Mar Biol (2007) 151:483–494 DOI 10.1007/s00227-006-0477-z
R E S E A R C H A RT I C L E
Scales of variation in otolith elemental chemistry of juvenile staghorn sculpin (Leptocottus armatus) in three Pacific Northwest estuaries J. A. Miller
Received: 3 January 2006 / Accepted: 29 August 2006 / Published online: 20 October 2006 Springer-Verlag 2006
Abstract Although distinct otolith elemental signatures are often observed in fish collected from different estuaries, significant differences are also observed among sites within estuaries. Variation at these smaller spatial scales is not well quantified and has the potential to lead to inappropriate interpretations of otolith elemental data. To quantify variation at multiple scales, the otolith elemental composition (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, and Pb:Ca) of juvenile staghorn sculpin (Leptocottus armatus Girard, 1854) collected from five sites within three estuaries, the Columbia River (two sites) and Coos Bay (one site), Oregon, and Humboldt Bay, California (two sites), was examined. Using laser ablation-inductively coupled plasma mass spectrometry, each otolith was sampled at three zones: (1) within the primordium, which represents the egg and early larval periods; (2) at the outer edge, which represents the juvenile period just prior to collection; and (3) midway between the primordial and edge samples, which represents the late larval and early juvenile period. There were significant differences in otolith metal-to-calcium ratios at all scales examined. Using multi-element otolith signatures, fish were clasCommunicated by J.P. Grassle, New Brunswick. Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s00227-006-0477-z and is accessible for authorized users. J. A. Miller (&) Coastal Oregon Marine Experiment Station, Hatfield Marine Science Center, Department of Fisheries and Wildlife, Oregon State University, 2030 SE Marine Science Drive, Newport, OR 97365, USA e-mail:
[email protected]
sified to estuary and site within estuary with relatively high levels of accuracy (av = 70–90%). The largest differences in metal-to-calcium ratios were observed between sites within estuaries ( 85 mm SL were collected in lower portions of Coos Bay. Therefore, a total of five sites were examined: Columbia River Channel (CRC 4612¢28¢¢N, 12355¢45¢¢W); Columbia River Old Young’s Bridge (CR-OYB 4609¢59¢¢N, 12354¢05¢¢W); Coos Bay South Slough, located in the South Slough National Estuarine Research Reserve (CB-SS 4316¢36¢¢N, 12419¢05¢¢W); Humboldt Bay South Jetty (HB-SJ 4044¢40¢¢N, 12313¢36¢¢W); and Humboldt Bay South Bay (HB-SB 4043¢40¢¢N, 12313¢20¢¢W) (Fig. 1). Fish were measured to the nearest mm (standard length, SL). Sagittal otoliths were removed within 24 h of collection for all except the CRC samples, which
485
were frozen for 30 days prior to otolith removal. The effects of freezing on otolith chemistry are equivocal (Milton and Chenery 1998; Proctor and Thresher 1998; Rooker et al. 2001) and were not examined directly in the present study. Sagittal otoliths were removed with acid-washed (10% OmniTrace UltraTM HNO3, VWRTM, Bristol, CT, USA) plastic forceps, cleaned, weighed to the nearest 0.0001 g (Mettler H20T, VWRTM), and measured to the nearest 0.05 mm (total length, rostrum to post-rostrum) with a Leitz dissecting microscope. Otoliths were cleaned ultrasonically for 15 min in NANOpure water (Barnstead International, Dubuque, IA, USA) and stored in acid-washed plastic vials. Left sagittal otoliths were embedded in resin (Spurr’s low viscosity resin, Electron Microscopy Sciences, Hatfield, PA, USA) and thin sections were prepared by grinding both sides of the otolith to expose the dorsal–ventral growth axis from core to edge. Otoliths were ground with Tri-m-ite wetordryTM paper (240–1,200 grit, 3MTM, St Paul, MN, USA), polished
Fig. 1 Leptocottus armatus. Collection locations of staghorn sculpin. a Region of study. b Columbia River Channel (CRC) and Old Young’s Bridge (CR-OYB) sites. c Coos Bay South Slough (CB-SS) site. d Humboldt Bay South Jetty (HB-SJ) and South Bay (HBSB) sites
