aquatic toxicity workshop-2008 - usask.cas new/atw-2008-presentation… · the reproduction system...

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.

University of Saskatchewan,Michigan State University, & ENTRIX

Evaluation of Environmental Endocrine Disrupting Chemicals Using the Medaka

HPG Axis Model

Xiaowei Zhang, Ph.D. Prof. John Giesy, Ph.D., Dr. Markus Hecker,

Dr. Paul Jones, Dr. John Newsted Dr. June-Woo Park, Ms. Amber Tompsett

University of Saskatchewan

35th Annual Aquatic Toxicology Workshop

Oct 5-8, 2008, Saskatoon, SK, Canada


AbstractA graphical system model was developed for the testing and evaluation of environmental EDCs using medaka (Oryzias latipes). The model illustrates the key pathways that are associated with the reproduction system (hypothalamic-pituitary-gonadal (HPG) axis). Real time RT-PCR array was developed to examine expression profiles of 36 genes in brain, liver and gonad. Evaluated by examining effects of five model compounds. The medaka HPG axis model provides a powerful tool

To delineate mechanism of toxicityTo quantitatively predict the adverse effects on reproduction.

1


Greatest concern is that exposure to endocrine disruptors duringcritical periods of development may predispose individuals to adverse health effects at later stages of life.A large number of environmental chemicals need to be tested for potential endocrine disrupting effectsMechanism of action (MOA) is required to evaluate the risk of chemical exposure.

Introduction IEndocrine 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)IPCS, 1998 (Global Assessment)

2 University of Saskatchewan,Michigan State University, & ENTRIX

• Small fish model (i.e. medaka, fathead minnow, zebrafish)– Small body size– Relatively rapid life-cycle – Standard, validated technique for culture in the lab– Literatures of basic biological/toxicological attributes

• Ecotoxicogenomics– Transcriptionomics, proteomics and metabolomics: maximize the

information collected from each animals;– Whole genome sequences are publicly available for medaka and

zebrafish;– System models can integrate high-dimensional data to aid in

mechanism understanding.

Introduction II

3


Medaka HPG axis

1. Hypothalamus 2. Pituitary

• LH: luteinizing hormone• FSH: follicle-stimulating

hormone

3. Gonad1. T: testosterone2. E2:17β-estradiol3. KT:11-ketotestosterone4. HDL: high-density lipid5. LDL: low-density lipid

4. Liver

4

Villeneuve et al, EST 2007


Methods

• SYBR Green technology • 384-well format /ABI system• 3 reference genes

PCR arrayPCR array

ExposureExposure • Fluorescence in situ hybridization (FISH)

• Fecundity (egg production)• BSI: brain-somatic index• HSI: hepatic-somatic index• GSI: gonadal-somatic index

• Animal: 4 month adult medaka• Exposure: 5 model chemicals • Sex: 5 male : 5 female per tank• Vehicle control: DMSO• RNA isolation: brain, liver & gonads

Other endpointsOther endpoints

5


Cumulative Fecundity

EE2: 17α-ethinylestradiol; TRB: 17β-trenbolone

n =3 n =3

6

* p < 0.05


(p < 0.05)17α-ethinylestradiol (EE2)

♂♂

• Increased VTG conc.• Feminization• Liver: Increased HSI for

males

1. Up-regulation of ER-α and egg precursor genes

2. Down-regulation of CYP173. Down-regulation of brain

androgen receptor (AR)(disordered male sexual behaviors)

7


♀♀Prochloraz exposure

• Pesticide• Inhibitor of CYP17

and CYP19

• Down-regulation of activin: retarded oocyte maturation

• T and E2 ↓ (Ankley et al 2005)

• Fecundity ↓

• Compensatory response: Up-regulation of CYP17 and CYP19A


Fluorescence in situ hybridization (FISH)

A: Control ovary hybridized with sense probe

B: Control ovary, antisense probeC: 7 day exposure of 500 ng EE2/L D: 7 day exposure of 5000 ng TRB/L

9

• 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.

CYP19A mRNA in the ovary


Fecundity v.s. Gene Expression


Summary

• 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

12


Application and Future Work

1. 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. Risk assessment1. Diagnostic (or retrospective) risk assessment2. Predictive risk assessment


Strategic to Achieve Results (STAR) grant from US EPA Computational Toxicology Program of the US EPA, ORD and OSCP,

and the US EPA ORD Service Center/NHEERL

Acknowledgements

14

The Toxicology Centre at the University of Saskatchewan

Prof. John P. Giesy, Ph.D.Dr. Markus Hecker Dr. Junewoo ParkMs Amber TompsettDr. Paul JonesDr. John Newsted

Dr. Doris Au (CityU HK)Prof. Rudolf Wu (CityU HK)

Dr. Daniel Villeneuve (US EPA)


Related Publications 1. 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)


Xiaowei Zhang, Ph.D.

