A Methodology for Quantifying Trace Elements in the 

Exoskeletons of Florida Stone Crab (Menippe mercenaria) 

Larvae Using Inductively Coupled Plasma Optical Emission 

Spectrometry (ICP–OES) 

By Philip M. Gravinese, Jennifer A. Flannery, and Lauren T. Toth

Open-File Report 2016–1148

U.S. Department of the Interior

U.S. Geological Survey
ii

U.S. Department of the Interior

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Director

U.S. Geological Survey, Reston, Virginia: 2016

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must be secured from the copyright owner.

Suggested citation:

Gravinese, P.M., Flannery, J.A., and Toth, L.T., 2016, A methodology for quantifying trace elements in the exoskeletons 

of the Florida stone crab (Menippe mercenaria) larvae using inductively coupled plasma optical emission spectrometry 

(ICP–OES): U.S. Geological Survey Open-File Report 2016–1148, 12 p., https://doi.org/10.3133/ofr20161148.

ISSN 2331-1258 (online)
iii

Acknowledgments

The authors thank Ian Enochs and Derek Manzello of the National Oceanic and Atmospheric Administration 

(NOAA) Atlantic Oceanographic and Meteorological Laboratory for their support in completing this work and Ilsa 

Kuffner and Julie Richey of the U.S. Geological Survey (USGS) St. Petersburg Coastal and Marine Science Center 

for discussions that improved this manuscript. This research was funded by a “Protect Our Reefs” grant from Mote 

Marine Laboratory awarded to Philip Gravinese and Richard Tankersley, both of Florida Institute of Technology. 
iv
v

Contents

Acknowledgments ...........................................................................................................................................................................................iii

Abstract ........................................................................................................................................................................................................... 1

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

Methods Used ................................................................................................................................................................................................. 2

Stone Crab Collection and Larval Rearing.................................................................................................................................................. 2

Chitinous Matrix Standard........................................................................................................................................................................... 4

Larval Crab Sample Cleaning ..................................................................................................................................................................... 4

Trace-Element Determinations ................................................................................................................................................................... 4

Results and Discussion ................................................................................................................................................................................... 5

Conclusions..................................................................................................................................................................................................... 9

References Cited .......................................................................................................................................................................................... 10

Appendix........................................................................................................................................................................................................ 12

Figures

1. Scanning electron microscope image of stage III stone crab larvae at 37x magnification ............................................................. 3

2. Scanning electron microscope image of stage V stone crab larvae at 37x magnification.............................................................. 3

3. Strontium-to-calcium and magnesium-to-calcium ratios for stage III stone crab larvae from 11 different broods raised at

30 degrees Celsius and 37 practical salinity units .......................................................................................................................... 7

4. Strontium-to-calcium and magnesium-to-calcium ratios for stage V stone crab larvae from 12 different broods raised at

30 degrees Celsius and 37 practical salinity units. ........................................................................................................................ 7

5. Strontium-to-calcium ratios for stone crab larval stage III and stage V ......................................................................................... 8

6. Magnesium-to-calcium ratios for stone crab larval stage III and stage V ....................................................................................... 8

Tables

1. Average strontium-to-calcium and magnesium-to-calcium values for Menippe mercenaria larval stages III and V and the

chitinous matrix standard with associated errors............................................................................................................................ 6
vi

Conversion Factors

International System of Units to U.S. customary units

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

kilometer (km) 0.6214 mile (mi)

Volume

microliter (μL) 3.38135e-05 ounce, fluid (fl. oz)

milliliter (mL) 0.034 ounce, fluid (fl. oz)

liter (L) 33.82 ounce, fluid (fl. oz)

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as °F = (1.8 × °C) + 32.

