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Bioaccumulation of heavy metals and radionuclides from seawater by encased embryos of the spotted dogfish Scyliorhinus canicula
Marine Pollution Bulletin 52 (2006) 1278–1286 www.elsevier.com/locate/marpolbul
Bioaccumulation of heavy metals and radionuclides from seawater by encased embryos of the spotted dogfish Scyliorhinus canicula Ross A. Jeffree *, Michel Warnau, Francois Oberhansli, Jean-Louis Teyssie Radioecology Laboratory, IAEA Marine Environment Laboratories, 4 Quai Antoine, MC 98000, Monaco
Abstract Encased embryos of spotted dogfish Scyliorhinus canicula absorbed six radio-isotopes (241Am, 109Cd, 57Co, 134Cs, 54Mn and 65Zn) directly from seawater during short-term experimental exposure, demonstrating the permeability of the egg-case to these contaminants. Embryo to water concentration factors (CFs) ranged from 0.14 for 134Cs to 7.4 for 65Zn. The 65Zn and 57Co CFs increased exponentially with embryo length, whereas the CF for 109Cd declined with length. Among different components of the encased embryo the egg case was the major repository (69–99%) of all six radio-isotopes that were distributed throughout its wall. Egg-case CFs were as high as 103 for 57 Co and 65Zn, making it the major source of gamma radiation exposure to the embryo and potentially of radio-isotopes for continued absorption by the embryo, following the uptake phase of the experiment. The patterns of uptake by the egg-case approximated linearity for most isotopes and loss rates were isotope-specific; egg-case biokinetics were not greatly affected by the viability of the contained embryo. Within the embryo initial data on radio isotopic distribution show that the skin is their major site of uptake, as previously demonstrated for juveniles. 2006 Elsevier Ltd. All rights reserved. Keywords: Dogfish; Scyliorhinus canicula; Heavy metals; Radionuclides; Egg; Embryo
1. Introduction A previous experimental study compared the uptake of seven radio-isotopes (241Am, 109Cd, 57Co, 51Cr, 134Cs, 54 Mn and 65Zn) from seawater by spotted dogfish Scyliorhinus canicula (Chondrichthys: Scyliorhinidae) and the turbot Psetta maxima (Actinopterygii: Teleostei) (Jeffree et al., in press). The results indicated the greater permeability of dogfish to most of these isotopes, with some isotopes attaining whole-body concentration factors (CFs) in dogfish of more than 102 during short-term exposures. These results also suggested the enhanced susceptibility of dogfish to such contaminants in seawater. Chondrichthyans are also characterized by low fecundity relative to Teleost fishes, that is also reflected in their smaller egg clutches (Camhi et al., 1998; Barker and Schl-
*
Corresponding author. Tel.: +377 97 97 72 78; fax: +377 97 97 72 76. E-mail address: R.Jeff[email protected] (R.A. Jeffree).
0025-326X/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2006.03.015
uessel, 2005). For the encased Chondrichthyan embryo that has been laid at a site chosen by the mother, its potential pathway of exposure to environmental contaminants is directly from seawater, apart from those contaminants that may be included in yolk and other egg components due to maternal transfer and subsequently absorbed by the embryo. A previous study has shown that developing embryos of S. canicula can increase their iron concentration when exposed to artificial seawater with added iron (Irwin and Davenport, 2002). However, the egg-case is strong and very protective of the embryo, which is reported to be particularly susceptible to seawater exposure during the earlier stages of embryogenesis (Ballard et al., 1993). This experimental study was undertaken to determine the extent to which the encased embryo of S. canicula is exposed to radio-isotopes of heavy metals and radionuclides (241Am, 109Cd, 57Co, 134Cs, 54Mn and 65Zn) in seawater and their subsequent patterns of bioaccumulation and depuration by the embryo and other components of the egg.
R.A. Jeffree et al. / Marine Pollution Bulletin 52 (2006) 1278–1286
2. Materials and methods 2.1. Acclimation and experimental exposure of organisms Two clutches of eggs of the spotted dogfish S. canicula were maintained for six weeks in 40 L Mediterranean sea water aquaria at 17.0 ± 0.5 C, salinity 38 psu, pH 8.05, with 10% water renewal h 1 and a light/dark cycle of 10 h/14 h. Viable eggs (n = 23) were distributed in a 70 L glass aquarium and acclimated for two weeks to experimental conditions, as described above, in a constantly 0.22 lm-aerated closed circuit. Eggs ranged in weight from 2.92 g to 6.44 g with a mean individual wet wt of 4.66 ± 0.98 g. Eggs were carefully sampled from the aquaria to avoid bubble formation in the case and were daily transferred in a counting tube within clean seawater for radio-spectrometric analysis. Two encased embryos that were sacrificed by exposure to low temperate were also included in the uptake experiment to initially assess any obvious effect of the embryo on total uptake of each radio-isotope. At the end of the 15 day uptake and 21 day loss experiments, eggs were dissected into the following compartments for determination of their radioisotope concentrations, following wet-weighing: egg case, jelly, yolk and embryo. One embryo also hatched from its egg cases at the 11th day of the uptake experiment and at the 13th day of the loss experiment. These were immediately dissected into liver, digestive tract, skin, kidneys, head, muscle + skeleton, for radioanalysis. 3. Radio-isotopes and their radioanalysis High-specific activity radio-isotopes were purchased from Amersham, UK (241Am, 57Co, 51Cr and 134Cs,) and Isotope Product Laboratory, USA (109Cd, 54Mn and 65Zn). Seawater spikes resulted in low activities of each selected radio-isotope: 241 Am (0.2 kBq l 1), 109Cd (1.3 kBq l 1), 57Co (0.5 kBq l 1), 134 Cs (1 kBq l 1), 54Mn (0.5 kBq l 1) and 65Zn (0.5 kBq l 1). No change in pH was detectable after radio-isotope addition. Seawater and spikes were renewed daily for 5 days, and then every second day in order to keep exposure activities constant. Activities of the radio-isotopes in seawater were checked daily, both before and after each seawater renewal, to determine their time-integrated activities. The speciation
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of these radio-isotopes in the experimental solution had been modeled for a previous study (Jeffree et al., in press). Radioactivity levels in seawater, dogfish eggs and their components were determined using a high-resolution cspectrometry system consisting of four coaxial Germanium (N- or P-type) detectors (EGNC 33-195-R, Intertechnique; 40–70% efficiency) that were connected to a multi-channel analyzer and a personal computer employing spectral analysis software (Interwinner 6, Intertechnique). The radioactivity levels of the samples were determined by comparison with known standards of appropriate geometry and were corrected for background and isotope physical decay. Counting times were adapted to obtain count rates with relative propagated errors less than 5%, viz. typically 10– 30 min for whole organism radioanalyses and 1–12 h for seawater and dissected body compartment samples. 4. Results 4.1. Uptake In Table 1 is shown the mean and median percentage distribution of each radio-isotope at the end of the uptake experiment in the dogfish embryo, its yolk, the egg case and the other contents of the egg case, i.e., the jelly. Ninety eight percent or more of the 241Am, 109Cd, 57Co, 54Mn and 65Zn are associated with the egg case, and with less than 1% associated with the embryo. For these five radio-isotopes very small percentages are also found in both the yolk and the jelly. In contrast to these results, 10% of the total activity of 134Cs is associated with the embryo and about 20% associated with the jelly, with again the majority of this radio-isotope associated with the egg case, and a very small percentage associated with the egg yolk. In Table 2 is given the mean and median CF for each isotope and dissected component at the end of the uptake phase of the experiment. With the exception of 134Cs the egg case has CFs of radio-isotopes that are 2–3 orders of magnitude higher than those of the embryo, which are more comparable to those of the jelly; the yolk is also low in its CFs compared to the jelly, with the exception of 109Cd. For 134Cs the CF for the egg case is only an order of magnitude greater than that of the embryo, that is com-
