strategies for mitigating risk to aquatic environments ... for mitigating risk to aquatic...

Elisabeth Henson, B.Sc. Ecotoxicology Team Leader

Lyriam Marques, Ph.D. Senior Scientist

Strategies for Mitigating Risk to Aquatic Environments Using

Biological Testing

WaterTech 2013 April 10-12th

OUTLINE

Introduction to Whole Effluent Toxicity Testing

Defined Key Toxicity Concepts Potential Applications Regulatory Requirements Methodologies for identifying and treating

toxic constituents

Branch of toxicology Toxic effects of chemicals and

physical agents on living organisms, especially on populations and communities with defined ecosystems

ECOTOXICOLOGY

http://fortox.org/forensic-toxicology-methods-of-analysis

TOXICITY TESTING

simple complex

Molecular, cellular, organs

Populations Ecosystem Single Organism

TOXICITY TESTING

Replicability - simplicity

Complexity - representativity

In situ studies

Outdoor ecotoxicological models (ponds, mesocosms, enclosures, artificial streams,

etc.)

Indoor ecotoxicological models (microcosms, experimental trophic chains, etc.)

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

Replicability - simplicity

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CHEMISTRY APPROACH

CHEMISTRY APPROACH Environmental samples are complex mixtures

parameter specific

CHEMISTRY APPROACH

Other toxicants may be missed

WET TESTING

integrated response

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)


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.


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)

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

ACUTE LETHALITY Acute Survival and

behaviour Duration: 96 & 48

Hours Screen

100% and Negative Control

Definitive 100%, 50%, 25%,

12.5%, 6.25% and Negative Control

Rainbow Trout

Daphnia magna

LC25/LC50 Calculations

Trout Results: LC50 = 20% (17-24)

Daphnia Results: LC50 =13% (10-16)

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Concentration (%) Mortality Immobility

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AQUATIC PLANTS

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

Pseudokirchneriella subcapitata

Lemna minor

IC25/IC50 Calculations

Algae Results: IC25= >91% (na->91%) IC50= >91% (65-na)

Lemna minor Results: Frond number: IC25= 2.7 (<1.5-4.4) IC50= 5.5 (3.6-8.4)

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STATIC RENEWAL TESTS CD Survival and

reproduction

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

Ceriodaphnia dubia

Fathead Minnows

IC25/IC50 Calculations

Ceriodaphnia Results: IC25= 5.8 (1.6-16) IC50= 22 (13-29) LC25= 30 (30-30) LC50= 35 (28-44)

Fathead Minnow Results: IC25= 60 (55-65) IC50= 71 (67-71) LC25= 57 (55-58) LC50= 67 (64-71)

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APPLICATIONS

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

APPLICATIONS

Alternative applications:

Aquatic baseline studies Environmental Impact Assessment Evaluate Treatment Options

(flocculants, coagulants) Model environmental impacts Test environmental models

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

WHAT DOES THIS MEAN?

We have the results…

…Now what? …How do these results help us?

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.

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

FISHERIES ACT

http://www.ec.gc.ca/default.asp?lang=En&n=714D9AAE-1&news=E5A11B36-398F-4AF1-8086-42AA9507C545

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


Environmental samples are complex mixtures

parameter specific

integrated response


separate identify manage


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


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


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)


pH 3 filter, sparge, C18

25%, 50%, 75%, 100% MeOH fractions off column

Ambient filter, sparge, C18


pH 9.0 filter, sparge, C18


detailed chemistry rationalize confirmatory testing

Retest

3 aliquots

fractionate chemical characterization

select biotests no effect - end

confirm effects

Approach

Look for patterns in effects

pH 3, ambient & 9


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

CONCLUSIONS

Introduction to Whole Effluent Toxicity Testing

Defined Key Toxicity Concepts Potential Applications Regulatory Requirements Methodologies for identifying and treating

toxic constituents

CONTACT INFORMATION: Elisabeth Henson, B.Sc.

Email: [email protected]

THANK YOU

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