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Individual-Specific Transgenerational Marking of Fish Populations Based on a Barium Dual-Isotope Procedure Gonzalo Huelga-Suarez,† Mariella Moldovan,† America Garcia-Valiente,‡ Eva Garcia-Vazquez,‡ and J. Ignacio Garcia Alonso*,† † ‡
Department of Physical and Analytical Chemistry, University of Oviedo, Julian Clavería 8, 33006-Oviedo, Spain Department of Functional Biology, University of Oviedo, Julian Clavería s/n, 33006-Oviedo, Spain ABSTRACT: The present study focuses on the development and evaluation of an individualspecific transgenerational marking procedure using two enriched barium isotopes, 135Ba and 137Ba, mixed at a given and selectable molar ratio. The method is based on the deconvolution of the isotope patterns found in the sample into four molar contribution factors: natural xenon (Xe nat), natural barium (Ba nat), Ba135, and Ba137. The ratio of molar contributions between Ba137 and Ba135 is constant and independent of the contribution of natural barium in the sample. This procedure was tested in brown trout (Salmo trutta) kept in captivity. Trout were injected with three different Ba137/Ba135 isotopic signatures ca. 7 months and 7 days before spawning to compare the efficiency of the marking procedure at long and short term, respectively. The barium isotopic profiles were measured in the offspring by means of inductively coupled plasma mass spectrometry. Each of the three different isotopic signatures was unequivocally identified in the offspring in both whole eggs and larvae. For 9 month old offspring, the characteristic barium isotope signatures could also be detected in the otoliths even in the presence of a high and variable amount of barium of natural isotope abundance. In conclusion, it can be stated that the proposed dual-isotope marking is inheritable and can be detected after both long-term and short-term marking. Furthermore, the dual-isotope marking can be made individual-specific, so that it allows identification of offspring from a single individual or a group of individuals within a given fish group.
ish marking methods are an essential tool for fisheries management and research because of their interest and usefulness for population studies, study of migratory routes, and/or definition of protected areas, among other purposes. Currently, a number of fish marking methods are available, including physical,1 genetic,2 or chemical3 tags and marks. Chemical marking can be divided into two main categories: fluorescent compound4,5 and elemental6 marking. Both types of chemical marking methods have been widely used, demonstrating their robustness and marking efficiency over a wide range of chemical compound/element concentrations, immersion times, and development stages.7 However, the use of chemical compounds can be associated with important drawbacks, such as limited success of marking,8 excessive stress and mortality,9 or the need to adjust the pH of the water to improve the solubility of the compounds.10 On the other hand, although strontium is the most used element for fish elemental marking, its main disadvantage is that it is found naturally in fish tissue. In this sense, large amounts of this element are needed to ensure a clearly artificial mark, as strontium concentration varies significantly in different environments as a function of water chemistry, temperature, and diet.11 Rare-earth elements have also been employed as chemical markers, because the presence of these elements in many fish at very low concentration makes them less ambiguous markers than strontium. The use of stable isotopes offers an alternative to the methods previously discussed. Natural variations in isotopic ratios have
F
r 2011 American Chemical Society
been primarily used to trace the origin and movement of different species by measuring isotopic ratios in scales, muscle tissue, and otoliths.13,14 Nonetheless, this method has been mainly limited to C, O, N, S, and Sr. In this respect, the use of enriched stable isotopes is gaining popularity as it permits creation of marks that cannot be mistaken for a natural signature. For example, Munro et al.15 employed 137Ba and 86Sr to mark juvenile golden perch by immersion in water enriched in such isotopes. Nevertheless, an important advance in the use of enriched stable isotopes as markers was proposed by Thorrold et al.,16 who described a new transgenerational marking procedure that relies on the transfer