Table 1 Mean (+ 1SD) and median (range) percentage radio-isotope distributions in dissected egg components at the end of the uptake experiment 54
57
65
109
134
241
Embryo
0.47 ± 0.85 0.13 (0.06–2.84)
0.28 ± 0.39 0.08 (0.02–1.00)
0.47 ± 0.60 0.21 (0.02–1.50)
0.11 ± 0.20 0.02 (0.01–0.62)
10.27 ± 12.03 6.18 (0.37–32.65)
0.77 ± 0.71 0.49 (0.02–2.19)
Yolk
0.08 ± 0.10 0.04 (0.00–0.14)
0.02 ± 0.01 0.01 (0.00–0.04)
0.04 ± 0.05 0.03 (0.00–0.16)
0.10 ± 0.16 0.03 (0.00–0.46)
1.62 ± 1.51 1.11 (0.00–3.77)
0.03 ± 0.03 0.03 (0.00–0.11)
Case
97.95 ± 2.06 98.89 (93.65–99.70)
99.39 ± 0.56 99.80 (98.46–99.90)
99.39 ± 0.69 99.74 (98.09–99.92)
99.65 ± 0.25 99.78 (99.18–99.94)
68.87 ± 19.19 75.08 (32.23–93.53)
98.72 ± 0.85 99.03 (97.23–99.42)
Jelly
1.50 ± 1.47 0.85 (0.16–3.36)
0.31 ± 0.33 0.11 (0.04–0.57)
0.10 ± 0.15 0.03 (0.00–0.41)
0.14 ± 0.14 0.10 (0.01–0.47)
19.24 ± 21.73 7.27 (1.88–66.22)
0.48 ± 0.37 0.33 (0.10–1.13)
Mn
Co
Zn
Cd
Cs
Am
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Table 2 Mean concentration factors (CFs) and median (range) for radio-isotope distributions in dissected egg components at the end of the uptake experiment 54
57
65
109
134
241
Embryo
2.98 ± 2.26 2.21 (0.95–7.89)
2.93 ± 2.53 1.48 (0.69–6.76)
7.38 ± 6.13 5.33 (1.60–16.45)
0.42 ± 0.32 0.32 (0.23–1.25)
0.14 ± 0.07 0.11 (0.07–0.23)
5.06 ± 2.61 4.72 (0.66–8.57)
Yolk
0.40 ± 0.37 0.27 (0.00–1.08)
0.16 ± 0.11 0.14 (0.00–0.38)
0.81 ± 0.79 0.71 (0.00–2.72)
1.15 ± 1.91 0.23 (0.00–5.49)
0.02 ± 0.01 0.01 (0.00–0.04)
0.16 ± 0.13 0.16 (0.00–0.41)
Case
873.33 ± 278.1 732.51 (602.99–1401.71)
1031.63 ± 213.2 1049.54 (641.93–1432.91)
1786.72 ± 442.3 1850.95 (825.97–2461.11)
957.87 ± 311.99 1061.92 (521.30–1538.69)
1.20 ± 1.10 0.87 (0.44–3.94)
539.89 ± 105.5 544.80 (375.21–729.89)
Jelly
9.31 ± 7.22 7.63 (1.99–25.29)
2.18 ± 2.02 1.55 (0.42–5.77)
1.32 ± 2.21 0.43 (0.04–7.07)
0.99 ± 1.01 0.69 (0.10–3.32)
0.26 ± 0.35 0.09 (0.03–1.11)
2.67 ± 2.43 2.08 (0.58–8.32)
Mn
Co
Zn
parable to the jelly, with the yolk being an order of magnitude lower than the jelly. Among the radio-isotopes the CFs of the egg case varied only by a factor of three, with exception of 134Cs that is 2–3 orders of magnitude lower than for the other five radio-isotopes. The CFs in the embryo were highest for 65Zn then 241Am and lowest by an order magnitude for 109Cd and 134Cs. These data confirm that the egg case is permeable to each water-born radio-isotope used in this experiment, particularly 134Cs, and indicate that the egg case itself is very sorptive of the majority of these radio-isotopes. The standard deviations of means and the ranges of values presented in Tables 1 and 2 for embryos also indicate that radio-isotope concentrations were highly variable among individuals sampled at the end of the uptake experiment. The embryos did vary considerably in length, from between 25 and 80 mm, which encompassed a phase of embryological development during which many physiological and anatomical changes were taking place. It also covered the period of development when the egg-case apertures, that were previously plugged, would be opened by excretions of the hatching gland (Mellinger et al., 1986). So we plotted for each radio-isotope the calculated embryo: water CF as a function of the total length of the embryo upon dissection, to discern whether some of their variability in radioisotopic content could be statistically explained. Seven eggs from one batch and two from the other, that were characterized by different size and colour, were included in this experiment. In Fig. 1 are plotted these relationships, and the fitted regression lines that were statistically significant (P < 0.05); their regression equations are given in Table 3. Our results show that the CFs of 65 Zn and 57Co significantly (P < 0.05) and appreciably increase with increasing embryo length. The data for both 241 Am and 134Cs show similar trends of increasing CF with increasing embryo length, although neither relationship is statistically significant (P > 0.05). In contrast to these results, the CF for 109Cd significantly (P < 0.001) declines with increasing embryo length. This inverse relationship is strongly influenced by one outlying point for the smallest embryo. To further assess the statistical validity of this regression, the CF values were log-transformed to further approximate a normal distribu-
Cd
Cs
Am
tion of CF values, and then a linear regression was fitted to these data. This regression explained 65% of the variance in the CFs and was also highly significant (P < 0.01), giving greater confidence in the initial finding of the inverse relationship shown in Fig. 1. There was no obvious clear separation between eggs from the two batches in these plots, however, the numbers are too small to satisfactorily evaluate statistically for differences between batches. 4.2. Loss In Table 4 is shown the mean and median percentage radio-isotope distributions in dissected egg components following 21 days of exposure to unlabelled seawater. The egg case still contains nearly all of each of the isotopes (99%), with 87.5% for 134Cs, a pattern very similar to that seen at the end of the uptake phase (Table 1). Similarly about 10% of the 134Cs is found in the embryo that contains much lower percentages of the other radio-isotopes. The yolk contains very low percentages of each radio-isotope, as it did at the end of the uptake experiment. The jelly shows a reduction in percentage activity of 134Cs by a factor of 10, compared to the end of the uptake phase. Table 5 shows the activity concentrations of radio-isotopes in dissected egg components at the end of both the uptake and loss phases that allows a measurement of the absolute amounts of depuration over 21 days. The case, with the highest activity concentrations among the dissected components, declines in these concentrations by the end of the loss phase by about a factor of 1.5–2 for 54 Mn, 109Cd, 134Cs and 241Am. Cobalt-57 and 65Zn show much smaller reductions in activity concentrations. The embryo also shows reductions in activity concentrations during the loss phase, with factors of reduction that are greatest for 57Co, 54Mn and 241Am, that range from 6– 10, and least for 134Cs and 109Cd, by factors of only about 2. If it is most simply assumed that the activity lost by the case diffuses equally in both directions, to the internal region of the egg case and back to seawater, then there are appreciable amounts of activities of all radio-isotopes
R.A. Jeffree et al. / Marine Pollution Bulletin 52 (2006) 1278–1286
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0.25 1.4
0.20
1.0
CF for 134Cs
CF for 109Cd
1.2
0.8 0.6
0.15
0.10
0.4 0.05 0.2 0.0 0
20
40
60
80
0.00
100
0
20
60
80
100
Length of embryo (mm)
10
10
8
8
CF for 241Am
CF for 57Co
Length of embryo (mm)
40
6
4
2
6
4
2
0
0 0
20
40
60
80
100
0
20
Length of embryo (mm)
40
60
80
100
Length of embryo (mm) 10
20
8
CF for 54Mn
CF for 65Zn
15
10
6
4
5 2
0
0 0
20
40
60
80
100
0
20
40
60
80
100
Length of embryo (mm)
Length of embryo (mm)
Fig. 1. Plots of embryo: water CFs versus length of embryo, as measured at the end of the uptake experiment, and the fitted significant (P < 0.05) regressions (equations given in Table 3).
