Strategies for Mitigating Risk to Aquatic Environments Using Biological Testing

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1 Strategies for Mitigating Risk to Aquatic Environments Using Biological Testing Elisabeth Henson, B.Sc. Ecotoxicology Team Leader Lyriam Marques, Ph.D. Senior Scientist WaterTech 2013 April 10-12th

2 OUTLINE Introduction to Whole Effluent Toxicity Testing Defined Key Toxicity Concepts Potential Applications Regulatory Requirements Methodologies for identifying and treating toxic constituents

3 ECOTOXICOLOGY Branch of toxicology Toxic effects of chemicals and physical agents on living organisms, especially on populations and communities with defined ecosystems

4 TOXICITY TESTING Molecular, cellular, organs Single Organism Populations Ecosystem simple complex

5 TOXICITY TESTING Complexity - representativity In situ studies Outdoor ecotoxicological models (ponds, mesocosms, enclosures, artificial streams, etc.) Indoor ecotoxicological models (microcosms, experimental trophic chains, etc.) Replicability - simplicity Replicability - simplicity Single Species Studies (bioassays mechanistic approaches) Mathematical models Principal methodologies in aquatic ecotoxicology showing the relationship between representativity-complexicity and reproducibility-simplicity Adapted from Boudou and Ribeyre, Environmental Health Perspectives 105(Suppl.1, February): 21-35

6 CHEMISTRY APPROACH PP TOX DAT GC/MS REG CMPDS ORG CMPDS

7 CHEMISTRY APPROACH Environmental samples are complex mixtures parameter specific

8 CHEMISTRY APPROACH Other toxicants may be missed

9 WET TESTING integrated response

10 TOXICITY TESTING DEFINITIONS Whole Effluent Toxicity (WET) test Used to measure, predict and control the discharge of materials that may be harmful to aquatic life Acute Toxicity Short duration exposure Immediate e.g. Lethal effects Chronic Toxicity Long (relative to the organism s life span) or repeated exposures Lethal or sublethal Effects (e.g. growth or reproduction)

11 TOXICITY TESTING DEFINITIONS Static tests The test organisms are exposed to the same test solution for the duration of the test Static renewal tests The test organisms are exposed to fresh solution of the same concentration of sample every 24h or other prescribed interval, either by transferring the test organisms from one test chamber to another, or by replacing all or a portion of solution in the test chambers.

12 TOXICITY TESTING DEFINITIONS Screening pass/fail toxicity test, 100% sample and negative control can only determine toxic vs. non-toxic Definitive or Multiple Concentration multiple-concentration test, allows you to determine statistical test endpoints Statistical Test Endpoints Used to determine the degree of toxicity of a sample (e.g. LC50, IC25)

13 INDICATOR ORGANISMS Microbes Luminescent bacteria, Vibrio fischeri Plants Green algae, Pseudokircherella subcapitata Duckweed, Lemna minor Invertebrates Water flea, Daphnia magna Water flea, Ceriodaphnia dubia Vertebrates (fish) Rainbow trout, Oncorhynchus mykiss (w/ph stabilization option) Fathead minnow, Pimephales promelas

14 ACUTE LETHALITY Rainbow Trout Daphnia magna Acute Survival and behaviour Duration: 96 & 48 Hours Screen 100% and Negative Control Definitive 100%, 50%, 25%, 12.5%, 6.25% and Negative Control

15 LC25/LC50 Calculations 100 Trout Results: Mortality (%) LC50 = 20% (17-24) 0 control Concentration (%) 100 Daphnia Results: Response (%) control LC50 =13% (10-16) Concentration (%) Mortality Immobility

16 AQUATIC PLANTS Pseudokirchneriella subcapitata Lemna minor Chronic Growth Inhibition & Stimulation Duration: 72 Hours & 7 Days Screen 91%; 97% and Negative Control Definitive 91, 46, 23, 11, 5.7, 2.8, and 1.4% and Negative Control; 97%, 49, 24, 12, 6.1, 3.1 and 1.6 Endpoints IC25, IC50, stimulation

17 IC25/IC50 Calculations Inhibition (% controls) control Algae Results: IC25= >91% (na->91%) IC50= >91% (65-na) Concentration (%) Response (% controls) control Concentration (%) frond number biomass Lemna minor Results: Frond number: IC25= 2.7 (< ) IC50= 5.5 ( )