of a Ba isotope spike from gravid females to the embryonic otoliths of their offspring. Almany et al.17 evaluated this procedure to study coral reef populations, showing its effectiveness. With this methodology it is possible to mark thousands of larvae with a single maternal parent injection. Once deposited in the core of the otolith, the isotope signature remains intact throughout the life of the fish. According to this, there is no doubt that this method offers a faster and a more efficient alternative to physical and fluorescent chemical/elemental marking. Unfortunately, this single-isotope procedure does not allow differential marking. In fact, in a recent study, Williamson et al.18 demonstrated the efficacy of this methodology when using two dose rates of Received: July 28, 2011 Accepted: November 21, 2011 Published: November 21, 2011 127
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Table 1. Natural and Enriched Barium Isotopic Abundances (atom %)a isotope
Xe nat
Ba nat
Ba137
130
4.0710 ( 0.0013
0.106 ( 0.001
0.0024 ( 0.0001
0.0017 ( 0.0001
132
26.9086 ( 0.0033
0.101 ( 0.001
0.0114 ( 0.0004
0.0098 ( 0.0002
134
10.4357 ( 0.0021
0.0324 ( 0.0009
135 8.8573 ( 0.0044
136
a
Ba135
2.417 ( 0.018
0.1973 ( 0.0002
6.592 ( 0.012
94.8488 ( 0.0014
0.084 ( 0.016
7.854 ( 0.024
2.9932 ( 0.0004
0.3182 ( 0.0034
137
11.232 ( 0.024
0.6752 ( 0.0002
82.234 ( 0.039
138
71.698 ( 0.042
2.2717 ( 0.0011
17.321 ( 0.020
The isotope composition of natural abundance xenon at the barium isotopes is also given.
Table 2. Instrument Settings and Acquisition Parameters for the Element2 SF-ICPMS and the Neptune MC-ICPMS Units
described here is based on the use of two barium-enriched isotopes mixed at a given and selectable molar ratio. The development of the data treatment procedure and its evaluation on brown trout (Salmo trutta) is described.
Instrument Settings for the Element2 SF-ICPMS Unit rf power
1275 W
cool gas flow rate
16 L min1
sample gas flow rate
0.98 L min1
’ EXPERIMENTAL SECTION
1
auxiliary gas flow rate
0.87 L min
Reagents and Materials. Two barium carbonates, enriched in
135
Ba (94.85%) and 137Ba (82.23%), respectively, were purchased from Isoflex (San Francisco, CA). These enriched tracers will be referred to as “Ba135” and “Ba137” throughout the paper. Barium standard stock solutions of natural isotope abundance were supplied by Merck (Darmstadt, Germany). The isotopic abundances of natural barium and the two isotopic profiles used for the marking of trout are presented in Table 1.21 The isotope composition of the two enriched barium spikes was determined by multicollector (MC) inductively coupled plasma mass spectrometry (ICPMS) using a natural abundance barium solution for mass bias correction. Ultrapure water was obtained from a Milli-Q Gradient A10 water purification system (Millipore, Molsheim, France). All material employed throughout this work was thoroughly cleaned before its use following a three-step cleaning procedure consisting in successive immersions (24 h each) in sub-boiled HCl (10%, v/v), sub-boiled HNO3 (10%, v/v), and Milli-Q water baths. Instrumentation. Egg and larva measurements were carried out on an Element2 sector field (SF)-ICPMS unit (Thermo Electron Corp., Bremen, Germany). This is a double-focusing magnetic sector mass spectrometer of reverse NierJohnson geometry. In the configuration used, the instrument was equipped with a Meinhard nebulizer, a double-pass Scott-type spray chamber operating at room temperature, and a secondary electron multiplier with a discrete dynode detector. All isotope ratio measurements were performed at low-resolution setting (m/Δm = 300) using the electrostatic scanning (E-scan) mode, so the accelerating voltage is scanned keeping the magnetic field constant. To obtain optimum precision and accuracy, the operating parameters were adjusted to the values listed in Table 2. Otolith measurements were performed using the Neptune multicollector ICPMS instrument (Thermo Electron). This instrument provides double-focusing with a NierJohnson geometry and was operated in low-resolution mode (m/Δm = 400). The sample introduction system consisted of an autoaspirating low-flow (100 μL min1) PFA nebulizer (ESI Scientific, Omaha, NE) mounted onto a combined cyclonic/double-pass spray chamber made of quartz glass. The instrument settings, cup configuration, and data acquisition parameters used for the determination of barium isotope ratios are also summarized in Table 2.
