Final Report presented to the Nisqually Indian Tribe 

Characterization of Estuary Use by Nisqually Hatchery 

Chinook Based on Otolith Analysis 

By Angie Lind-Null, Kim Larsen, and Reg Reisenbichler 

Open-File Report 2008-1102 

U.S. Department of the Interior 

U.S. Geological Survey 
U.S. Department of the Interior 

DIRK KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia: 2008 

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Suggested citation: 

Lind-Null, A.M., Larsen, K.A., and Reisenbichler, Reg, 2008, Characterization of estuary use by Nisqually 

Hatchery Chinook based on otolith analysis: U.S. Geological Survey Open-File Report 2008-1102, 12 p. 

Any use of trade, product, or firm names is for descriptive purposes only and does not imply 

endorsement by the U.S. Government. 

Although this report is in the public domain, permission must be secured from the individual 

copyright owners to reproduce any copyrighted material contained within this report. 

ii
iii

Contents 

Introduction .........................................................................................................................................................................1

Objective ..............................................................................................................................................................................2

Methods ...............................................................................................................................................................................2

Results ..................................................................................................................................................................................5

Discussion..........................................................................................................................................................................10

Acknowledgments............................................................................................................................................................11

References.........................................................................................................................................................................11

Figures 

Figure 1: Nisqually field sampling sites. ........................................................................................................................3

Figure 2: Relationship between fish fork length (mm) and otolith radial distance (microns) .............................5

Figure 3: Representative sample of FW growth...........................................................................................................6

Figure 4: The tidal delta check (TDCK) was seen on samples collected in the EFT and EEM 

in mid to late May ...............................................................................................................................................................7

Figure 5: The delta-flats check (DFCK) was seen on samples collected in DF beginning in early July ............9

Figure 6: Mean Increment width (microns) for FW, tidal delta, and DF/NS residence within each habitat...10

Tables 

Table 1: Number of marked juvenile Chinook providing otoliths ..............................................................................2

Table 2: Number of otoliths (one per fish) analyzed / processed.............................................................................4

Table 3: Number of otoliths (one per fish) with a tidal delta check (TDCK) or delta-flats check (DFCK)..........6
Characterization of Estuary Use by Nisqually 

Hatchery Chinook Based on Otolith Analysis 

By Angie Lind-Null, Kim Larsen and Reg Reisenbichler 

Introduction 

The Nisqually Fall Chinook population is one of 27 stocks in the Puget Sound 

evolutionarily significant unit listed as threatened under the federal Endangered Species Act (ESA). 

Preservation and extensive restoration of the Nisqually delta ecosystem are planned to assist in 

recovery of the stock. A pre-restoration baseline including life history types, estuary residence 

time, growth rates, and habitat use are needed to evaluate the potential response of hatchery and 

wild Chinook salmon to restoration. 

Otolith analysis has been selected as a means to examine Chinook salmon life history, 

growth, and residence in the Nisqually estuary. Over time, the information from the otolith 

analyses will be used to: 1) determine if estuary restoration actions cause changes to the population 

structure (i.e. frequency of the different life history trajectories) for Nisqually River Chinook, 2) 

compare pre- and post- restoration residence times and growth rates, 3) suggest whether estuary 

restoration yields substantial benefits for Chinook salmon through (1) and (2), and 4) compare 

differences in habitat use between hatchery and wild Chinook to further protect ESA listed stock. 

Otoliths are calcium carbonate structures in the inner ear that grow in proportion to the 

overall growth of the fish. Daily growth increments can be measured so date and fish size at 

various habitat transitions can be back-calculated. Careful analysis of otolith microstructure can be 

used to determine the number of days that a fish resided in the estuary as a juvenile (increment 

counts), size at entrance to the estuary, size at egress, and the amount that the fish grew while in the 

estuary. Juvenile hatchery Chinook salmon are generally released as smolts that move quickly 

through the delta with much shorter residence times than for many wild fish and are not dependent 

on the delta as nursery habitat (Myers and Horton 1982; Mace 1983; Levings et al. 1986). 

