ECOTOXICOLOGY: Toxicity testing for environmental effects and their endpoints Bangkok Ecotoxicology III p.1 Ecological risk assessment: exposure and effects assessment objective of ecological risk assessment: supply information to protect our environment for adverse effects of chemicals chemical environmental fate effects at ecosystem level concentration in a certain compartment PEC no-effect concentration in a compartment PNEC risk Bangkok Ecotoxicology III p.2 Ecotoxicity Topics Test systems Modifying factors Sensitivity of organisms Bangkok Ecotoxicology III p.3 Effects of chemicals in the environment direct effects: indirect effects: • mortality • inhibition of growth • inhibition of reproduction • food webs • avoidance • disturbance of processes single species ecosystem level Bangkok Ecotoxicology III p.4 Test systems used in ecological risk assessment single species tests: acute effects (mortality) single species tests: semi-chronic, sublethal effects single species test with effect at population dynamics microcosms ecosystems Bangkok Ecotoxicology III p.5 Acute test with Daphnia and fish duration: effects: test systems: 2-4 days survival static, daily renewal Bangkok Ecotoxicology III p.6 Semi-chronic tests with algae, Daphnia and fish duration: 4, 15, 32 days effects: survival, growth, reproduction, outcome of eggs, development, behaviour test systems: static, daily renewal or flow through Example: early life stage test with fish (ELS test) Bangkok Ecotoxicology III p.7 Question What endpoints you consider most relevant for ecotoxicological risk assessment? What endpoints would you suggest for top predators in ecosystems? What is a significant difference between human and ecotoxicological risk assessment? Bangkok Ecotoxicology III p.8 Test systems system: in vivo (whole organisms) or in vitro (cells, protein) dosing systems: static daily renewal flow through system (continuous renewal) Bangkok Ecotoxicology III p.9 Log concentration in water (mg/L) Log concentration in fish (mg/kg) Test system: concentration of chemicals Fish Flow-through problems: Renewal Static Water decrease of concentration during test making “good” test solutions for: low soluble or low boiling test chemicals Flow-through Renewal Static Time (h) from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.10 Modifying factors Toxicity (effect concentration) may depend on: exposure duration environmental parameters: pH hardness oxygen concentration presence of suspended particles Bangkok Ecotoxicology III p.11 LC50 Influence of exposure time on effect concentration A Reaching steady state will depend on: LC50 - kinetic behavior - other phenomena B Time (d) from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.12 Influence of pH on effect concentration Relative bioavailability (%) Bioavailability of heavy metals in soil 1200 1000 Lead 800 600 400 200 Cadmium 3.5 4.0 4.5 5.0 5.5 6.0 6.5 pH soil from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.13 Influence of hardness on LC50 in rainbow trout 100 48-h LC50 (m of m g etal/L) 50 Nickel 20 10 Zinc Lead 0.5 Cadmium 0.2 Copper 0.1 0.05 0.02 0.01 10 20 50 100 200 500 Total hardness (mg/L CaCO3) from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.14 Speciation of aluminium as a function of pH Speciation of heavy metals depends on: Al fraction 1.0 pH hardness salinity 0.8 + Al(OH) Al 3+ - Al(OH) 0.6 0.4 + AlSO 0.2 0.0 AlOH 2+ 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Water pH from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.15 LC50 based on concentration in: - interstitial water - sediment 1.0 30 0.9 0.8 0.7 Sediment 25 20 0.6 0.5 15 0.4 0.3 0.2 Water 10 48-h LC 50 Based on sediment kepone concentration (mg/kg) 48-h LC50 of kepone to the midge larve 48-h LC 50 Based on sediment interstitial water kepone concentration (mg/L) Influence of organic carbon content on bioavailability 0.1 0 0.5 1.0 1.5 2.0 Sediment organic carbon content (%) from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.16 Differences in sensitivity - Organisms have different sensitivities to a chemical substance - Variation in