZMDFe-QBU598rfeCvHryOWorunBunH oi4zpA 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Chemicals in the environment Do you believe that chemicals in products sold to consumers have been proven safe? Think again Most chemicals in modern use have simply not been tested for their impacts on human, even very basic effects. … what about the effects in nature, then ? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Chemicals in the environment •Rats exposed in the womb to a single low dose of a widespread brominated flame retardant become hyperactive and have decreased sperm counts… • •Experiments with dioxin and similar compounds provide support for the assumption that cancer risks mediated by the aryl hydrocarbon receptor are additive. Previously untested for cancer, this assumption underpins a standard way of estimating exposure risks to these compounds. The results reinforce the need to focus health standards on mixtures rather than single compounds. • •At exposure levels within the range experienced by the general public, the phthalate DBP reduces expression of genes necessary for testosterone synthesis in fetal rats… • •Eutrophication of frog ponds is linked to epidemics of frog deformities, because it creates conditions that lead to higher rates of parasitic infections of tadpoles. The parasitic infections in turn disrupt normal development of the tadpoles' limb buds during metamorphosis. • OSF%20Hardback%20Large 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Major anthropogenic threats – example: waters Impacts 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Loss of biodiversity – Major impacts 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Changes in biodiversity 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Changes in biodiversity NATURE (2012) 482: 20 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Loss of biodiversity Major impacts •Impairment of ecosystem services –Unbalanced water cycles •Water scarcity •Draughts/floods –Impaired water quality •Drinking waters •Bathing waters •Toxicants in food chain –Shrinking of food supplies •Direct à lowering fish amounts •Indirect à crop yield 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Impacts on fish à decreased crop yields C:\Documents and Settings\Ludek\Dokumenty\katedra\vyuka\ObecEkotox\Nature-Ecosystem\nature03962-f1.jpg NATURE (2005) 437: 880 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Impacts on biota à global effects Mixing oceans à cooling the atmosphere [Nature 447, p.522, May 31, 2007] Marine life supplies up to 50% of the mechanical energy required worldwide to mix waters from the surface to deeper cool layers [Dewar, Marine Res 64:541 (2006)] [Katija a Dabiri, Nature 460:624 (2009)] 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Ecotoxicology - from molecules to ecosystems … and backwards 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1962 © Patuxent Wildlife Refuge, MA, USA http://www2.ucsc.edu/scpbrg/ 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Bitman et al. Science 1970, 168(3931): 594 Biochemistry bird carbonate dehydratase In vivo: shell thinning In situ: bioaccumulation -> bird population decline 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Aim: to maintain the natural structure and function of ecosystems • •Definitions: §ecotoxicology is concerned with the toxic effects of chemical and physical agents on living organisms, especially on populations and communities within defined ecosystems; it includes the transfer pathways and their interactions with the environment §science of contaminants in the biosphere and their effect on constituents of the biosphere, including humans’ (Newman & Unger, 2002) §science that provides critical information on effects of toxic compounds on living organisms which SERVE various practical aims (environmental protection) ECOTOXICOLOGY by definition 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Ecotoxic effects Escher, B. I., Behra, R., Eggen, R. I. L., Fent, K. (1997), "Molecular mechanisms in ecotoxicology: an interplay between environmental chemistry and biology", Chimia, 51, 915-921. 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1) From molecules to individuals http://www.epa.gov/ncer/rfa/2010/comptox_001.jpg MECHANISMS OF TOXICITY 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 2) From molecules to individuals - AOPs ADVERSE OUTCOME PATHWAYS Models for ecotoxicology and risk assessment. Toxicokinetics are "what the organism does with the chemical" and toxicodynamics are "what the chemical does to the organism". 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Ethinylestradiol Binds to ESTROGEN RECEPTOR https://www-pls.llnl.gov/data/assets/images/science_and_technology/life_sciences/cancer_diet/kulp1. jpg Target genes - Proliferation/Apoptosis (sexual organs) - Synthesis of egg yolk (fish, amphibia) Effects - Females: reproduction regulation - Males: feminization (+ e.g. cancer promotion, development, immunomodulation) AOP Example: ethinylestradiol 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Kidd, K.A. et al. 2007. Collapse of a fish population following exposure to a synthetic estrogen. Proceedings of the National Academy of Sciences 104(21):8897-8901 Controls +Ethinylestradiol 5 ng/L (!) 