Abbreviations

C Celsius

Ca calcium

CaCO3 calcium carbonate

H2O2 hydrogen peroxide

HNO3 nitric acid

ICP–OES Inductively coupled plasma optical emission spectrometry

IGS internal gravimetric standard

M molarity

Mg magnesium

mmol mol–1 millimole per mole

NaOH sodium hydroxide

NIST National Institute of Standards and Technology

N normal

ppm parts per million

psu practical salinity units

RPM revolutions per minute

RSD relative standard deviation

SEM scanning electron microscope

Sr strontium

USGS U.S. Geological Survey

V volume
1

A Methodology for Quantifying Trace Elements in the 

Exoskeletons of Florida Stone Crab (Menippe mercenaria) 

Larvae Using Inductively Coupled Plasma Optical Emission 

Spectrometry (ICP–OES) 

By Philip M. Gravinese,1 Jennifer A. Flannery,2 and Lauren T. Toth2

Abstract

The larvae of the Florida stone crab, Menippe mercenaria, migrate through a variety of habitats 

as they develop and, therefore, experience a broad range of environmental conditions through ontogeny. 

Environmental variability experienced by the larvae may result in distinct elemental signatures within 

the exoskeletons, which could provide a tool for tracking the environmental history of larval stone crab 

populations. A method was developed to examine trace-element ratios, specifically magnesium-tocalcium (Mg/Ca) and strontium-to-calcium (Sr/Ca) ratios, in the exoskeletons of M. mercenaria larvae. 

Two developmental stages of stone crab larvae were analyzed—stage III and stage V. Specimens were 

reared in a laboratory environment under stable conditions to quantify the average ratios of Mg/Ca 

and Sr/Ca of larval stone crab exoskeletons and to determine if the ratios differed through ontogeny. 

The elemental compositions (Ca, Mg, and Sr) in samples of stage III larvae (n = 50 per sample) from 

11 different broods (mean Sr/Ca = 5.916 ± 0.161 millimole per mole [mmol mol−1]; mean Mg/Ca = 

218.275 ± 59.957 mmol mol−1) and stage V larvae (n = 10 per sample) from 12 different broods (mean 

Sr/Ca = 6.110 ± 0.300 mmol mol−1; mean Mg/Ca = 267.081 ± 67.211 mmol mol–1) were measured 

using inductively coupled plasma optical emission spectrometry (ICP–OES). The ratio of Sr/Ca significantly increased from stage III to stage V larvae, suggesting an ontogenic shift in Sr/Ca ratios between 

larval stages. The ratio of Mg/Ca did not change significantly between larval stages, but variability 

among broods was high. The method used to examine the trace-element ratios provided robust, highly 

reproducible estimates of Sr/Ca and Mg/Ca ratios in the larvae of M. mercenaria, demonstrating that 

ICP–OES can be used to determine the trace-element composition of chitinous organisms like the 

Florida stone crab. 

Introduction

Benthic coastal crustaceans brood their embryos for several weeks before larvae hatch. Upon 

hatching, planktonic larvae are released into the water column where behavioral responses to certain environmental cues will help facilitate dispersal (Dibacco and Levin, 2000). The specific dispersal strategy 

of a given species is dependent on how the species synchronizes the timing of larval release with certain 

environmental cues (for example, tidal cues) and (or) if the larvae can regulate their vertical position 

throughout ontogeny (that is, endogenous or exogenous responses to environmental stimuli). Synchronic 

behaviors, such as larval release and vertical swimming, to certain environmental stimuli advect larvae away from parent populations where visual predators are abundant and transport larvae to habitats 

where conditions are more favorable for continuing development (Sulkin, 1984). As a result, larvae are 

1

Florida Institute of Technology

2

U.S. Geological Survey
2

exposed to a wide range of environmental conditions through ontogeny (Dibacco and Levin, 2000), 

which may drive shifts in the elemental composition of their exoskeletons. Quantifying trace-element 

composition within larval exoskeletons could, therefore, be useful in tracking the environmental history 

of marine larvae among different populations (Anastasia and others, 1998). 