Table 3 Regression equations for CFs of radio-isotopes in embryos at the end of the uptake experiment (y) as function of embryo length (x) Radio-isotope 109
Cd Co 65 Zn 57
Regression equation 0.04x
y = 3.47e y = 0.24e0.04x y = 0.65e0.04x
P value for regression
R2
n
0.0004 0.036 0.012
0.85 0.49 0.62
9 9 9
that would still be available for absorption by the embryo during the loss phase of the experiment. If this interpretation is correct then the degrees of reduction in their concentrations in the embryo measured during the loss phase may be an underestimate of the embryo’s true rates of depuration of these radio-isotopes.
These sets of data on the distributions of radio-isotopes in components of the encased embryos that were dissected at the end of both the uptake and loss experiments have demonstrated that the egg case is the repository of nearly all activity of each radio-isotope, with the exception of 134 Cs. Thus, the measurements of total activity in the encased embryo that were taken during the experiments were used to determine the patterns of uptake and loss in the egg-case for each isotope, not including 134Cs. These plots of CFs calculated for the total encased embryo are shown in Fig. 2 for egg cases containing live embryos and control egg cases. The uptake data show patterns of uptake that approximate to linear rates that indicate that equilibrium concentration factors have not been attained within 15 days of exposure to radio-isotopes. The overlaps
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Table 4 Mean (+ 1SD) and median (range) percentage radio-isotope distributions in dissected egg components at the end of the loss experiment 54
57
65
Embryo
0.19 ± 0.08 0.20 (0.06–0.29)
0.05 ± 0.03 0.05 (0.00–0.09)
Yolk
0.04 ± 0.06 0.02 (0.00–0.17)
Case
97.94 ± 3.01 98.96 (91.16–99.93)
Jelly
1.82 ± 2.96 0.81 (0.38–8.49)
Mn
109
134
241
0.30 ± 0.23 0.23 (0.03–0.74)
0.13 ± 0.18 0.06 (0.00–0.53)
9.09 ± 9.62 4.08 (1.97–30.12)
0.44 ± 0.24 0.47 (0.01–0.79)
0.01 ± 0.01 0.02 (0.00–0.03)
0.02 ± 0.03 0.01 (0.00–0.09)
1.51 ± 1.69 0.73 (0.00–5.09)
0.01 ± 0.01 0.01 (0.00–0.02)
99.87 ± 0.05 99.85 (99.80–99.95)
99.67 ± 0.23 99.70 (99.24–99.94)
99.83 ± 0.18 99.89 (99.44–99.98)
87.50 ± 9.57 89.39 (67.89–96.19)
99.19 ± 0.50 99.32 (98.24–99.74)
0.07 ± 0.05 0.06 (0.02–0.14)
0.01 ± 0.01 0.01 (0.00–0.03)
0.02 ± 0.01 0.02 (0.01–0.03)
1.90 ± 1.29 1.28 (0.75–4.21)
0.36 ± 0.39 0.23 (0.08–1.19)
Co
Zn
Cd
Cs
Am
Table 5 Mean (+1SD) and median (range) activity concentrations (Bq/g) of radio-isotope in dissected eggs components at (a) the end of 15 days uptake experiment and (b) at the end of 21 day loss experiment 54
57
65
109
134
241
3.93 ± 4.46 1.96 (0.84–14.56)
2.06 ± 1.69 1.19 (0.56–5.44)
6.17 ± 5.02 4.55 (1.36–14.03)
1.99 ± 2.70 0.84 (0.60–8.81)
0.29 ± 0.15 0.23 (0.14–0.50)
1.94 ± 0.95 1.87 (0.26–3.39)
Yolk
0.36 ± 0.30 0.24 (0.14–1.03)
0.15 ± 0.14 0.09 (0.04–0.46)
0.87 ± 1.10 0.49 (0.06–3.51)
2.50 ± 3.50 0.70 (0.29–9.94)
0.04 ± 0.03 0.02 (0.02–0.09)
0.06 ± 0.04 0.06 (0.01–0.11)
Case
735.89 ± 324.48 641.88 (421.51–1292.17)
765.82 ± 212.90 757.92 (546.27–1259.37)
1407.40 ± 461.95 1461.50 (749.39–2430.75)
2325.53 ± 821.27 2163.12 (1346.93–4029.27)
2.38 ± 1.97 1.46 (0.90–6.14)
199.49 ± 60.81 184.37 (122.91–316.90)
Jelly
7.25 ± 5.91 5.39 (2.03–19.22)
1.73 ± 1.95 0.89 (0.38–5.10)
1.41 ± 2.35 0.31 (0.04–6.63)
3.07 ± 3.44 1.49 (0.20–9.65)
0.52 ± 0.45 0.32 (0.06–1.28)
1.18 ± 1.27 0.58 (0.30–3.62)
0.59 ± 0.34 0.48 (0.14–1.06)
0.23 ± 0.13 0.25 (0.04–0.37)
2.35 ± 1.30 2.46 (0.50–4.38)
1.09 ± 1.07 0.74 (0.09–3.26)
0.22 ± 0.43 0.04 (0.02–1.19)
0.27 ± 0.13 0.31 (0.02–0.41)
Yolk
0.43 ± 0.61 0.24 (0.05–1.50)
0.07 ± 0.06 0.05 (0.03–0.16)
0.46 ± 0.19 0.50 (0.22–0.68)
1.28 ± 1.82 0.61 (0.13–4.52)
0.05 ± 0.01 0.05 (0.04–0.06)
0.02 ± 0.01 0.02 (0.01–0.04)
Case
325.40 ± 112.31 290.34 (260.53–503.80)
607.37 ± 96.83 614.52 (458.87–763.06)
1014.57 ± 194.86 1029.66 (742.55–1238.40)
1344.71 ± 280.78 1289.32 (1015.45–1767.40)
1.32 ± 0.61 1.26 (0.66–2.58)
84.11 ± 29.70 79.54 (42.02–132.74)
Jelly
8.25 ± 7.35 5.19 (1.65–23.29)
0.80 ± 0.41 0.83 (0.29–1.36)
0.25 ± 0.12 0.29 (0.03–0.44)
0.69 ± 0.59 0.48 (0.17–1.69)
0.05 ± 0.02 0.05 (0.02–0.08)
1.57 ± 3.45 0.24 (0.16–9.40)
Mn
Panel (a) Embryo
Panel (b) Embryo
Co
Zn
in the mean (+ 1SD) values between viable eggs and controls show no significant differences (P < 0.05) between them in the accumulatory capacities of their egg-cases for any of the five radio-isotopes. The data also indicate that the rates of accumulation by the egg case are highest for 65 Zn, followed by 54Mn, 57Co and 109Cd, and lowest for 241 Am. The loss data plotted as percentage of activity retained as a function of period of exposure to tracer-free water also shows that there are no significant differences between egg-cases of viable embryos and controls over 20 days. Rates of loss are highest for 241Am, lowest for 57Co and 65Zn, with 54Mn and 65Zn being intermediate. 4.3. Tissue distributions of radio-isotopes In Fig. 3 is shown the percentage distribution of each of the six isotopes among the following six dissected body
Cd
Cs
Am
components for one individual embryo sampled during the uptake and loss experiments; liver, digestive tract, skin, kidney, head, and muscle/skeleton. During the uptake phase the skin is the major repository (in descending order) of 241Am, 57Co, 134Cs, 65Zn, 54Mn and 109Cd that range from about 90% down to 30%. These percentage distributions in the skin are similar during the loss phase among all radio-isotopes, with the exception of 134Cs that declines from 55% during uptake down to about 20% during the loss phase. Other body components that are prominent repositories of radio-isotopes were the head for 57Co during both the uptake and loss phases, 241Am in the kidney during loss, 134Cs in the head during uptake and in the muscle/skeleton during loss. With regard to the patterns of distribution of individual isotopes among the six body components they range in contrast between 241Am, that is predominantly associated with skin during both uptake
R.A. Jeffree et al. / Marine Pollution Bulletin 52 (2006) 1278–1286
LOSS
UPTAKE 100
CF
600
54
Mn
400
200
% activity retained
800
10
0 0
5
10
15
20
5
10
15
20
25
57
400
Co
200
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
% activity retained
600
CF
0 100
800
10
0 0
5
10
15
20
800
65
400
Zn
200
% activity retained
100
600
CF
1283
0
10 0
5
10
15
20
500
400
CF
300
109
Cd
200
100
% activity retained
100
0
10 0
5
10
15
20
500
400
CF
300
241
Am
200
100
% activity retained
100
0
10 0
5
10
15
20
DAYS
DAYS
Control eggs Live eggs Fig. 2. Patterns of uptake and loss of radio-isotopes in the egg cases of control and live embryos. Error bars represent 1 SD. The data are expressed as CFs for the total encased embryo.
