Alternative methods for marking otoliths: enriched stable isotopes

and fluorescent dyes

Andrew R. Munro

> 60 million native fish stocked in MDB over past 30 years

Fate of stocked fish unknownsuccess of stockingeffects on ecology

Lack of suitable methods for marking hatchery fish

CWTAlizarin complexone

Murray-Darling Basin

Native fish stocking - Australia

Enriched stable isotopesOtolith marking experiments

Fingerling immersion Larval immersionBroodstock injection (transgenerational marking)

Osmotic induction of fish with fluorescent compounds

Developing methods for marking hatchery fish

Stable isotopes

Barium

natural relative abundances

137Ba = 11.30% 138Ba = 71.70%

137Ba 11.30

138Ba 71.70= = 6.38

Non radioactive

Enriched stable isotopes

0

20

40

60

80

100

Natural Enriched

% a

bu

nd

ance

137Ba

138Ba

81.90%11.30%

BaCO3 – enriched in 137Ba

Enriched Isotopes

Hypothesis: can alter otolith isotopic ratios by exposing fish to specific isotopes

Enriched Ba added (µg/L)

0 5 10 15 20

138 B

a/13

7 Ba

0

2

4

6

8

Fingerling immersion

Reared juvenile golden perch in varying levels of enriched Ba for different lengths of time

1d 4d 8d 24d

0 µg/L 1 µg/L 5 µg/L 15 µg/L

Munro et al. (2008) CJFAS

Analysis

Otolith analysis: either whole or sectioned otoliths

Transects/spot analyses

Measured isotopes of interest (e.g.137Ba & 138Ba)

LA-ICPMS (single collector)

8 d immersion – 15 µg137Ba/L

Fingerling immersion results

Distance from edge (m)

020406080100120140

13

8 Ba

/13

7 Ba

0

2

4

6

8

Ba rationatural ratio

Length of immersion (d)

control 1 4 8 24

13

8 Ba/1

37 B

a

0

2

4

6

1 µg/L5 µg/L Ba15 µg/L Ba

Fingerling immersion results

Significant mark 1 d @ 15 µg/L

100% marked 8 d @ 15 µg/L

Base water concentration ~ 15 µg/L Munro et al. (2008) CJFAS

Fingerling immersion summary

• Altered otolith Ba isotope ratio

• 100% mark success – 8 d @ 15µg/L

• Unambiguous mark – not natural

• Stress free

• Requires extended holding time

0 h 1 d 21 d

20 µg/g 2 fish 2 fish X

40 µg/g 2 fish 2 fish 2 fish

Maternal dose rate (137Ba)

Length of time prior to hormone injection

Brood stock injection

Munro et al. (2009) JFB

Distance from edge (µm)

0 10 20 30 40 50 60

138 B

a/13

7 Ba

0

2

4

6

8

Ba ratio

natural Ba ratio

Maternal parent injected with 40 µg/g of enriched 137Ba at same time hormone

Brood stock injection results

nucleus

Conc. (µg/g)

Injection (d)

4020Treatment

Control 0 1 0 1 21

138 B

a/137 B

a

0

1

2

3

4

5

6

7

Brood stock injection results

100%

Munro et al. (2009) JFB

• Altered otolith Ba isotope ratio

• 40 µg/g at time of hormone 100% mark

• Fits in with standard hatchery practices

• Variable spawning success of injected fish

Brood stock injection summary

Larval immersion

Reared larval golden perch in varying levels of enriched Ba for different lengths of time

1d 2d 3d 5d

0 µg/L 30 µg/L 60 µg/L 90 µg/L

Woodcock et al. (2011) EFF

Larval immersion results

Days exposure

Control 1 2 3 5

138 B

a/13

7 Ba

0

1

2

3

4

5

6

7

30 µg/L60 µg/L 90 µg/L

100 % mark success

Base water concentration ~ 10 µg/L Woodcock et al. (2011) EFF

Larval immersion summary

• compatible with hatchery procedures

• 100% mark success

• high density = less isotope

• mark location known

• most cost effective

• variable survival to stocking• 30–50%

Calcein marked golden perch otoliths

Method developed for marking Atlantic salmon with calcein (Mohler 2003)