Toxicology CentreUniversity of Saskatchewan44 Campus Drive, Saskatoon, SK, S7N5B3, CanadaTel: 306-966-1204Fax: 306-966-4796Email: [email protected]

Lab Web Site: http://www.usask.ca/toxicology/jgiesy/

16

Thank you!Thank you!


Fecundity v.s. Gene Expression

Chemical Conc. Effects (%)

EE25 ng/L 91.0%

50 ng/L 92.4%

500 ng/L 65.1%

TRB50 ng/L 99.8%

500 ng/L 46.1% (*)

5000 ng/L 26.0% (*)

Prochloraz3 ug/L 95.5%

30 ug/L 49.8% (*)

300 ug/L 18.0% (*)

Ketoconazole3 ug/L 89.7%

30 ug/L 84.2%

300 ug/L 20.3% (*)

Fadrozole 50 ug/L 20.4% (*)

♀♀

5 ng/L

50 ng /L

500 ng/L

1.0 ug/L

10.0 ug/L

100 ug/L

3 ug/L

30 ug /L

30 ug /L

3 ug/L

30 ug /L

300 ug/L

50 ng /L

500 ng/L

5000 ng/L

E E 2

F A D

K TC

P RO

TRB

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1

Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan

White sturgeon hatch and survival after exposure to Columbia River surface water at two sites in British Columbia, Canada

Amber TompsettAquatic Toxicity Workshop

Saskatoon, SKOctober 6, 2008


Background

• Poor recruitment of white sturgeon in the trans-boundary region of the Columbia

• Adult sturgeon spawn and lay fertilized eggs, which hatch • 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


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

• May act either alone or in combination


Possible Sources of Pollution

• Metal smelter-Liquid effluent-Granular slag/sediment

• Pulp and paper mill


Project Objectives• 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


Site Location

2



Site Locations

Reference Site

Downstream Site

Canada

U.S.A.

Trail

Rossland

Fruitvale

Smelter Site


Experiment Setup

• Retrofitted commercial trailers

• Identical trailer at each site

• Continuous river water supply

• Controls at upstream site


Exposure Systems

85L Reser-

voir

Recirculating System (205 L)

Overflow to River

Fully Replace Every 6h (205 L)

River Intake

40 L Streams


Experimental Design

• 4 replicates per treatment

• 3 chambers per replicate

• Maintenance of WQ-adequate flow rates-chilled water


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

3


Methods: Exposure Termination

• Survival

• Weight and length

• Abnormal morphology

• Preservation for:-histology-molecular biology-contaminant analysis


C D UTreatment

60

70

80

90

Perc

ent H

atch

Results: Survival to Hatch

• No significant treatment differences

• Lower hatch rates in river water treatments-insufficient flow in hatching jars-fungal growth


Exposure MortalityCumulative Mortality

0

10

20

30

40

50

60

70

80

90

12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60

Day of Exposure

% D

ead

ControlUpstreamDownstream

% M

orta

lity


Day 1 7 20 60

Egg Yolk Sack Larvae

Exogenous Feeding Larvae

Fertilization Hatch Transition to exogenous feeding

Transition to juvenile

Exposure initiation Exposure termination

Why do the fish die?


Cumulative Mortality: Yolk-sac Larvae

0

5

10

15

20

25

30

35

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Day of Exposure

% M

orta

lit ControlUpstreamDownstream

% M

orta

lity


Cumulative Mortality: Exogenous Feeding Larvae

0

2

4

6

8

10

12

14

16

18

3839 4041424344 4546474849 5051525354 5556575859 6061

Day of Exposure

% M

orta

lit ControlUpstreamDownstream

% M

orta

lity

4


Why do more control fish die?

R2=0.983!!!


Absolute Survival: A Better Measure?

• No significant treatment differences

• Similar survival even with different stocking rates


Tentative Conclusions

• 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


What next?• Further analysis of survival and termination data

• Analysis of water samples -Trace metals-Organic carbon

• Histological and molecular analysis

• Expansion to new sites in 2009


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


Questions?

In Vitro Assessment of the Endocrine Modulating Potential of Oil Sands Processed Water

and Naphthenic Acids

Xiaowei Zhang Steve Wiseman Paul D. Jones

Markus HeckerJohn P Giesy

Mahamed Gamel El-DinJohnathan Martin

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

Contain an estimated 1.7 – 2.5 trillion barrels of bitumen - 1/3 of known global oil reserves.

Could supply Canada’s energy needs for more than 475 years, or total world needs for up to 15 years!