sensitivity strongly depends on mode of action LC50 (mg/L) 1,3,5trimethylbenzene chlorothion Daphnia Guppy Algae Pond snail Midge larvae 4.4 5.8 2.7 0.0062 0.192 1.3 0.031 data from Verhaar, 1991 and Legierse, 1997 Bangkok Ecotoxicology III p.17 1,3,5-trimethylbenzene versus chlorothion 1,3,5-trimethylbenzene: non specific mode of action in acute tests chlorothion: interaction with a receptor of an active metabolite OCH3 OCH3 O P=S OCH3 oxidation O (NADPH) P=O OCH3 Cl Cl NO2 NO2 de-alkylation GSH specific mode of action: inhibition of Acetylcholin-esterase by oxon metabolite OCH3 O P=S OH Cl NO2 Bangkok Ecotoxicology III p.18 Reasons for differences in sensitivity Environmental factors (external) pH, hardness, salinity, suspended particles experimental artifacts ? Internal factors uptake / accumulation kinetics biotransformation capacity (in case of bio-activation) presence of target (example: neurotoxicants, herbicides) sensitivity of the target or receptor Bangkok Ecotoxicology III p.19 Setting safe concentrations at the ecosystem level Impossible to test many different species number of different protozoa: 30.000 number of different crustaceans: 25.000 In practice: data for 3-6 test organisms How to set safe concentrations: - use of application factors - use of extrapolation techniques Bangkok Ecotoxicology III p.20 Extrapolation techniques Assumption: Probability certain distribution for differences in sensitivity 0.50 0.40 0.30 0.20 - triangular - normal - logistic 0.10 -4 -3 -2 -1 Number of standard deviations Kooijman, van Straalen, Wagner and Lokke, Aldenberg and Slob from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.21 HC5: hazerdous concentration for % of the species chosen as safe concentration at ecosystem level input: NOECs for at least species Number of species Extrapolation technique: logistic distribution to calculate HC5 p% Log NOEC Uncertainty Log HCp Mean Log NOEC Extrapolation from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.22 Extrapolation techniques - Assumption of certain distribution which is not valid for several chemicals (example: specific mode of action) - Certain organisms are key species Number of species Strengths and weaknesses: Log NOEC - Takes into account that differences in sensitivity depend on the chemical (mode of action) from van Leeuwen and Hermens, 1995: Risk Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.23 ECOTOXICOLOGY: Estimation methods in ecotoxicology Bangkok Ecotoxicology III p.24 Activity of a chemical depends on: Cl hydrophobicity / solubility in water Cl Cl OH methanol Cl size tetrachlorobiphenyl electronic parameters: charge Bangkok Ecotoxicology III p.25 Quantitative structure-activity relationship: properties Hydrophobic parameters Aqueous solubility Octanol-water partition coefficient (Kow) Total Surface Area (TSA) Total Molecular Volume (TMV) Electronic parameters Atomic charges (q) Energy of molecular orbitals (Elumo or Ehomo) Delocalizability (D) Hardness (n) Hammett sigma substituent constants (s) Reduction potential (E1/2) Steric parameters Total Surface Area (TSA) Total Molecular Volume (TMV) Taft substituent constant (Es) Bangkok Ecotoxicology III p.26 Question Which chemical or environmental properties you consider to be most important for environmental occurrence of the following compounds? Chlorinated biphenyls (PCBs) Organophospate esters (e.g parathion) Pyrethroids (e.g permethrin) Mercury Bangkok Ecotoxicology III p.27 Prediction of fate and effects via quantitative structureactivity relationships (QSARs) chemical structure or properties fate or effect properties X Y Cl Cl OH O O biodegradation rates no-effect concentrations soil sorption bioaccumulation Cl quantitative model relating Y to X (QSAR) Y = f (X) prediction of fate or effect properties from chemical structure for related chemicals Bangkok Ecotoxicology III p.28 QSAR for ecotoxicity bioconcentration / partitioning effect concentrations endocrine disruption Bangkok Ecotoxicology III p.29 Bioconcentration uptake via food or particles uptake via gills (aqueous phase) concentration in fish concentration