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
0
20000000
40000000
60000000
80000000
100000000
PP
TOX
DA
T
GC
/MS
REG
CM
PDS
OR
GC
MPD
S
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)
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.
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)
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)
0
25
50
75
100
control 6.3 13 25 50 100Res
po
nse
(%
)
Concentration (%) Mortality Immobility
0
25
50
75
100
control 6.3 13 25 50 100
Mo
rtal
ity
(%)
Concentration (%)
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)
-75-50-25
0255075
100
control 1.4 2.8 5.7 11 23 46 91
Inh
ibit
ion
(% c
on
tro
ls)
Concentration (%)
0
25
50
75
100
125
150
control 1.5 3.0 6.1 12 24 49 97
Res
po
nse
(%
co
ntr
ols
)
Concentration (%) frond number biomass
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)
0
25
50
75
100
125
ctl 1.6 3.2 6.25 12.5 25 50 100
Res
po
nse
(%
)
Concentration (%) mortality (%) biomass (dry weight % controls)
0
25
50
75
100
125
ctl 1.6 3.1 6.3 13 25 50 100
Res
po
nse
(%
)
Concentration (%) mortality (%) reproduction (% controls)
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
TOXICITY IDENTIFICATION EVALUATION
Environmental samples are complex mixtures
parameter specific
integrated response
TOXICITY IDENTIFICATION EVALUATION
separate identify manage
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
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
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)
TOXICITY IDENTIFICATION EVALUATION
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
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
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
CONCLUSIONS
Introduction to Whole Effluent Toxicity Testing
Defined Key Toxicity Concepts Potential Applications Regulatory Requirements Methodologies for identifying and treating
toxic constituents