Aquatic Toxicity Workshop-2008
Prof Giesy was on the organizing committee of the 35th Annual Aquatic Toxicity
Workshop held October 5-8, 2008 in Saskatoon, Saskatchewan. In addition to helping
organize the meeting, he and his students and post docs presented 7 papers at the meeting.
“Application of a medaka HPG axis real time PCR array method to chemical screening”.
With X. Zhang, M. Hecker, A. Tompsett, J. Newsted, and P. Jones.
“White sturgeon growth, morphology, and survival after exposure to Columbia River
surface water at two sites in British Columbia, Canada. With A. Tompsett, D. Vardy, M.
Hecker, S. Wiseman, H. Zhang, and K. Liber.
“In vitro evaluation of the toxic effects and endocrine disrupting potential of oil sands
processed water and naphthenic acids. With X. Zhang, S. Wiseman, E. Higley, P. D.
Jones, M. Hecker, M. Gamel El Din, and J. W. Martin.
“Aquatic toxicology of perfluoroctanesulfonate and related fluorochemicals With J. Naile,
J. Khim, J. Newsted, and P. Jones.
“Toxicity of perfluorooctane sulfonate (PFOS) to avian wildlife: ambient safe water
value derivation and uncertainty analysis. With J. Newsted, J. Naile, J. Khim, and P.
Jones.
“Sensitivity of early white sturgeon (Acipenser transmontana) life-stages to copper,
cadmium, and zinc”. With D. Vardy, A. Tompsett, M. Hecker, J. Duquette, D. Janz, K.
Liber, and M. Adzic.
“Assessment of toxicity of upper Danube River sediments using a combination of
chemical fractionation, the Danio rerio embryo assay and the Ames fluctuation test.
With E. Higley, S. Grund, T. Seiler, U. Vare, W. Brack, T. Schulz, J. Wolz, H. Zielke, H.
Hollert and M. Hecker.
“Medaka : an in vivo model for molecular ecotoxicology. With D.W.T. Au.
35th Annual Aquatic Toxicology Workshop
Abstract
Oct 5-8, 2008, Saskatoon, SK, Canada
¾ A graphical system model was developed for the testing and
evaluation of environmental EDCs using medaka (Oryzias latipes).
Evaluation of Environmental Endocrine
Disrupting Chemicals Using the Medaka
HPG Axis Model
¾ The model illustrates the key pathways that are associated with
the reproduction system (hypothalamic-pituitary-gonadal (HPG)
axis).
¾ Real time RT-PCR array was developed to examine expression
profiles of 36 genes in brain, liver and gonad.
Xiaowei Zhang, Ph.D.
¾ Evaluated by examining effects of five model compounds.
Prof. John Giesy, Ph.D., Dr. Markus Hecker,
Dr. Paul Jones, Dr. John Newsted
Dr. June-Woo Park, Ms. Amber Tompsett
¾ The medaka HPG axis model provides a powerful tool
¾ To delineate mechanism of toxicity
¾ To quantitatively predict the adverse effects on reproduction.
University of Saskatchewan
University of Saskatchewan,
Michigan State University, & ENTRIX
University of Saskatchewan,
Michigan State University, & ENTRIX
Introduction I
Introduction II
• Small fish model (i.e. medaka, fathead minnow, zebrafish)
¾ Endocrine Disruptor: Definition
An exogenous substance that alters function(s) of the
endocrine system and consequently causes adverse health effects
in an intact organism, or its progeny, or (sub)populations.
--- IPCS, 1998 (Global Assessment)
– Small body size
– Relatively rapid life-cycle
– Standard, validated technique for culture in the lab
– Literatures of basic biological/toxicological attributes
• Ecotoxicogenomics
¾ Greatest concern is that exposure to endocrine disruptors during
critical periods of development may predispose individuals to
adverse health effects at later stages of life.
– Transcriptionomics, proteomics and metabolomics: maximize the
information collected from each animals;
– Whole genome sequences are publicly available for medaka and
zebrafish;
¾ A large number of environmental chemicals need to be tested for
potential endocrine disrupting effects
– System models can integrate high-dimensional data to aid in
mechanism understanding.
¾ Mechanism of action (MOA) is required to evaluate the risk of
chemical exposure.
University of Saskatchewan,
Michigan State University, & ENTRIX
2
University of Saskatchewan,
Michigan State University, & ENTRIX
Hypothalamus
2.
Pituitary
3.
4.
PCR array
•
SYBR Green technology
•
LH: luteinizing hormone
•
384-well format /ABI system
•
FSH: follicle-stimulating
hormone
•
3 reference genes
•
Fluorescence in situ
hybridization (FISH)
•
Fecundity (egg production)
•
BSI: brain-somatic index
•
HSI: hepatic-somatic index
•
GSI: gonadal-somatic index
Other endpoints
Gonad
1.
Exposure
T: testosterone
2.
E2:17β-estradiol
3.
KT:11-ketotestosterone
4.
HDL: high-density lipid
5.
LDL: low-density lipid
Liver
3
Methods
Medaka HPG axis
1.
1
•
•
•
•
•
Villeneuve et al, EST 2007
University of Saskatchewan,
Michigan State University, & ENTRIX
4
Animal: 4 month adult medaka
Exposure: 5 model chemicals
Sex: 5 male : 5 female per tank
Vehicle control: DMSO
RNA isolation: brain, liver & gonads
University of Saskatchewan,
Michigan State University, & ENTRIX
5
17α-ethinylestradiol
(EE2)
Cumulative Fecundity
n =3
(p < 0.05)
♂
• Increased VTG conc.
• Feminization
• Liver: Increased HSI for
males
n =3
* p < 0.05
1.
Up-regulation of ER-α and egg
precursor genes
2.
Down-regulation of CYP17
3.
Down-regulation of brain
androgen receptor (AR)
(disordered male sexual behaviors)
EE2: 17α-ethinylestradiol; TRB: 17β-trenbolone
6
University of Saskatchewan,
Michigan State University, & ENTRIX
Prochloraz exposure
♀
Fluorescence in situ hybridization (FISH)
CYP19A mRNA in the ovary
•
•
Pesticide
Inhibitor of CYP17
and CYP19
•
Fecundity
•
↓
T and E2 ↓ (Ankley et al 2005)
•
Compensatory response: Upregulation of CYP17 and CYP19A
•
Down-regulation of activin:
retarded oocyte maturation
University of Saskatchewan,
Michigan State University, & ENTRIX
7
University of Saskatchewan,
Michigan State University, & ENTRIX
A: Control ovary hybridized with sense
probe
B: Control ovary, antisense probe
C: 7 day exposure of 500 ng EE2/L
D: 7 day exposure of 5000 ng TRB/L
•
•
8
Localization of a specific gene at
the tissue and/or cellular level
Change of CYP19A expression in a
whole tissue basis was due to a
combination of increase of
CYP19A-containing cells and an
increase of mRNA amount per cell.
9
University of Saskatchewan,
Michigan State University, & ENTRIX
Summary
Fecundity v.s. Gene Expression
• Application of the medaka HPG PCR array facilitated
mechanistic understanding of environmental EDCs
• The gender-, organ-, time- and concentration –specific
gene expression profiles provide systematic information
to delineate chemical-induced modes of action.
• Molecular response at mRNA has potential to
quantitatively evaluate chemical induced adverse effects
on reproduction.
• The medaka HPG axis model has potential to be an
effective ecotoxicological screening tool for EDCs
University of Saskatchewan,
Michigan State University, & ENTRIX
11
University of Saskatchewan,
Michigan State University, & ENTRIX
12
Application and Future Work
1.
Acknowledgements
¾
¾
¾
¾
¾
¾
Among -species comparison
–
Fish, mammalian, and human
–
Sequences alignments, transcriptional regulations, pathways,
sensitivities.
