SWASSW | Jenifer McIntyre | Dec. 4 2013

David Baldwin, Nat Scholz - NOAA-Fisheries, NWFSC

The influence of water chemistry on copper neurotoxicity in fish

Ph

oto

by

Mo

rgan

Bo

nd

Copper is neurotoxic

Peripheral sensory system

Mechanosensation (lateral line)

Olfaction (smell)

Gustation (taste)

Olfaction begins at olfactory rosettes

Kuhlia sandvicensis

Anguilla anguilla

Lepisosteus platostomus Eleotris sandwicensis

Hansen &

Zielinski. 2005. J.Neurocytol. 34

Olfactory Sensory Neurons

Microvillous

Ciliated Non-

sensory

Hansen & Zielinski. 2005. J.Neurocytol. 34

Cross-Species Cu Toxicity

All sensitive to olfactory toxicity at

low ppb dissolved Cu

Bioavailability

Is copper in PNW salmon streams bioavailable to the salmon nose?

• Biotic Ligand Model (BLM) = classic metals toxicity in fish

Cu+

Cations

Anions

DOM Na+ transporter

Gill

• Water chemistry determines bioavailability

water tissue

Copper Bioavailability - Fish Gill

• Metals compete with cations at the ‘biotic ligand’

• Metals complex with anions & DOM

Copper Bioavailability – Fish Nose

Odour Receptor Golf

AC

Cation channel

Cl-

Odour molecules

2+

Na+ K+

Do ions & DOM ‘protect’ nose against Cu ?

? Cu+

Cations

Anions

DOM

Nose

ATP

Cl channel

water tissue

0.2 mM Ca 0.2 mM HCO3 0 mg/L DOM

Dissolved Organic Matter Hardness

[Ca] 0.4 0.8 1.6

Alkalinity

[HCO3] 0.8 1.6 3.2

[Fulvic Acid] 2.5 5

10

[Natural Organic Matter]

10 Hi pH Low pH

Low-ion Control

+

One of 3 increases

Copper Bioavailability in Different Waters

• pre-exposure to artificial test water (24-h)

Odour exposure •L-serine, TCA: 10 s each •Alternating odour pulses every 2.5 min.

Fish placed on ‘rig’

• Test water over nose • Measure electrical

response to odours • 15 min acclimation • 30 min Cu exposure

(20 μg/L)

Bioavailability Study Design

Water Quality on Copper Neurotoxicity

•20 μg/L Cu (30 min) significantly inhibits

olfaction of L-ser (and TCA)

•Olfaction improves with ↑ calcium,

bicarbonate, and DOC

•DOC has strongest effect

[Calcium] mM0.0 0.4 0.8 1.2 1.6

020406080

100120 Hardness

[HCO3-] mM0 1 2 3

020406080

100120 Alkalinity

Normal pHLow pH

[DOC] mg/L0 2 4 6

020406080

100120 Dissolved Organic Carbon

Fulvic AcidNOM

% R

elat

ive

Olfa

ctor

y R

espo

nse

No-copper control 95% L.C.L. control

McIntyre et al. 2008. ES&T. 42

Fathead minnow corroboration

0 0.1

10 0.1

ppb Cu mM Ca

10 0.5

10 1.0

1.2 1.0 0.8 0.6 0.4 0.2

0

Green et al. 2010. ES&T 44

Hardness & Alkalinity in PNW Streams

Only 1 stream sample (<1%) had enough bicarbonate for 50% protection

No streams contain enough calcium for even 50% protection

Puget Sound

Cal

cium

(mM

)

0

1

2

3

4

5SacramentoYakimaWillamette

Puget Sound

Bic

arbo

nate

(mM

)

0

2

4

6

8

10

12

14

95% SER

50% SER

Willamette Yakima Sacramento

Copper toxicity reduced 50%

Copper nontoxic

Copper toxicity reduced 50%

Copper nontoxic

McIntyre et al. 2008. ES&T. 42

HC

O3- (

mM

)

C

alci

um (m

M)

DOC in PNW Streams

Puget Sound

DO

C (m

g/L)

0

2

4

6

8

10

12Willamette Yakima Sacramento

DOC should be measured along with dissolved copper concentrations in streams of concern

Copper toxicity reduced 50%

Copper nontoxic

19% of samples

6% of samples

McIntyre et al. 2008. ES&T. 42

DOC protective at: • Fish gill Less protective at: • Nose

Hardness effect similar at: • Gill • Nose

Little protection at: • Nose

Alkalinity • Very protective at fish gill

Water Chemistry Comparison: Gill vs Nose

McIntyre et al. 2008. EST 42

A

B

C

Copper Toxicity to Lateral Line

Linbo et al. 2006. ET&C

Control Fish Copper-exposed

Danio rerio larva

Hair cells

No hair cells

Copper Toxicity to Lateral Line

1. Hardness • CaCl2 • MgSO4 • CaCl2:MgSO4 2. Sodium • NaCl • NaHCO3

3. Dissolved organic matter (DOC)

?

