Lecture 2 Toxicity Testing (i) terms and endpoints_large format.pdf

BIOL 2162 Ecotoxicology TOXICITY TESTING (I): types of tests, endpoints and performance criteria.  Objectives 1. Describe the dose-response relationship; 2. Define terms and describe different toxicity test types and endpoints; 3. Discuss the advantages and disadvantages of d...

BIOL 2162 Ecotoxicology TOXICITY TESTING (I): types of tests, endpoints and performance criteria.  Objectives 1. Describe the dose-response relationship; 2. Define terms and describe different toxicity test types and endpoints; 3. Discuss the advantages and disadvantages of different types of tests; 4. Discuss the importance of good experimental design; 5. Discuss data analysis and interpretation of results.  The Dose and Response Relationship  Fundamental to ecotoxicology is the relationship between dose and response (cause and effect)  The dose refers to the amount of toxicant to which the organism is exposed  The response is the observed effect, or endpoint that is measured  Traditional toxicity testing uses rats and other animals (endpoint = death)  A series of concentrations are tested ranging from no deaths to 100% deaths  A dose-response curve can then be generated  From that the LC50/LD50 is calculated  are the results relevant to the environment? Dose-response curve Connell, Lam, Richardson and Wu (1999) Introduction to Ecotoxicology, 1st Ed.  Response to Increasing Concentration  Essential (1)  Substance is essential for growth and survival  Without this substance the organism cannot survive eg. some trace metals Cu, Mg, Zn salt  Non-essential (2)  Substance is not required for growth and survival  As concentration/dose increases, adverse effects occur eg. DDT, parathion, pesticides Connell, Lam, Richardson and Wu (1999) Introduction to Ecotoxicology, 1st Ed.  Endpoints (Response)  Death (lethality tests)  Growth inhibition  Changes in metabolism  Cellular stress (induction/inhibition)  Changes in respiration  Developmental toxicity  Reproductive effects  embryo production and viability (animals)  germination (plants)  Immunotoxicity  Genotoxicity  Carcinogenicity  Mutagenicity  Problems With These Endpoints  May not be a statistically significant effect so in order to be accurate need to have appropriate:  sample size and replication  number of endpoints observed  number of dosages or concentrations  ability to measure endpoints  little variability of endpoints  statistical methodology Effects on a Population Connell, Lam, Richardson and Wu (1999) Introduction to Ecotoxicology, 1st Ed. Interaction with Ecosystems Connell, Lam, Richardson and Wu (1999) Introduction to Ecotoxicology, 1st Ed.  Types of Toxicity Tests  Acute Toxicity Tests: The tested concentration/dose is severe enough to cause a rapid response (eg. mortality)  usually within 24 h to 4 days (96 h).  Chronic Toxicity Tests: The tested concentration/dose causes a response after a longer period of exposure  usually signifies a period greater than 1/10 of the life span of an organism. eg. 21 days for some fish species.  Lifecycle / Partial Lifecycle Toxicity Tests: Entire lifecycle (embryo- adult), or critical life stages (eg. embryonic period) are studied.  Sublethal Toxicity Tests: The tested concentration/dose is below the level which directly causes death.  Microcosm and Mesocosm Tests: Used to simulate field conditions.  Field Studies: Measure effects at the population level.  Toxic Effects  Toxic effect depends on:  Dose/concentration  Length of exposure (eg. hours, days, weeks)  Lifestage (embryos and juveniles generally more sensitive than adults)  Previous exposure and toxicological interactions  Reference toxicants Connell, Lam, Richardson and Wu (1999) Introduction  A known toxicant is used as a to Ecotoxicology, 1st Ed. positive control  Used to determine viability of test organisms and test consistency  Classification of Toxicity Tests Field studies Mesocosms Microcosms Multispecies Chronic Single Acute Species Proportion of lifespan & system complexity Landis and Yu (2004) Introduction to Environmental Toxicology, 3rd Ed.  