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Fig. 3. Distributions of radio-isotopes among six body components of a dogfish embryo that hatched during the uptake and loss experiment.
and loss with small proportions in most other body regions, and 109Cd that is more evenly distributed throughout the body during both experimental phases. The association of the radio-isotopes with the skin of these hatched individuals may be explained as due to the external presence of the placoid scales, as these are reported to erupt from the skin just before hatching (Ballard et al., 1993). Between the uptake and loss experiments our preliminary data may suggest the following redistributions of radioisotopes among components of the body: 134Cs reduces in the skin and head and increases in the muscle/skeleton; 241 Am declines in the skin and increases in the kidney. 5. Discussion 5.1. Patterns of radio-isotope accumulation in embryos The patterns of radio-isotope bioaccumulation shown in Fig. 1 need to be assessed and interpreted in relation to two factors; the permeability of the egg-case and the biokinetic processes of the embryo itself.
The wall of the egg case of this species is primarily composed of a highly crystalline and laminated collagen. Experimental studies have shown its high permeability for water and urea to pass from the interior of the egg-case to the external environment (Luong et al., 1998; Foulley and Mellinger, 1980) and for oxygen to pass symmetrically in both directions (Diez and Davenport, 1987). It consists of collagen molecules that are aggregated in three dimensional networks that confers strength and indicated filtering properties (Knupp et al., 1998). To make an initial assessment of the permeability of the wall of the egg-case to the isotopes used in this experiment, material was scraped from both the internal and external wall of an egg-case retained from the end of the uptake experiment and they, in addition to the remaining middle case layer, were weighed and analysed for their radioisotopic contents. These results showed activity concentrations of the six radio-isotopes were similar among these three components of the egg-case, consistent with the egg-case material being permeable to the six radio-isotopes in its external aquatic medium. Egg-case permeability in this species is known to be related to the stage of its embryological development and preparation for hatching from the egg-case. The eggcase will appreciably increase its access to the seawater and radio-isotopes through the opening of its apertures at embryo lengths of about 25–30 mm reported by Mellinger et al. (1986) and 40 mm by Ballard et al. (1993) due to the activity of the secretions of the hatching gland that digest the jelly content of the egg case and the cement that seals the slits in the egg case (Ballard et al., 1993). Our experimental observations suggest that even in embryos of smaller sizes (22 mm, Fig. 1) these apertures were already open. Together, our results are consistent with the egg case being porous to radio-isotopes during the experimental exposure, through both the apertures and the wall itself. However, there are distinct contrasts in the relationships between embryo size and radio-isotope CF, i.e., 109Cd reduces whereas 57Co and 65Zn increase with embryo size. These contrasts in time-dependent bioaccumulation patterns are therefore inconsistent with them being determined by an increasing permeability of the egg-case as the embryo increased in size. If it is concluded that the permeability of the egg-case was not unduly related to embryo size, then the relationships between radio-isotope CFs in the embryo and its size can be interpreted as being predominantly driven by its metabolic processes. The elemental metabolism of the embryo could also be expected to be appreciably influenced by the developmental changes that take place over this size range of S. canicula (Mellinger et al., 1986; Ballard et al., 1993), up to hatching stage that did take place in one specimen during the uptake experiment. The pattern of reduction in the CF for 109Cd with increasing embryo size (Fig. 1) is very comparable in shape to that previously determined experimentally for weight-specific oxygen consumption by embryonic S. canicula (Diez and Davenport,
R.A. Jeffree et al. / Marine Pollution Bulletin 52 (2006) 1278–1286
5.2. Distribution of radio-isotopes in the encased embryo The results show the dominating bioaccumulatory nature of the egg-case relative to the other egg components for each of the heavy metals and radionuclides, both in its elevated CFs and very high percentages of total activity associated with it (Tables 1, 2 and 5). Its capacity for bioaccumulation does not appear to be greatly influenced by the viability the embryo (Fig. 2). The permeability of the egg case to ions has been demonstrated in previous studies (Irwin and Davenport, 2002; Foulley and Mellinger, 1980), results that are consistent with our findings of the presence of all tested radio-isotopes in the embryo and other egg components. Together, these results would indicate that • It is a potential source of these heavy metals and radionuclides to be absorbed by the embryo, when they are no longer elevated in seawater. If this is a correct interpretation, then the presence of such a large reservoir of radio-isotopes in the case may have also confounded and led to an underestimate of the reduction of the CFs of radio-isotopes in the embryo during the loss experiment due to its depuration processes (Table 5). • The egg case represents a source of radiation exposure to the embryo from these gamma-emitting radioisotopes, which is much greater than its exposure from those internally accumulated in the embryo, which has CFs that can be lower by two orders of magnitude compared to the egg case. The duration of the radiation exposure would depend on their retention rates by the egg-case over the period until hatching, which is reported to range from 145 to 175 days at 16 C (Ballard et al., 1993). The patterns of loss shown in Fig. 2 indicate that they are variable among radio-isotopes and may be retained for considerable periods, e.g., 57Co and 65Zn.
longer periods of exposure. Our initial data also indicate that the patterns of uptake and loss of radio-isotopes by the egg-case are not greatly affected by the activities of the viable embryo. These results suggest that the egg-case material may have potential as an accumulating monitor of these elements. 5.4. Comparison of radio-isotope CFs in embryos and juveniles of S. canicula In Fig. 4 is shown a comparison of the mean (+ SD) CFs for six radio-isotopes for comparison between embryos and juveniles (mean wet weight 6.08 g) that were also determined after 15 days of uptake under experimental conditions (Jeffree et al., in press). The CFs are very similar between embryos and juveniles for 54Mn, 57Co and 109Cd but higher in juveniles by a factor of 3 for 134Cs, a factor of about 10 for 65Zn, and a factor of about 30 for 241 Am. Cadmium-109, 57Co and 54Mn, that show comparable CFs for embryos and juveniles, have uptake patterns in juveniles that approximate to equilibrium after the 15 days of uptake. In contrast, the radio-isotopes (134Cs, 65 Zn and 241Am) that show enhancement in juveniles, relative to embryos, also showed patterns of uptake in juveniles indicating that equilibrium CFs had not been reached in 15 days (Jeffree et al., in press). The increased CFs of 65Zn in juveniles, compared to their mean CFs in embryos, are also consistent with its increasing CF in embryos as they increase in size (Fig. 1). This phenomenon of increasing CF with increasing size indicates an expanding exchangeable pool for this radioisotope, in this species. With regard to potential chemotoxic effects the enhanced CFs for juveniles would make them more exposed to contaminant impacts, compared to embryos. However, with regard to radiation exposure from gamma-emitting isotopes the absorptive capacity of the egg-case (Table 1) would result in the potential for radio1000 Embryo Juvenile 100
CF for radioisotopes
1987). This would be consistent with 109Cd bioaccumulation being correlated with general metabolic rate and accord with 109Cd more even distribution throughout the body (Fig. 3). The contrasting increases in 57Co and 65Zn with increasing embryo length may be associated with the qualitative changes in anatomy and associated physiology that are taking place in the embryo over this size range, possibly with scale development in the skin, where these radio-isotopes are mainly located (Fig. 3) (Ballard et al., 1993). Clearly, more investigation is required to understand these contrasting patterns of bioaccumulation in the embryo.
1285
10
1
0.1
5.3. Radio-isotope kinetics in the egg case 0.01
Our results have shown that the egg case has not approached equilibrium concentrations (Fig. 2) during 15 days of exposure to these radio-isotopes and therefore is likely to have a much greater capacity to absorb them over
54-Mn
57-Co
65-Zn
109-Cd
134-Cs
241-Am
Fig. 4. Comparison of mean (+ SD) whole body to water CFs for six radionuclides in dogfish embryos (this study) and juveniles (from Jeffree et al., in press).