Acquisition Parameters for the Element2 SF-ICPMS Unit settling time
0.001 s
mass window
5%
sample time
0.001 s
number of points per peak
200
number of runs
10
number of passes
600
Instrument Settings for the Neptune MC-ICPMS Unit rf power
1200 W
plasma gas flow rate
15 L min1
nebulizer gas flow rate
1.0 L min1
auxiliary gas flow rate
0.8 L min1
Acquisition Parameters for the Neptune MC-ICPMS Unit integration time
4.2 s
number of cycles
50 per block
number of blocks
1
L3 Ba+
Cup Configuration for the Neptune MC-ICPMS Unit L2 L1 C H1 H2
130
132
134
130
132
134
Xe+
Ba+ Xe+
Ba+ Xe+
135
Ba+
136
Ba+
137
Ba+
H3 138
Ba+
136
Xe+
enriched barium isotopes to produce unequivocal marks on the otoliths of brown-marbled groupers (Epinephelu fuscoguttatus). However, as they stated, “it was not possible to assign eggs produced from each treatment group to individual females”. Recently, a dual-isotope marking procedure for manufactured goods and living organisms was developed in our laboratory19 and first applied for the labeling of black powder by using two enriched tin isotopes.20 The method is based on the use of molar fraction ratios of two enriched isotopes and the automatic correction of the natural element isotope contribution. In this study, such an alternative marking procedure was applied to mark fish so that individuals could be specifically marked, allowing both marking and codification with a single injection. The procedure 128
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Analytical Chemistry This instrument was also employed for the measurement of the isotope composition of the spikes given in Table 1. Trout eggs and larvae were freeze-dried in a Lyolab 3000 instrument (Heto-Holten A/S, Allerod, Denmark). For their digestion, a CEM automated research microwave reactor, purchased from CEM Explorer (Matthews, NC), was used. Procedures. Fish Marking. Twelve adult brown trout (S. trutta) reared at El Esmerillon fisherman society (Asturias, Spain) were used for this study. Trout were injected ca. 7 months and 7 days before spawning to compare the efficiency of the marking procedure at long and short term, respectively. Three different mixtures of Ba135 and Ba137 were prepared so that each mixture had a different molar ratio between both enriched isotopes. Thus, different Ba137/Ba135 isotopic signatures (hereafter A, B, and C, in which the isotopic mixtures had a molar ratio of ca. 3:1, 1:1, and 1:3, respectively) were deliberately prepared. Trout were first anesthetized in a bucket containing ethylene glycolmonophenyl ether diluted in water and then weighed to determine the injection volume of the marking isotopic mixture for each fish so that a 0.3 mg of Ba/kg of body mass dosage was delivered. The barium solution was intramuscularly administered using an insulin syringe. Each fish was afterward gently revived in its initial tank, where they were kept until the spawning period. In this sense, nine trout (three A + three B + three C) were marked in July 2009 (long-term study). During the spawning period, it was determined that only one A + two B + two C were female trout as, during the marking phase, it was not possible to distinguish the gender of the trout. For the short-term study three female trout (one A + one B + one C) were marked in January 2010, 1 week before spawning. During spawning (carried out several times between Dec 22, 2009, and Jan 26, 2010), eggs from each individual female trout were collected separately. Before fertilization with the sperm of two to three nonmarked trout males, a certain amount of eggs were