Alberta’s Oil Sands

Production has steadily increased sincethe first oil sands development in 1967.

2004 - Oil sands production accounted for 62 percent of Alberta’s total oil.

2015 - Expected to be 87 percent.

Alberta’s Oil Sands ... Mining

Two Mining Practises

1.Surface / Open Pit Mining- Truck and shovel operations- Oil sands transported to processing plants

2. In Situ Mining- SAG-D (Steam Assisted Gravity Drainage). - CSS – Cyclic Steam Stimulation.- HSAGD – Hybrid Steam Assisted Gravity drainage

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 ... Extraction

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.

Waste water is stored in tailings ponds.> For each barrel of oil recovered, 2.5 barrels of waste are generated.

Alberta’s Oil Sands ... Tailings Ponds

Tailings Ponds Chemistry

NA are a complex mixture of alkyl-substitutedcycloaliphatic carboxylic acids and acyclic aliphatic acids.

Non-volatile and chemically stable.

Naturally components of petroleum.

Produced by biodegradation of petroleum.

Basin

MLSB 1978 50 25 600 20 350 350

WIP 1995 77 15 910 15 510 370

Year [NA] [NH4] [NA] [Ca] [Cl] [SO4]

Clemente and Fedorak, 2005

In Vitro Assessment of the Endocrine Modulating Potential of Sediment-Free OSPW and Commercial

NA Using the H295R Cell Line

The H295R Cell Line

Derived from a human female adrenocortical carcinoma.

Produces a variety of hormones- androgens and estrogens- mineralcorticoids- glucocorticoids

Cell line has been used to investigate endocrine disruption in response to: - PBDEs- PCBs- Pesticides- Pharmaceuticals

Effects on Steroidogenesis have been measured at the level of- mRNA abundance- Enzyme activity- Steroid hormone concentrations

The H295R Cell LineCholesterol

Pregnenolone

Progesterone

11-Deoxy-Corticosterone

Corticosterone

Aldosterone

17α-OH-Pregnenolone

11-Deoxycortisol

Cortisol

17α-OH-Progesterone

DHEA

Androstenedione

Testosterone Estrone

17β-Estradiol

CYP11A

3β-HSD

CYP21

CYP17

CYP11B2

CYP19

CYP11B2

CYP17

CYP21

CYP11B1

3β-HSDCYP17

CYP17

3β-HSD

17β-HSD

17β-HSD CYP19

Endocrine Modulation Protocol

Seed Plate

Incubate 24h

Change MediaDose Cells

Incubate 48h

Extract MediaEstradiol and Testosterone ELISATreatment groups:

- OSPW- Sigma NA- Merichem NA

Impact of OSPW on Estradiol Production

% OSPW

*

****

Significant (p< 0.05) increase in estradiol synthesis.

Impact of NA on Estradiol Production

Sigma*

* *

Merichem

* *

* ** *

Significant (p< 0.05) increase in estradiol synthesis.

Merichem NA stimulate greater increase in estradiol production.

% NA% NA

Impact of OSPW on Testosterone Production

% OSPW

Testosterone *

Very little impact on testosterone synthesis.

Impact of NA on Testosterone Production

Sigma

**

*

Merichem

*

* *

**

Significant (p< 0.05) increase in testosterone synthesis.

Merichem NA have greater impact on testosterone synthesis than sigma NA.

% NA% NA

Summary

OSPW and NA impact estradiol production.

Testosterone production is less impacted by OSPW exposure.

NA have greater impact on testosterone production than OSPW.

OSPW as an EDC

Goldfish Reduced plasma testosterone and estradiol(Carassius auratus) in goldfish exposed to OSPW.

> Based on hCG challenge assay steroidogenesis(Lister et al., 2008) remained functionally intact.

> Exposure to a NA extract failed to give the same response.

Slimy Sculpin Reduced in vitro production of estradiol and(Cottus cognatus) testosterone by ovarian and testicular tissues.

(Tetreault et al., 2003)

Pearl Dace No difference in the in vitro production of (Semotilus margarita) testosterone and estradiol by ovarian tissue

in individuals collected from reference sites (Tetreault et al., 2003) and OSPW.

Goldfish Reduced plasma testosterone and estradiol(Carassius auratus) in goldfish exposed to OSPW.

> Based on hCG challenge assay steroidogenesis(Lister et al., 2008) remained functionally intact.

> Exposure to a NA extract failed to give the same response.

Slimy Sculpin Reduced in vitro production of estradiol and(Cottus cognatus) testosterone by ovarian and testicular tissues.

(Tetreault et al., 2003)

Pearl Dace No difference in the in vitro production of (Semotilus margarita) testosterone and estradiol by ovarian tissue

in individuals collected from reference sites (Tetreault et al., 2003) and OSPW.