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toxic effects being much greater for the encased embryo. If the contaminants depurated from the egg case (Table 5) are also subsequently available for absorption by the embryo then their radiotoxic and chemotoxic effects may be more comparable. Acknowledgements The IAEA is grateful for the support provided to its Marine Environment Laboratory by the Government of the Principality of Monaco. The Oceanographic Museum, Monaco, is thanked for the provision of dogfish embryos used in this experiment. References Ballard, W.W., Mellinger, J., Lechenault, H., 1993. A series of normal stages for development of Scyliorhinus canicula, the lessed spotted dogfish (Chondrichthyes: Scyliorhinidae). J. Exp. Zool. 267, 318–336. Barker, M.J., Schluessel, V., 2005. Managing global shark fisheries: suggestions for prioritizing management strategies. Aquatic Conserv.: Mar. Freshw. Ecosyst. 15, 325–347.
Camhi, M., Fowler, S.L., Musick, J.A., Brautigam, A., Fordham, S.V., 1998. Sharks and their Relatives – Ecology and Conservation. IUCN/ SSC Shark Specialist Group. IUCN, Gland, Switzerland and Cambridge, UK. iv+39 pp. Diez, J.M., Davenport, J., 1987. Embryonic respiration in the dogfish (Scyliorhinus canicula L.). J. Mar. Biol. Assoc. UK 67, 249–261. Foulley, M., Mellinger, J., 1980. La diffusion de l’eau tritiee, de l’uree-14C and d’autres substances a travers la coque de l’oeuf de Roussette Scyliorhinus canicula. CR Acad. Sci. D, Paris t 290, 427–430. Irwin, S., Davenport, J., 2002. Iron status of adult dogfish (Scyliorhinus canicula) tissue and sources of iron during embryonic development. J. Mar. Biol. Assoc. UK 82, 4141/1-5. Jeffree, R.A., Warnau, M., Teyssie, J.-L., in press. Experimental comparison of the bioaccumulation of radio-isotopes from seawater by the spotted dogfish Scyliorhinus canicula (Chondrichthys) and the turbot Psetta maxima (Actinopterygii: Teleostei). Sci. Total Environ. Knupp, C., Chew, M., Squire, J., 1998. Collagen packing in the dogfish egg case wall. J. Struct. Biol. 122, 101–110. Luong, T.-T., Boutillon, M.-M., Garrone, R., Knight, D.P., 1998. Characterisation of Selachian egg case collagen. Biochem. Biophys. Res. Commun. 250, 657–663. Mellinger, J., Wrisez, F., Alluchon-Gerard, M.-J., 1986. Developmental biology of an oviparous shark, Scyliorhinus canicula. In: Uyeno, T., Arai, R., Taniuchi, T., Matsuura, K. (Eds.), Proc. Second Int. Conf. On Indo-Pacific Fishes, Tokyo, pp. 310–332.
Bioaccumulation of heavy metals and radionuclides from seawater by encased embryos of the spotted dogfish Scyliorhinus canicula Ross A. Jeffree *, Michel Warnau, Francois Oberhansli, Jean-Louis Teyssie Radioecology Laboratory, IAEA Marine Environment Laboratories, 4 Quai Antoine, MC 98000, Monaco
Abstract Encased embryos of spotted dogfish Scyliorhinus canicula absorbed six radio-isotopes (241Am, 109Cd, 57Co, 134Cs, 54Mn and 65Zn) directly from seawater during short-term experimental exposure, demonstrating the permeability of the egg-case to these contaminants. Embryo to water concentration factors (CFs) ranged from 0.14 for 134Cs to 7.4 for 65Zn. The 65Zn and 57Co CFs increased exponentially with embryo length, whereas the CF for 109Cd declined with length. Among different components of the encased embryo the egg case was the major repository (69–99%) of all six radio-isotopes that were distributed throughout its wall. Egg-case CFs were as high as 103 for 57 Co and 65Zn, making it the major source of gamma radiation exposure to the embryo and potentially of radio-isotopes for continued absorption by the embryo, following the uptake phase of the experiment. The patterns of uptake by the egg-case approximated linearity for most isotopes and loss rates were isotope-specific; egg-case biokinetics were not greatly affected by the viability of the contained embryo. Within the embryo initial data on radio isotopic distribution show that the skin is their major site of uptake, as previously demonstrated for juveniles. 2006 Elsevier Ltd. All rights reserved. Keywords: Dogfish; Scyliorhinus canicula; Heavy metals; Radionuclides; Egg; Embryo
1. Introduction A previous experimental study compared the uptake of seven radio-isotopes (241Am, 109Cd, 57Co, 51Cr, 134Cs, 54 Mn and 65Zn) from seawater by spotted dogfish Scyliorhinus canicula (Chondrichthys: Scyliorhinidae) and the turbot Psetta maxima (Actinopterygii: Teleostei) (Jeffree et al., in press). The results indicated the greater permeability of dogfish to most of these isotopes, with some isotopes attaining whole-body concentration factors (CFs) in dogfish of more than 102 during short-term exposures. These results also suggested the enhanced susceptibility of dogfish to such contaminants in seawater. Chondrichthyans are also characterized by low fecundity relative to Teleost fishes, that is also reflected in their smaller egg clutches (Camhi et al., 1998; Barker and Schl-
*
Corresponding author. Tel.: +377 97 97 72 78; fax: +377 97 97 72 76. E-mail address: R.Jeff[email protected] (R.A. Jeffree).
0025-326X/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2006.03.015
uessel, 2005). For the encased Chondrichthyan embryo that has been laid at a site chosen by the mother, its potential pathway of exposure to environmental contaminants is directly from seawater, apart from those contaminants that may be included in yolk and other egg components due to maternal transfer and subsequently absorbed by the embryo. A previous study has shown that developing embryos of S. canicula can increase their iron concentration when exposed to artificial seawater with added iron (Irwin and Davenport, 2002). However, the egg-case is strong and very protective of the embryo, which is reported to be particularly susceptible to seawater exposure during the earlier stages of embryogenesis (Ballard et al., 1993). This experimental study was undertaken to determine the extent to which the encased embryo of S. canicula is exposed to radio-isotopes of heavy metals and radionuclides (241Am, 109Cd, 57Co, 134Cs, 54Mn and 65Zn) in seawater and their subsequent patterns of bioaccumulation and depuration by the embryo and other components of the egg.