sampled for analytical purposes. Furthermore, fertilized eggs were kept in frames until the hatching of larvae. Finally, larvae (9 weeks old) and juveniles (9 months old) were collected to monitor the behavior in time of the proposed methodology. Egg and Larva Preparation. Eggs and larvae were freeze-dried for 8 h. Subsequently, 1 mL of sub-boiled HNO3, 0.5 mL of Suprapur H2O2 (30%, v/v), and 50 μL of Suprapur HF (40%, v/v) were added as an acid mixture to digest these samples. The microwaveassisted digestion program employed was the following: (a) 2 min ramp to 50 °C, (b) 2 min hold at 50 °C, (c) 2 min ramp to 70 °C, (d) 1 min hold at 70 °C, (e) cooling. Steps a and b were performed at 50 W of power, while a power of 70 W was used for steps c and d. Samples were diluted with Milli-Q water prior to their measurement by SF-ICPMS. Otolith Preparation. Sagittal otoliths were extracted at the Department of Functional Biology of the University of Oviedo. Upon extraction, otoliths were thoroughly rinsed with Milli-Q water to remove possible adhering tissue, dried on filter paper, and stored in 1.5 mL microcentrifuge tubes. Full otoliths were dissolved in 100 μL of sub-boiled HNO3 at room temperature and diluted with Milli-Q water for their measurement by means of MC-ICPMS. Measurement of Barium Isotope Compositions in the Spikes and Enriched Mixtures by ICPMS. Barium has seven stable isotopes of masses 130, 132, 134, 135, 136, 137, and 138. Their natural isotope abundances, as tabulated by IUPAC, are given in Table 1. In ICPMS measurements, isobaric interferences at masses 130, 132, 134, and 136 may occur due to xenon impurities in the argon plasma gas. The natural isotope composition of xenon is also
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given in Table 1 for comparison purposes. Therefore, isotopes 135, 137, and 138 are free from isobaric interferences (the interference from low-abundance 138La and 138Ce on 138Ba was considered negligible in fish tissues). This fact explains why enriched 135Ba and 137Ba isotopes were selected for fish marking. The isotope composition of the enriched spikes used in this study was measured by MC-ICPMS using a bracketing procedure in which a blank, a natural abundance barium, the enriched spike, and a second natural abundance barium were measured in succession. The blank was employed to subtract the xenon contribution to all barium masses measured, while natural barium was used for mass bias correction (exponential model). The final isotope compositions of both Ba135 and Ba137 are shown in Table 1. The same procedure was employed for the measurement of the isotope composition of the enriched mixtures. Calculation of the Molar Contributions of Each Isotope Signature in the Samples. In this case, all isotope ratios Ri = i Ba/138Ba were measured in the samples. Then the isotope composition in the sample was calculated using the equation Ais ¼
Ri
ð1Þ
n
∑
i¼1
Ri
Finally, the measured isotope abundances were then deconvoluted by multiple linear regression to calculate the molar contributions of each isotope signature in the blend using the following matrix equation: 2
A130 s A132 s A134 s A135 s A136 s
6 6 6 6 6 6 6 6 6 6 137 6 As 4 A138 s
3
2
A130 Xe nat 7 6 6 A132 7 6 Xe nat 7 6 A134 7 6 Xe nat 7 6 135 7 A nat 7¼6 6 Xe 7 6 A136 7 Xe nat 6 7 6 A137 7 4 Xe nat 5 A138 Xe nat
A130 Ba nat A132 Ba nat A134 Ba nat A135 Ba nat A136 Ba nat A137 Ba nat A138 Ba nat
A130 Ba137 A132 Ba137 A134 Ba137 A135 Ba137 A136 Ba137 A137 Ba137 A138 Ba137