2.
Chemical classification
–
Database buildingup
–
Classification of chemical based on mechanisms of toxicity
3.
¾ Dr. Doris Au (CityU HK)
¾ Prof. Rudolf Wu (CityU HK)
¾ Dr. Daniel Villeneuve (US EPA)
Risk assessment
1.
Diagnostic (or retrospective) risk assessment
2.
Predictive risk assessment
13
Zhang, X *., Hecker,M., Park, J., Tompsett, A.R., Jones, P.D., Newsted, J.L. and Giesy, J.P.
(2008). Development and validation of a medaka brain-gonadal-liver axis model and a real
time-PCR array method to facilitate the mechanistic classification of endocrine-disrupting
chemicals (EDCs). Aquatic Toxicol. 88, 173–182.
2.
Zhang, X*., Hecker, M. Park, J., Tompsett, A.R., Newsted, Jones, P. D., Newsted, J. L., Wu, R.
S. S., Kong, R. Y. C., and Giesy, J. P. (2008). Responses of the Medaka HPG axis PCR array
and reproduction to prochloraz and ketoconazole. Environ Sci Technol 42, 6762-6769.
3.
Zhang, X*., Hecker, M. Park, J., Tompsett, A.R., Newsted, J.L., Jones, P.D., Wu, R.S.S., Giesy,
J. P. (2008). Time-dependent transcriptional profiles of hypothalamic-pituitary-gonadal (HPG)
axis to fadrozole and 17beta-trenbolone in medaka (O. latipes). Environ Toxicol Chem. (Article
In press)
4.
Park, J.-W., A.R. Tompsett, Zhang, X., P.D. Jones, J.L Newsted, D. Au, R. K., R. S.S. Wu, J.P.
Giesy, M. Hecker. (2008) Fluorescence in situ hybridization techniques (FISH) to detect
changes in CYP19a gene expression of Japanese medaka (Oryzias latipes). Toxicol. Appl.
Pharmacol (Article In press).
5.
Tompsett, A. R.; Park, J.; Zhang, X.; Jones, P. D.; Newsted, J. L.; Au, D. W. T.; Chen, E. X. H.;
Yu, R. M. K.; Wu, R. S. S.; Kong, R. Y. C.; et al. (2008). Development and validation of an in situ
hybridization system to detect gene expression along the HPG axis in Japanese medaka,
Oryzias latipes. Toxicol. Sci. (submitted)
Fecundity v.s. Gene Expression
TRB
Prochloraz
Ketoconazole
Fadrozole
46.1% (*)
5000 ng/L
26.0% (*)
3 ug/L
95.5%
30 ug/L
49.8% (*)
300 ug/L
18.0% (*)
3 ug/L
89.7%
30 ug/L
84.2%
300 ug/L
20.3% (*)
50 ug/L
20.4% (*)
ERB_L
VTG.I_L
CHGH_L
VTG.II_L
♀
nnexinM2_L
99.8%
500 ng/L
AR_L
65.1%
50 ng/L
ERA_L
92.4%
500 ng/L
HGHminor_L
50 ng/L
CHGL_L
EE2
Effects (%)
91.0%
-4096
-1024
-256
-64
-16
-4
1
4
16
Fo ld Ch a ng e
Conc.
5 0 0 0 n g /L
TR B
5 0 0 n g /L
5 0 n g /L
3 0 0 u g /L
P RO
3 0 u g /L
3 u g /L
3 0 u g /L
K TC
3 0 u g /L
FA D
1 0 .0 u g /L
3 u g /L
1 0 0 u g /L
1 .0 u g /L
5 0 0 n g /L
EE2
5 0 n g /L
5 n g /L
University of Saskatchewan,
Michigan State University, & ENTRIX
14
Xiaowei Zhang, Ph.D.
15
University of Saskatchewan,
Michigan State University, & ENTRIX
University of Saskatchewan,
Michigan State University, & ENTRIX
Thank you!
Related Publications
1.
5 ng/L
The Toxicology Centre at
the University of
Saskatchewan
¾ Strategic to Achieve Results (STAR) grant from US EPA
¾ Computational Toxicology Program of the US EPA, ORD and OSCP,
and the US EPA ORD Service Center/NHEERL
University of Saskatchewan,
Michigan State University, & ENTRIX
Chemical
Prof. John P. Giesy, Ph.D.
Dr. Markus Hecker
Dr. Junewoo Park
Ms Amber Tompsett
Dr. Paul Jones
Dr. John Newsted
10
Toxicology Centre
University of Saskatchewan
44 Campus Drive,
Saskatoon, SK, S7N5B3, Canada
Tel: 306-966-1204
Fax: 306-966-4796
Email: xiaowei.zhang@usask.ca
Lab Web Site: http://www.usask.ca/toxicology/jgiesy/
University of Saskatchewan,
Michigan State University, & ENTRIX
16
White sturgeon hatch and survival after
exposure to Columbia River surface water
at two sites in British Columbia, Canada
Background
• Poor recruitment of white sturgeon in the trans-boundary
region of the Columbia
• Adult sturgeon spawn and lay fertilized eggs, which hatch
Amber Tompsett
Aquatic Toxicity Workshop
Saskatoon, SK
October 6, 2008
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Background
• Possible causes for poor recruitment:
– Lack of suitable habitat
– Flow regime
– Alteration of water quality
–
–
–
–
–
–
Nutrition
Genetic bottlenecks or inbreeding depression
Predation by introduced species such as walleye
Interspecies competition
Pathogens/disease
Pollution
• However, few young of the year (YOY) have been found in
habitats considered suitable for this life stage
• Hatchery reared juveniles released to the river exhibit good
survival and growth
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Possible Sources of Pollution
• Metal smelter
-Liquid effluent
-Granular slag/sediment
• Pulp and paper mill
• May act either alone or in combination
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Project Objectives
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Site Location
• Exposure of white sturgeon early life stages to
Columbia River surface water at 2 sites
-Upstream of metal smelter
-Downstream of metal smelter
-Filtered city water control
• Evaluation of biological endpoints at each site
-Survival
-Growth
-Morphology
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
1
Site Locations
Reference Site
Fruitvale
Trail
Smelter Site
Rossland
Downstream Site
Canada
U.S.A.
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Experiment Setup
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Exposure Systems
• Retrofitted commercial trailers
River
Intake
• Identical trailer at each site
Overflow
to River
Fully Replace
Every 6h (205 L)
85L
Reservoir
• Continuous river water supply
Recirculating
System (205 L)
• Controls at upstream site
40 L Streams
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Experimental Design
• 4 replicates per treatment
• 3 chambers per replicate
• Maintenance of WQ
-adequate flow rates
-chilled water
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Methods: Exposure
• Fertilized eggs obtained from wild
broodstock
• Eggs hatched and reared to ~60 d
post-fertilization
• Dead fish collected and counted daily
-basic morphometrics
• Subsampling and water sampling
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
2
Methods: Exposure Termination
Results: Survival to Hatch
90
• Survival
Percent Hatch
• Weight and length
80
• Abnormal morphology
•
No significant treatment differences
•
Lower hatch rates in river water
treatments
-insufficient flow in hatching jars
-fungal growth
70
• Preservation for:
-histology
-molecular biology
-contaminant analysis
60
C
D
U
Treatment
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Exposure Mortality
Why do the fish die?