Also trialed alizarin red S as a cheaper alternative

5% Salt Freshwater Calcein

3.5 min 5 minRinse

Osmotic induction marking

Crook et al. (2007, 2009) NAJFM

Control Calcein treatment ARS treatment

Dissecting microscope - white light

Dissecting microscope - fluorescence filters

9 months post-marking

Control Calcein treatment ARS treatment

Dissecting microscope - white light

Dissecting microscope - fluorescence filters

9 months post-marking

Control Calcein treatment

Dissecting microscope - white light

Dissecting microscope - fluorescence filters

ARS treatment

9 months post-marking

Practical and objective way of identifying

marks on live fish in the field

Non-lethal field detection

GFP portable fluorometer

Calcein marked fish – 26 months

Billabong Ck Edward R Narrandera

Sam

ple

:ref

eren

ce f

luo

resc

ence

rat

io

Unmarked fish

Reference

0

0.5

1

1.5

2 Signal overload

Calcein marked

n=12

n=12n=32

n=20

Non-lethal field detection

Advantages:

Marking procedure is easy and quick (15 min)

Detectable on live fish in the field

Excellent accuracy (100% after 18 months in field, 26 months in lab)

Disadvantages:

Adjustments to hatchery protocols required

Chemicals must be disposed of appropriately

Unknown longevity of external marks

Osmotic induction marking summary

Method $ per 1,000 fingerling Notes

Isotope immersion (137Ba)

fingerlings 9.80 15 µg/L @ 10 fish/L

larvae 1.60 30 µg/L @ 250 fish/L

Isotope injection (137Ba) 0.66 - 19.14 20 µg/g

Osmotic induction

Calcein 37.00 0.5% @ 800 fish/L

ARS 0.50 0.05% @ 800 fish/L

Marking Costs

Cost comparison for marking

Method $ per 1,000

Immersion

fingerling 9.80

larvae 1.60

Injection 0.66 - 19.14

Calcein (OI) 37.00

ARS (OI) 0.50

Thermal 6.25

CWT 83

ALC (10-400 mg/L) 11.48 - 459

Cost comparison for reading marks

reading cost/fish

Enriched isotopes $14.50 - $45.00

Calcein & field detector $0.00

Thermal marking $5.00 - $14.00

Coded wire tags $2.14

Alizarin complexone $3.75

marking/1,000

$1.60

$37.00

$6.25

$83.00

$11.48

Scaling up

Osmotic induction Calcein marking

• 60,000 fish• batches ~4,000 – 5,000 fish

Larval marking w/isotopes

- ~100,000 fish- few ml isotope solution

Multiple enriched stable isotopes4 Ba isotopes 15 unique combinations

Multiple unique batch marks

138Ba/136Ba

0 10 20 30 40

138 B

a/1

37 B

a

0

5

10

15

20

25

NaturalControl136Ba137Ba138Ba136Ba & 137Ba136Ba & 138Ba137Ba & 138Ba136Ba, 137Ba & 138Ba

3 isotopes7 unique marks

Woodcock et al. (2011) EFF

Many more if include isotopes of other elements (e.g. Sr, Mg)

Multiple unique batch marks

88Sr/86Sr

3 4 5 6 7 8

138 B

a/13

7 Ba

3

4

5

6

7

n = 6/treatment

error bars: 95% CI

0 µg/L100 µg/L 25 µg/L

0 µg/L

1 µg/L

15 µg/L

8 unique marks; 96% mark success Munro et al. (2008) CJFAS

All methods able to produce distinctive mark in fish otoliths

Most cost effective method larval immersion in enriched isotopes

Combine with osmotic induction at fingerling stage

external & internal mark

Investigate effects on growth & survival

Investigate survival & dispersal of stocked fish, & impacts of stocking

Methods have potential for use in other areas (e.g. larval dispersal)

Summary

Acknowledgements

Co-investigators: Bronwyn M. GillandersDavid A. CrookSkye H. WoodcockAndrew C. Sanger

Funding: Australian Research CouncilMurray-Darling Basin Authority

And many others who have helped.Photo: NSW Fisheries - The Fisherman, 1962