The Florida stone crab, Menippe mercenaria, inhabits nearshore, subtidal coastal habitats from 

North Carolina to Texas, with isolated populations occurring throughout the Caribbean. Stone crabs support an important commercial fishery throughout the Southeastern United States. For example, in Florida, commercial stone crab fisheries contribute approximately $25 million per year to the local economy 

(Florida Fish and Wildlife Conservation Commission, undated) and this estimate does not account for 

active, recreational stone crab fisheries within the region. During the summer reproductive season, stone 

crabs brood larvae on their abdomen for about 2 weeks before larval release. Larval release occurs on a 

diel cycle and can be synchronized with tidal cues in some areas (Krimsky and others, 2009). Like most 

other brachyuran crabs, stone crabs release their larvae in nearshore coastal habitats, but the larvae are 

immediately transported to offshore areas where they complete larval development. Larval development 

takes about 28 days and consists of five planktonic larval stages and one post-larval (meglopae) stage, 

after which individuals recruit back into nearshore environments (Porter, 1960; Mootz and Epifanio, 

1974). 

The purpose of this study was to develop a method to efficiently quantify trace-element ratios, 

specifically magnesium to calcium (Mg/Ca), in the stage III and stage V exoskeletons of M. mercenaria

larvae (figs. 1 and 2, respectively). The exoskeleton of newly hatched crustacean larvae are lightly mineralized (Whiteley, 2011); therefore, we analyzed the exoskeletons of mid- to late-stage stone crab larvae (stages III and V), which are primarily built of high-Mg calcite (Ries and others, 2009; Ries 2011). 

Our goals were to quantify a baseline estimate of Mg/Ca and Sr/Ca ratios for stone crab larvae reared 

under controlled laboratory conditions and determine if the elemental composition of their exoskeletons 

changes through development (that is, from stage III to stage V). 

Methods Used

Stone Crab Collection and Larval Rearing

 Ovigerous female M. mercenaria were collected by the Florida Fish and Wildlife Conservation Commission using commercial stone crab traps located 17 kilometers from the Everglades coast in 

Florida Bay. Newly hatched larvae from each female were transferred into separate experimental rearing chambers (modified plastic containers; 18.9 liters). Each container was kept in its own water bath to 

maintain temperatures within a narrow range (30.0 ± 0.23 degrees Celsius (°C) [mean ± standard deviation]). Salinity was also maintained within a narrow range (37.7 ± 0.47 practical salinity units [psu]). 

Each day, larvae were fed 0.5 per milliliter (mL–1) Artemia spp. (brine shrimp) enriched with self-emulsifying lipid concentrate (SELCO) and rotifers (Rotigrow® Onestep). Larvae were kept on a 14-hourslight to 10-hours-dark photoperiod that approximated natural conditions during the time of collection. 

To minimize buildup of nutrients, dead larvae and wastes were removed from each chamber twice daily. 

Samples were harvested from the experimental rearing chambers when larvae reached stage III (n = 50 

larvae) and stage V (n = 10 larvae) of development (figs. 1 and 2; based on the descriptions of Porter, 

1960). The larval samples were flash frozen using liquid nitrogen and kept at −80 °C until they were 

prepared for trace-element analysis.
3

Figure 2. Scanning electron microscope (SEM) image of stage V stone crab larvae at 37x magnification. According to Porter (1960), stage V stone crab larvae can be identified by the presence of dorsal 

and rostral spines that are three times the size of the lateral spines (observed prior to placement on the 

SEM stub). Additionally, the first four abdominal segments contain elongated pleopods (labeled as “b”). 

Figure 1. Scanning electron microscope (SEM) image of stage III stone crab larvae at 37x magnification. According to Porter (1960), stage III stone crab larvae can be identified by the presence of a sixth 

abdominal segment (labeled as “a”), whereas stage II larvae only have five abdominal segments. 
4