Cumulative Mortality
90
% Mortality
% Dead
80
Day 1
70
60
7
Egg
50
60
20
Yolk Sack
Larvae
Exogenous Feeding
Larvae
40
Fertilization
30
Control
20
Upstream
Downstream
Hatch
Transition to
exogenous feeding
Exposure initiation
10
Transition to
juvenile
Exposure termination
60
57
54
51
48
45
42
39
36
33
30
27
24
21
18
15
12
0
Day of Exposure
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Cumulative Mortality: Yolk-sac Larvae
35
Cumulative Mortality: Exogenous Feeding
Larvae
18
30
16
14
Control
20
Upstream
15
Downstream
10
Mortalit
%%
Mortality
Mortalit
%%
Mortality
25
12
Control
10
Upstream
8
Downstream
6
4
5
2
0
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Day of Exposure
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
0
38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Day of Exposure
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
3
Why do more control fish die?
Absolute Survival: A Better Measure?
R2=0.983!!!
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
•
No significant treatment differences
•
Similar survival even with different
stocking rates
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Tentative Conclusions
What next?
• Further analysis of survival and termination data
• Downstream river water had no adverse effects on hatching
success
• Mortality rates of white sturgeon in culture were highly
dependent upon initial fish density
• Downstream river water probably did not have adverse
effects on survival of larval white sturgeon to 60 d
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
• Analysis of water samples
-Trace metals
-Organic carbon
• Histological and molecular analysis
• Expansion to new sites in 2009
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Thanks to…
•
•
•
•
•
•
•
•
•
David Vardy
Markus Hecker
John Giesy
Marco Adzic
Howard Zhang
Marcie Allan
Hanne Smith
Adam Jonas
Kootenay Trout Hatchery
-Ron Ek
• Karen Smyth
• Eric Higley
• Jonathan Naile
• TeckCominco Metals Ltd.
-Bill Duncan
-Rick Brown
• Selkirk College
• US EPA
• University of Saskatchewan
-Environmental Tox Lab
-Shanda Sedgwick
-Jacinda Duquette
-Liber Lab
-Dube Lab
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Questions?
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
4
In Vitro Assessment of the Endocrine Modulating
Potential of Oil Sands Processed Water
and Naphthenic Acids
Xiaowei Zhang
Steve Wiseman
Paul D. Jones
Markus Hecker
John P Giesy
Mahamed Gamel El-Din
Johnathan Martin
Alberta’s Oil Sands
Contain an estimated 1.7 – 2.5 trillion barrels of bitumen
- 1/3 of known global oil reserves.
Alberta’s Oil Sands
“…The banks of the Athabasca would furnish an in exhaustible supply of fuel..[they] have
found it to contain from 12-15 per cent of bitumen. Although this proportion may appear small,
yet the material occurs in such enormous quantities that a profitable means of extracting
oil…may be found.”
- Robert Bell; Geological Survey of Canada; 1884
Alberta’s Oil Sands ... Mining
Two Mining Practises
1.Surface / Open Pit Mining
Could supply Canada’s energy needs for more than 475 years, or total world needs
for up to 15 years!
- Truck and shovel operations
- Oil sands transported to processing plants
2. In Situ Mining
Production has steadily increased since
the first oil sands development in 1967.
SAG-D (Steam Assisted Gravity Drainage).
CSS – Cyclic Steam Stimulation.
- HSAGD – Hybrid Steam Assisted Gravity drainage
2004 - Oil sands production accounted
for 62 percent of Alberta’s total oil.
2015 - Expected to be 87 percent.
Alberta’s Oil Sands ... Extraction
Clarke Hot Water Extraction
> Hot water and caustic soda added to sand.
> Resulting slurry is piped to the extraction plant
> It is agitated and the oil skimmed from the top.
> Combination of hot water and agitation releases
bitumen from the oil sand.
Alberta’s Oil Sands ... Tailings Ponds
Waste water is stored in tailings ponds.
> For each barrel of oil recovered, 2.5 barrels of waste are generated.
Tailings ponds estimated at more than 550 cubic kilometres and growing.
- Syncrude Mildred Lake site contains over 600 x 10(6) cubic maters of water.
Replacing conventional crude with oil sands to meet the world's energy demands
would require about 700 additional plants.
- Generate a tailings pond the size of Lake Ontario.
Tailings Ponds Chemistry
Basin
Year
[NA]
[NH4]
[NA]
[Ca]
[Cl]
[SO4]
MLSB
1978
50
25
600
20
350
350
WIP
1995
77
15
910
15
510
370
NA are a complex mixture of alkyl-substituted
cycloaliphatic carboxylic acids and acyclic aliphatic
acids.
In Vitro Assessment of the Endocrine Modulating
Potential of Sediment-Free OSPW and Commercial
NA Using the H295R Cell Line
Non-volatile and chemically stable.
Naturally components of petroleum.
Produced by biodegradation of petroleum.
Clemente and Fedorak, 2005
The H295R Cell Line
The H295R Cell Line
Cholesterol
Derived from a human female adrenocortical carcinoma.
CYP11A
CYP17
CYP17
Produces a variety of hormones
Pregnenolone
- androgens and estrogens
- mineralcorticoids
- glucocorticoids
17α-OH-Pregnenolone
3β-HSD
Progesterone
Cell line has been used to investigate endocrine disruption in response to:
- PBDEs
- PCBs
- Pesticides
- Pharmaceuticals
CYP21
11-DeoxyCorticosterone
DHEA
3β-HSD
3β-HSD
CYP17
CYP17
17α-OH-Progesterone
Androstenedione
17β-HSD
CYP21
11-Deoxycortisol
Estrone
CYP19
17β-HSD
CYP11B1
CYP11B2
17β-Estradiol
Cortisol
Corticosterone
Effects on Steroidogenesis have been measured at the level of
- mRNA abundance
- Enzyme activity
- Steroid hormone concentrations
CYP11B2
Aldosterone
Endocrine Modulation Protocol
Incubate 24h
Impact of OSPW on Estradiol Production
Incubate 48h
*
*
Seed Plate
*
Change Media
Dose Cells
% OSPW
Treatment groups:
- OSPW
- Sigma NA
- Merichem NA
CYP19
Testosterone
Estradiol and Testosterone
ELISA
Extract Media
Significant (p< 0.05) increase in estradiol synthesis.
*
*
Impact of NA on Estradiol Production
Merichem
Impact of OSPW on Testosterone Production
Sigma
*
*
*
*
*
*
*
Testosterone
*
% NA
*
*
% NA
% OSPW
Significant (p< 0.05) increase in estradiol synthesis.
Very little impact on testosterone synthesis.
Merichem NA stimulate greater increase in estradiol production.
Summary
Impact of NA on Testosterone Production
OSPW and NA impact estradiol production.
Merichem
Sigma
*
*
*
*
Testosterone production is less impacted by OSPW exposure.
*
*
*
NA have greater impact on testosterone production than OSPW.
*
% NA
% NA
Significant (p< 0.05) increase in testosterone synthesis.
Merichem NA have greater impact on testosterone synthesis than sigma NA.
OSPW as an EDC
Goldfish
Goldfish
(Carassius
(Carassiusauratus)
auratus)
(Lister
(Listeretetal.,
al.,2008)
2008)
Slimy
SlimySculpin
Sculpin
(Cottus
(Cottuscognatus)
cognatus)
Future Directions
Reduced
Reducedplasma
plasmatestosterone
testosteroneand
andestradiol
estradiol
in
ingoldfish
goldfishexposed
exposedto
toOSPW.
OSPW.
>>Based
on
hCG
challenge
assay
steroidogenesis
Based on hCG challenge assay steroidogenesis
remained
remainedfunctionally
functionallyintact.
intact.
>>Exposure
Exposureto
to aaNA
NAextract
extractfailed
failedto
togive
givethe
thesame
same
response.
response.
Reduced
Reducedininvitro
vitroproduction
productionof
ofestradiol
estradioland
and
testosterone
testosteroneby
byovarian
ovarianand
andtesticular
testiculartissues.
tissues.