7 years 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Molecular §Nonspecific effects §Hydrophobic interactions with phospholipid membranes (baseline = narcotic toxicity) §Direct reactivity: electrophilic compounds à nucleophilic organism (e.g. oxidation of PROTEINS, lipids (membranes), DNA …) §Specific effects §Activation of ER, AR and other „nuclear receptors“ §Inhibition of enzymes (e.g. CN- inhibits hemes in mitochondria/hemoglobin, insecticides …) § Effects at different levels https://encrypted-tbn2.google.com/images?q=tbn:ANd9GcSG1cHajHLtmVDjpZ78t4F7TH0Se4qLRsuMTInEY17Eupfv mEa4LQ 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Cellular §Effects on structure §Effects on metabolism (maintenance) §Effects on regulation àChanges in functions (e.g. Ethinylestradiol) àRepair, survival, growth àDeath (apoptosis or necrosis) àProliferation àDifferentiation Effects at different levels http://www.helmholtz-muenchen.de/uploads/pics/stem-cell-fate-options_09.gif 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Metabolism Control, Interactions with environment Defence against pathogens predators … Defence against toxicants Energy hv food Losses heat faeces Life (maintenance) Growth to sexual maturity Reproduction Chemical stress 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Metabolism Control, Interactions with environment Defence against pathogens predators … Defence against toxicants Energy hv food Losses heat faeces Life (maintenance) Growth to sexual maturity Reproduction Chemical stress Chemical stress à energy re-allocation à „insufficient“ resourses elsewhere 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Metabolism Control, Interactions with environment Defence against pathogens predators … Defence against toxicants Energy hv food Losses heat faeces Life (maintenance) Growth to sexual maturity Reproduction Chemical stress Chemical stress + ... another stress (food scarcity) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Organism §Effects on structure §Effects on metabolism (maintenance) §Effects on regulation § àChanges in functions (e.g. Ethinylestradiol) àRepair, survival, growth àDeath àProliferation = Reproduction Effects at different levels 3 key apical endpoints (reflected e.g. in regulations) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Population –(… all the organisms that both belong to the same group or species (i.e. can sexually reproduce) and live in the same time within the same geographical area) §Effects on structure §elderly vs. young, males vs. females §Effects on maintenance & growth §Natality, mortality, reproduction fitness § Effects at different levels https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcRy8TZLY8w9X0x--nqSFW4CqPPTUX3RdYucJtb5pGmKOWt- n2r_ 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Community & Ecosystem –(… a group of interacting living organisms sharing a populated environment) §Effects on structure §Loss of species, loss of biodiversity §Effects on functioning §(including „ecosystem functions“) § § § Effects at different levels http://www.combat-fishing.com/LowestReachesOZStreamSFW.JPG 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif (Eco)toxicology – science of „doses“ •‘What is there which is not a poison? • „Cause-effect paradigm“ •All things are poison and nothing without poison. •Solely the dose determines that a thing is not a poison. • 2 Paracelsus (1493 - 1541) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif To identify (or predict) safe vs hazardous levels Ecotoxicology – ultimate goal ? https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcSWXP3_J8AL3Ztuuywtr6xnr5JbJCEbihf-ufB3aAuq_jnS VovH http://www.blog.joelx.com/wp-content/uploads/2008/02/passed-out-drunk-toast.gif 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif RETROSPECTIVE PROSPECTIVE Bioassessment Field assessment Monitoring Bioassessment Field assessment Monitoring Lab studies Lab studies Simulated small ecosystems DISASTERS PREDICTIONS for future Time: NOW ! Ecotoxicology: problems and approaches Most common in practice 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1 Bioassays - single / multiple species - acute / chronic effects - standardized (practical) vs. experimental (research) Simulation of the ecosystem - major trophic levels - producers - consumers - decomposers figure4 Eisenia_fetida scenedesmus_quadricauda_upr Testing ecotoxicity – basics 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif auto0 AcuteToxScheme_RM HaF-adult arenic~4 Daphnia Dania pict Cu addition Effect concentrations expressed in total/dissolved Cu ??? Safe concentrations ??? Ecotoxicology methods 1. - standardized assays 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 50 100 LC50 [concentration] in mg/L or % effluent Threshold: No Observed Effect Concentration (NOEC) Laboratory ecotoxicology – data and results 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Expensive & time consuming (e.g. Pesticide testing) Variable results (natural variability …) Higher ecological relevancy 1 1 figure4 Ecotoxicology – methods 2. Micro & Mesocosms 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif - complex issue (geology, climate, chemistry, biology ..) Ecotoxicology mixes with Ecology - comparing „contaminated“ with „control“ sites figure4 figure4 figure4 figure4 Ecotoxicology – methods 3. Field assessment / biomonitoring 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Notes on practical testing •Testing chemicals –Traditional / bioassays developed to assess individual chemicals –Advantages: Standardized approaches –Disadvantage: Limited ecological relevance •often acute tests only •„too standardized…“ (? Less representative ?) •does not assess/consider bioavailability •no consideration of mixture effects •no consideration of specific modes of action •no consideration of ecological situation – •Example: Acute (96h) fish toxicity assay with ethanol –No deaths (but fish are passive – slow swimming) à OK ? –Real life: easy prey à population decline • 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Notes on practical testing •Testing toxicity of natural contaminated matrices – –Rather new in ecotoxicology – many open challenges •Whole effluent toxicity testing (WET) •Contact soil toxicity assays – –More complex and more complicated •„cause-effects“ often not clear –Natural variability in matrices –Algal tests - nutrients (Nitrogen, Phosporus) >> Toxic compounds – 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Ecotoxicology in current practice •Most legislations on chemicals) (e.g. REACH, Pharmaceuticals, Pesticides) –have very simple (basic) requirements •EC50 from acute toxicity •Of 3 basic assays –Algae –Daphnia –Fish – –Ecotox database: –www.epa.gov/ecotox 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Ecotoxicology in current practice •How to extrapolate 3 (or few more) EC50 values to get legally binding safe concentration, which is protecting virtually all organisms? • • PNEC • (Predicted No Effect Concentration) • “value recommended by scientists” • • • EQS • (Environmental Quality Standard) • “value that occurs in legislation” • ? ? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Atmospheric Deposition Erosion & Runoff Untreated discharges Predicted Environmental Concentration (PEC) bosmina FISH Laboratory (and field) studies Ecotoxicity tests sample56 lab2 faCTORY1 WWTP j0173962 effective concentrations (PNEC) Exposure (resulting from load) Effects (what exposures cause effects ?) Cause – effect à Risk assessment 2 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Extrapolation approaches Data Assessment factor L(E)C50 short-term toxicity tests NOEC for 1 long-term toxicity test NOEC for additional long-term toxicity tests of 2 trophic levels NOEC for additional long-term toxicity tests of 3 species of 3 trophic levels 1000 100 50 10 Ecotoxicological data Assessment / Extrapolation factors PNEC 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Extrapolation approaches Data Assessment factor L(E)C50 short-term toxicity tests NOEC for 1 long-term toxicity test NOEC for additional long-term toxicity tests of 2 trophic levels NOEC for additional long-term toxicity tests of 3 species of 3 trophic levels 1000 100 50 10 Protection level: 95% Ecotoxicological data Species sensitivity distribution (SSD) HC5 = 95% protection level [C] Assessment / Extrapolation factors PNEC 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Environmental quality standards / criteria Risk assessment & management ‘Hazard’ identification Data compilation Exposure assessment PEC Effect assessment PNEC Risk characterisation HQ = PEC/PNEC < 1 > 1 Hazard quotient HQ Risk management 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Results of ecotoxicology WHAT IS IT GOOD FOR ? SOLVING PRACTICAL PROBLEMS 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif http://www.scotland.gov.uk/Resource/Img/237458/0069907.jpg EQS in reality – example EU Water Framework Directive 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif List of priority compounds EU WFD (selection/examples) Most recent (2015) 44 priority compounds (table here) + additional “watch list” à see further 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=uriserv:OJ.L_.2015.078.01.0040.01.ENG 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Another example where ecotoxicology results are used European strategy how to deal with chemicals 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif EU and chemicals EU%2520Flag •± 40 Directives or Regulations concerning the evaluation and management of the dangers/risks associated with chemical substances –Regulation EEC 793/93 –Existing substances –Dir. 67/548/EEC – New substances –Dir. 98/8/EC – Biocides / Plant Protection Products –Further Directives – E.R.A. of new pharmaceuticals 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Existing substances –> 95,000,000 known chemicals (…and counting http://www.cas.org/) –100,000 substances