2 3 e130 A130 Ba135 6 7 72 3 6 e132 A132 Ba135 7 6 7 xXe nat 6 e134 A134 7 6 Ba135 76 6 7 x 7 6 135 135 76 Ba nat 7 6e ABa135 6 þ 7 7 6 136 x Ba137 5 74 6e A136 Ba135 7 6 137 137 7 xBa135 6e ABa135 5 4 138 e138 ABa135
3 7 7 7 7 7 7 7 7 7 7 7 5
ð2Þ It is worth stressing that eq 2 takes into account the contribution of natural abundance barium and natural abundance xenon to the final isotope abundances measured, so blank correction was not necessary here. Mass bias correction was performed internally by minimizing the square sum of residuals of the multiple linear regression using the SOLVER application in Excel. A full explanation of the theoretical background of the multiple linear regression marking procedure employed here can be found elsewhere.19,20
’ RESULTS AND DISCUSSION A 0.3 mg of Ba/kg of body mass dosage was delivered intramuscularly to each female trout as said in the Procedures. This amount of barium was selected according to what was observed in previous studies. In this sense, Williamson et al.22 stated that a single injection of barium solution (up to 4 mg of Ba/kg) has no significant effects on the physiology of Plectropomus leopardus (coral-reef grouper). Furthermore, the risk to humans who may consume treated fish is minimal as defined by the International Programme for Chemical Safety (IPCS)23 and the U.S. Department of Health and Human Services.24 Thus, enriched barium solutions can be used at low dosages to mark female fish and, therefore, to mark larvae of fish subject to potential human consumption. 129
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mixed solutions, which contained NBa137/NBa135 at ca. 3:1, 1:1, and 1:3 molar ratios (mixtures A, B, and C, respectively), were prepared for the marking of fish. The actual isotope composition of the mixed spike solutions (A, B, and C) was measured as described in the Procedures. The molar ratio NBa137/NBa135 in all mixtures was obtained from the molar fraction ratios xBa137/ xBa135 determined from eq 2. Final results are given in Table 3. Please note that the results are given here both as molar ratios and as isotope ratios in agreement with the two alternative evaluation procedures employed for the samples as described below. Use of Isotope Ratios (138Ba/137Ba vs 138Ba/135Ba) for Sample Characterization. Classical double-isotope procedures rely on isotope ratio plots for source assignment.25 For example, double-isotope plots of 207Pb/206Pb vs 208Pb/206Pb are typically employed for lead isotope sourcing of archeological samples.26 In the present work, we have evaluated first the double-isotope plots of 138Ba/137Ba vs 138Ba/135Ba for sample characterization. All the analytical results for all the measured samples after mass bias correction are shown in Figure 1. As can be observed, all samples follow three distinct curves depending on the original isotope signature of the marking used. On the basis of the known molar composition of each enriched mixture (A, B, and C) (see Table 3) and their possible blends with natural abundance barium in the samples, theoretical mixing curves can be computed. It is possible to observe that the experimental results clearly follow the theoretical mixing curves from the pure spike mixtures (low values of 138Ba/137Ba and 138Ba/135Ba) to the natural barium composition (high values of both 138Ba/137Ba and 138 Ba/135Ba). The position of each point in the mixing curve depends both on the spike mixture used and on the amount of natural abundance barium present in the sample. Anyway, once the theoretical mixing curves are computed, source assignment is straightforward with this approach.