Chitinous Matrix Standard

To our knowledge, no laboratory/instrument matrix standard for crab samples currently exists for 

elemental (that is, calcium [Ca], strontium [Sr], and magnesium [Mg]) analyses. It is important to have a 

standard with a similar elemental matrix as the unknown samples; therefore, we created a matrix standard using the chitinous exoskeleton (that is, a claw) of an adult M. mercenaria. Soft tissues were physically removed from the claw exoskeleton by using tweezers and a scalpel, and the remaining exoskeletal 

material was sonicated in 30 percent trace-metal-grade hydrogen peroxide (H2O2) for 20 minutes to 

remove any organic material. This was followed by sonication in type-2 analytical water to remove any 

remaining H2O2 and (or) particulate material. The remaining claw material was pulverized with a mortar and pestle to obtain a fine white powder. An aqueous stock solution of the standard was created by 

adding 0.5 gram (g) of the pulverized powder to 1,000 milliliters (mL) of 2 percent nitric acid (HNO3) 

and sonicated in a water bath at 80 °C for an hour to fully dissolve all of the calcium carbonate (CaCO3) 

within the exoskeleton. This procedure yielded a concentration of 1,000 parts per million (ppm) Ca in 

the solution. An aqueous working standard solution was created by diluting the stock solution to a concentration of 20 ppm on the basis of the solution’s molarity (M) and volume (V) using the formula 

M1V1=M2V2.

Larval Crab Sample Cleaning

Estimates of trace-metal concentrations were determined by analyzing samples of stage III (n = 

50) and stage V larvae (n = 10) from unique female broods (n = 11 and n = 12, respectively). Prior to 

analysis, each sample of larvae underwent an oxidation cleaning treatment to remove any organic material within the exoskeleton by using a modified procedure developed for oxidizing planktonic foraminifera tests (Russell and others, 2004). Each sample was cleaned three times using hot-buffered hydrogen 

peroxide (1:1 solution of 0.1 normal [N] sodium hydroxide [NaOH] and 30 percent H2O2) held at 80 °C 

for 30 minutes and rinsed five times with type-2 analytical water. All samples were cleaned and analyzed at the U.S. Geological Survey Coastal and Marine Science Center in St. Petersburg, Fla. 

Trace-Element Determinations

Once each larval-crab sample was cleaned and oxidized, 100 microliters (μL) of concentrated 

trace-metal-grade 70 percent nitric acid (HNO3, 16 molar [M]) was added to each sample. Acidified 

samples underwent sonication in a hot water bath for 15 minutes to fully dissolve all CaCO3 into aqueous solution. Samples were then centrifuged at 7,000 revolutions per minute (RPM) for 5 minutes 

to drive any remaining organic/non-soluble material to the bottom of the vial. Seventy μL of 70 percent HNO3 liquid containing dissolved CaCO3

 was transferred into a 17 mL test tube and was diluted 

with 2.45 mL of type-2 analytical water to bring each liquid sample concentration to 2 percent HNO3

(0.45 M), consistent with the HNO3 concentration of all analytical standards used. 

A PerkinElmer 7300 dual view ICP–OES was used to measure the elemental composition of 

Ca, Sr, and Mg. At the beginning of each run, the machine was calibrated using a mixture of PlasmaCal (traceable to the National Institute of Standards and Technology [NIST] factory standards) with 

a Sr/Ca ratio of 0.00795 (3.635 millimole per mole [mmol mol−1]) and a Mg/Ca ratio of 0.12915 

(212.916 mmol mol−1) with calcium concentrations at 8, 20, 30, and 40 ppm. A test run on the ICP–OES 

(using ~1 mL of aqueous solution) was used to estimate the Ca concentrations of each larval-crab 
5

sample and, if necessary, 2 percent HNO3 was used to dilute each sample to a target Ca concentration of 

20 ppm (the concentration yielding the highest linear calibration on the instrument). The full run on the 

instrument had an internal gravimetric standard (IGS) run before and after every sample. The IGS solution was also prepared from single-element PlasmaCal solutions with a 20 ppm Ca concentration and 

Sr/Ca ratios of 0.00790511 (3.61 mmol mol−1) and Mg/Ca ratios of 0.12804106 (211.145 mmol mol−1). 