(Tetreault
(Tetreaultetetal.,
al.,2003)
2003)
Pearl
PearlDace
Dace
(Semotilus
(Semotilusmargarita)
margarita)
(Tetreault
(Tetreaultetetal.,
al.,2003)
2003)
No
Nodifference
differencein
inthe
theininvitro
vitroproduction
productionof
of
testosterone
testosteroneand
andestradiol
estradiolby
byovarian
ovariantissue
tissue
in
inindividuals
individualscollected
collectedfrom
fromreference
referencesites
sites
and
andOSPW.
OSPW.
- Studies need repeating!!!
- Determine Mechanism of Action
- mRNA Abundance
- Aromatase Activity
- Coexposure studies
- Other hormones ???
Impact of OSPW fractions on hormone production.
Aquatic Toxicology of Perfluoroctanesulfonate and Related Chemicals
Authors
Jonathan Naile, Jong Seong Khim,
John Newstead, Paul Jones, and John Giesy Toxicology Centre
University of Saskatchewan Saskatoon, SK, Canada
Aquatic Toxicity Workshop
Saskatoon, SK
October 7th, 2008
Jonathan.naile@usask.ca
Website: http://www.usask.ca/toxicology
What are they?
Background and History
z
z
F
F F
F F
F
O
F
z
O
F F
F
F F
F F
F
F
F F
F F F O
O
S
F
F
F F
F F
F F
F
O
Perfluorooctane sulfonate (PFOS)
Physical/Chemical Properties
• PFOS is a fatty acid analogue
• Log Kow is not useful due to Amphiphilic
properties
• Resistant to hydrolysis, photolysis, and biodegradation
• Preferentially retained in liver and blood
Fundamentally different from traditional organic pollutants
Previously thought to be chemically stable and biologically inert in the environment
F
Perfluorooctanoate (PFOA)
F
Previous research primarily focused on brominated and/or chlorinated halogenated compounds
z
z
Globally distributed in matrices varying form human blood to polar bear tissue
Many uncertainties from analytical methods for quantification, to toxicity to wildlife and humans
Bioaccumulation and concentration
tion
(Laboratory)
• BAF for trout was calculated to be 0.32 ± 0.05, therefore based on lab studies diet does not appear to be the major source for PFOS accumulation in fish
• Enterohepatic recirculation may cause Kow to under predict accumulation
Apparent
Species
Tissue
Bluegill
Edible
Unnedible
Whole
Rainbow trout Carcass
Blood
Liver
a
a
Ku
Kinetic Parameters
Kd
BCFK b Half‐life
BCF 484
1124
(L/kg*d)
(1/d)
(L/kg)
(d)
8.9
22
0.0047
0.0052
1866
4312
146
133
856
‐
‐
‐
16
53
240
260
0.0045
0.048
0.057
0.05
3614
1100
4300
5400
152
15
12
14
Apparent BCF was calculated as the concentration in fish at the end of the exposure phase divided by the average water concentration
BCFK was estimated as Ku/Kd
b
Bioconcentration and Accumulation
(Field)
Acute Ecotoxicology (Fresh water)
Y In general based on the laboratory toxicity studies, PFOS is known to be moderately acutely toxic to aquatic organisms
• However big differences exist between laboratory and field measured results
Trophic level
• Bioaccumulation calculated in the field ranges greatly (6,300 to 125,000 for the common shiner), and is often much higher than what is predicted in the laboratory
• Reasons for the difference include: interspecies variability, sex‐dependent variables, diet over the entire life span, and precursors being metabolized to PFOS
Lemna gibba
Invertebrate
Daphnia magna
Amphibians
Acute Ecotoxicology (Marine)
AS
Invertebrate
Fish
Test organism/Species
Artemia salina
Test Duration
Endpoint
LOEC
(mg/L)
(mg/L)
EC50/LC50/IC50
Robertson 1986
48 h
Survival
9.4
Robertson 1986
Survival
96 h
2nd generation survival
Crassostrea virginica
96 h
96 h
Oncorhynchus mykiss
96 h
Survival
96 h
Survival
96 h
Survival
1.1
8.9
Robertson 1986
3.6
Drottar and Krueger 0.53
Shell growth
<15
>3.0
Drottar and Krueger 13.7
Robertson 1986
13.7
Robertson 1986
>15
Palmer et al. 2002
Chronic Ecotoxicology (Marine) y
Desjardins et al. 2001
7 d
Frond number
29.2
59.1
Desjardins et al. 2001
7 d
48 h
Biomass
Survival
6.6
33.1
31.1
130
Boudreau et al. 2003
48 h
Immobility
0.8
67.2
48 h
Survival/immobility
32
61
48 h
2nd generation survival 12
96 h
Growth
Survival
96 h
Survival
Trophic level
96 h 96 h
Survival
96 h
Survival
Test Species
Test Duration
Endpoint
NOEC
LOEC
(mg/L) (mg/L)
EC50/LC50/IC50 (mg/L)
Test Species
Skeletonema costatum
Mysidopsis bahia
Reference
96 h
Growth (cell density)
93.8
131
Desjardins et al. 2001
96 h
Inhibition of growth rate
93.8
176
Desjardins et al. 2001
96 h
35 d
Growth (cell density)
Growth, # young produced
>3.2
0.24
>3.2
Drottar and Krueger 7.97
5.4
6.3
13
Palmer and Krueger 15.6
9.1
Drottar and Krueger 7.8
Robertson 1986
9.9
Robertson 1986
22
Palmer et al. 2002
Test Duration
Endpoint
NOEC
LOEC
(mg/L)
(mg/L)
Desjardins et al. 2001
Drottar and Krueger 2000
EC50/LC50/IC50 (mg/L)
Reference
96 h
Respiratory inhibition
Microalgae
Selenastrum capricornutum
96 h
Growth (cell density)
96 h
Inhibition of growth rate
42
96 h
Growth (cell density)
150
263
Sutherland and Krueger 2001
96 h
Inhibition of growth 206
305
Sutherland and Krueger 2001
Chlorella vulgaris
96 h
Growth (cell density)
8.2
81.6
Boudreau et al. 2003
Zooplankton community
35 d
Community structure
3
Myriophyllum spicatum
42 d
Biomass, dw
11.4
12.5
42 d
Root length, cm
11.4
16.7
42 d
Biomass, dw
2.9
3.4
Hanson et al. 2005
42 d
Root length, cm
0.3
2.4
Hanson et al. 2005
Daphnia magna
21 d
Adult survival
5.3
42.9
Boudreau et al. 2003
Chironomus tentans
10 d
>0.15
MacDonald et al. 2004
Macroalgae
Myriophyllum sibiricum
Invertebrate
42
>870
Schaefer and Flaggs 2000
68
Drottar and Krueger 2000
121
Drottar and Krueger 2000
Boudreau et al. 2003
Hanson et al. 2005
Hanson et al. 2005
Survival
0.05
10 d
Growth (chlorophyll a)
0.05
Amphibians
Rana pipiens
16 wk
Partial life cycle
0.3
3
6.21
Ankley et al. 2004
Fish
Pimephales promelas
28 d
Microcosm
0.3
3
7.2
Oakes et al. 2005
47 d
Early life stage
0.29
0.58
0.087
MacDonald et al. 2004
Drottar and Krueger 2000
Ecotoxicology for Perfluorobutanesulfonate (PFBS) PFBS was chosen because it one of the main replacement chemicals now used instead of PFOS
Organism
Genus/Species
Acute
Water flea
Fathead minnow
Daphnia magna
Pimephales promelas
Bluegill
Algae Microalgae
Invertebrate
Boudreau et al. 2003
Drottar and Krueger 4.82
3.2
Test
Trophic level
Boudreau et al. 2003
Microorganism community
There is limited chronic marine toxicological data available, but in general it appears that marine microorganisms and invertebrates behave similarly to their freshwater relatives
Microorganisms Anabaena flos‐aquae
Reference
(mg/L)
Microorganisms
Drottar and Krueger 1.8
EC50/LC50/IC50
108
Pimephales promelas
Navicula pelliculosa
9.4
Survival
LOEC
(mg/L) (mg/L)
15
Oncorhynchus mykiss
Reference
Survival
48 h
NOEC
Endpoint
Frond number
Based on the laboratory toxicity studies, PFOS is known to be slightly chronically toxic to aquatic organisms
(mg/L)
48 h
Mysidopsis bahia
Cyprinodon variegatus
NOEC
Test Duration
7 d
Chronic Ecotoxicology (Fresh water)
Limited marine toxicology data exists, and the Sheepshead minnow
(Cyprinodon variegatus) study reports a value above the solubility of PFOS in salt water because they added 0.05% methanol to increase PFOS
solubility.