Once the different enriched mixtures of barium were injected into the female trout, they were kept in frames until the spawning period, in which 10 eggs of each mark (A, B, and C), randomly collected before the addition of sperm, were analyzed by using an Element2 SF-ICPMS unit. For all marks, a comparison between short (7 days) and long (ca. 7 months) term marking was done. After spawning, and subsequent fertilization with the sperm of nonmarked male trout, eggs were kept in frames until they hatched. Eggs that had the same mark were mixed together. It is worth mentioning that all the larvae whose mothers were injected with the A mixture died due to technical problems in the hatchery, so only B and C offspring remained to continue with this study. Thus, 9 week old larvae, reared in indoor tanks, were collected and analyzed to monitor the behavior of the mark. Larvae of B and C marks (five of each one) were analyzed by SFICPMS for this purpose. After this experiment, B and C larvae were mixed together. Eventually, a last experiment to control the mark evolution was performed with 9 month old juveniles. Otoliths of five juveniles randomly selected were extracted and analyzed by MC-ICPMS. Characterization of the Enriched Mixtures. By using the enriched spikes, whose abundances are shown in Table 1, three Table 3. Molar Ratios, NBa137/NBa135, and Isotope Ratios, 138 Ba/137Ba and 138Ba/135Ba, Obtained for the Enriched Mixtures A, B, and Ca parameter
mixture A
mixture B
mixture C
NBa137/NBa135
2.9645 ( 0.0087
0.9905 ( 0.0022
0.3317 ( 0.0013
138
0.2303 ( 0.0006
0.2478 ( 0.0010
0.2983 ( 0.0007
138
0.5972 ( 0.0024
0.2164 ( 0.0011
0.0887 ( 0.0003
Ba/137Ba Ba/135Ba
a
Uncertainty is given as 1 standard deviation. For comparison, the natural isotope ratios are 138Ba/137Ba = 6.3834 and 138Ba/135Ba = 10.877.21
Figure 1. Isotope ratios 138Ba/137Ba and 138Ba/135Ba found in the eggs (squares and triangles correspond to long-term and short-term marking, respectively), larvae (circles), and otoliths of juveniles (tilted squares) spawned from injected females. Each curve represents the theoretical isotope ratios 138Ba/137Ba and 138Ba/135Ba for the three different enriched mixtures: A (black), B (white), and C (gray). 130
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Figure 2. Molar fraction ratios xBa137/xBa135 found in the eggs (squares and triangles correspond to long-term and short-term marking, respectively), larvae (circles), and otoliths of juveniles (tilted squares) spawned from injected females. The y axis is given on the logarithmic scale. The horizontal lines represent the NBa137/NBa135 molar ratios in the enriched mixtures: A (black), B (white), and C (gray). Error bars correspond to combined uncertainties for each molar fraction ratio.
Use of Molar Fraction Ratios (xBa137/xBa135) for Sample Characterization. As an alternative, we have applied eq 2 to all
contribution can be found in each sample. In this sense, according to Figure 2, it is clear that samples with the same mark (A, B, or C) show an excellent agreement among them irrespective of such a difference in terms of natural barium contribution, which is a great advantage of the proposed methodology. Table 4 compiles the average of all molar fraction ratios xBa137/xBa135 for each type of sample (eggs, larvae, and otoliths) that are represented in Figure 2. In this case, the uncertainties are shown as total combined uncertainties (n = 10 for eggs and n = 5 for larvae and otoliths). As previously explained, larvae of B and C marks were mixed together, so the collection of five juveniles for the analyses of otoliths resulted in individuals that only were marked with the C enriched mixture. For this reason, no data from B otoliths are displayed. On the basis of what is shown in Figure 2 and Tables 3 and 4, it can be concluded that a good correlation does exist between the ratios of the molar fractions of the enriched mixtures and the different samples, which, in turn, were unambiguously identified from each other when having different marks. It is also worth emphasizing that there is good agreement between long-term and short-term marking. Comparison of Data Treatment Procedures. When comparing Figures 1 and 2, it is clear that molar fraction ratios offer a better data treatment in double-isotope marking procedures. When using traditional double-isotope ratio plots, a theoretical mixing curve needs to be built based on the known isotope compositions of natural barium and the two enriched spikes used. If the theoretical mixing curve is not calculated, the graphical representation of isotope ratios does not allow the identification of the mark at first sight. Additionally, another important disadvantage when using isotope ratio plots is the fact that distinguishing samples of different marks might be very difficult when a high natural barium concentration is present as all of the theoretical curves converge to the same values.