Classification of Toxicity Tests Laboratory Field (small scale) (large scale)  Acute Toxicity Tests  Acute toxicity tests are usually short term tests (days)  Endpoints:  Mortality, immobilisation, loss of equilibrium (fish and invertebrates)  Mortality, growth inhibition (algae and vascular plants)  Terms:  LC50 – lethal exposure concentration to 50% of tested organisms  LD50 – lethal exposure dose to 50% of tested organisms  LT50 – lethal time of exposure for a given concentration to 50% of tested organisms  The HIGHER the LC value, the MORE TOLERANT an organism is to the tested toxicant Recently updated animal ethics regulations prohibit the use of decapod crustaceans and post-embryonic vertebrates for such tests (therefore some standard guidelines no longer valid).  Chronic Toxicity Tests  Chronic toxicity tests are usually longer term tests involving a full or partial life cycle.  Full life cycle tests = expose for entire reproductive cycle (egg to egg).  Partial life cycle tests = exposing critical life stages (embryos, larvae, early juveniles).  Endpoints:  Reproduction (gamete production, egg viability, hatching success, germination)  Growth and mortality  Terms:  LC50 – lethal exposure concentration to 50% of tested organisms Measured over a longer time period than in acute tests (eg. 21 days)  EC50 – effective concentration causing adverse effects in 50% of tested organisms (endpoint other than mortality – eg. growth inhibition) IC50 – the concentration that causes a 50% reduction (inhibition) of growth  No-observed-effect-concentration (NOEC): “highest concentration tested at which no significant effect is observed relative to controls.”  Lowest-observed-effect-concentration (LOEC): “lowest concentration tested at which a significant effect was observed relative to controls.”  Lifecycle / Partial Lifecycle Toxicity Tests  Early life stages are very sensitive  Exposure to toxicants during the early life stages may have carry over effects to older life stages Connell, Lam, Richardson and Wu (1999) Introduction to Ecotoxicology, 1st Ed.  Sublethal Toxicity Tests  Sublethal toxicity tests may be short term or long term and measure effects at dose/concentrations lower than those that cause mortality.  Endpoints:  Biochemical/physiological – induction/inhibition of enzymes and proteins, up/down regulation of genes, hematology, respiration  Histological – tissue necrosis, tumour formation, pathology  Behavioural – locomotion, swimming ability, avoidance, predator-prey interactions, courtship (reproductive behaviour)  Terms:  EC50 - effective concentration causing adverse effects in 50% of tested organisms  NOEC – No observed effect concentration  LOEC – Lowest observed effect concentration  Advantages and Disadvantages of Tests Acute Chronic Sublethal Short term (rapid results) Longer term Short/long term High dose exposure Environmentally relevant Environmentally relevant (environmental relevance?) exposure dose exposure dose Single endpoint Single/multiple endpoints Multiple endpoints Simple laboratory setup More complex laboratory More complex laboratory setup setup No identification of toxic Can identify mechanisms of Can identify mechanisms of mechanisms toxic action toxic action Individual effects Lifecycle/partial lifecycle Lifecycle/population effects effects Simple data analysis More complex data analysis More complex data analysis  Microcosms, Model Aquatic Systems Rand, G.M. (Ed) (1995) Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment Chapter 4: 154-160, Chapter 9: 257-264.  Microcosms and mesocosms used to simulate field conditions.  Most chemicals are less toxic in field because of reduced persistence and bioavailability.  But some chemicals (eg. PAHs, anthracene) show photoenhanced toxicity and are more toxic under field conditions.  Single Species Tests versus Microcosms Single species tests Microcosms/ Mesocosms Most sensitive species in lab Complex multispecies ecosystems Single finite endpoints Complex effects, population dynamics Single environmental factors Multitude of environmental factors Easy to resolve/calculate Need complex analysis (multivariate statistics etc.)  Microcosms and Mesocosms  Classified based on size  Microcosms - small to large tanks. Mainly outdoors in field locations. Field benthos and water introduced – allowed to colonise.  Mesocosms – same idea but larger scale. Generally earthenware ponds 0.01-0.1 ha in size. Ponds are allowed to colonise for some time, then stocked with fish and invertebrates as required.  