IGS statistics were computed for every run, and the runs were accepted if the relative standard deviation 

(RSD, 1σ standard deviation/average) for the IGS solution was <0.2 percent for Sr/Ca and <1.5 percent 

for Mg/Ca. The chitinous matrix standard (see above section) was run every five samples to test for 

any potential matrix effects caused by small-scale variability in the composition of the chitinous exoskeleton. Ratios of Sr/Ca and Mg/Ca were corrected for instrumental drift and noise following the IGS 

method devised by Schrag (1999). The average corrected IGS precision for Sr/Ca was 0.0030 mmol 

mol−1 (0.08 percent, 1σ, n = 35) and Mg/Ca was 1.739 mmol mol−1 (0.81 percent, 1σ, n = 35). The matrix standard had an average corrected precision of Sr/Ca 0.0067 mmol mol−1 (0.12 percent, 1σ, n = 56) 

and Mg/Ca was 1.473 mmol mol−1 (1.04 percent, 1σ, n =5 6). Sr/Ca and Mg/Ca values were accepted if 

they ranged from 10 to 30 ppm Ca concentration. Ratios were converted to millimole per mole using the 

formula

Rmmol/mol = r*(molar mass of calcium (40.078)/molar mass of metal (Sr = 87.62; Mg = 24.305)*1,000.

The Sr/Ca and Mg/Ca ratios of each sample represent average values across the total number of 

individuals in each sample (n = 50 for stage III and n = 10 for stage V) (figs. 3–6). 

Results and Discussion

Sr/Ca values were similar among broods (figs. 3 and 4) and between the two larval stages 

(fig. 5). The average Sr/Ca values for all stage III and stage V larvae were 5.916 ± 0.161 mmol mol−1

and 6.110 ± 0.300 mmol mol−1, respectively (table 1). Although there was a statistically significant 

increase in Sr/Ca from stage III to stage V (paired t-test: t = –2.67, p = 0.028), Sr/Ca values were lower 

(5.571 ± 0.007) in the adult stone crab exoskeleton that was used to develop the matrix standard. This 

suggests that there may be ontogenetic shifts in the trace-element concentration of stone crab exoskeletons, but the direction of those shifts may change between larval and adult stages of development. 

To our knowledge, no other studies have been conducted to determine the Sr/Ca ratio of larval crabs, so direct comparison of our results to published data from other crustaceans is not possible. 

Instead, we compare the Sr/Ca ratios from this study to other well-studied marine calcifiers. Currently 

[2016], the accepted range of Sr/Ca ratios in coral aragonite varies between 8 and 10 mmol mol−1 depending on the coral species (that is, Siderastrea siderea, 8.96–8.99 mmol mol−1, DeLong and others, 

2011; Porites lutea, 9.01 mmol mol−1, Wu and others, 2013, Orbicella faveolata, 9.21 mmol mol−1, 

Flannery and Poore, 2013; Flannery and others, 2017). Sr/Ca values for biogenic calcite tend to be much 

lower than values for aragonite (for example, 0.8–1.56 mmol mol−1 in planktic foraminifera, Edgar and 

others, 2015; 3.5 mmol mol−1 in the coralline red algal species Sporolithon durum, Darrenougue and 

others, 2014). The carbonate incorporated into the chitinous skeleton of crabs is primarily high-Mg calcite (Ries and others, 2009; Ries, 2011). This may explain why the Sr/Ca ratio determined in this study 

from M. mercenaria larvae fell between the accepted values for biogenic low-Mg calcite and aragonite. 

Mg/Ca ratios were much more variable than Sr/Ca ratios both among broods within each stage 

(figs. 3 and 4) and between the two stages (fig. 6). The average Mg/Ca values for all stage III and stage 

V larvae were 218.275 ± 59.957 mmol mol−1 and 267.081 ± 67.211 mmol mol−1, respectively (table 1). 
6

Because of the high variability among broods, the apparent increase in Mg/Ca ratios between stages was 

not significant (paired t-test: t = –1.75, p = 0.118). 