R.A. Jeffree et al. / Marine Pollution Bulletin 52 (2006) 1278–1286
2. Materials and methods 2.1. Acclimation and experimental exposure of organisms Two clutches of eggs of the spotted dogfish S. canicula were maintained for six weeks in 40 L Mediterranean sea water aquaria at 17.0 ± 0.5 C, salinity 38 psu, pH 8.05, with 10% water renewal h 1 and a light/dark cycle of 10 h/14 h. Viable eggs (n = 23) were distributed in a 70 L glass aquarium and acclimated for two weeks to experimental conditions, as described above, in a constantly 0.22 lm-aerated closed circuit. Eggs ranged in weight from 2.92 g to 6.44 g with a mean individual wet wt of 4.66 ± 0.98 g. Eggs were carefully sampled from the aquaria to avoid bubble formation in the case and were daily transferred in a counting tube within clean seawater for radio-spectrometric analysis. Two encased embryos that were sacrificed by exposure to low temperate were also included in the uptake experiment to initially assess any obvious effect of the embryo on total uptake of each radio-isotope. At the end of the 15 day uptake and 21 day loss experiments, eggs were dissected into the following compartments for determination of their radioisotope concentrations, following wet-weighing: egg case, jelly, yolk and embryo. One embryo also hatched from its egg cases at the 11th day of the uptake experiment and at the 13th day of the loss experiment. These were immediately dissected into liver, digestive tract, skin, kidneys, head, muscle + skeleton, for radioanalysis. 3. Radio-isotopes and their radioanalysis High-specific activity radio-isotopes were purchased from Amersham, UK (241Am, 57Co, 51Cr and 134Cs,) and Isotope Product Laboratory, USA (109Cd, 54Mn and 65Zn). Seawater spikes resulted in low activities of each selected radio-isotope: 241 Am (0.2 kBq l 1), 109Cd (1.3 kBq l 1), 57Co (0.5 kBq l 1), 134 Cs (1 kBq l 1), 54Mn (0.5 kBq l 1) and 65Zn (0.5 kBq l 1). No change in pH was detectable after radio-isotope addition. Seawater and spikes were renewed daily for 5 days, and then every second day in order to keep exposure activities constant. Activities of the radio-isotopes in seawater were checked daily, both before and after each seawater renewal, to determine their time-integrated activities. The speciation
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of these radio-isotopes in the experimental solution had been modeled for a previous study (Jeffree et al., in press). Radioactivity levels in seawater, dogfish eggs and their components were determined using a high-resolution cspectrometry system consisting of four coaxial Germanium (N- or P-type) detectors (EGNC 33-195-R, Intertechnique; 40–70% efficiency) that were connected to a multi-channel analyzer and a personal computer employing spectral analysis software (Interwinner 6, Intertechnique). The radioactivity levels of the samples were determined by comparison with known standards of appropriate geometry and were corrected for background and isotope physical decay. Counting times were adapted to obtain count rates with relative propagated errors less than 5%, viz. typically 10– 30 min for whole organism radioanalyses and 1–12 h for seawater and dissected body compartment samples. 4. Results 4.1. Uptake In Table 1 is shown the mean and median percentage distribution of each radio-isotope at the end of the uptake experiment in the dogfish embryo, its yolk, the egg case and the other contents of the egg case, i.e., the jelly. Ninety eight percent or more of the 241Am, 109Cd, 57Co, 54Mn and 65Zn are associated with the egg case, and with less than 1% associated with the embryo. For these five radio-isotopes very small percentages are also found in both the yolk and the jelly. In contrast to these results, 10% of the total activity of 134Cs is associated with the embryo and about 20% associated with the jelly, with again the majority of this radio-isotope associated with the egg case, and a very small percentage associated with the egg yolk. In Table 2 is given the mean and median CF for each isotope and dissected component at the end of the uptake phase of the experiment. With the exception of 134Cs the egg case has CFs of radio-isotopes that are 2–3 orders of magnitude higher than those of the embryo, which are more comparable to those of the jelly; the yolk is also low in its CFs compared to the jelly, with the exception of 109Cd. For 134Cs the CF for the egg case is only an order of magnitude greater than that of the embryo, that is com-
Table 1 Mean (+ 1SD) and median (range) percentage radio-isotope distributions in dissected egg components at the end of the uptake experiment 54
57
65
109
134
241
Embryo
0.47 ± 0.85 0.13 (0.06–2.84)
0.28 ± 0.39 0.08 (0.02–1.00)
0.47 ± 0.60 0.21 (0.02–1.50)
0.11 ± 0.20 0.02 (0.01–0.62)
10.27 ± 12.03 6.18 (0.37–32.65)
0.77 ± 0.71 0.49 (0.02–2.19)
Yolk
0.08 ± 0.10 0.04 (0.00–0.14)
0.02 ± 0.01 0.01 (0.00–0.04)
0.04 ± 0.05 0.03 (0.00–0.16)
0.10 ± 0.16 0.03 (0.00–0.46)
1.62 ± 1.51 1.11 (0.00–3.77)
0.03 ± 0.03 0.03 (0.00–0.11)
Case
97.95 ± 2.06 98.89 (93.65–99.70)
99.39 ± 0.56 99.80 (98.46–99.90)
99.39 ± 0.69 99.74 (98.09–99.92)
99.65 ± 0.25 99.78 (99.18–99.94)
68.87 ± 19.19 75.08 (32.23–93.53)
98.72 ± 0.85 99.03 (97.23–99.42)
Jelly
1.50 ± 1.47 0.85 (0.16–3.36)
0.31 ± 0.33 0.11 (0.04–0.57)
0.10 ± 0.15 0.03 (0.00–0.41)
0.14 ± 0.14 0.10 (0.01–0.47)
19.24 ± 21.73 7.27 (1.88–66.22)
0.48 ± 0.37 0.33 (0.10–1.13)
Mn
Co
Zn
Cd
Cs
Am
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Table 2 Mean concentration factors (CFs) and median (range) for radio-isotope distributions in dissected egg components at the end of the uptake experiment 54
57
65
109
134
241
Embryo
2.98 ± 2.26 2.21 (0.95–7.89)
2.93 ± 2.53 1.48 (0.69–6.76)
7.38 ± 6.13 5.33 (1.60–16.45)
0.42 ± 0.32 0.32 (0.23–1.25)
0.14 ± 0.07 0.11 (0.07–0.23)
5.06 ± 2.61 4.72 (0.66–8.57)
Yolk
0.40 ± 0.37 0.27 (0.00–1.08)
0.16 ± 0.11 0.14 (0.00–0.38)
0.81 ± 0.79 0.71 (0.00–2.72)
1.15 ± 1.91 0.23 (0.00–5.49)
0.02 ± 0.01 0.01 (0.00–0.04)
0.16 ± 0.13 0.16 (0.00–0.41)
Case
873.33 ± 278.1 732.51 (602.99–1401.71)
1031.63 ± 213.2 1049.54 (641.93–1432.91)
1786.72 ± 442.3 1850.95 (825.97–2461.11)
957.87 ± 311.99 1061.92 (521.30–1538.69)
1.20 ± 1.10 0.87 (0.44–3.94)
539.89 ± 105.5 544.80 (375.21–729.89)
Jelly
9.31 ± 7.22 7.63 (1.99–25.29)
2.18 ± 2.02 1.55 (0.42–5.77)
1.32 ± 2.21 0.43 (0.04–7.07)
0.99 ± 1.01 0.69 (0.10–3.32)
0.26 ± 0.35 0.09 (0.03–1.11)
2.67 ± 2.43 2.08 (0.58–8.32)
Mn
Co
Zn
parable to the jelly, with the yolk being an order of magnitude lower than the jelly. Among the radio-isotopes the CFs of the egg case varied only by a factor of three, with exception of 134Cs that is 2–3 orders of magnitude lower than for the other five radio-isotopes. The CFs in the embryo were highest for 65Zn then 241Am and lowest by an order magnitude for 109Cd and 134Cs. These data confirm that the egg case is permeable to each water-born radio-isotope used in this experiment, particularly 134Cs, and indicate that the egg case itself is very sorptive of the majority of these radio-isotopes. The standard deviations of means and the ranges of values presented in Tables 1 and 2 for embryos also indicate that radio-isotope concentrations were highly variable among individuals sampled at the end of the uptake experiment. The embryos did vary considerably in length, from between 25 and 80 mm, which encompassed a phase of embryological development during which many physiological and anatomical changes were taking place. It also covered the period of development when the egg-case apertures, that were previously plugged, would be opened by excretions of the hatching gland (Mellinger et al., 1986). So we plotted for each radio-isotope the calculated embryo: water CF as a function of the total length of the embryo upon dissection, to discern whether some of their variability in radioisotopic content could be statistically explained. Seven eggs from one batch and two from the other, that were characterized by different size and colour, were included in this experiment. In Fig. 1 are plotted these relationships, and the fitted regression lines that were statistically significant (P < 0.05); their regression equations are given in Table 3. Our results show that the CFs of 65 Zn and 57Co significantly (P < 0.05) and appreciably increase with increasing embryo length. The data for both 241 Am and 134Cs show similar trends of increasing CF with increasing embryo length, although neither relationship is statistically significant (P > 0.05). In contrast to these results, the CF for 109Cd significantly (P < 0.001) declines with increasing embryo length. This inverse relationship is strongly influenced by one outlying point for the smallest embryo. To further assess the statistical validity of this regression, the CF values were log-transformed to further approximate a normal distribu-