in EINECS (i.e. commercial use) –2747 HPVCs (High Production Volume Chemicals) •14% minimum data-set (base-set) •65% less than base-set •21% no toxicity data –Various priority lists •Aquatic hazard (EU Water framework directive) •Endocrine disruptors •…. EU and chemicals 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •REACH Registration, Evaluation and Authorisation •of Chemicals –27-2-2001: White Paper on the Strategy for Future Chemicals Policy –23-10-2003: Commission’s proposal REACH –December 2008: Pre-registration mandatory (all chemicals in EU must be registered at ECHA European Chemicals Agency (http://echa.europa.eu) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif REACH legislation in EU Registration, Evaluation and Authorisation and Restriction of Chemicals 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif REACH EU%2520Flag http://www.sigmaaldrich.com/content/dam/sigma-aldrich/safc/safc-reach-timeline.jpg 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif REACH: aims & timing EU%2520Flag •Major goals –Protection of man and the environment –Increase competiveness of EU chemical industry –Increase transparency –Avoid fragmentation of market –Integration with international policies –Reduction use of test animals • •Approach –Industry is responsible – provides data • •30000 existing substances •0-3 year (2010): all HPVC and CMR substances (~ 3000) •4-6 year (2013): all 100-1000 t/y substances •7-11 year (2018´): all 10-100 and 1-10 t/y substances 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif REACH: data type? EU%2520Flag •Physico-chemical properties, e.g.: –Vapour pressure, boiling point, Kow,… • •Human toxicology, e.g.: –Acute and chronic toxicity, skin irritation, carcinogenity,… • •Environment/ Ecotoxicological information, e.g.: –Acute and/or chronic toxicity for aquatic organisms, biodegradation, … • • 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif REACH: testing EU%2520Flag 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif REACH: implications EU%2520Flag •Total: 2,8 to 5,6 billion € • •Industry pays •Test costs (50-60% of total cost): •86% for HH tests •14% for environment tests •0% for analyses •Manpower and expertise? •Tests •Risk assessments •Evaluations – •Financial and time pressure: danger for ‘hazard-based’ instead of ‘risk-based’ approach 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Risks of chemicals: a balancing act …. between perception, uncertainties, science and pragmatism? Final considerations 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif janus • – dna Wetlands janus Cellular and molecular effects Population/community effects Effects of chemicals 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif • – Local Global Value Other Technologies Other Laws and Regulations Other Social Needs janus Other Sciences Risks vs. Benefits 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Do we still need ecotoxicologists? •All scientific questions answered ..., and •We have standard tools and good legislations ... • 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Problems of ecotoxicology – pharmaceuticals •Unexpected effects at “non-target” organisms • - nephrotoxicity at vultures • - relevant also in EU (ESP, EL,CY) • • • • • • • • • http://img2.allvoices.com/thumbs/image/609/480/103574038-diclofenac-drug.jpg http://www.painstopanswers.com/images/diclofenac.jpg 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Ivermectin – antiparasitics for large herds nUsed 2-times per season per sheep/cow nKills 100% parasites in sheep nReleased in dung - kills 80-90% larvae of dung flies nHigh concentrations in dung (released 2 days post application) nPersistent in the soil (half-life 30 days) nCan be washed into adjacent streams (highly toxic to water insects) https://encrypted-tbn1.google.com/images?q=tbn:ANd9GcQDHBMH2T3nCNNs0D0dL3i9DAZFsRcqXDrax6t3C410Rx3E OLYBWg http://www.aphotofauna.com/images/flies/fly_scathophagidae_scathophaga_stercoraria_yellow_dung_15-0 4-06.jpg Moxidectin – small animals: spot-on https://encrypted-tbn0.google.com/images?q=tbn:ANd9GcQYuav5FXXhPNb_O-2Bpl0rfQjaDBUqZl867IJ47-6yMdCb b36UkA ? Avermectin antiparasitics Problems of ecotoxicology – pharmaceuticals 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif ECONOMIC importance of BATS ... http://www.sciencemag.org/content/332/6025/41/F1.large.jpg http://www.sciencemag.org/content/332/6025/41.figures-only 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif j0173962 Greenpeace International faCTORY1 EU%2520Flag Scientists Society is a balancing act … 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Closing remarks •Ecotoxicology is exciting science! •Interface: science and society •Many opportunities •Sometimes hard work –10% inspiration and 90% „perspiration“ • •Be creative: move frontiers •Keep the purpose in mind •Be critical: do not accept perceptions as facts •Speak up: you have something to say! • unclesam 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Introduction to ecotoxicology Ludek Blaha blaha@recetox.muni.cz http://www.recetox.cz