These values are slightly higher than previously published values for high-Mg calcite determined for crustose coralline algae Clathromorphum spp. from the Aleutian Islands in the North Pacific Ocean (120–160 mmol mol−1, Williams and others, 2014) and from the North Atlantic Ocean 

(~132 mmol mol−1, Gamboa and others, 2010), which may be because the coralline algae came from 

cold-water environments whereas stone crabs live in warmer, subtropical areas. The Mg/Ca values 

determined in this study are lower than Mg/Ca values (290–310 mmol mol−1) determined in coralline 

red algal species Sporolithon durum samples from New Caledonia (Darrenougue and others, 2014), 

where ocean waters of the South Pacific are warmer than waters near the Florida Keys. As expected, the 

Mg/Ca values from high-Mg calcite M. mercenaria larvae are much greater than accepted Mg/Ca values of foraminiferal low-Mg calcite, which typically range from ~3 to 5mmol mol−1 (that is, 4.4 mmol 

mol−1, Richey and others, 2007). The Mg/Ca values of M. mercenaria larvae are also greater than the 

Mg/Ca of the chitinous matrix standard developed in this study. Temperature influences the partitioning 

of Mg from seawater into the CaCO3 matrix (Williams and others, 2014) for crustose coralline algae. 

Because stone crab larvae generally live in the upper portion of the water column, the greater values 

found in M. mercenaria larvae could reflect warmer water temperatures at the time of growth (controlled 

at 30 °C). Alternatively, the difference between the Mg/Ca ratios of the matrix standard and the larval 

samples could reflect long-term changes in the incorporation of Mg through ontogeny, but further sampling (replication of adults) would be required to test this hypothesis.

Table 1. Average strontium-to-calcium (Sr/Ca) and magnesium-to-calcium (Mg/Ca) values for Menippe mercenaria larval 

stages III and V and the chitinous matrix standard with associated errors.

[1σ standard deviation. mmol mol–1, millimole per mole; +, plus or minus]

Larval stage Sr/Ca mmol mol–1 ± standard deviation Mg/Ca mmol mol–1 ± standard deviation

III 5.916 ± 0.161 218.275 ± 59.957

V 6.11 ± 0.300 267.081 ± 67.211

Chitinous matrix standard 5.571 ± 0.007 138.389 ± 1.473
7

Magnesium-to-calcium ratio, in millimole per mole 

Strontium-to-calcium ratio, in millimole per mole 

Female (Brood)

Sr/Ca Mg/Ca 

0 

50 

100 

150 

200 

250 

300 

350 

400 

450 

500 

0 

1 

2 

3 

4 

5 

6 

7 

1 2 4 5 6 7 8 10 11 15 16 

Female (Brood) 

0 

50 

100 

150 

200 

250 

300 

350 

400 

450 

500 

0 

1 

2 

3 

4 

5 

6 

7 

8 

1 2 3 4 5 6 7 8 9 10 11 12 

Sr/Ca Mg/Ca 

Strontium-to-calcium ratio, in millimole per mole 

Magnesium-to-calcium ratio, in millimole per mole 

Figure 3. Strontium-to-calcium (Sr/Ca; blue) and magnesium-to-calcium (Mg/Ca; red) ratios for 

stage III stone crab larvae from 11 different broods raised at 30 degrees Celsius and 37 practical 

salinity units. Each sample contained 50 individuals. Error bars represent standard error of the 

mean. Error is so small in Sr/Ca that error bars are obscured by the symbols marking data points. 

Figure 4. Strontium-to-calcium (Sr/Ca; light blue) and magnesium-to-calcium (Mg/Ca; pink) ratios 

for stage V stone crab larvae from 12 different broods raised at 30 degrees Celsius and 37 practical 

salinity units. Each sample contained 10 individuals. Error bars represent standard error of the mean. 