The purpose of this study is to use and evaluate otolith microstructure analysis as a tool for 

assessing the role of the estuary in the life history of hatchery Chinook salmon in the Nisqually 

River before and after restoration efforts at the Nisqually National Wildlife Refuge (Nisqually 

NWR). This tool is used to quantify changes in rate of growth, length of residence and habitat use 

to help predict restoration benefits to the federally threatened Nisqually River hatchery and wild 

Chinook salmon populations. 

Analysis of otolith microstructure typically is superior to the alternative of traditional markrecapture methods. The latter are extremely expensive or inadequate in estuary habitats, typically 

are biased and substantially underestimate use, and do not directly reveal the importance or 

contribution to adult recruitment (i.e., they do not account for any differential survival afterward in 

Puget Sound or the ocean). Analysis of otolith microstructure for these purposes is proving 

successful for the Nisqually wild Chinook stock as well as a similar study that USGS and partners 

are conducting in the Skagit River estuary system located in northern Puget Sound. This work is 

based on research by Neilson et al. (1985). We expect to use the Skagit River as a reference for the 

before/after restoration comparison in the Nisqually River. 

1
Objective 

Characterize the importance of the Nisqually estuary to hatchery Chinook salmon by 1) 

estimating growth rates, 2) residence times, and 3) size at entry to the tidal delta and nearshore 

habitats. 

Methods 

Unmarked and marked juvenile Chinook salmon were collected by the Nisqually Tribe and 

the U.S. Fish and Wildlife Service – Nisqually NWR in March through October of 2004 from 

various sites in the lower Nisqually River, the tidally influenced region of the estuary near the 

river’s mouth (hereafter referred to as tidal delta), and the shallow sub-tidal and intertidal areas 

(accessible by beach seine; hereafter referred to as nearshore) outside of Nisqually delta complex 

(Table 1). Most fish were collected by beach seining in the following distinct habitat zones 

(Cowardin et al. 1979; Figure 1): 

1. Freshwater (FW) – forested slow water habitat on the mainstem Nisqually River without 

tidal influence. 

2. Forested Riverine Tidal (FRT) – riparian forest, mud/silt substrate, and tidal influence 

(uppermost portion of the tidal delta). 

3. Emergent Forested Transition (EFT) – scrub/shrub and marsh vegetation, mud/silt 

substrate, and tidal influence (tidal delta). 

4. Estuarine Emergent Marsh (EEM) – low and high salt marsh vegetation, mud substrate, and 

full tidal influence (lowermost portion of the tidal delta). 

5. Delta Flats (DF) – sparse to no vegetation, mud or gravel/cobble substrate, and large tidal 

fluctuations. 

6. Nearshore (NS) – saltwater, shallow sub-tidal and intertidal areas, vegetation and substrate 

variable. 

7. Pocket Estuary (PE) – sand-spit enclosed estuary with salt marsh vegetation, sand and mud 

substrate, and forested bluffs. 

A few sites within the EEM habitat were sampled with fyke nets. 

Table 1: Number of marked juvenile Chinook providing otoliths. All fish were collected in 2004. Only 

marked fish were used in this study. 

March April May June July August September October TOTAL 

FW 0 0 19 7 0 0 0 0 26 

FRT 0 0 5 14 1 0 0 0 20 

EFT 0 0 31 5 0 0 0 0 36 

EEM 0 0 107 61 24 0 0 0 192 

DF 0 4 15 25 9 0 1 0 54 

NS 0 0 7 7 0 0 0 0 14 

PE 0 0 1 0 0 0 0 0 1 

TOTAL 0 4 185 119 34 0 1 0 343 

2
Figure 1: Nisqually field sampling sites. 

3
Each fish was euthanized and measured for length and weight. The fish were preserved in 

alcohol and sent to USGS where the sagittal otoliths of marked fish were extracted. 