123
486
Mar Biol (2007) 151:483–494
with AlO2 powder (12.5 and 3.0 lm, Buehler, Lake Bluff, IL, USA), and again cleaned ultrasonically in NANOpure water for 15 min. Otolith elemental analysis Elemental analysis was completed at Oregon State University’s WM Keck Collaboratory for Plasma Spectrometry in Corvallis, OR, using a VG PQ ExCell inductively coupled plasma mass spectrometer with a DUV193 excimer laser (New Wave Research, Fremont, CA, USA). The laser was set at a pulse rate of 15 Hz with a 40-lm ablation spot size. All samples were taken in the anterior–dorsal quadrant of left otoliths. No significant differences in analyte concentrations were observed between duplicate ablation series in the anterior–dorsal quadrant (paired t tests, n = 9, P > 0.20, df = 16). Background levels of all analyte isotopes (25Mg, 43 Ca, 55Mn, 86Sr, 138Ba, and 208Pb) were measured for 45 s prior to otolith ablation and subtracted from those measured during otolith ablation. Count rates for each analyte isotope were normalized to 43Ca to account for variations in instrument sensitivity and ablation rate (Campana et al. 1997). Normalized ion ratios were converted to elemental concentrations using measurements of NIST 612 standard glass and Eq. 1 (Kent and Ungerer 2006). N N O N N O O CO i =CCa ¼ Ci =CCa = Iij =ICa;k Iij =ICa;k
ð1Þ
where, COi = concentration of element i in otolith O, COCa = concentration of internal standard, i.e., calcium, in otolith O; CNi/CNCa = the ratio of known concentrations of element i to internal standard, i.e., calcium, in reference standard NIST 612 N; INij/INCa, k = normalized ion yield for isotope j of element i and isotope k of internal standard element in NIST 612 N; IOij/ IOCa,k = normalized ion yield for isotope j of element i and isotope k of the internal standard element in otolith, O. Average elemental concentrations (lg g–1) in NIST 612 are reported in Pearce et al. (1997). Eggins and Shelley (2002) reported that element concentrations in NIST 612 slides are homogenous for all of the elements used in this study except manganese (often > 10%) and lead (usually < 10%). Otolith metal-to-calcium data are reported in mg g–1 for Sr:Ca and lg g–1 for all other ratios. The mean relative standard deviations (%RSD) for NIST 612 glass during data collection were: 25 Mg = 11.0%, 43Ca = 3.7%, 55Mn = 4.4%, 86Sr = 6.3%, 138 Ba = 5.1%, 208Pb = 7.5% (n = 24). Limits of detection (LOD) (lg g–1) were calculated as three standard deviations of the average blank values and were:
123
25
Mg = 1.1, 43Ca = 0.17, 55Mn = 0.32, 86Sr = 0.06, Ba = 0.01, 208Pb = 0.008. For elemental analysis, six otoliths from a site were placed on each slide and slides were processed randomly. On every otolith, data were collected from three transects, each 120-lm in length, oriented 90 to the growth axis. The 40 · 120-lm transects were located: (1) within the primordium, (2) midway between the primordium and outer edge, and (3) at the outer edge. The transect within the otolith primordium (1) represents the egg and early larval periods and will be referred to hereafter as the ‘‘early larval’’ zone. The transect at the otolith edge (3) represents the juvenile period just prior to collection of the fish and will be referred to hereafter as the ‘‘juvenile’’ zone. The transect midway between the primordium and edge (2) represents the late larval and early juvenile period and will be referred to hereafter as the ‘‘late larval’’ zone. Analyte measurements were averaged across each transect using time-resolved software (PlasmaLab International, Everett, WA, USA). Otolith increment analysis was not completed on all fish due to frequently poor increment resolution. Otolith increment width ranged from 4 to 13 lm and averaged 7.6 ± 2.1 lm (±SD). Therefore, each transect represented an average of 16 ± 2.1 (±SD) increments. Tasto (1975) reported an average growth rate, based on cohort anlaysis, of 0.45 mm day–1 for staghorn sculpin in March and April in Anaheim Bay, California while fish maintained in the laboratory