Future Directions

- Studies need repeating!!!

- Determine Mechanism of Action- mRNA Abundance- Aromatase Activity- Coexposure studies- Other hormones ???

Impact of OSPW fractions on hormone production.

Aquatic Toxicity WorkshopSaskatoon, SK

October 7th, 2008

Aquatic Toxicology of Perfluoroctanesulfonate and Related Chemicals

Authors

Jonathan Naile, Jong Seong Khim,John Newstead, Paul Jones, and John Giesy

Toxicology CentreUniversity of Saskatchewan Saskatoon, SK, Canada

[email protected]: http://www.usask.ca/toxicology

What are they?

Perfluorooctanoate (PFOA)

Perfluorooctane sulfonate (PFOS)

FO

O

F F

F F

F F

F F

F F

F F

F F

SO

O

F

FF

F F

F F

F F

F F

F F

F F

F F O

Background and History

Previous research primarily focused on brominated and/or chlorinated halogenated compounds

Fundamentally different from traditional organic pollutants

Previously thought to be chemically stable and biologically inert in the environment

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

Physical/Chemical Properties

• PFOS is a fatty acid analogue

• Log Kow is not useful due to Amphiphilicproperties

• Resistant to hydrolysis, photolysis, and biodegradation

• Preferentially retained in liver and blood

tionBioaccumulation and concentration

(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 Ku Kd BCFK b Half‐life

Species Tissue BCF a (L/kg*d) (1/d) (L/kg) (d)

Edible 484 8.9 0.0047 1866 146Unnedible 1124 22 0.0052 4312 133

Whole 856 16 0.0045 3614 152Carcass ‐ 53 0.048 1100 15Blood ‐ 240 0.057 4300 12Liver ‐ 260 0.05 5400 14

b BCFK was estimated as Ku/Kd

Kinetic Parameters

Bluegill

Rainbow trout

a Apparent BCF was calculated as the concentration in fish at the end of the exposure phase divided by the average water concentration

Bioconcentration and Accumulation(Field)

• However big differences exist between laboratory and field measured results

• 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

• More data is needed to evaluate bioconcentration and bioaccumulation under environmental conditions

Y Acute Ecotoxicology (Fresh water)

NOEC LOEC EC50/LC50/IC50

(mg/L) (mg/L) (mg/L)

Macroalgae Lemna gibba 7 d Frond number 15 108 Desjardins et al. 2001

7 d Frond number 29.2 59.1 Desjardins et al. 2001

7 d Biomass 6.6 31.1 Boudreau et al. 2003

Invertebrate Daphnia magna 48 h Survival 33.1 130 Boudreau et al. 2003

48 h Immobility 0.8 67.2 Boudreau et al. 2003

48 h Survival/immobility 32 61 Drottar and Krueger

48 h 2nd generation survival 12 Drottar and Krueger

Amphibians Xenopus laevis 96 h Growth 4.82 7.97 15.6 Palmer and Krueger

Fish Pimephales promelas 96 h Survival 3.2 5.4 9.1 Drottar and Krueger

Oncorhynchus mykiss 96 h Survival 7.8 Robertson 1986

96 h Survival 9.9 Robertson 1986

96 h Survival 6.3 13 22 Palmer et al. 2002

Trophic level Test organism/Species Test Duration

Endpoint Reference

In general based on the laboratory toxicity studies, PFOS is known to be moderately acutely toxic to aquatic organisms

ASAcute Ecotoxicology (Marine)

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 PFOSsolubility.

NOEC LOEC EC50/LC50/IC50

(mg/L) (mg/L) (mg/L)

Invertebrate Artemia salina 48 h Survival 9.4 Robertson 1986



Mysidopsis bahia 96 h Survival 1.1 3.6 Drottar and Krueger

96 h 2nd generation survival 0.53 Drottar and Krueger

Crassostrea virginica 96 h Shell growth 1.8 >3.0 Drottar and Krueger

Fish Oncorhynchus mykiss 96 h Survival 13.7 Robertson 1986


Cyprinodon variegatus 96 h Survival <15 >15 Palmer et al. 2002

Trophic level Test organism/Species Test Duration

Endpoint Reference

Chronic Ecotoxicology (Fresh water)

Test Species NOEC LOEC

(mg/L) (mg/L)

Microorganisms Microorganism community 96 h Respiratory inhibition >870 Schaefer and Flaggs 2000

Microalgae Selenastrum capricornutum 96 h Growth (cell density) 42 68 Drottar and Krueger 2000

96 h Inhibition of growth rate 42 121 Drottar and Krueger 2000

Navicula pelliculosa 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 Boudreau et al. 2003