A total of 343 pairs of otoliths were collected from marked fish. All fish otoliths (one from 

each pair), excluding those with a coded wire tag from other than the Nisqually River (n=31) and 

the single fish collected from PE habitat (n=1) were processed according to the Western Fisheries 

Research Center’s (WFRC) standard protocols (Table 2). An additional 59 samples were not 

suitable for analysis because of: (i) presence of vaterite (a morph of the calcium carbonate 

structure), (ii) poor initial quality, (iii) uneven microstructural growth along the radial axis or (iv) 

processing error. In total, 252 samples were analyzed out of the 296 processed marked fish. 

Fish collected from FW showed a consistently recognizable pattern which was used as a 

reference pattern for all fish otoliths collected below FW habitat. This reference pattern had no 

checks beyond the recognizable button-up and first feed checks. A check is a consistently 

prominent mark or pattern on the otolith which interrupts the normal sequence of otolith deposition 

(Campana 1983). Each increment was interpreted as one day’s growth for the fish (Stevenson and 

Campana 1992). Otoliths from fish collected in all other habitat types were visually analyzed for 

additional patterns, checks, or increased growth beyond the identifiers observed on the FW 

residence portion of the otoliths. 

Daily growth increments and checks in the otolith microstructure were measured with the 

aid of a digital imaging system, Image-Pro. We selected a standardized radial axis for 

measurements at 85 ± 5 degrees ventral of the longitudinal axis passing through an identifiable and 

preferred nucleus. Distances along the radial axis and individual increment widths between checks 

or an increase in growth representing change in habitat, were recorded for each fish. 

Growth rates (mm/day) in the tidal delta and DF/NS habitats were calculated from lengths 

based on the Fraser-Lee method (DeVries and Frie 1996): 

aS

S

aL L i

c

c

i + − =

where is the back-calculated length of the fish at the beginning of a habitat transition, is the 

length of the fish at capture, is the radius of the otolith at capture, is the radius of the otolith 

at the beginning of a habitat transition, and is the intercept from the overall regression of capture 

fork length verses otolith radius (Figure 2). Average growth rate and mean increment widths 

(MIW) were determined for all habitat zones. Residence time and fork lengths upon entry to the 

tidal delta and DF/NS habitat zones were also calculated. 

Li Lc

Sc Si

a

Table 2: Number of otoliths (one per fish) analyzed / processed. 

March April May June July August September October TOTAL 

FW 0 / 0 0 / 0 17 / 18 7 / 7 0 / 0 0 / 0 0 / 0 0 / 0 24 / 25 

FRT 0 / 0 0 / 0 5 / 5 12 / 14 1 / 1 0 / 0 0 / 0 0 / 0 18 / 20 

EFT 0 / 0 0 / 0 21 / 28 4 / 5 0 / 0 0 / 0 0 / 0 0 / 0 25 / 33 

EEM 0 / 0 0 / 0 85 / 101 40 / 47 12 / 15 0 / 0 0 / 0 0 / 0 137 / 163 

DF 0 / 0 0 / 2 12 / 13 19 / 22 5 / 5 0 / 0 0 / 1 0 / 0 36 / 43 

NS 0 / 0 0 / 0 7 / 7 5 / 5 0 / 0 0 / 0 0 / 0 0 / 0 12 / 12 

PE 0 / 0 0 / 0 0 / 0 0 / 0 0 / 0 0 / 0 0 / 0 0 / 0 0 / 0 

TOTAL 0 / 0 0 / 2 147 / 172 87 / 100 18 / 21 0 / 0 0 / 1 0 / 0 252 / 296 

4
y = 0.1477x + 2.1703

R2

 = 0.4809

45

65

85

105

125

425 525 625 725

Otolith radius (microns)

Fish FL at capture (mm)

Figure 2: Relationship between fish fork length (mm) and otolith radial distance (microns). 