Linbo et al. 2009. ETC

DOC protective at: • Fish gill Less protective at: • Nose

Hardness effect similar at: • Gill • Nose

Little protection at: • Nose

Alkalinity • Very protective at fish gill

Chemistry Comparison

• Lateral line

• Lateral line (Na effect)

• Lateral line McIntyre et al. 2008. EST 42

Mg

Ca

Linb

o et

al.

2009

. ETC

28

Freshwater Cu Bioavailability

Cu+ DOC

Alkalinity

DOC

Alkalinity

Cu+

Cu Toxicity at Fish Gill Cu Toxicity at Fish Nose (and LL)

Hardness Hardness

Important to measure DOC and alkalinity in receiving waters

Fresh vs Seawater Cu Bioavailability

Does saltwater protect against copper sensory toxicity?

DOC Cu+

Saltwater Cu Toxicity

CO32- OH-

HCO3- SO4

2-

Ca2+ Mg2+

DOC Cu+

Freshwater Cu Toxicity

CO32- OH-

HCO3- SO4

2-

Ca2+ Mg2+

Na+ Hardness

Alkalinity

Na+

Important uncertainties relative to toxicity in freshwater

• Salinity - changes in copper complexation, etc.

• DOC influence - changes in copper/DOC interaction

• Physiology - changes in fish upon smolting

Seawater Cu Bioavailability

Olfactory toxicity of copper to seawater-phase salmon

No olfactory toxicity up to 100 ppb in seawater (35 ppt)

Seawater Cu Bioavailability Baldwin et al. Unpublished results.

Linbo et al. 2009. ETC 28

Sodium protective against Cu?

436 mM

Copper toxicity to lateral line neurons

EC50 >500 ppb

Cop

per t

oxic

ity (E

C50

ppb

) (NaCl, NaHCO3)

Measuring Olfactory Response: EOG & EEG

Electro-olfactogram (EOG)

Perfusion Recording electrode

EEG measured at olfactory bulb

Measures changed in electrical current at olfactory epithelium in perfusion water

Measures changed in electrical current at olfactory bulb – no water chemistry interference with recording

Olfaction important to salmon behaviors Cu Reproductiv

e priming & behaviour

Cu

And/or fry emergence

Olfactory predation cues can alter egg hatch timing

Cu

May interfere with imprinting Cu

Copper may be developmentally neurotoxic Cu

Olfaction required for natal stream

Cu homing

Cu Sensory Toxicity: Research Needs

• What are current water chemistry conditions (esp. DOC)? • What changes in water chemistry are predicted? • Is olfactory toxicity in adult salmon similar to juveniles? • Species specific measurements for SOC (e.g. sockeye) • Consequences of Cu toxicity to other olfactory behaviors

– Homing – Reproductive priming and behaviors

• Better understanding of mechanosensory toxicity – Testing in salmonids – Functional impairment vs cell death – Survival consequences of mechanosensory impairment

Acknowledgements

Technical & Field Assistance James Meador (NOAA Fisheries - Montlake) Julann Spromberg NOAA Fisheries – Montlake) Dave Rose (University of Washington) Gordy George (University of Washington) Matt Gilman (WA Dept. Fish & Wildlife) Chris Tatara (NOAA Fisheries - Manchester) Barry Berejikian (NOAA Fisheries - Manchester) Sarah McCarthy (King County – DNR) Evan Malczyk (King County Environmental Lab)

Funding Sources:

NOAA Coastal Storms Program

EPA S.T.A.R. Graduate Fellowship

NOAA Oceans & Human Health

Olfactory Neuroanatomy

ciliated microvillous crypt

Olfactory Sensory Neuron Types

Bile salts Amino acids Steroids Stimulated By:

Feeding Reproductive Alarm Behaviours:

4 hr

(Hansen et al. 1999. E

TC 18)

Num

ber o

lfact

ory

neur

ons

Copper impacts olfactory receptors

Epithelium

Copper can destroy olfactory receptors

Rosette

Dendrites with odour receptors

Axon reaching olfactory bulb

Inhibition of olfaction: • Dose-dependent • Short exposures • Low concentrations

30 min in 10 μg/L Cu

Bal

dwin

et a

l. 20

03.

ET&

C. 2

2:22

66

San

dahl

et a

l. 20

07. E

S&

T

Copper inhibits olfaction Recording electrode

70% inhibition Relative EOG = 0.3

Electro-olfactogram (EOG)