in water time bioconcentration factor in fish (BCF) BCF = Cf / Ca Bangkok Ecotoxicology III p.30 Uptake: bioconcentration in fish Relation between bioconcentration factors and octanol-water partition coefficeints (Kow) models are developed for relatively simple chemicals: • no biotransformation • uptake only via aqueous phase log BCF several chemcials bioconcentrate less than predicted due to biotransformation 2 log Kow Bangkok Ecotoxicology III p.31 Question Can explain what other possible reasons there could be (besides biotransformation) that causes deviation from the observed QSAR ? Think of at least two groups of compounds that might deviate from this QSAR due to the effect of biotransformation Bangkok Ecotoxicology III p.32 Prediction of ecotoxicity Four classes in acute toxicity class 1: chemicals with base-line toxicity (non-polar narcosis) class 2: chemicals that act by polar narcosis class 3: alkylating agents (reactive towards nucleophiles: SH, OH, NH) leading to cytotoxicity, membrane irritation, mutagenicity or carcinogenicity class 4: chemicals with specific modes of action Verhaar, 1992 / Veith, 1990 / Bradbury, 1990 / Hermens, 1989 Bangkok Ecotoxicology III p.33 Class 1: chemicals that act by narcosis LC50 to fish OH Cl log LC50 Cl Cl CH3 O Cl -2 -1 log Kow data from Könemann, 1981 alcohols, ethers, (chorinated) aromatic hydrocarbons Bangkok Ecotoxicology III p.34 Class 1: chemicals that act by narcosis NOEC early life stage test (reproduction, growth) log C (umol/L) LC50 NOEC: early life stage test -2 -1 log Kow data from Call, 1985 / van Leeuwen, 1990 Bangkok Ecotoxicology III p.35 Narcosis cel membrane phospholipids sodium channel (protein) sodium channel (protein) is blocked phospholipids xenobiotics blocking of protein channel: disturbance of transport of nerve impulses narcosis Bangkok Ecotoxicology III p.36 Class 2: chemicals that act by polar narcosis NH2 NH2 NH2 Cl CH3 OH OH OH log LC50 Cl CH3 O + N O O + N O O + N O CH3 -2 -1 Cl anilines, phenols and nitroaromatics log Kow data from Könemann, 1981 / Hermens, 1984 / Deneer, 1987 Bangkok Ecotoxicology III p.37 Reactive chemicals Examples: epoxides, aldehydes, unsaturated chorinated hydrocarbons These chemicals may react with nucleophiles (NH, OH and SH) in for example DNA, proteins, glutathion, leading to: irritation cytoxiciity DNA damage (carcinogens, mutagens) more toxic than narcosis Bangkok Ecotoxicology III p.38 Class 3: reactive chemicals Cl CH2Cl allylchloride Br I benzylic chlorides O O O log LC50 Cl aldehydes O epoxides -2 -1 epoxides, unsaturated chlorohydrocarbons, aldehydes, etc log Kow data from Hermens, 1985 / Deneer, 1988 Bangkok Ecotoxicology III p.39 Class 4: chemicals with a specific mode of action Cl Cl Cl Cl DDT OH CCl3 Cl lindane log LC50 Cl Cl Cl OCH3 Cl Cl -2 -1 Cl O P=S OCH3 Cl Cl pentachlorophemol organophosphates -2 -3 log Kow data from Hermens, 1982 / De Bruijn, 1992 Bangkok Ecotoxicology III p.40 Chemical domain of models: example Acute toxicity to fish: log Kow LC50 (mol/L) 1-chloro-2,4-dinitrobenzene 3,5-dichloro-nitrobenzene 2.20 3.20 6.5 10-7 2.3 10-5 1-chloro-2,4-dinitrobenzene 3,5-dichloro-nitrobenzene alkylating agent “polar narcotic” Bangkok Ecotoxicology III p.41 Application of QSARs predictions of effect concentration for risk assessment classifying chemicals into “mode of action” classes (100.000 chemicals on EINECS) mechanistic studies (receptor mediated effects) priority setting evaluation of effects of mixtures Bangkok Ecotoxicology III p.42 ... Assessment of Chemicals Chapter (van Leeuwen) Bangkok Ecotoxicology III p.23 ECOTOXICOLOGY: Estimation methods in ecotoxicology Bangkok Ecotoxicology III p.24 Activity of a chemical depends on:... Leeuwen) Bangkok Ecotoxicology III p.14 Speciation of aluminium as a function of pH Speciation of heavy metals depends on: Al fraction 1.0 pH hardness salinity 0.8 + Al(OH) Al 3+ - Al(OH) 0.6 0.4 +. .. microcosms ecosystems Bangkok Ecotoxicology III p.5 Acute test with Daphnia and fish duration: effects: test systems: 2-4 days survival static, daily renewal Bangkok Ecotoxicology III p.6 Semi-chronic