Results 

A check coincident with release from the Clear Creek (6 May – 4 June 2004) and Kalama 

(14 May – 1 June 2004) hatcheries was difficult to determine because the fish were volitionally 

released or perhaps a release check does not exist. Lack of a release check might reflect direct and 

rapid movement to the tidal delta once fish leave the hatchery; however we have no direct evidence 

for this possibility. Since a release check was not identified, FW growth included for analysis 

represents time spent in the hatchery prior to release and time spent in the river after hatchery 

release. 

The increments on all otoliths became more legible and consistent across the radial axis 

beyond the first-feed check (Figure 3). An interruption in the microstructure pattern, designated as 

a tidal delta check (TDCK), was detected on samples collected within tidal delta habitats EFT and 

EEM beginning in mid to late May indicating transition to tidal delta habitat following volitional 

release from the hatcheries (Table 3, Figure 4). The check was abbreviated in some samples 

(n=16), possibly because the fish were caught immediately upon entrance to the habitat. 

Increments were consistently thin with narrow spacing across the radial axis until the TDCK 

appeared. At this point, the increments became consistently thicker with wider spacing, indicating 

an increase in growth with habitat transition from FW habitat to tidal delta habitat. No TDCK or 

increase in growth was seen on otoliths from fish collected in FW or FRT. Hereafter, the tidal delta 

is in reference to EFT and EEM habitats only. 

5
20x objective 

Figure 3: Representative sample of FW growth. The letters 

below represent: H = hatch, B = button-up, FF = first feed, 

FW = FW residence. 

Table 3: Number of otoliths (one per fish) with a tidal delta check (TDCK) or delta-flats check 

(DFCK). Dashes indicate where a check should not be expected 

May June July 

TDCK DFCK TDCK DFCK TDCK DFCK 

FRT 0 - 0 - 0 - 

EFT 3 - 4 - 0 - 

EEM 10 - 36 - 12 - 

DF 0 0 14 0 4 3 

NS 6 0 4 0 0 0 

TOTAL 19 0 58 0 16 3 

6
20x objective 

40x objective 

Figure 4: The tidal delta check (TDCK) was seen on samples 

collected in the EFT and EEM in mid to late May. The check was 

bold and prominent consisting of two thick dark bands with a wider 

white space between. Beyond the TDCK, increments were 

consistently wider indicating an increase in growth. The letters 

below represent: H = hatch, B = button-up, FF = first feed, FW = FW 

residence, TDCK = tidal delta check, and TD = tidal delta residence. 

7
In addition to the TDCK, an additional check was seen on some otoliths collected in DF 

habitat beginning in early July. We called this check a delta-flats check (DFCK). It indicated the 

fish’s transition from tidal delta habitat to the DF/NS habitat (Figure 5). This check looked 

identical to the NS check located on Chinook in the Skagit River system (Beamer et al. 2000). Due 

to classification of sites, we called this check a DFCK instead of a NS check. The number of 

samples containing a DFCK that were collected in the DF habitat was considerably low (3 out of 

36). A DFCK was not seen on samples collected in NS habitat possibly due to the lack of samples 

from later in the season. 

No difference was visually observed in the microstructure pattern between EFT and EEM. 

To further validate this observation, a one-way ANOVA was run to test for significant differences 

between EFT and EEM. No significant difference occurred in the MIW (P>.05) and therefore the 

MIW data were combined and classified as “tidal delta.” There was a significant difference 

(P<.05) in the growth rate and therefore the growth rate data were not combined. This apparent 

discrepancy seems to have resulted because the otoliths from EFT fish were small for the size of 

the fish (5 out of 7). Any given increment width of these fish corresponded to a greater growth on 

body size compared to the EEM fish. FRT was not included as part of the tidal delta habitat for 

analysis because visually the microstructure pattern did not differ from FW samples nor was an 

additional check or an increase in growth ever observed on FRT samples as stated previously. 

We tested for differences in MIW in the FW and tidal delta portions of the otoliths (Figure 

6). One-way ANOVA showed a significant difference (P<.05) across habitats for the FW portion 

and no significant difference (P>.05) for the tidal delta portion. On average, fish from FRT and 

FW habitats had the smallest FW MIW. In general, the MIW of the FW portion of all otolith 

samples was smallest followed by the tidal delta and DF/NS habitats, respectively. 