Macroalgae Myriophyllum spicatum 42 d Biomass, dw 11.4 12.5 Hanson et al. 2005

42 d Root length, cm 11.4 16.7 Hanson et al. 2005

Myriophyllum sibiricum 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

Invertebrate Daphnia magna 21 d Adult survival 5.3 42.9 Boudreau et al. 2003

Chironomus tentans 10 d Survival 0.05 >0.15 MacDonald et al. 2004

10 d Growth (chlorophyll a) 0.05 0.087 MacDonald et al. 2004

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 Drottar and Krueger 2000

Trophic level Test Duration

Endpoint EC50/LC50/IC50 (mg/L)

Reference

Based on the laboratory toxicity studies, PFOS is known to be slightly chronically toxic to aquatic organisms

yChronic Ecotoxicology (Marine)

There is limited chronic marine toxicological data available, but in general it appears that marine microorganisms and invertebrates

behave similarly to their freshwater relatives

Test Species NOEC LOEC

(mg/L) (mg/L)

Microorganisms Anabaena flos‐aquae 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

Microalgae Skeletonema costatum 96 h Growth (cell density) >3.2 >3.2 Desjardins et al. 2001Invertebrate Mysidopsis bahia 35 d Growth, # young

produced0.24 Drottar and Krueger 2000

Trophic level Test Duration

Endpoint EC50/LC50/IC50 (mg/L)

Reference

Ecotoxicology for Perfluorobutanesulfonate (PFBS)

PFBS was chosen because it one of the main replacement chemicals now used instead of PFOS

Test NOEC LOEC LC50Organism Genus/Species duration Media (mg/L) (mg/L) (mg/L) Reference

AcuteWater flea Daphnia magna 48 h FW 886 1707 2183 WLI 2001Fathead minnow Pimephales promelas 96 h FW 888 1655 1938 WLI 2001

Bluegill Lepomis macrochirus 96 h FW 2715 5252 6452 WLI 2001

Algae a Selenastrum capricornutum

96 h FW 1077 2216 2347 WLI 2001

Mysid Mysidopsis bahia 96 h SW 127 269 372 WLI 2001

Chronic

Water flea b Daphnia magna 21 d FW 502 995 WLI 2001a Reported data are based on biomass measurementsb Reported data based on reproduction and length measurements

Water Quality Criteria for PFOS

• 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

Water Quality Criteria (PFCs)

21 ng/L‐CMC for PFOS

47 ng/L‐AWV for PFOS

2.9 mg/L‐CCC for PFOA

25 mg/L‐CMC for PFOA121 mg/L‐ CMC for PFBS

17 ng/L‐AWV for PFBS

5.1 mg/L‐CCC for PFOS

24 mg/L‐CCC for PFBS

Log Scale

CCC: criteria continuous concentration CMC: criteria maximum concentration

AWV: avian wildlife value

• 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

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


0.1

1

10

100

1000

10000

100000

0 2 4 6 8 10 12 14

Perfluorinated Carbons

Fish

Bio

conc

entr

atio

nFa

ctor

s

PFASPFCA

0.1

1

10

100

1000

10000

100000

0 2 4 6 8 10 12 14

Perfluorinated Carbons

Fish

Bio

conc

entr

atio

nFa

ctor

s

PFASPFCA

Chain‐length not functional group makes the difference


• 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

• There are big differences between BCF calculated in the field and what has been calculated in the Laboratory

• There are still many knowledge gaps and more aquatic toxicity data is needed

Conclusions

Thank You!

1

Toxicity of Perfluorooctane Sulfonate (PFOS) to Avian Wildlife: Ambient Safe Water Derivation and Uncertainty Analysis

J.L. Newsted1, J. Naile2, J. Khim2, P.D. Jones2, J.P. Giesy2,3

1ENTRIX, Inc., East Lansing, MI, USA2Department of Biomedical and Veterinary Biosciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada3 Biology and Chemistry Department, City University of Hong Kong, Kowloon, Hong Kong, SAR, China

2

Structure of Perfluorooctane sulfonate (PFOS)

The compound is a mixture of isomers and homologues. Commercial PFOS is approx. 70 % straight chain and 30 %

branched

O

C8F17 S - O-

O

C C C C C C C C SO

O OF

FF

F F

FF

F F

FF

F F

F F

F F

PFOS is the ultimate degradation product of POSF-based compounds and the compound

found in the environment

*

*

3

Global Sampling Locations

4

PFOS concentrations in North American Waters

0

0.2

0.4

0.6

0.8

1

1.2

0.001 0.01 0.1 1 10 100 1000 10000

PFOS Concentration (ng/L)

Cum

ulat

ive

Prob

abili

ty

Wildlife Value=50 ng/L

5

Where did this number come from?