Trophic level
Xenopus laevis
Fish
• More data is needed to evaluate bioconcentration and bioaccumulation under environmental conditions
Test organism/Species
Macroalgae
Mysid
a
NOEC
LOEC
LC50
duration
Media
(mg/L)
(mg/L)
(mg/L)
Reference
48 h
96 h
FW
FW
886
888
1707
1655
2183
1938
WLI 2001
WLI 2001
Lepomis macrochirus
96 h
FW
2715
5252
6452
WLI 2001
Selenastrum capricornutum
Mysidopsis bahia
96 h
FW
1077
2216
2347
WLI 2001
96 h
SW
127
269
372
WLI 2001
502
995
Chronic
Daphnia magna
21 d
FW
Water flea b
a
Reported data are based on biomass measurements
b
Reported data based on reproduction and length measurements
WLI 2001
Water Quality Criteria for PFOS
Water Quality Criteria (PFCs)
Log Scale
• Purpose: To derive water quality values for those perfluorinated compounds (PFCs) that have sufficient and appropriate toxicity data
• Used the US EPA Great Lakes Initiative methodology because it provided specific procedures and methodologies for utilizing toxicity data to derive water quality values protective of aquatic life
• OVERALL GOAL: To derive toxicity reference values that are protective of aquatic life Quantitative Structure Activity Relationship (QSAR)
• Limited Toxicological data available for many PFCs, so the use a Quantitative Structure Activity Relationship was developed to estimate toxicological data where no measured data is available
• Results show that chain‐length is the most important factor in determining toxicity, although functional head group and the addition of an amide group can also be important
24 mg/L‐CCC for PFBS
121 mg/L‐ CMC for PFBS
25 mg/L‐CMC for PFOA
2.9 mg/L‐CCC for PFOA
21 ng/L‐CMC for PFOS
5.1 mg/L‐CCC for PFOS
47 ng/L‐AWV for PFOS
17 ng/L‐AWV for PFBS
CMC: criteria maximum concentration CCC: criteria continuous concentration AWV: avian wildlife value Quantitative Structure Activity Relationship (QSAR)
• Shorter than 6 or 7 carbons do not tend to accumulate and bioconcentration factors are usually less than 1.0
• Bioconcentration tends to go up by a factor of about 100 with the addition of 2 carbons for PFCs C4 to C8
• Chain‐lengths greater than 12 appear to have reduced toxicity
• Length does not appear to be as important for fluorotelomer alcohols
Quantitative Structure Activity Relationship (QSAR)
Chain‐length not functional group makes the difference
100000
PFAS
Fish Bioconcentration
Factors
10000
Conclusions
• Based on the GLI a protective water concentration of PFOS was calculated to be 0.46mg PFOS/L for chronic exposure and 0.78mg PFOS/L for acute exposure.
• In most cases chain‐length appears to be the most important factor determining PFC toxicity
PFCA
1000
100
• There are big differences between BCF calculated in the field and what has been calculated in the Laboratory
10
1
• There are still many knowledge gaps and more aquatic toxicity data is needed
0.1
0
2
4
6
8
10
Perfluorinated Carbons
12
14
Thank You!
Structure of Perfluorooctane sulfonate (PFOS)
Toxicity of Perfluorooctane Sulfonate (PFOS) to Avian
Wildlife: Ambient Safe Water Derivation and Uncertainty
Analysis
O
C8F17 S - O
PFOS is the ultimate degradation product of
POSF-based compounds and the compound
found in the environment
-
O
J.L. Newsted1, J. Naile2, J. Khim2, P.D. Jones2, J.P. Giesy2,3
F
1
F
ENTRIX, Inc., East Lansing, MI, USA
2
Department of Biomedical and Veterinary Biosciences and Toxicology Centre, University of
Saskatchewan, Saskatoon, Saskatchewan, Canada
C
C
F F
F F
F F
C
FF
C
C
FF
C
F O
C
S O
FO
*
* The compound is a mixture of isomers and homologues.
Biology and Chemistry Department, City University of Hong Kong, Kowloon, Hong Kong, SAR, China
Commercial PFOS is approx. 70 % straight chain and 30 %
branched
2
Global Sampling Locations
PFOS concentrations in North American Waters
1.2
Cumulative Probability
3
F F
F
C
1
0.8
0.6
0.4
Wildlife Value=50 ng/L
0.2
0
0.001
0.01
0.1
1
10
100
1000
10000
PFOS Concentration (ng/L)
3
4
Assessment Approach
˜ Great Lakes Water Quality Initiative or GLI (USEPA 1995)
˜ Comprehensive approach used by regulatory agencies
Where did this number come from?
9 Utilizes environmental properties of chemical
9 Utilizes Environmental Fate Properties
9 Toxicological data for both humans and ecological receptors
˜ Approach assumes primary exposure pathways to receptors of concern
is from water through species specific food chains
5
6
1
Derivation of Safe Water Concentrations for the
Protection of Wildlife
Wildlife Value =
Test Dose
x BW
Uncertainty Factor
W +∑(FTLi xBAF
WL
TLi
Establishing a Toxicological Dose
)
• WV = Wildlife Value in milligrams of PFOS per liter (mg/L)
• TD = Test dose or threshold dose in mg of PFOS per kg per day (mg/kg
body weight-day).
• UF = Overall Uncertainty factor interspecies, toxicological endpoint and exposure
duration extrapolations.
• BW = Average body weight in kilograms (kg) for the representative species.
• FTLi = Species specific average daily amount of food consumed (kg/day) for
trophic level I
• W = Species specific average daily amount of water consumed (L/day)
• BAFWLTLi =Bioaccumulation factor for wildlife food in trophic level i. For
consumption of piscivorous birds by other birds, the BAF is derived by
multiplying the Trophic Level 3 BAF by the biomagnification factor (BMF).
7
8
Thresholds for Avian Species Exposed to PFOS in the Diet
Relevant Avian Toxicological Studies
1000
603: 8-day LC50 juvenile mallard
PFOS Concentration
(mg PFOS/kg in diet)
Laboratory Studies
In vitro
Cwinn, MA et al 2008-Chicken hepatocyte study
In ovo
Molina ED et al. 2007-Chicken embryo toxicity study
In vivo
Newsted JL et al. 2006. Mallard and Bobwhite Quail acute dietary study
Newsted JL et al. 2007. Mallard and Bobwhite quail chronic dietary study
Newsted JL et al. 2005. Derivation of an Avian Toxicity Reference Value
Field Studies
Hoff PD et al. 2005. Biochemical evaluation of song birds collected
from organo-halogen contaminated site
212: 8-day LC50 juvenile bobwhite quail
100
10
141: No mortality concentration mallard acute
70.3 No mortality concentration quail acute
50: LOAEL for mortality in mallard and
quail in chronic dietary study
10: LOAEL reduced survival of quail
offspring from exposed adults
10: LOEL reduced testes size in adult
mallard and quail
1
Arrows indicate various toxicity thresholds for avian species that have been
tested in laboratory studies. Values are dietary PFOS concentrations.