The equivalent results for growth rate were that the FW growth rates (mean= .40 mm/day) 

were lower compared to the tidal delta growth rates for fish residing in the EFT (mean = .67 

mm/day), EEM (mean = .59 mm/day), DF (mean = .61 mm/day), and NS (mean = .55 mm/day) 

habitats, with a 50% increase in growth from FW habitat to tidal delta habitat. No significant 

difference was found between tidal delta and DF/NS growth rates (one-way ANOVA, P>.05). The 

increase in growth from the tidal delta habitat to the DF habitat was 2%, however sample size was 

small (n=3). 

The average fork length upon entry to the tidal delta was 86.3 mm. Fish caught in the tidal 

delta spent an average of 9 days with a minimum residence time of 4 days and a maximum of 20. 

These samples provided a minimum estimate of residence because the fish were sacrificed prior to 

entering the Sound. Evaluation of those fish caught in the DF habitats exhibited an average 

residence time of 8 days in the tidal delta (n = 3). Normally, this value would represent a truer 

estimate of residence time in the tidal delta, however sample size was small (n=3). Fish caught in 

the DF were on average 88 mm when they entered the tidal delta whereas fish caught in the NS 

were 83.1 mm upon entrance to the tidal delta. We were able to measure size at entrance (78.8 

mm) and exit (82.4 mm) for the three fish caught in the DF with a DFCK. 

8
20x objective 

40x objective 

Figure 5: The delta-flats check (DFCK) was seen on samples collected in DF beginning in early July. 

The check was bold and prominent consisting of two thin dark bands encompassing two wide bright 

bands containing a thick dark band between them. Beyond the DFCK, increments were consistently 

wider indicating an increase in growth. The letters below represent: H = hatch, B = button-up, 

FF = first feed, FW = FW residence, TDCK = tidal delta check, TD = tidal delta residence, 

DFCK = delta-flats check, and NS = DF/NS residence. 

9
2.25

2.75

3.25

3.75

4.25

4.75

0123456

Location of Capture 

Mean Increment Width (microns)

FW

Tidal Delta (EFT and EEM)

DF/NS

(24)

(18)

(64)

(162)

(3)

(18)

(36)

(10)

(12)

FW FRT Tidal 

Delta 

(EFT and EEM)

DF NS

Figure 6: Mean Increment width (microns) for FW, tidal delta, and DF/NS residence within each 

habitat. One sample collected in the tidal delta was excluded from the tidal delta portion of the MIW 

analysis because residence time was only one day. The number of samples are represented in 

parentheses. Error bars represent ±1 standard deviation. 

Discussion 

A check associated with release from the Clear Creek and Kalama hatcheries could not be 

identified on samples possibly due to volitional releases. Collection of samples directly from the 

hatchery prior to ponding events may help us to identify the release check. Presence of a release 

check will allow us to determine how quickly hatchery fish move to the delta and estimate FW 

growth in the river following release. 

We characterized a Nisqually-specific signature of otolith microstructure growth patterns 

and checks for hatchery Chinook that allowed us to distinguish entry into the tidal delta and DF/NS 

habitats following volitional release from the hatcheries. The TDCK first appeared in mid to late 

May on samples from EFT and EEM habitats whereas the DFCK first appeared in early July on DF 

samples. It is unclear whether a DFCK appeared on DF samples in April due to limited sample size 

(n=2). A DFCK was never seen on samples collected in the NS habitat possibly due to the lack of 

samples from later in the season. No check or increase in growth was visible on any FW or FRT 

samples regardless of time of year. 

The MIW generally increased as fish moved from FW habitat to tidal delta to NS. The 

magnitude of the difference in MIW between the tidal delta and DF/NS habitats probably is 

underestimated and may be an artifact of low sample size (n=3). 