measured samples, and the molar fraction ratios xBa137/xBa135 were computed for all of them. Please note that this alternative procedure should show constant molar fraction ratios xBa137/ xBa135 regardless of the contribution of natural barium in the samples (xBa nat) for each isotope mixture. Additionally, the molar fraction ratios should be equal to the molar ratios computed for each isotope mixture as given in Table 3. All the analytical results obtained are represented in Figure 2, in which xBa137/xBa135 molar fraction ratios are plotted versus the normalized molar fractions of natural barium. The horizontal lines correspond to the molar ratios NBa137/NBa135 obtained for the enriched mixtures (A, B, and C) shown in Table 3. Please note that combined uncertainties were calculated for each measurement (error bars in Figure 2). These are based on the molar fraction uncertainties obtained by the multiple linear regression approach. When using eq 2, standard uncertainties can be determined on the basis of the variancecovariance matrix. Detailed information in this regard can be found elsewhere.27 As can be observed, all molar fraction ratios determined can be easily assigned to a given isotope mixture irrespective of the contribution of natural barium in each sample. Thus, different samples from the same isotope signature could be compared without difficulty. It can be observed that the natural barium contribution for short-term and long-term marking samples are different in some cases, especially in the C enriched mixture. Short-term and long-term marking samples belong to different mothers, so the assimilation of the enriched mixture could have been different depending on various aspects, such as the quality of the Milli-Q water used for sample dilutions, the assimilation of the enriched mixture by the trout, or the efficacy of the injection, among many others. As a result, a different natural barium 131
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Table 4. Average Molar Fraction Ratios xBa137/xBa135 Obtained for the Different Samples (Eggs, Larvae, and Otoliths)a sample
xBa137/xBa135
are not gravid yet) produced a 100% successful marking of the offspring from the first spawning after injection in all samples investigated. Given the increasing interest of fisheries in identifying individuals or groups of fish for assessing essential issues, such as mortality, abundance, migration movements, or stocking success, among many others, a methodology in which an individual fish can be specifically marked was urgently required. All the characteristics and results shown throughout this work would make this individual-specific transgenerational marking procedure very attractive and easy to employ. In this work, two data treatment procedures were compared. Although both procedures were able to clearly identify the artificial mark, the molar fraction ratio approach was more intuitive and provided direct identification of the mark regardless of the contribution of barium of natural origin. In comparison to previous transgenerational isotope marking procedures, the possibility of differential marking of individuals is a clear advantage of the proposed procedure, which provides all the advantages attributed only to genetic markers until now.29 Moreover, it is also important to bear in mind that despite the fact that enriched isotopes are relatively expensive, their use as markers is not more expensive than other tags or marks, as one single maternal parent injection with a given Ba137/Ba135 mixture permits marking for life more than 1000 larvae (i.e., trout) for ca. $5.00. Thus, the methodology described in this paper represents a new and important advance in the transgenerational isotopic marking and should instill confidence in the use of this technique for future fish population studies.
uncertainty
A eggs (long-term marking)
3.000
0.053
A eggs (short-term marking) B eggs (long-term marking)
3.037 0.976
0.132 0.016
B eggs (short-term marking)
0.984
0.008
C eggs (long-term marking)
0.341
0.013
C eggs (short-term marking)
0.322
0.014
B larvae
1.006
0.021
C larvae
0.330
0.004
C otoliths
0.341
0.038
a
Uncertainties are given as combined uncertainties (n = 10 for eggs and n = 5 for larvae and otoliths).