6

Assessment Approach

Great Lakes Water Quality Initiative or GLI (USEPA 1995)

Comprehensive approach used by regulatory agencies

Utilizes environmental properties of chemical

Utilizes Environmental Fate Properties

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

2

7

• 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)• BAFWL

TLi =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).

Derivation of Safe Water Concentrations for the Protection of Wildlife

∑( )+=

WLTLiTLi xBAFFW

FactortyUncertainDoseTest

ValueWildlifeBWx

8

Establishing a Toxicological Dose

9

Relevant Avian Toxicological Studies

Laboratory StudiesIn vitro

Cwinn, MA et al 2008-Chicken hepatocyte studyIn ovo

Molina ED et al. 2007-Chicken embryo toxicity studyIn vivo

Newsted JL et al. 2006. Mallard and Bobwhite Quail acute dietary studyNewsted JL et al. 2007. Mallard and Bobwhite quail chronic dietary studyNewsted JL et al. 2005. Derivation of an Avian Toxicity Reference Value

Field StudiesHoff PD et al. 2005. Biochemical evaluation of song birds collected

from organo-halogen contaminated site

10

1

10

100

1000Thresholds for Avian Species Exposed to PFOS in the Diet

Arrows indicate various toxicity thresholds for avian species that have been tested in laboratory studies. Values are dietary PFOS concentrations.

PFO

S Co

ncen

trat

ion

(mg

PFO

S/kg

in d

iet)

603: 8-day LC50 juvenile mallard

212: 8-day LC50 juvenile bobwhite quail

141: No mortality concentration mallard acute70.3 No mortality concentration quail acute50: 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

11

Derivation of Toxicant Reference Values (TRVs)

Toxicity data based on whole-life in vivo studies with bobwhite and mallards

Application of uncertainty factors

12

Uncertainty Factors for a Generic Trophic Level 4Predator Exposed to PFOS

UNCERTAINTY FACTORS (UF) Values

Inter-taxon Extrapolation (UFA) 6Exposure Duration (UFL) 2

Toxicological Endpoint (UFS) 2

UF for TRV UF= (6 x 2 x 2) = 24

US EPA Great Lakes Initiative (GLI)

3

13

Bioaccumulation Factors (BAFs)

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

14

Bioaccumulation Factors Derived from Field Studies

0.00.10.20.30.40.50.60.70.80.91.0

0 5000 10000 15000 20000 25000 30000

Bioaccumulation Factors

Cum

ulat

ive

Prob

abili

ty

BAF~13,000

BAF~3,500

15

1

10

100

1000

10000

100000

0.0001 0.001 0.01 0.1 1 10 100

Log PFOS Water Concentraton (ug/L)

Log

BAF

BAF and Water PFOS Concentration Association

16

PFOS BCFs in Aquatic Vertebrates Species

27.7 to 200-WholeNorthern Leopard Frog

-300 to 600LiverFathead Minnow

--

13004300

WholeLiver

Carp

11005400

--

CarcassLiver

Rainbow trout

2796859WholeBluegill

Kinetic BCF(L/kg)

ApparentBCF

TissueSpecies

17

Biomagnification Factor for PFOS in Avian Species

- 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

- BMF values calculated from the dietary chronic studies

Species Feed(ug PFOS/g)

Liver(ug PFOS/g)

BMF

Mallard 10 61 6.1

Quail 10 88 8.8

Geometric mean 7.3

18

Surrogate Avian Species Used in Wildlife Value Estimates

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

Belted Kingfisher (Ceryle alcyon)• Order Coraciidormes, Family Alcedinidales• Generally feeds only on fish but when available, will also consume

crayfish.

4

19

Exposure Parameters for Three Surrogate Avian Species Identified for Deriving Wildlife Values

Herring gull 1.1 0.063 TL3: 0.192

TL4: 0.0480

Other: 0.0267

Fish: 90 (TL3: 80; TL4: 20)

Other: 10

Species

AdultBody

wt. (kg)

Wateringestion rate

(L/day)

Food ingestion rate ofeach prey in each

trophic level (kg/day)

Trophic level of prey

(% diet)

Bald Eagle 4.6 0.160 TL3: 0.371

TL4: 0.0929

PB: 0.0283

Other: 0.0121

Fish: 92 (TL3: 80; TL4: 20)

Birds: 8 (PB: 70; other: 30)

BeltedKingfisher

0.15 0.017 TL3: 0.0672 TL3: 100

Note: TL3 or TL4 = trophic level 3 or 4 fish; PB= piscivorous birds; Other = non-aquatic birds andmammals

20

MODEL ASSUMPTIONS

Bioaccumulation FactorsTrophic Magnification Factor (TFM): 2.0Trophic Level 3 BAF*: 2000Trophic Level 4 BAF: 4000Biomagnification Factor (BMF): 7.0