Error is so small in Sr/Ca that error bars are obscured by the symbols marking data points. 
8

Strontium-to-calcium ratio, in millimole per mole 

Female (Brood) 

0 

1 

2 

3 

4 

5 

6 

7 

8 

1 2 3 4 5 6 7 8 9 10 11 12 15 16 

 Stage III 

 Stage V

Female (Brood) 

0 

50 

100 

150 

200 

250 

300 

350 

400 

450 

500 

1 2 3 4 5 6 7 8 9 10 11 12 15 16 

 Stage III 

 Stage V 

Magnesium-to-calcium ratio, in millimole per mole 

Figure 5. Strontium-to-calcium (Sr/Ca) ratios for stone crab larval stage III (dark blue) 

and stage V (light blue). Error bars represent standard error of the mean. Error is so 

small in Sr/Ca that error bars are obscured by the symbols marking data points. 

Figure 6. Magnesium-to-calcium (Mg/Ca) ratios for stone crab larval stage III (red) and 

stage V (pink). Error bars represent standard error of the mean.
9

Conclusions

The method used in this study provided credible estimates of trace Sr/Ca and Mg/Ca ratios in 

the larval exoskeletons of M. mercenaria, demonstrating that ICP–OES can be used to determine the 

trace-metal composition of chitinous organisms like the Florida stone crab. We demonstrated that these 

methods could be used to measure trace elements in field-collected larvae. No field data exist on the 

larval dispersal and migration patterns of stone crabs, which is surprising considering the importance of 

the commercial fishery. Trace-element ratios in larval exoskeletons could, therefore, be used as a management tool for identifying potential source-sink populations/habitats and determining larval trajectories (that is, correlating larval signatures with environmental signatures to help describe dispersal in the 

field).

We found that there was a significant difference in the concentration of Sr/Ca between stage III 

and stage V stone crab larvae, but the difference in Mg/Ca was not significant. The difference in the 

Sr/Ca and Mg/Ca ratios between the stone crab larvae and the matrix standard (adult crab) suggests that 

ontogenetic shifts may also occur over longer periods in the life history of these crustaceans. Additionally, our results showed that there was a high degree of variability among broods of stone crab larvae. 

Future work could attempt to determine the magnitude and causes of among-brood variability, including 

the potential influence of the brooding female, on the elemental composition of stone crab larvae. 

This study lays the groundwork for future research such as focusing on the skeletal composition 

of larval stone crabs throughout ontogeny when exposed to multiple stressors such as elevated partial 

pressure of carbon dioxide (pCO2) levels and temperature. Changes in the skeletal composition during 

exposure to elevated pCO2 and temperature could make larvae more susceptible to predation or affect 

larval swimming behaviors that mediate vertical positioning in the water column. These changes could 

ultimately affect larval transport and the sustainability of future stone crab populations. 
10

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Appendix

Florida stone crab (Menippe mercenaria) larval trace-metal data.

[Sr/Ca, strontium-to-calcium ratio; Mg/Ca, magnesium-to-calcium ration; mmol mol–1, millimole per mole]

Brood Stage Sr/Ca (mmol mol-1) Mg/Ca (mmol mol-1)

F1 III 5.856 145.540

F2 III 5.956 182.001

F4 III 5.730 220.434

F5 III 6.150 275.597

F6 III 6.233 322.400

F7 III 5.795 159.566

F8 III 5.770 249.495

F10 III 5.805 266.556

F11 III 6.023 127.893

F15 III 5.848 235.156

F16 III 5.910 216.384

F1 V 6.009 274.591

F2 V 6.691 432.760

F3 V 6.256 259.305

F4 V 6.359 322.922

F5 V 6.120 200.995

F6 V 6.457 298.697

F7 V 6.154 197.350

F8 V 5.972 231.113

F9 V 5.932 243.803

F10 V 5.913 255.762

F11 V 5.905 297.372

F12 V 5.555 190.304
Gravinese and others—A Methodology for Quantifying Trace Elements in the Exoskeletons of Florida Stone Crab (Menippe mercenaria) Larvae Using Inductively Coupled 

Plasma Optical Emission Spectrometry (ICP–OES)—Open-File Report 2016–1148

ISSN 2331-1258 (online)

https://doi.org/10.3133/ofr20161148