Cd
Cs
Am
tion of CF values, and then a linear regression was fitted to these data. This regression explained 65% of the variance in the CFs and was also highly significant (P < 0.01), giving greater confidence in the initial finding of the inverse relationship shown in Fig. 1. There was no obvious clear separation between eggs from the two batches in these plots, however, the numbers are too small to satisfactorily evaluate statistically for differences between batches. 4.2. Loss In Table 4 is shown the mean and median percentage radio-isotope distributions in dissected egg components following 21 days of exposure to unlabelled seawater. The egg case still contains nearly all of each of the isotopes (99%), with 87.5% for 134Cs, a pattern very similar to that seen at the end of the uptake phase (Table 1). Similarly about 10% of the 134Cs is found in the embryo that contains much lower percentages of the other radio-isotopes. The yolk contains very low percentages of each radio-isotope, as it did at the end of the uptake experiment. The jelly shows a reduction in percentage activity of 134Cs by a factor of 10, compared to the end of the uptake phase. Table 5 shows the activity concentrations of radio-isotopes in dissected egg components at the end of both the uptake and loss phases that allows a measurement of the absolute amounts of depuration over 21 days. The case, with the highest activity concentrations among the dissected components, declines in these concentrations by the end of the loss phase by about a factor of 1.5–2 for 54 Mn, 109Cd, 134Cs and 241Am. Cobalt-57 and 65Zn show much smaller reductions in activity concentrations. The embryo also shows reductions in activity concentrations during the loss phase, with factors of reduction that are greatest for 57Co, 54Mn and 241Am, that range from 6– 10, and least for 134Cs and 109Cd, by factors of only about 2. If it is most simply assumed that the activity lost by the case diffuses equally in both directions, to the internal region of the egg case and back to seawater, then there are appreciable amounts of activities of all radio-isotopes
R.A. Jeffree et al. / Marine Pollution Bulletin 52 (2006) 1278–1286
1281
0.25 1.4
0.20
1.0
CF for 134Cs
CF for 109Cd
1.2
0.8 0.6
0.15
0.10
0.4 0.05 0.2 0.0 0
20
40
60
80
0.00
100
0
20
60
80
100
Length of embryo (mm)
10
10
8
8
CF for 241Am
CF for 57Co
Length of embryo (mm)
40
6
4
2
6
4
2
0
0 0
20
40
60
80
100
0
20
Length of embryo (mm)
40
60
80
100
Length of embryo (mm) 10
20
8
CF for 54Mn
CF for 65Zn
15
10
6
4
5 2
0
0 0
20
40
60
80
100
0
20
40
60
80
100
Length of embryo (mm)
Length of embryo (mm)
Fig. 1. Plots of embryo: water CFs versus length of embryo, as measured at the end of the uptake experiment, and the fitted significant (P < 0.05) regressions (equations given in Table 3).
Table 3 Regression equations for CFs of radio-isotopes in embryos at the end of the uptake experiment (y) as function of embryo length (x) Radio-isotope 109
Cd Co 65 Zn 57
Regression equation 0.04x
y = 3.47e y = 0.24e0.04x y = 0.65e0.04x
P value for regression
R2
n
0.0004 0.036 0.012
0.85 0.49 0.62
9 9 9
that would still be available for absorption by the embryo during the loss phase of the experiment. If this interpretation is correct then the degrees of reduction in their concentrations in the embryo measured during the loss phase may be an underestimate of the embryo’s true rates of depuration of these radio-isotopes.
These sets of data on the distributions of radio-isotopes in components of the encased embryos that were dissected at the end of both the uptake and loss experiments have demonstrated that the egg case is the repository of nearly all activity of each radio-isotope, with the exception of 134 Cs. Thus, the measurements of total activity in the encased embryo that were taken during the experiments were used to determine the patterns of uptake and loss in the egg-case for each isotope, not including 134Cs. These plots of CFs calculated for the total encased embryo are shown in Fig. 2 for egg cases containing live embryos and control egg cases. The uptake data show patterns of uptake that approximate to linear rates that indicate that equilibrium concentration factors have not been attained within 15 days of exposure to radio-isotopes. The overlaps
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Table 4 Mean (+ 1SD) and median (range) percentage radio-isotope distributions in dissected egg components at the end of the loss experiment 54
57
65
Embryo
0.19 ± 0.08 0.20 (0.06–0.29)
0.05 ± 0.03 0.05 (0.00–0.09)
Yolk
0.04 ± 0.06 0.02 (0.00–0.17)
Case
97.94 ± 3.01 98.96 (91.16–99.93)
Jelly
1.82 ± 2.96 0.81 (0.38–8.49)
Mn
109
134
241
0.30 ± 0.23 0.23 (0.03–0.74)
0.13 ± 0.18 0.06 (0.00–0.53)
9.09 ± 9.62 4.08 (1.97–30.12)
0.44 ± 0.24 0.47 (0.01–0.79)
0.01 ± 0.01 0.02 (0.00–0.03)
0.02 ± 0.03 0.01 (0.00–0.09)
1.51 ± 1.69 0.73 (0.00–5.09)
0.01 ± 0.01 0.01 (0.00–0.02)
99.87 ± 0.05 99.85 (99.80–99.95)
99.67 ± 0.23 99.70 (99.24–99.94)
99.83 ± 0.18 99.89 (99.44–99.98)
87.50 ± 9.57 89.39 (67.89–96.19)
99.19 ± 0.50 99.32 (98.24–99.74)
0.07 ± 0.05 0.06 (0.02–0.14)
0.01 ± 0.01 0.01 (0.00–0.03)
0.02 ± 0.01 0.02 (0.01–0.03)
1.90 ± 1.29 1.28 (0.75–4.21)
0.36 ± 0.39 0.23 (0.08–1.19)
Co
Zn
Cd
Cs
Am
Table 5 Mean (+1SD) and median (range) activity concentrations (Bq/g) of radio-isotope in dissected eggs components at (a) the end of 15 days uptake experiment and (b) at the end of 21 day loss experiment 54
57
65
109
134
241
3.93 ± 4.46 1.96 (0.84–14.56)
2.06 ± 1.69 1.19 (0.56–5.44)
6.17 ± 5.02 4.55 (1.36–14.03)
1.99 ± 2.70 0.84 (0.60–8.81)
0.29 ± 0.15 0.23 (0.14–0.50)
1.94 ± 0.95 1.87 (0.26–3.39)
Yolk
0.36 ± 0.30 0.24 (0.14–1.03)
0.15 ± 0.14 0.09 (0.04–0.46)
0.87 ± 1.10 0.49 (0.06–3.51)
2.50 ± 3.50 0.70 (0.29–9.94)
0.04 ± 0.03 0.02 (0.02–0.09)
0.06 ± 0.04 0.06 (0.01–0.11)
Case
735.89 ± 324.48 641.88 (421.51–1292.17)
765.82 ± 212.90 757.92 (546.27–1259.37)
1407.40 ± 461.95 1461.50 (749.39–2430.75)
2325.53 ± 821.27 2163.12 (1346.93–4029.27)
2.38 ± 1.97 1.46 (0.90–6.14)
199.49 ± 60.81 184.37 (122.91–316.90)
Jelly
7.25 ± 5.91 5.39 (2.03–19.22)
1.73 ± 1.95 0.89 (0.38–5.10)
1.41 ± 2.35 0.31 (0.04–6.63)
3.07 ± 3.44 1.49 (0.20–9.65)
0.52 ± 0.45 0.32 (0.06–1.28)
1.18 ± 1.27 0.58 (0.30–3.62)
0.59 ± 0.34 0.48 (0.14–1.06)
0.23 ± 0.13 0.25 (0.04–0.37)
2.35 ± 1.30 2.46 (0.50–4.38)
1.09 ± 1.07 0.74 (0.09–3.26)
0.22 ± 0.43 0.04 (0.02–1.19)
0.27 ± 0.13 0.31 (0.02–0.41)
Yolk
0.43 ± 0.61 0.24 (0.05–1.50)
0.07 ± 0.06 0.05 (0.03–0.16)
0.46 ± 0.19 0.50 (0.22–0.68)
1.28 ± 1.82 0.61 (0.13–4.52)
0.05 ± 0.01 0.05 (0.04–0.06)
0.02 ± 0.01 0.02 (0.01–0.04)
Case
325.40 ± 112.31 290.34 (260.53–503.80)
607.37 ± 96.83 614.52 (458.87–763.06)
1014.57 ± 194.86 1029.66 (742.55–1238.40)
1344.71 ± 280.78 1289.32 (1015.45–1767.40)
1.32 ± 0.61 1.26 (0.66–2.58)
84.11 ± 29.70 79.54 (42.02–132.74)
Jelly
8.25 ± 7.35 5.19 (1.65–23.29)
0.80 ± 0.41 0.83 (0.29–1.36)
0.25 ± 0.12 0.29 (0.03–0.44)
0.69 ± 0.59 0.48 (0.17–1.69)
0.05 ± 0.02 0.05 (0.02–0.08)
1.57 ± 3.45 0.24 (0.16–9.40)
Mn
Panel (a) Embryo
Panel (b) Embryo
Co
Zn
in the mean (+ 1SD) values between viable eggs and controls show no significant differences (P < 0.05) between them in the accumulatory capacities of their egg-cases for any of the five radio-isotopes. The data also indicate that the rates of accumulation by the egg case are highest for 65 Zn, followed by 54Mn, 57Co and 109Cd, and lowest for 241 Am. The loss data plotted as percentage of activity retained as a function of period of exposure to tracer-free water also shows that there are no significant differences between egg-cases of viable embryos and controls over 20 days. Rates of loss are highest for 241Am, lowest for 57Co and 65Zn, with 54Mn and 65Zn being intermediate. 4.3. Tissue distributions of radio-isotopes In Fig. 3 is shown the percentage distribution of each of the six isotopes among the following six dissected body