Artificial streams constructed in forested areas. Water used from a nearby creek. Stream bed introduced.  Often used to study pesticide applications, spray drift etc.  Advantages of Simulated Mesocosms  Advantages:  Tests system is a complete functioning ecosystem in roughly the appropriate proportions  System can be characterised and maintained for a defined time  Conditions within system can be monitored before and after addition of toxicant  Replicated in appropriate statistical design  Components of the system can be isolated, removed or supplemented to observe effects on these components. Eg: can start with sterilized benthos and then look at colonisation  Environmental conditions and therefore exposure more realistic than in lab  Effects of chemicals on many species, with different sensitivities investigated simultaneously  Ecosystem level effects and interactions among species can be investigated. (From Rand, pg. 263)  Limitations of Simulated Mesocosms  Limitations:  Difficult to establish a realistic community especially for the higher trophic levels like fish and associated predator/prey relationships  Very expensive to construct and maintain  The replicated systems change differently with time and diverge  Scaling factors must be carefully considered. Are the scales appropriate with the natural system? In enclosed systems different species tend to dominate compared to natural open field  Difficult to interpret results and characterize endpoints. (From Rand, pg. 263) Therefore several single species tests (both acute and chronic) have been the mainstay of ecological risk assessment.  Field Studies  Natural environment is complex, dynamic and changeable (unlike the controlled laboratory environment)  Provide a more realistic evaluation of the concentration- response relationship  Interactive effects of physico-chemical factors (salinity, temperature, pH, hardness, dissolved oxygen, etc) with toxicants  Can measure indirect effects – predator/prey interactions, biotic and abiotic interactions  Field Studies  Field studies are usually complex and require careful design  What are the specific objectives of the study?  How are sites selected?  Cost, accessibility  Presence of desired test species  What endpoints will be measured?  Can these effects be detected in field studies?  Laboratory validation (acute and chronic tests) Relationships Between Different Levels of Biological Organisation Connell, Lam, Richardson and Wu (1999) Introduction to Ecotoxicology, 1st Ed.  Types of Test Organisms Aquatic systems Terrestrial (freshwater, estuarine and marine)  Microorganisms  Microorganisms  Primary producers  Primary producers  phytoplankton  plants  macro-algae (seaweed)  aquatic plants  Invertebrates (insects,  Invertebrates (molluscs, spiders, mites) crustaceans)  Reptiles  Fishes  Birds  Amphibians  Sediments  Mammals  Criteria for Selection 1. Organisms with a range of sensitivities should be tested; 2. Tested species should be widely available and abundant; 3. Tested species should be native (indigenous) or representative of tested environment; 4. Tested species should be recreationally, commercially or ecologically important; 5. Tested species should be adaptable to laboratory conditions; 6. Background information on the physiology and ecology of the tested species should be known.  Experimental Design  Replication  Randomisation  Adequate number of treatments  Realistic treatment levels?  Testing guidelines provide standardised test methods  OECD Guidelines  ASTM Guidelines  Test Guidelines  Provide detailed instructions on experimental design  principles of the test  test validity  description of methods  choice of species, holding vessels  preparation of test chemicals  required observations  data analysis and interpretation American Society for Testing and Organisation for Economic Co-operation Materials (ASTM) Guidelines and Development (OECD) Guidelines  Important to Understand the LOGARITHMIC Nature of Biological Responses  Organisms respond to a proportional increase in toxicant concentration, not to an absolute increase in concentration.  