10
There was a corresponding increase in growth rate as the fish migrated from FW to tidal 

delta to NS habitats. Fish grew 50% faster in the tidal delta than in FW. Fish grew only slightly 

faster (2%) in the DF compared to the tidal delta, however this estimate was based on a very small 

sample size (n=3). 

Our results suggest that otolith microstructure analysis can be a valuable tool to establish a 

baseline for use of the Nisqually River estuary habitats by juvenile hatchery Chinook salmon under 

existing conditions. However, this study provides limited information due to small sample sizes in 

some months. Collection and analysis of additional hatchery Chinook especially from DF and NS 

habitat zones later in the season (July – October) should be addressed. Furthermore, these 

collections should occur over several years to allow adequate evaluation of inter-annual variation in 

microstructure growth patterns and checks, and may reveal additional life history types. Of course, 

further work should include analysis of hatchery adults because they show the proportions and 

numbers of adults that reared in the delta as juveniles. 

Acknowledgments 

This research was a collaborative effort between the Nisqually Indian Tribe and the U.S. 

Geological Survey, Western Fisheries Research Center. A special thanks to Sayre Hodgson, Chris 

Ellings, Jeanette Dorner, Jean Takekawa, Marian Bailey, Jennifer Cutler, Nano Perez, Craig Smith, 

John Kerwin, NNWR volunteers, and WDFW staff at Lakewood/Garrison Springs, Clear Creek, 

Kalama, Tumwater Falls Acclimation Pond, Hupp Springs, Minter Creek, Soos Creek, Voights 

Creek, White River, and Clark’s Creek Hatcheries, including Jim Jenkins, Bill St. Jean, Mary 

Evans, John Lovrak, Wayne Tran, Sherman Davis, Richard Johnson, and Blake Smith. 

The use of trade, firm, or corporation names in this publication is for the information and 

convenience of the reader. Such use does not constitute an official endorsement or approval by the 

United States Department of Interior or the United States Geological Survey of any product or 

service to the exclusion of others that may be suitable. 

References 

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report number 3 [online]. Available from http://www.skagitcoop.org/documents.html [accessed 

18 September 2007]. 

Campana, S.E. 1983. Calcium deposition and otolith check formation during periods of stress in 

coho salmon, Oncorhynchus kisutch. Comp. Biochem. Physiol. 75A(2): 215-220 

Cowardin, L.M., V. Carter, F.C. Golet, and E.T. LaRoe. 1979. Classification of wetlands and 

deepwater habitat of the United States. Department of the Interior, Fish and Wildlife Service, 

Washington, D.C. 79 p. 

DeVries, D.R. and R.V. Frie. 1996. Determination of age and growth. In Fisheries Techniques, 

2nd edition. Edited by B.R. Murphy and D.W. Willis. American Fisheries Society, Bethesda, 

Maryland. pp. 483-512. 

Levings, C.D., C.D. McAllister, and B.D. Chang. 1986. Differential use of the Campbell River 

estuary, British Columbia, by wild and hatchery-reared juvenile Chinook salmon (Oncorhynchus 

tshawytscha). Can. J. Fish. Aquat. Sci. 43: 1386-1397. 

Mace, P.M. 1983. Bird predation on juvenile salmonids in the Big Qualicum estuary, Vancouver 

Islands. Can. Tech. Rep. Fish. Aquat. Sci. 1176: 79 p. 

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12

Myers, K.W., and H.F. Horton. 1982. Temporal use of an Oregon estuary by hatchery and wild 

juvenile salmon. In Estuarine Comparisons. Edited by V.S. Kennedy. Academic Press, New 

York, NY. pp. 377-392. 

Neilson, J.D., G.H. Geen, and D. Bottom. 1985. Estuarine growth of juvenile Chinook salmon 

(Oncorhynchus tshawytscha) as inferred from otolith microstructure. Can. J. Fish. Aquat. Sci. 

42: 899-908. 

Stevenson, D.K., and S.E. Campana (Editors). 1992. Otolith microstructure examination and 

analysis. Can. Spec. Publ. Fish. Aquat. Sci. 117: 130 p.