18 STATIC RENEWAL TESTS CD Survival and reproduction Ceriodaphnia dubia Fathead Minnows FM Survival and Growth Duration: ~7 days Screen 100% and Negative Control Definitive 100%, 50%, 25%, 12.5%, 6.25%, 3.0%, 1.5% and Negative Control Endpoints: Survival: LC25/LC50 Reproduction: IC25/IC50 Growth: IC25/IC50

19 IC25/IC50 Calculations Response (%) ctl Concentration (%) mortality (%) reproduction (% controls) Ceriodaphnia Results: IC25= 5.8 (1.6-16) IC50= 22 (13-29) LC25= 30 (30-30) LC50= 35 (28-44) Response (%) ctl Fathead Minnow Results: IC25= 60 (55-65) IC50= 71 (67-71) LC25= 57 (55-58) Concentration (%) mortality (%) biomass (dry weight % controls) LC50= 67 (64-71)

20 APPLICATIONS Common applications of whole effluent toxicity testing: Environmental effects monitoring Regulatory compliance Due diligence

21 APPLICATIONS Alternative applications: Aquatic baseline studies Environmental Impact Assessment Evaluate Treatment Options (flocculants, coagulants) Model environmental impacts Test environmental models

22 Benefits of Standard Testing Benefits for use in alternative applications: Prescribed standard methods Results are scientifically and legally defensible Following precedents already set in some industries Standard tests are less costly than custom We have received feedback that regulators prefer laboratory-derived data using standard methods over data from modelling May make the permitting process faster

23 WHAT DOES THIS MEAN? We have the results Now what? How do these results help us?

24 REGULATIONS Fisheries Act Subsection 36(3) no person shall deposit or permit the deposit of a deleterious substance of any type in water frequented by fish or in any place under any conditions where the deleterious substance or any other deleterious substance that results from the deposit of the deleterious substance may enter any such water.

25 REGULATIONS Fisheries Act Subsection 36(3) reduce the threats to fish, fish habitat and human health from fish consumption decreasing the level of deleterious and harmful substances Acute Lethality Test Fisheries Act

26 FISHERIES ACT

27 TOXICITY IDENTIFICATION EVALUATION If a toxic result is observed in a test species of interest you may need to determine what is causing the toxicity Toxicity Identification Evaluations (TIEs) Process for identifying the bioactive constituents or properties of a sample Involves confirmation, isolation, identification, and confirmation of effects Customized for each sample type

28 TOXICITY IDENTIFICATION EVALUATION Environmental samples are complex mixtures parameter specific integrated response

29 TOXICITY IDENTIFICATION EVALUATION separate identify manage

30 TOXICITY IDENTIFICATION EVALUATION Many tools that can be used: Sample fractionation ph adjustment Size exclusion testing Simulated sample Standard additions Success is a result of experience, collaboration (biological testing and chemical analysis) and taking a weight of evidence approach

31 TOXICITY IDENTIFICATION EVALUATION TIE is organized into 3 phases Phase 1 Characterization Identification of broad classes of chemicals causing toxicity Phase 2 Identification Chemicals of concern are further narrowed down Phase 3 Confirmation Verification of chemicals of concern through additions/spiking toxicity experiments

32 TOXICITY IDENTIFICATION EVALUATION Our approach is consistent with US EPA Standards incorporates standard wastewater treatments that can be readily scaled up to remove toxicity from effluent streams (manipulation of ph, aeration, filtration, solid phase extraction)

33 TOXICITY IDENTIFICATION EVALUATION Approach confirm effects select biotests no effect - end fractionate chemical characterization 3 aliquots ph 3, ambient & 9 ph 3 filter, sparge, C18 25%, 50%, 75%, 100% MeOH fractions off column Ambient filter, sparge, C18 25%, 50%, 75%, 100% MeOH fractions off column ph 9.0 filter, sparge, C18 25%, 50%, 75%, 100% MeOH fractions off column Retest Look for patterns in effects detailed chemistry rationalize confirmatory testing

34 TOXICITY IDENTIFICATION EVALUATION Treatments not limited to these manipulations a TIE is an effects based investigation and other treatments may be included depending on the properties of the toxic constituent Success is dependent on experience

35 CONCLUSIONS Introduction to Whole Effluent Toxicity Testing Defined Key Toxicity Concepts Potential Applications Regulatory Requirements Methodologies for identifying and treating toxic constituents

36 THANK YOU CONTACT INFORMATION: Elisabeth Henson, B.Sc.