On the other hand, when using molar fraction ratios, it is not necessary to calculate a theoretical curve and direct interpretation of the data is possible (Figure 2, Tables 3 and 4). In addition, the graphical representation of the data is much clearer, as the determination of molar fraction ratios is independent of the concentration of natural barium, which appeared to be up to 98% in some samples, most of which were otoliths. Due to this high concentration of natural barium against the enriched mixture, otoliths were measured by MC-ICPMS because the Element2 unit did not have enough precision to clearly identify the isotopic mark. In this sense, although MC-ICPMS was used for the analyses of otoliths, the precision obtained was worse than expected for this kind of instrument as the concentration of barium in the analyzed solutions was below 2 ng L1 while the standard concentration for MC-ICPMS measurements should be at least 100 times higher. Ideally, the direct analysis of the otoliths by laser ablation ICPMS would have avoided the dilution of the mark with natural barium, and a less precise ICPMS instrument could have been employed. Unfortunately, no laser ablation system was available when these studies were performed. Furthermore, mass bias in the samples was internally corrected by minimizing the regression sum of squared residuals,28 so the measurement of natural abundance barium solutions could be avoided. As barium contains seven stable isotopes, eq 2 is overdetermined (seven equations with four unknowns) and one of the extra degrees of freedom can be used to calculate the mass bias factor which best fits the data. Also, together with the values of the unknown xBa nat, xBa137, and xBa135, their uncertainties can be easily obtained by multiple linear regression. Further information regarding this mathematical development for traceability purposes can be found elsewhere.19,28 To sum up, the measurement of the ratio of the molar fractions between those two isotopic profiles in the offspring by means of ICPMS provides a new alternative to traditional isotope ratios and permits the unequivocal identification of each isotopic signature.20
’ AUTHOR INFORMATION Corresponding Author
*Phone: +34 985103484. Fax: +34 985103125. E-mail: jiga@ uniovi.es.
’ ACKNOWLEDGMENT We thank Jeronimo de la Hoz and the Direccion General de Recursos Naturales y Proteccion Ambiental (Gobierno del Principado de Asturias) for their collaboration. El Esmerillon fisherman society is acknowledged for their great assistance. Special thanks are due to Juan Corzo for his daily care of the marked trout. This research was supported by the Ministerio de Ciencia e Innovacion (MICINN; Project CTQ-2009-12814) and the Fundacion para el Fomento en Asturias de la Investigacion Científica Aplicada y la Tecnología (FICYT; Project PCTI-IB09-089). ’ DEFINITIONS OF SYMBOLS USED Ba135 Ba137 Ba nat NBa135 NBa137 Ais
’ CONCLUSIONS In the present study, a new individual-specific transgenerational marking procedure using enriched 135Ba and 137Ba mixed at a given and selectable molar ratio has been presented. In this sense, it has been demonstrated that the dual-isotope marking is inheritable. The injection of 0.3 mg of Ba/kg of body mass of a Ba137/Ba135 mixture into the body of female trout (even if they
AiBa nat AiBa135 AiBa137 132
tracer enriched in 135Ba tracer enriched in 137Ba natural abundance Ba number of moles of tracer Ba135 in the enriched mixture or sample number of moles of tracer Ba137 in the enriched mixture or sample isotope abundance of isotope i in the sample (measured) isotope abundance of isotope i in the natural Ba (tabulated) isotope abundance of isotope i in the Ba135 tracer (measured) isotope abundance of isotope i in the Ba137 tracer (measured) dx.doi.org/10.1021/ac201946k |Anal. Chem. 2012, 84, 127–133
Analytical Chemistry 138 138
Ba/137Ba Ba/135Ba
xBa135 xBa137 xBa nat xBa nat normalized xBa137/xBa135
ARTICLE
isotope ratio isotope ratio molar fraction of tracer Ba135 in the sample (calculated using eq 2) molar fraction of tracer Ba137 in the sample (calculated using eq 2) molar fraction of natural Ba in the sample (calculated using eq 2) normalized molar fraction of natural Ba in the sample (xBa nat/(xBa nat + xBa135 + xBa137)) molar fraction ratio in the samples (equal to NBa137/NBa135 in the enriched mixtures)
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dx.doi.org/10.1021/ac201946k |Anal. Chem. 2012, 84, 127–133