9
10
Uncertainty Factors for a Generic Trophic Level 4
Predator Exposed to PFOS
Derivation of Toxicant Reference Values (TRVs)
US EPA Great Lakes Initiative (GLI)
˜ Toxicity data based on whole-life in vivo studies with
bobwhite and mallards
˜ Application of uncertainty factors
UNCERTAINTY FACTORS (UF)
Values
Inter-taxon Extrapolation (UFA)
Exposure Duration (UFL)
6
2
Toxicological Endpoint (UFS)
2
UF for TRV
11
UF= (6 x 2 x 2) = 24
12
2
Bioaccumulation Factors Derived from Field Studies
Bioaccumulation Factors (BAFs)
Cumulative Probability
1.0
Potential Sources of BAFs
• Field derived values
• Should include multiple trophic level species (TL3 and 4)
• Laboratory based values
• Need to include Food Chain Magnification (FCM) factors
• Values based on Kow
• Structure-Activity
0.9
0.8
0.7
0.6
BAF~13,000
0.5
0.4
0.3
0.2
BAF~3,500
0.1
0.0
0
5000
10000
15000
20000
25000
30000
Bioaccumulation Factors
13
BAF and Water PFOS Concentration Association
PFOS BCFs in Aquatic Vertebrates Species
100000
10000
Log BAF
14
1000
Species
Tissue
Apparent
BCF
Kinetic BCF
(L/kg)
Bluegill
Whole
859
2796
Rainbow trout
Carcass
Liver
-
1100
5400
Carp
Whole
Liver
1300
4300
-
Fathead Minnow
Liver
300 to 600
-
Northern Leopard
Frog
Whole
-
100
10
1
0.0001
0.001
0.01
0.1
1
10
100
Log PFOS Water Concentraton (ug/L)
27.7 to 200
15
Biomagnification Factor for PFOS in Avian Species
Species
Feed
Liver
(ug PFOS/g) (ug PFOS/g)
10
61
6.1
Quail
10
88
8.8
Geometric mean
Surrogate Avian Species Used in Wildlife Value
Estimates
BMF
Mallard
16
Herring Gull (Larus argentatus)
• Order Charadriiformes, Family Laridae
• Feeds on a variety of foods including fish, crustacea, molluscs,
insects, small mammals and birds, and garbage
Bald Eagle (Haliaeetus leucocephalus)
• Order Falconiformes, Family Accipitridae
• Opportunistic feeder that consumes fish, birds, and small mammals
depending on availability
7.3
- BMF values calculated from the dietary chronic studies
Belted Kingfisher (Ceryle alcyon)
• Order Coraciidormes, Family Alcedinidales
• Generally feeds only on fish but when available, will also consume
crayfish.
- Geometric mean of mallard and bobwhite quail BMFs used in the
calculation of wildlife values. BMF = 7.3
- All measured values reported on a wet weight basis
17
18
3
Exposure Parameters for Three Surrogate Avian
Species Identified for Deriving Wildlife Values
Adult
Water
Food ingestion rate of
Body ingestion rate
each prey in each
wt. (kg)
(L/day)
trophic level (kg/day)
Species
Herring gull
1.1
0.063
MODEL ASSUMPTIONS
Bioaccumulation Factors
Trophic level of prey
(% diet)
TL3: 0.192
Fish: 90 (TL3: 80; TL4: 20)
TL4: 0.0480
Other: 10
Trophic Magnification Factor (TFM):
Trophic Level 3 BAF*:
Trophic Level 4 BAF:
Biomagnification Factor (BMF):
Other: 0.0267
Bald Eagle
4.6
0.160
TL3: 0.371
Fish: 92 (TL3: 80; TL4: 20)
TL4: 0.0929
Birds: 8 (PB: 70; other: 30)
Toxicity Data
PB: 0.0283
Test Dose (mg PFOS/kg bw/day):
Total Uncertainty Factor:
Other: 0.0121
Belted
Kingfisher
0.15
0.017
TL3: 0.0672
2.0
2000
4000
7.0
TL3: 100
Note: TL3 or TL4 = trophic level 3 or 4 fish; PB= piscivorous birds; Other = non-aquatic birds and
mammals
0.77
24
*Based on use of the bluegill (2796) and rainbow trout (1100) BCF
19
20
Effects Ranges: Water
PFOS Wildlife Values Concentration for
Avian Species
70,000 ng/L – Lethal to Juvenile birds (LOAEL)
Species
Wildlife Value
(μg PFOS/L)
Herring Gull
0.048
Bald Eagle
0.078
Kingfisher
Geometric Mean
1,500 ng/L – Subtle effects on testes without any effects on survival,
growth or reproduction of quail (LOAEL)
0.029
0.046
TRV: 50 ng/L – No effects, includes safety factor of 24 (EPA GLI)
15 ng/L - Average Great Lakes Water PFOS concentration
21
Comparison of PFOS Concentrations in
Laurentian Great Lakes to its Chronic Value
22
General Conclusions
˜ Concentrations less than 100 ng PFOS/L pose no
environmental hazard to birds
Lake Erie
WV = 50 ng/L
Lake Huron
˜ Values greater than 1,000 would require site-specific
assessments, including sampling to confirm exposures and
population-level effects
Lake Ontario
Mich. Waters
Niagara River
˜ Concentrations greater than 5,000 ng PFOS/L are likely to
cause adverse effects to fish-eating birds
1000
0.10
1.0
10
100
Log PFOS Concentrations (ng/L)
23
24
4
Thank You
˜
John L. Newsted
˜
Entrix,
Entrix, Inc.
˜
Okemos, Michigan 48864 USA
˜
Tel: (517) 381381-1434
˜
Fax: (517) 432432-1984
˜
Email: jnewsted@entrix.com
25
5
Sensitivity of early lifelife-stages of white sturgeon Acipenser transmontana
to copper, cadmium, and zinc
David Vardy1, Amber Tompsett1, Jacinda Duquette1, John Giesy1 , Markus Hecker1,2
1 Department
• A sensitive transition period from yolk sac to exogenous feeding (~day 20-35) was
discovered within the controls and all treatment groups (except the high metal doses where
100% mortality occurred prior to feeding) that promoted fish mortality (Fig 2, 3, 4.)
• The drastic increase in mortalities across all groups during the transition feeding stage has
raised the question of whether it may be more appropriate to test early life-stages of white
sturgeon at independent time intervals and thereby excluding this period of time that is
characterized by a naturally greater mortality.
• All treatment groups experienced greater mortality throughout the exposure period
compared to controls (Fig 6.)
Results
• 100% mortality occurred between hatch and day 10 for the two highest doses of Cu
and the highest dose of Cd and Zn.
• Copper affects sodium regulation across the gills and appears to drastically impact
early life-stages of white sturgeon during the first few days of exposure, especially at
the higher doses (Fig 2,5).
Cumulative Copper Mortality
100
% Mortality
% Mortality
100
80
60
Cu 0.2 ug/L
Cu 1.2 ug/L
Cu 7.2 ug/L
40
Cu 43.2 ug/L
Cu3
Cu4
Cu5
60
10
20
30
60
Cd2
40
Cd3
30
Cd4
20
Cd5
10
Zn1
0
Zn2
40
50
60
Cd3
Cd4
Cd5
Zn1
Zn2
Zn3
Doses
Zn4
Zn5
Zn4
Zn5
Fig 6. Cumulative mortalities for Cu, Cd
and Zn for duration of exposure.