Toxicity DataTest Dose (mg PFOS/kg bw/day): 0.77Total Uncertainty Factor: 24

*Based on use of the bluegill (2796) and rainbow trout (1100) BCF

21

PFOS Wildlife Values Concentration for Avian Species

SpeciesWildlife Value

(μg PFOS/L)

Herring Gull 0.048

Bald Eagle 0.078

Kingfisher 0.029

Geometric Mean 0.046

22

Effects Ranges: Water

70,000 ng/L – Lethal to Juvenile birds (LOAEL)

1,500 ng/L – Subtle effects on testes without any effects on survival,growth or reproduction of quail (LOAEL)

TRV: 50 ng/L – No effects, includes safety factor of 24 (EPA GLI)

15 ng/L - Average Great Lakes Water PFOS concentration

23

Comparison of PFOS Concentrations in Laurentian Great Lakes to its Chronic Value

Lake Erie

Lake Huron

Lake Ontario

Mich. Waters

Niagara River

Log PFOS Concentrations (ng/L)1.0 10 100 10000.10

WV = 50 ng/L

24

General Conclusions

Concentrations less than 100 ng PFOS/L pose no environmental hazard to birds

Values greater than 1,000 would require site-specific assessments, including sampling to confirm exposures and population-level effects

Concentrations greater than 5,000 ng PFOS/L are likely to cause adverse effects to fish-eating birds

5

25

Thank You

John L. NewstedJohn L. Newsted

EntrixEntrix, Inc., Inc.

Okemos, Michigan 48864 USAOkemos, Michigan 48864 USA

Tel: (517) 381Tel: (517) 381--14341434

Fax: (517) 432Fax: (517) 432--19841984

Email: Email: [email protected]@entrix.com

AbstractAbstractPoor 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.

1 Department of Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada.2ENTRIX, Inc., Saskatoon, SK, Canada

Acknowledgments: 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.

David Vardy1, Amber Tompsett1, Jacinda Duquette1, John Giesy1 , Markus Hecker1,2

MethodsMethods• 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.• Further morphological analyses are currently being conducted at the University of

Saskatchewan Toxicology Centre.

Sensitivity of early lifeSensitivity of early life--stages stages of of white sturgeon white sturgeon Acipenser transmontanaAcipenser transmontanato copper, cadmium, and zincto copper, cadmium, and zinc

IntroductionIntroductionThere 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. 07-1480-0031F: 28p.)

. 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).

Figure 1. A: Flow-through exposure system; B: Exposure chamber design.

ResultsResults• 100% mortality occurred between hatch and day 10 for the two highest doses of Cu

and the highest dose of Cd and Zn.• 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).

DiscussionDiscussion• 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).

• 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).

• 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).

Cumulative Copper Mortality

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Day

% M

orta

lity

Cu 0.2 ug/LCu 1.2 ug/LCu 7.2 ug/LCu 43.2 ug/LCu 259.2 ug/LControl

Cumulative Cadmium Mortality

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Day

% M

orta

lity

Cd 0.02 ug/LCd 0.16 ug/LCd 1.28 ug/LCd 10.24 ug/LCd 81.92 ug/LControl

Fig 2. Copper cumulative mortalities Fig 3. Cadmium cumulative mortalities

Cumulative Zinc Mortality

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Day

% M

orta

lity

Zn 1.0 ug/LZn 6 ug/LZn 36 ug/LZn 216 ug/LZn 1296 ug/LControl

Fig 4. Zinc cumulative mortalities

• 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.)

• 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.

% Mortality after 96h - Cu

0

25

50

75

100

125

1 10 100 1000ug/L

% M

orta

lity

Cu Lab

Cu In Situ

% Mortality after 96h - Cd

0

25

50

75

100

125

1 10 100 1000ug/L

% M

orta

lity

Cd Lab

Cd In Situ

% Mortality after 96h - Zn

0

25

50

75

100

125

10 100 1000 10000ug/L

% M

orta

lity

Zn Lab

Zn In Situ

Figure 7. 96hr Acute LC50 tests for Columbia River Water and Standard Laboratory Water (Conducted through a parallel study with Amber Tompsett)

LC50 (µg/L) Lab In Situ WERCu 74.29 38.675 0.520595Cd 15.786 62.53603 3.961486Zn 156.2893 646.2608 4.135029

Table 1. LC50 values and WER for early life-stages of white sturgeon exposed to Cu, Cd and Zn in laboratory and UCR water

Future and upcoming work:Future and upcoming work:--Histology and bioHistology and bio--energetic analyses. energetic analyses. --Metal speciation modelMetal speciation model--Benthic invertebrate experiments.Benthic invertebrate experiments.-Slag exposure experiments.