at 17C and fed ad libitum grew an average of 0.30 mm day–1. These rates are comparable with estimates for other species in temperate estuaries, i.e., 0.27–0.77 mm day–1 for juvenile Chinook (Oncorhynchus tshawytscha) in Oregon and Washington estuaries based on scales, otolith analysis, and mark-recapture studies (Reimers 1973; Shreffler et al. 1990) and 0.19–0.32 mm day–1 for juvenile English sole (Pleuronectes vetulus) based on otolith increment analysis (Rosenberg 1980). Given potential geographic variation in spawn timing and growth rate, the more conservative laboratory average value ± 10% (i.e., 0.3 ± 0.03 mm day–1) was used to estimate a range of potential hatch dates. The reported average size at hatch (4.5 mm) was subtracted from the length of each fish at capture and then divided by growth rates of 0.27–0.33 mm day–1. These approximations were used only to provide an estimate for the period represented by each of the three life histories transects. 138
Physical information Salinity and water temperature (C) data were obtained from various sources. In the Columbia River
Mar Biol (2007) 151:483–494
estuary, data came from the National Oceanic and Atmospheric Administration’s (NOAA) Columbia River Estuary Real-Time Observation and Forecasting System (http://www.ccalmr.ogi.edu/CORIE/). In Coos Bay, data came from NOAA’s National Estuarine Research Reserve’s System Wide Monitoring Program (http://www.cdmo.baruch.sc.edu/). In Humboldt Bay, data were collected by the Center for Integrative Coastal Observation, Research and Education (CICORE) (http://www.cicore.humboldt.edu/), but only temperature data were available for the time period of interest. Fish size and growth In an effort to minimize potential age-related effects on elemental incorporation, only immature staghorn sculpin (30) were greater than at Columbia River and Coos Bay sites.
Otolith elemental analysis
Fish size and growth The collection date, sample size (n), and range and average fish size (SL) are reported for each site
Temperature (°C)
16
a
14 12 10 8 6
35 30
Salinity
Twenty linear regression analyses between fish size (SL) and otolith metal-to-calcium ratios (4 elements · 5 sites) yielded one significant relationship using Bonferroni adjusted alpha-level for multiple analyses (a = 0.009). There was a significant positive relationship between fish size and Sr:Ca ratios at the CRC site (P = 0.004). Otolith element data were log10-transformed to obtain normality and homogeneity of variance. The assumption of homogeneity of variance (Fmax < 7; Tabachnick and Fidell 2001) was met in all cases except for Ba:Ca ratios in 2002 due to a relatively small variance at the otolith edge of CB-SS fish (n = 23). MANOVA was still used because it is fairly robust with respect to deviations in homogeneity of variance when sample sizes are relatively large, i.e., > 10 (Quinn and Keough 2002).
b Spatial variation: estuaries
25 20 15 10 5 0
Jun 02
May 02
Apr 02
M a r 02
Feb 02
Jan 02
Dec 01
Nov 01
Oct 01
-5
Fig. 2 Temperature (C) and salinity. a Average water temperature (C) ± SD. b Mean salinity ± SD. CRC is represented by filled circles, CR-OYB by open circles, CB-SS by filled triangles, and HB-SB by open squares. Shaded areas indicate estimated time range associated with early larval, late larval, and juvenile otolith zones as determined by back-calculation. See text for site abbreviations
123
(Table 1). Length data were log10-tranformed to obtain normality and homogeneity of variance. There was no significant difference in the size of fish collected from Coos and Humboldt Bay. However, Columbia River fish were significantly larger (df = 79, P < 0.001) than fish collected in Coos or Humboldt Bay. ANCOVA was used to determine if there were differences in sizeat-age, using otolith weight as a proxy for age, among estuaries. A separate-slopes model was used because the assumption of homogeneity-of-slopes was not met (F2,76 = 10.5, P < 0.001). Columbia River fish were significantly larger, i.e., 8%, after correction for otolith weight (F2,76 = 35.9, P < 0.001).