% Mortality after 96h - Cu
Cd 81.92 ug/L
% Mortality after 96h - Cd
% Mortality after 96h - Zn
Control
Cu Lab
125
20
30
40
50
60
70
• Cadmium is known to disrupt calcium uptake but has also been found to
bioaccumulate within the kidneys and liver. In the present study cadmium appears to
have a pronounced acute effect at the highest dose at an early stage (Fig 5) and a
more chronic effect in the second to highest dose towards the end of the exposure
period (Fig 3).
75
50
25
100
75
50
25
0
100
1000
75
50
25
0
10
0
1
ug/L
• Zinc is an essential nutrient and most fish can tolerate relatively high
concentrations. In this study only the highest dose of zinc (1296 µg/L) had a
pronounced effect early in the experiment. A slight increase in mortality was
experienced in the second to highest does near the end of the exposure period
(Fig 4).
Zn In Situ
100
1
Zn Lab
125
Cd In Situ
100
Fig 3. Cadmium cumulative mortalities
Cd Lab
125
Cu In Situ
Day
10
100
1000
10
ug/L
100
1000
LC50 (µg/L) Lab
In Situ
WER
Cu
74.29
38.675 0.520595
Cd
15.786 62.53603 3.961486
Zn
156.2893 646.2608 4.135029
100
Table 1. LC50 values and WER for early life-stages of white sturgeon
exposed to Cu, Cd and Zn in laboratory and UCR water
80
60
Zn 1.0 ug/L
Zn 6 ug/L
Zn 36 ug/L
40
Zn 216 ug/L
Zn 1296 ug/L
Control
20
0
0
10
20
30
40
50
60
70
Day
10000
ug/L
Figure 7. 96hr Acute LC50 tests for Columbia River Water and Standard Laboratory Water (Conducted
through a parallel study with Amber Tompsett)
Cumulative Zinc Mortality
% Mortality
Cd2
0
Cd 0.16 ug/L
10
120
Future and upcoming work:
-Histology and biobio-energetic analyses.
-Metal speciation model
-Benthic invertebrate experiments.
-Slag exposure experiments.
Fig 4. Zinc cumulative mortalities
Acknowledgments:
Cd1
20
Cd 1.28 ug/L
0
Fig 2. Copper cumulative mortalities
Cu5
60
40
• Early-life stages of white sturgeon appear to be less sensitive to Cd and Zn in UCR waters
compared to standard laboratory water and more sensitive to Cu (Fig 7).
• Complexation of metals with organic materials decreases bioavailability and in turn toxicity
to fish and could explain the lower toxicity of Cd and Zn in river water.
• The decrease in Cu toxicity in laboratory water compared to river water is surprising and
merits further investigation.
Cd 10.24 ug/L
20
70
Cu4
80
Cd 0.02 ug/L
40
Day
B
Figure 1. A: Flow-through exposure system; B: Exposure chamber design.
Cd1
0
Cu3
100
Fig 5. Cumulative mortalities for Cu,
Cd and Zn during the first 20 days
0
0
• Further morphological analyses are currently being conducted at the University of
Saskatchewan Toxicology Centre.
80
Cu 259.2 ug/L
Control
0
• Continuous flow-through exposure systems were designed and used to test 5
different exposure concentrations per metal based upon environmentally relevant
concentrations found in the Columbia River and concentrations expected to
produce toxic effects (Fig 1.):
Cu 0.2 µg/L (ppb)—260 µg/L
Cd 0.02 µg/L (ppb)—82 µg/L
Zn 1 µg/L (ppb)—1300 µg/L
• Fertilized white sturgeon eggs were obtained from the Kootenay Trout Hatchery,
Fort Steele, B.C.
• Embryos, larvae and juveniles were exposed for 65 days and the surviving
juveniles were euthanized, measured, weighed and fixed in formalin.
• 96 hr static renewal LC50 tests were conducted with 8-day old larva.
A
Cumulative Cadm ium Mortality
120
20
Methods
Cu2
Zn3
Discussion
120
2. Collect information that will be used along with metal speciation models to predict
thresholds for effects of these metals on eggs and larvae of white sturgeon under
field conditions
Cu2
Cu1
90
80
70
50
Control
Cu1
Control
100
Doses
Objectives
1. Develop a species-specific dose-response relationship that will be used to establish
metal toxicity threshold values for white sturgeon for Cu, Cd and Zn.
Total Cumulative Mortalities
Cumulative Mortalities-Swim
Up Phase (Day 1-20)
• Cd 4 (10.24 µgL) treatment experienced greater mortality near the end of the
exposure period (day 40) than all other treatments.
• 96hr LC50 values for Cu, Cd and Zn were 74.29 µg/L, 15.29 µg/L and 156.29 µg/L,
respectively.
• Water effects ratios (WER) indicate a 4 fold factor for Cd and Zn and a 0.5 fold factor
for Cu between UCR waters and standard laboratory water for early life-stages of
white sturgeon (Table 1).
% Mortality
. It has been reported, however, that juveniles released into the UCR as part of a recovery
initiative exhibit good survival, growth rates and body condition. Habitat alteration, varying
flow regime, poor nutrition, genetic bottlenecks, predation and pollution have all been
suggested as possible explanations. Presently, little toxicity data exist characterizing the
sensitivity of white sturgeon to metals of concern (Cu, Cd, Zn).
Inc., Saskatoon, SK, Canada
% Mortality
There is evidence that adult white sturgeons are spawning and depositing viable eggs in
only certain areas of the Canadian reach of the Columbia River, especially at Waneta
Eddy located just north of the U.S.-Canada border, but only limited numbers of young of
the year (YOY) have been found in habitats considered suitable for this life stage (Golder
Associates Ltd. 2007. White sturgeon spawning at Waneta, 2007 investigations. Report
prepared for Teck Cominco Metals Ltd. Trail Operations. Golder Report No. 07-14800031F: 28p.)
2ENTRIX,
% Mortality
Introduction
of Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada.
% Mortality
Poor recruitment of white sturgeon Acipenser transmontana in the Upper Columbia River
(UCR) has been documented since the 1970s. There are many possible causes for this
phenomenon, including exposure to metals that may influence survival of eggs and or
juveniles. In general, little is known about the potential toxicity of metals such as Cu, Cd,
and Zn to white sturgeon. The purpose of this study was to establish baseline laboratory
toxicity data for the exposure of early life-stages of white sturgeon to Cu, Cd, and Zn that
can be used in risk assessments, and, in combination with field experiments conducted in
a parallel study, to assess the potential toxicity of these metals in waters of the Columbia
River. Embryos, larvae and fry were exposed to increasing concentrations of dissolved
Cu, Cd, and Zn for 65 days using laboratory based flow-through exposure systems. In
addition, 96hr LC50 static toxicity tests were conducted for each metal in order to gather
information to calculate water effect ratios (WER) between laboratory and separate
concurrent field studies (see Amber Tompsett et al.;metal coal and diamond mining
session, ATW). Preliminary results indicate that early life-stages of white sturgeon are
more sensitive to Cu and Zn during the first 20 days post hatch compared to Cd which
had a greater impact during prolonged exposure.
% Mortality
Abstract
Funding for this project was provided by Teck Metals Ltd. Thanks to the Kootenay Trout Hatchery, Dr. Liber’s Lab, Eric Higley, Jonathan Naile, the UofS undergraduate team and the US-EPA for their advise during the planning stage of the studies.
Assessment of Toxicity of Upper Danube River Sediments Using a Combination of Chemical Fractionation,
the Danio rerio Embryo Assay and the Ames Fluctuation Test
Eric Higley1, Stefanie Grund3, Thomas B.-Seiler2, Urte Lubcke-von Varel6 ,Werner Brack6, Tobias Schulz6, Jan Wölz2, Hanno Zielke2, John Giesy1,5, Henner Hollert2, Markus Hecker4
1. University of Saskatchewan, Saskatoon, SK, 2 RWTH, Aachen, Germany, 3 University of Heidelberg, Heidelberg, Germany, 4 ENTRIX, Inc., Saskatoon, SK, 5 City University of Hong Kong, Hong Kong, China, 6 UFZ Leipzig, Germany.