A B

ObjectivesObjectives1. 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.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

Total Cumulative Mortalities

0

20

40

60

80

100

Doses

% M

orta

lity

Control

Cu1

Cu2

Cu3

Cu4

Cu5

Cd1

Cd2

Cd3

Cd4

Cd5

Zn1

Zn2

Zn3

Zn4

Zn5

Cumulative Mortalities-Swim Up Phase (Day 1-20)

0

10

20

30

40

50

60

70

80

90

100

Doses

% M

orta

lity

Control

Cu1

Cu2

Cu3

Cu4

Cu5

Cd1

Cd2

Cd3

Cd4

Cd5

Zn1

Zn2

Zn3

Zn4

Zn5

0

Fig 6. Cumulative mortalities for Cu, Cdand Zn for duration of exposure.

Fig 5. Cumulative mortalities for Cu, Cd and Zn during the first 20 days

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

Assessment of Toxicity of Upper Danube River Sediments Using a Combination of Chemical Fractionation,

the Danio rerio Embryo Assay and the Ames Fluctuation Test

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.

IntroductionThe 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.

MethodsSampling and extraction•Sediments were sampled (top 5cm) at four locations along the Upper Danube River using a Van Veenen grabber in January 2006 (Figure 1)

•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 sampleinto 18 fractions according to their polarity, planarity and the size of their aromatic system

•Crude sediment extracts and all 18 fractions were analyzed for their toxicity using the Ames fluctuation assay and Danio rerio egg assay

Zebra fish egg + ISO

water

+Sediment sample in

DMSO

Incubate for 48 hours in 96 well plate

Record lethal

and sub-lethal

effects after 48 hours

Zebrafish are breed overnight

Viable eggs less than 1 hour old

are collected

HistidinedeficientBacteria culture

Sediment sample in

DMSO+

Incubate for 90 minutes with histidine

Add pH indicator

media without

histidine

Place histidine deficient bacteria into 384 well plate without histidine

Incubate at 37 ° C for 48 hours

After 48 hours, any bacteria that have backmutated and can produce histidine will live and grow and turn the media from purple to yellow

Count # of wells that are yellow

Ames Fluctuation Assay Danio rerio Embryo Assay

Objectives1. 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.

Conclusions • 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 Lauchertin 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 six-ringed 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.

0.02.04.06.08.0

10.012.014.016.0

SC 12.5 25.0 50.0 100.0 200.0 400.0 PC

Sediment Equivalent Concentrations (mg/ml)

Mut

agen

ic E

ffec

t (#

of rev

erta

nt)

Lauchert reference LauchertÖpfingen Sigmaringen

0.0

1.0

2.0

3.0

4.0

5.0

SC 12.5 25.0 50.0 100.0 200.0 400.0 PC

Sediment Equivlaent Concentrations (mg/ml)

Mut

agen

ic E

ffec

t (#

of rev

erta

nts)

Lauchert reference Lauchert

Öpfingen Sigmaringen

0.02.04.06.08.0

10.012.014.016.0

SC 12.5 25.0 50.0 100.0 200.0 400.0 PC

Sediment Equivalent Concentration (mg/ml)

Mut

agen

ic E

ffec

t (#

of rev

erta

nts)



0.0

5.0

10.0

15.0

SC 12.5 25.0 50.0 100.0 200.0 400.0 PC

Sediment Equivalent Concentrations (mg/ml)

Mut

agen

ic E

ffec

t (#

of rev

erta

nts)



TA98 Bacteria Strain +S9 TA98 Bacteria Strain -S9

TA100 Bacteria Strain +S9 TA100 Bacteria Strain -S9

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.

Results

Figure 3. Dose response of four sediment extracts analyzed with the Danio rerio embryo assay

010203040506070

100502512.5SCSediment equivalents concentration (mg/ml)

Mor

talit

y %

ÖpfingenSigmaringenLauchertLauchert Reference

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.

Figure 1: Map of Germany and sediment sampling locations

Chemical fractions that showed effects TA100 Strain

-XX+Lau ref

-XX+Lau

-XXXX+Opf

-XXXX+Sig

1311108With or

without S9Sample Location

14

X

Chemical fractions that showed effectsTA98 Strain

-+Lau

ref

X-XXXX+Lau

-XX+Opf

X-XXX+Sig

13111093With or without

S9

Sample Location

XXX

XX

XXX

XXXX

15

XX

X

16

XX

17

******

******

*******

**

*

**

*

*

** *

*

Danio rerio embryo assay on whole extracts

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

aquatic toxicity workshop-2008 - usask.cas new/atw-2008-presentation… · the reproduction system...

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