Otolith composition varied significantly among estuaries for all metal-to-calcium ratios examined (multivariate F10,458 = 63.2, P < 0.001) and among otolith zones except Pb:Ca (multivariate F10,458 = 29.4, P < 0.001; Fig. 3; Appendix). There were significant estuary-by-otolith zone interactions for Mn:Ca, Sr:Ca, and Ba:Ca. The significant interactions indicate that the metal:calcium ratios varied with otolith zone and, in some cases, varied to different degrees in each estuary. Fish from the Columbia River estuary had greater Mg:Ca, Mn:Ca, and Ba:Ca than fish from Coos and Humboldt Bays (Scheffe´’s, df = 233, P < 0.001), which were not statistically different. Fish from Humboldt Bay had significantly greater Pb:Ca than the other two estuaries (Scheffe´’s, df = 233, P < 0.001), which did not differ.
Mar Biol (2007) 151:483–494
489
Table 1 Leptocottus armatus. Collection site and date, sample size (n), size range and average size ± SD (SL, mm) Collection site
Collection date
n
Size range (mm)
Average size (± SD mm)
Columbia River, Channel Columbia River, Old Young’s Bridge Coos Bay, South Slough Humboldt Bay, South Jetty Humboldt Bay, South Bay
15 15 24 23 23
17 18 23 17 7
39–77 54–79 38–63 27–58 27–66
52.9 68.2 44.9 41.7 50.0
May June March March March
Spatial variation: sites
20
120
16
80 60
18
140
12 8 4
120 100 80 60
12
6
80
Ear
ly la r va l Lat e la rva l Ju v eni le
0.0
Fig. 3 Leptocottus armatus. Mean metal-to-calcium ratios ± SE for early larval, late larval, and juvenile otolith zones, separated by estuary. Columbia River estuary (n = 35, open circles), Coos Bay (n = 23, filled triangles), and Humboldt Bay (n = 24, open squares)
Ba:Ca (µg g-1)
20
1.5
HB-SJ
HB-SB
CB-SS
CRC
2.0
1.0 0.5 0.0
HB-SJ
0.5
40
0
CRC
1.0
4.6 4.4
Pb:Ca (µg g-1)
1.5
4.8
60
CR-OYB
Ju v eni le
Lat e
Ear
2.0
larv al
0
2.5
5.0
HB-SB
20
ly la r va l
4.0
40
0 80
CB-SS
4.8
40 5.2
CR-OYB
5.6
60
Sr:Ca (mg g-1)
Ba:Ca (µg g-1)
Sr:Ca (mg g-1)
6.4
Pb:Ca (µg g-1)
Variation in composition among otolith zones was examined with all fish combined (Fig. 5) and with fish
0
40
Temporal variation: otolith zone
Mg:Ca (µg g-1)
100
12.1 6.0 6.9 7.02 14.8
was due to higher ratios at the otolith edge for all sites except CR-OYB, where ratios were relatively similar across the growth axis. Mg:Ca and Ba:Ca varied significantly between sites in both the Columbia River estuary and Humboldt Bay (Scheffe´’s, df = 227, P < 0.001). Mn:Ca ratios were significantly different between sites only in Humboldt Bay (Scheffe´’s, df = 227, P £ 0.03). Pb:Ca ratios were similar at Humboldt Bay sites and significantly greater than Columbia River sites, which did not differ.