12 Additive therefore arithmetic scale  100 101  100 200 = geometric scale  THE PROBIT  a probit is one unit of standard deviation. To Linearise Response  X axis – use a log scale  Log of concentration  Y axis – use probit scale Probit of % mortality Log of exposure concentration  Probit Analysis  GRAPHICAL METHOD OF PROBIT ANALYSIS  Rainbow trout 96 h LC50 for Zinc = 6.4 mg/L 95% confidence interval (5.8 – 7.0 mg/L)  COMPUTER METHODS ** Preferred Method  SPSS PROBIT ANALYSIS - These generally provide estimates of LC1 LC99 with respective confidence limits  LC50 ANALYSIS  Conc. (log) VERSUS % MORTALITY (probit)  LT50 ANALYSIS  Time (log) VERSUS % MORTALITY (probit)  TOXRAT computer program used in lab for toxicity test results analysis Walker, Hopkin, Sibly and Peakall (2006) Principles of Ecotoxicology, 3rd Ed. Types of Data that can be Analysed by Probit Analysis QUANTAL CONTINUOUS ALL OR NONE GRADED RESPONSE lethality growth germination oxygen consumption tumour induction disease resistance change in enzyme level YES NO  Data Analysis  Statistical interpretation of ecotoxicological data is important  Variety of tests may be used, depending on  Type of data  Quality/quantity of data collected  Question being asked  Useful texts  Quinn and Keough (2002) Experimental Design and Data Analysis for Biologists. Cambridge University Press. 537 pp.  Sparks (Ed) (2000) Statistics in Ecotoxicology. John Wiley & Sons, Ltd. 320 pp. (Especially Ch. 4)  Data Analysis  Exploratory data analysis  Histograms, box plots, dot plots, scatterplots  Homogeneity of variance, normality, independence  Data transformation (log, arcsine, square root, probit)  Hypothesis testing  Analysis of variance (ANOVA)  Parametric/non-parametric data analysis  Type I/Type II error  Correlation and regression analysis  Multivariate analysis  Data Analysis  Examples Percentage of emerged Chironomus in eight different salinity Effect of Nodularia spumigena (2x105 cells/l and 2x106 cells/l) treatments. All larvae came from a single egg mass. Values are on glutathione (GSH) concentrations in Perna viridis gill (b). Means ± s.e.m. (N=10–15); different letters indicate statistically Significant differences (P=0.05) between treatments. (Davies, Siu, Jack, Wu, Lam and Nugegoda (2005) Marine Pollution Bulletin 51, 1026-1033) (Hassell, Kefford and Nugegoda (2006) Journal of Experimental Biology 209, 4024-4032)  Performance Criteria  Relevance  must be ecologically realistic, depends on complexity & variation between ecosystems  Reliability  test organisms and materials must be readily available and of consistent quality  Robustness  tests must be able to be performed relatively easily (by trained personnel)  Responsiveness  tests should produce significant differences between the test material and controls (clear dose-response relationship)  Reproducibility  consistent results both within and between laboratories - standard methods  Conclusions  Toxicity testing  Determines the relationship between dose and response  Different types of tests are used – laboratory or field based  Variety of endpoints can be measured  Tested concentrations should be of environmental relevance  Standard guidelines available  Experimental design  Data analysis  Selected References  Connell, D., Lam, P., Richardson, B. and Wu, R. (1999) Introduction to Ecotoxicology. First Edition. Blackwell Science. 170 pp.  Davies, W. R., Siu, W. H. L., Jack, R. W., Wu, R. S. S., Lam, P. K. S. and Nugegoda, D. (2005) Comparative effects of the blue green algae Nodularia spumigena and a lysed extract on detoxification and antioxidant enzymes in the green lipped mussel (Perna viridis). Marine Pollution Bulletin, 51, 1026- 1033.  Hassell, K. L., Kefford, B. J. and Nugegoda, D. (2006). Sub-lethal and chronic salinity tolerances of three freshwater insects: Cloeon sp. and Centroptilum sp. (Ephemeroptera: Baetidae) and Chironomus sp. (Diptera: Chironomidae). The Journal of Experimental Biology, 209, 4024-4032.  Landis, W. G. and Yu, M-H. (2004) Introduction to Environmental Toxicology: Impacts of Chemicals Upon Ecological Systems, 3rd Ed. 484 pp.  Quinn, G. P. and Keough, M. J. (2002) Experimental Design and Data Analysis for Biologists. Cambridge University Press. 537 pp.  Rand, G.M. (Ed) (1995) Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment Second Edition. Taylor & Francis. 1125 pp.  Sparks, T. (Ed) (2000) Statistics in Ecotoxicology. John Wiley & Sons, Ltd. 320 pp.  Walker, C.H., Hopkin, S.P., Sibly, R.M. and Peakall, D.B. (2006) Principles of Ecotoxicology. Third Edition. Taylor & Francis. 315 pp.