Introduction
Objectives
The world’s river systems provide fresh water to people and support thousands of
species. However, many of the great rivers have been polluted in the past decades.
Possible sources of such pollution include effluents from domestic sewage pants (i.e.
urine and feces, detergents, pharmaceuticals), industry (i.e. PCBs, dioxins, and
metals), agricultural runoff (i.e. pesticides and fertilizers), and storm water runoff from
urban areas (i.e. salts, oil, and antifreeze). Severely contaminated sediments from
many rivers and lakes have been shown to be acutely and chronically toxic to fish and
benthic invertebrate species. For example, sediment samples from the Upper Danube
River that were analyzed in six separate assays were found to have considerable
geno-toxic, cytotoxic, mutagenic, embryo-toxic and estrogenic effects. It has been
hypothesized that decline in fish stocks in the Upper Danube River since the early
1990s may be associated with this pollution. Here, we report on the results of a study
conducted to determine the toxicity of extracts from sediments of the Danube River by
means of the Danio rerio embryo assay, and by assessing lethal and sub lethal
endpoints. In addition, mutagenicity was assessed using the Ames fluctuation assay.
For the sediment samples that revealed toxicity, fractionation of each sample was
performed by separating compounds according to their polarity, planarity, and the size
of the aromatic ring system. 18 fractions for each sediment sample were tested
separately in the Ames fluctuation assay and Danio rerio embryo assay to assess
which group of chemicals within the sediment sample caused the original toxicity.
1. Assess the toxicity of raw sediment extracts from four locations along the Upper
Danube River using the Danio rerio Embryo Assay and Ames Fluctuation Assay
2. Evaluate which groups of chemicals caused the measured toxicities using new
chemical fractionation techniques that separate the raw sediment extracts into 18
different chemical fractions.
3. Analyze all 18 chemical fractions using the Danio rerio Embryo Assay and Ames
Fluctuation Assay.
Results
Figure 1: Map of Germany and sediment sampling locations
Methods
Lauchert reference
Öpfingen
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
***
***
SC
12.5 25.0
***
* ****
***
***
*
50.0 100.0 200.0 400.0
Danio rerio Embryo Assay
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
Sigmaringen
TA98 Strain
*
2.0
Lauchert reference
Lauchert
Öpfingen
Sigmaringen
*
*
12.5
25.0
50.0 100.0 200.0 400.0
Sig
PC
Lauchert reference
Lauchert
Öpfingen
Sigmaringen
Lau
**
15.0
Lau
ref
*
*
Zebrafish are
breed overnight
Add pH
indicator
media
without
histidine
12.5
25.0 50.0 100.0 200.0 400.0
PC
0.0
12.5 25.0 50.0 100.0 200.0 400.0
Incubate at 37 ° C for 48 hours
After 48 hours, any bacteria that have back
mutated and can produce histidine will live and
grow and turn the media from purple to yellow
Count # of wells
that are yellow
14
15
16
X
XXX
X
XXX
X
X
XX
17
XX
X
X
X
XXX
XX
Opf
Lau
Lau ref
SC
12.5
70
60
50
40
30
20
10
0
25
50
+
+
+
+
-
Chemical fractions that showed effects
8
X
10
XX
11
X
XX
X
13
XX
X
X
X
Conclusions
Incubate for 48 hours
in 96 well plate
Record
lethal
and sublethal
effects
after 48
hours
13
XX
PC
Sediment Equivalent Concentrations (mg/ml) Öpfingen
Sigmaringen
Lauchert
Lauchert Reference
in
+ sample
DMSO
Zebra fish
egg + ISO
water
11
X
Sig
SC
Danio rerio embryo assay on whole extracts
Sediment
Place histidine deficient bacteria into 384 well
plate without histidine
10
XX
TA100 Strain
Mortality %
Viable eggs less
than 1 hour old
are collected
9
X
X
With or
Sample
Location without S9
5.0
Figure 2. Dose response of four whole sediment extracts ran in the Ames Fluctuation
Assay with and without the liver enzyme S9 mix and on two different bacteria strains
(TA98 and TA100). * indicates significant difference from control.
Incubate for 90 minutes with histidine
+
+
+
+
-
10.0
Sediment Equivalent Concentration (mg/ml) Sediment
sample in
DMSO
3
S9
0.0
TA100 Bacteria Strain -S9
*
Chemical fractions that showed effects
Sample With or
Location without
1.0
Sediment Equivlaent Concentrations (mg/ml) *
SC
+
Öpfingen
Table 1. Fractions (3 – 17) showing significant increases in the number of mutations
compared to the controls as determined by the Ames Fluctuation Assay. TA98 Bacteria
measures frame shift mutations and TA100 Bacteria measures base pair substitutions.
Sig=Sigmaringen, Opf=Opfingen, Lau=Lauchert, Lau ref=Lauchert Reference.
X = less than a 3-fold increase; XX = 3- to 10-fold increase; XXX = greater than 10 fold
increase.
3.0
SC
M u t a g e n ic E f f e c t ( # o f r e v e r t a n t s )
•Crude sediment extracts and all 18 fractions were analyzed for their toxicity using the
Ames fluctuation assay and Danio rerio egg assay
Bacteria culture
Lauchert
4.0
PC
TA100 Bacteria Strain +S9
M u t a g e n ic E f f e c t ( # o f r e v e r t a n t s ) •Samples were extracted and fractionated into different chemical groups using a new
technique by Varel et al., 2008 that uses 3 HPLC columns and separates the sample
into 18 fractions according to their polarity, planarity and the size of their aromatic
system
deficient
Lauchert reference
Opf
•Sediments were sampled (top 5cm) at four locations along the Upper Danube River
using a Van Veenen grabber in January 2006 (Figure 1)
Histidine
5.0
Sediment Equivalent Concentrations (mg/ml) Sampling and extraction
Ames Fluctuation Assay
TA98 Bacteria Strain -S9
Lauchert
Sigmaringen
M u t a g e n ic E f f e c t ( # o f r e v e r t a n t s )
M u t a g e n ic E f f e c t ( # o f r e v e r t a n t )
TA98 Bacteria Strain +S9
100
Sediment equivalents concentration (mg/ml)
Figure 3. Dose response of four sediment extracts analyzed with the Danio rerio embryo assay
• Mortality of Danio rerio embryos increased in a dose-dependent manner when
exposed to whole sediments collected at Öpfingen and Sigmaringen, but none of the
fractionated samples were toxic. These results indicate that the observed toxicity was
likely due to the combination of groups of chemicals in the whole sediment samples.
• Toxicity was observed for whole sediments from Sigmaringen, Öpfingen and Lauchert
in the Ames Fluctuation Assay only when TA98 bacteria with S9 were tested. Toxicity
was also found in the fractionated samples in both bacterial strains, although the
pattern was inconsistent.
• However, toxicity was measured in fractions 10 and 15 of every sediment sample
except Lauchert reference. Previous work has found that fraction 10 can contain sixringed PAHs (i.e. benzo(a)pyrene or benzo(k)fluoranthene) and fraction 15 can
contain more non-polar chemicals like benzocarbazole and benzanthrone. Further
work using other analytical techniques may identify which chemicals caused the
observed toxicity.
Reference:
Varel U, Streck G, Brack W. 2008. Automated fractionation procedure for polycyclic aromatic compounds in sediment
extracts on three coupled normal-phase high-performance liquid chromatography columns. Journal of
Chromatography A. 1185:31-42