Mn:Ca (µg g-1)
140
Mn:Ca (µg g-1)
Mg:Ca (µg g-1)
Otolith composition varied significantly among sites within estuaries for all metal-to-calcium ratios examined (multivariate F20,740 = 51.0, P < 0.001) and among otolith zones except for Pb:Ca (multivariate F10,446 = 32.9, P < 0.001; Fig. 4; Appendix). There were significant site-by-otolith zone interactions for Mg:Ca, Mn:Ca, Sr:Ca, and Ba:Ca. These were due to significant differences among sites in metal-to-calcium ratios in only one or two otolith zones. For example, for Sr:Ca, the interaction between site and otolith zone
± ± ± ± ±
Fig. 4 Leptocottus armatus. Mean otolith metal-to-calcium ratios ± SE grouped by site for all otolith zones combined. Sites within an estuary within ovals. See text for sample sizes and site abbreviations
123
490
Mar Biol (2007) 151:483–494
separated by estuary (Fig. 3). When all fish were combined, there were significant differences among otolith zones for all metal-to-calcium ratios except Pb:Ca (multivariate F12,468 = 19.7, P < 0.001). Mn:Ca ratios consistently declined while Ba:Ca ratios consistently increased across the growth axis (Scheffe´’s posthoc comparison, df = 227, P < 0.001; Fig. 5). Mg:Ca ratios were greater in the early larval zone (Scheffe´’s post-hoc comparison, df = 227, P < 0.02). Sr:Ca ratios in the juvenile zone were significantly greater than the earlier two zones. However, the patterns were different when otolith zones were examined separately for each estuary (Fig. 3). There were no differences in composition among otolith zones that were consistently observed in all estuaries. In other words, patterns that potentially appeared to be ontogenetic differences when all fish were combined, such as the consistent decline in Mn:Ca ratios across the growth axis, were not observed in all estuaries. Scales of variation All calculations were completed on log10-transformed data. The maximum percent difference in metal-tocalcium ratios ranged from 103.3 to 208.0% (Table 2). Differences among otolith zones were consistently the smallest while site differences were always the greatest. The largest differences were observed in Ba:Ca ratios (av = 166.4% ± 36.4 SD) and the smallest were observed in Sr:Ca ratios (av = 113.3% ± 1.9 SD).
100
5.6
a Sr:Ca (mg g-1)
Element:Ca (µg g-1)
120
80 60 40 20
4.8
1.00
b Pb:Ca (µg g-1)
Mn:Ca (µg g-1)
5.2
4.4
0 12
c
10 8
d
0.25
nile
rva l
Juv e
l rva
e la Lat
ly la Ear
le
l rva
eni Juv
rva
l
e la Lat
ly la
NOAA status and trends program mussel watch project Mussel tissue manganese and lead concentrations (mg g–1, dry weight) were log10-transformed to obtain normality and homogeneity of variance. Tissue concentrations were significantly different among bays for both elements (multivariate F12,468 = 19.7, P < 0.001). Relative differences in tissue concentrations among estuaries were similar to the differences observed in staghorn sculpin otolith metal-to-calcium ratios (Fig. 6). Concentrations of manganese in mussel tissue were the greatest in the Columbia River, followed by Humboldt and Coos Bay (Scheffe´’s, df = 16, P < 0.001; Fig. 6). Concentrations of lead in mussel tissue were greater in Humboldt Bay (Scheffe´’s, df = 16, P < 0.001) than either the Columbia River or Coos Bay, which did not differ.
Discussion
0.50
0.00
Ear
A model was developed to classify fish to estuary using each otolith zone, i.e., the early larval, late larval, and juvenile. Mn:Ca, Ba:Ca, and Pb:Ca were significant variables in the estuary classification model (F > 20.0, P < 0.01) and yielded the most accurate classifications (Table 3). Overall, 75–100% of the fish were accurately classified to collection estuary. The greatest classification success (av = 95%) occurred using the recently deposited juvenile zone. For the model to classify fish to sites within estuaries, Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, and Pb:Ca were significant variables (F > 9.0, P < 0.01) and yielded the most accurate classifications (Table 3). Overall, 14– 100% of the fish were accurately classified to collection site. The greatest classification success (av = 87%) occurred using the juvenile zone. Site classification success was consistently the best at CR-OYB (‡ 88%) and the worst at HB-SB (14–57%), which was the site with the smallest sample size (n = 7) (Table 3).
0.75
6
Fig. 5 Leptocottus armatus. Mean metal-to-calcium ratios ± SE for early larval, late larval, and juvenile otolith zones for all fish combined (n = 82). a Mg:Ca (open squares) and Ba:Ca (open circles). b Mn:Ca, c Sr:Ca, d Pb:Ca
123
Classification
There were significant differences in otolith elemental composition of staghorn sculpin at all scales examined, including among estuaries, sites within estuaries, and otolith zones. An important finding is that differences in otolith elemental composition were the largest between sites and smallest between otolith zones. Gillanders and Kingsford (2003) found that variation in otolith chemistry (Li, Mn, and Ba) of juvenile snapper (Pagrus auratus) was smaller among sites
Mar Biol (2007) 151:483–494
491
Table 2 Leptocottus armatus. Maximum differences for pair-wise comparisons of otolith metal-to-calcium ratios Scale
Mg
Maximum metal: calcium differences Estuaries 120.1 Sites 126.6 Otolith zone 105.0 Average 117.2 ± 11.1 Ranks Estuaries 2 Sites 3 Otolith zone 1
Mn
Sr
Ba
Pb
Average (SD)
126.6 135.4 110.3 124.1 ± 12.7
114.1 114.7 111.1 113.3 ± 1.9
150.7 208.0 140.4 166.4 ± 36.4
127.5 130.6 103.3 120.5 ± 15.0
127.8 ± 13.9 143.1 ± 37.1 114.0 ± 15.1
2 3 1
2 3 1
2 3 1
2 3 1
2 3 1
Averages (±SD) for each spatial scale and element examined are presented. Scale differences are ranked from smallest (1) to largest (3). Data were log10-transformed
Estuary (n) Columbia River (35) Coos Bay (23) Humboldt Bay (24) Site (n) Columbia River Channel (17) Columbia River, Old Young’s Bridge (18) Coos Bay South Slough (23) Humboldt Bay South Jetty (17) Humboldt Bay South Bay (7)
Early larval
Late larval
Juvenile
78 84 75
83 94 75
96 100 88
81 88
88 94
94 100
70 71 14
70 88 57
83 100 57
Sample sizes (n) and percent correctly classified are presented
25
a
40
20
30
15
20
10
10
5
0
0
2.0
Mn:Caotolithl (µg g-1)
Mnmussel (mg g-1)
50
2.0
b
1.5
1.5
1.0
1.0
0.5
0.5
0.0
Pb:Caotolithl (µg g-1)
Table 3 Leptocottus armatus. Results of jackknifed classification of juvenile fish to estuary (Mn:Ca, Ba:Ca, and Pb:Ca) and site (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, and Pb:Ca) using multi-element otolith signatures from early larval, late larval, and juvenile otolith zones
argues against the presence of significant ontogenetic effects on otolith chemistry in this species. These data indicate that variation in the otolith elemental composition of fish collected from different geographic areas may not be easily obscured by ontogenetic differences, particularly if fish of similar age are used. Furthermore, there was no significant effect of fish size on otolith chemistry, which is in agreement with growth-rate effects in juvenile menhaden (Brevoortia patronus) (Chesney et al. 1998) and juvenile spot (Leiostomus xanturus) (Martin et al. 2004; Martin and Thorold 2005).
Pbmussel (mg g-1)
within estuaries than among estuaries. Although their ability to discriminate among estuaries was high in all cases, the discrimination among sites was typically as good or better, with one exception. Gillanders and Kingsford (2003) noted that to use otolith chemistry to assess connectivity between coastal and estuarine areas and/or identify estuary of origin, all potential source areas must be identified. Given that the largest differences observed in this study occurred at the smallest spatial scales, 40,000) than the other two estuaries and thus may have slower flushing times and greater anthropogenic inputs of lead. Columbia River mussel tissue and staghorn sculpin otoliths had the highest levels of manganese. Elevated levels of manganese have been observed in the south channel of the Columbia River estuary, which is where both the mussels and fish were collected. The high levels of manganese are proposed to be related to in situ production associated with microbial remineralization of particulate organic carbon (Klinkhammer et al. 1997; Klinkhammer and McManus 2001).
123
Mar Biol (2007) 151:483–494
In conclusion, I documented significant variation in the otolith chemistry of staghorn sculpin (Leptocottus armatus) at multiple scales and provided a quantitative comparison across those scales. Variation in otolith chemistry was the largest between sites within estuaries (