Cd
Cs
Am
components for one individual embryo sampled during the uptake and loss experiments; liver, digestive tract, skin, kidney, head, and muscle/skeleton. During the uptake phase the skin is the major repository (in descending order) of 241Am, 57Co, 134Cs, 65Zn, 54Mn and 109Cd that range from about 90% down to 30%. These percentage distributions in the skin are similar during the loss phase among all radio-isotopes, with the exception of 134Cs that declines from 55% during uptake down to about 20% during the loss phase. Other body components that are prominent repositories of radio-isotopes were the head for 57Co during both the uptake and loss phases, 241Am in the kidney during loss, 134Cs in the head during uptake and in the muscle/skeleton during loss. With regard to the patterns of distribution of individual isotopes among the six body components they range in contrast between 241Am, that is predominantly associated with skin during both uptake
R.A. Jeffree et al. / Marine Pollution Bulletin 52 (2006) 1278–1286
LOSS
UPTAKE 100
CF
600
54
Mn
400
200
% activity retained
800
10
0 0
5
10
15
20
5
10
15
20
25
57
400
Co
200
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
% activity retained
600
CF
0 100
800
10
0 0
5
10
15
20
800
65
400
Zn
200
% activity retained
100
600
CF
1283
0
10 0
5
10
15
20
500
400
CF
300
109
Cd
200
100
% activity retained
100
0
10 0
5
10
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500
400
CF
300
241
Am
200
100
% activity retained
100
0
10 0
5
10
15
20
DAYS
DAYS
Control eggs Live eggs Fig. 2. Patterns of uptake and loss of radio-isotopes in the egg cases of control and live embryos. Error bars represent 1 SD. The data are expressed as CFs for the total encased embryo.
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Fig. 3. Distributions of radio-isotopes among six body components of a dogfish embryo that hatched during the uptake and loss experiment.
and loss with small proportions in most other body regions, and 109Cd that is more evenly distributed throughout the body during both experimental phases. The association of the radio-isotopes with the skin of these hatched individuals may be explained as due to the external presence of the placoid scales, as these are reported to erupt from the skin just before hatching (Ballard et al., 1993). Between the uptake and loss experiments our preliminary data may suggest the following redistributions of radioisotopes among components of the body: 134Cs reduces in the skin and head and increases in the muscle/skeleton; 241 Am declines in the skin and increases in the kidney. 5. Discussion 5.1. Patterns of radio-isotope accumulation in embryos The patterns of radio-isotope bioaccumulation shown in Fig. 1 need to be assessed and interpreted in relation to two factors; the permeability of the egg-case and the biokinetic processes of the embryo itself.
The wall of the egg case of this species is primarily composed of a highly crystalline and laminated collagen. Experimental studies have shown its high permeability for water and urea to pass from the interior of the egg-case to the external environment (Luong et al., 1998; Foulley and Mellinger, 1980) and for oxygen to pass symmetrically in both directions (Diez and Davenport, 1987). It consists of collagen molecules that are aggregated in three dimensional networks that confers strength and indicated filtering properties (Knupp et al., 1998). To make an initial assessment of the permeability of the wall of the egg-case to the isotopes used in this experiment, material was scraped from both the internal and external wall of an egg-case retained from the end of the uptake experiment and they, in addition to the remaining middle case layer, were weighed and analysed for their radioisotopic contents. These results showed activity concentrations of the six radio-isotopes were similar among these three components of the egg-case, consistent with the egg-case material being permeable to the six radio-isotopes in its external aquatic medium. Egg-case permeability in this species is known to be related to the stage of its embryological development and preparation for hatching from the egg-case. The eggcase will appreciably increase its access to the seawater and radio-isotopes through the opening of its apertures at embryo lengths of about 25–30 mm reported by Mellinger et al. (1986) and 40 mm by Ballard et al. (1993) due to the activity of the secretions of the hatching gland that digest the jelly content of the egg case and the cement that seals the slits in the egg case (Ballard et al., 1993). Our experimental observations suggest that even in embryos of smaller sizes (22 mm, Fig. 1) these apertures were already open. Together, our results are consistent with the egg case being porous to radio-isotopes during the experimental exposure, through both the apertures and the wall itself. However, there are distinct contrasts in the relationships between embryo size and radio-isotope CF, i.e., 109Cd reduces whereas 57Co and 65Zn increase with embryo size. These contrasts in time-dependent bioaccumulation patterns are therefore inconsistent with them being determined by an increasing permeability of the egg-case as the embryo increased in size. If it is concluded that the permeability of the egg-case was not unduly related to embryo size, then the relationships between radio-isotope CFs in the embryo and its size can be interpreted as being predominantly driven by its metabolic processes. The elemental metabolism of the embryo could also be expected to be appreciably influenced by the developmental changes that take place over this size range of S. canicula (Mellinger et al., 1986; Ballard et al., 1993), up to hatching stage that did take place in one specimen during the uptake experiment. The pattern of reduction in the CF for 109Cd with increasing embryo size (Fig. 1) is very comparable in shape to that previously determined experimentally for weight-specific oxygen consumption by embryonic S. canicula (Diez and Davenport,
R.A. Jeffree et al. / Marine Pollution Bulletin 52 (2006) 1278–1286
5.2. Distribution of radio-isotopes in the encased embryo The results show the dominating bioaccumulatory nature of the egg-case relative to the other egg components for each of the heavy metals and radionuclides, both in its elevated CFs and very high percentages of total activity associated with it (Tables 1, 2 and 5). Its capacity for bioaccumulation does not appear to be greatly influenced by the viability the embryo (Fig. 2). The permeability of the egg case to ions has been demonstrated in previous studies (Irwin and Davenport, 2002; Foulley and Mellinger, 1980), results that are consistent with our findings of the presence of all tested radio-isotopes in the embryo and other egg components. Together, these results would indicate that • It is a potential source of these heavy metals and radionuclides to be absorbed by the embryo, when they are no longer elevated in seawater. If this is a correct interpretation, then the presence of such a large reservoir of radio-isotopes in the case may have also confounded and led to an underestimate of the reduction of the CFs of radio-isotopes in the embryo during the loss experiment due to its depuration processes (Table 5). • The egg case represents a source of radiation exposure to the embryo from these gamma-emitting radioisotopes, which is much greater than its exposure from those internally accumulated in the embryo, which has CFs that can be lower by two orders of magnitude compared to the egg case. The duration of the radiation exposure would depend on their retention rates by the egg-case over the period until hatching, which is reported to range from 145 to 175 days at 16 C (Ballard et al., 1993). The patterns of loss shown in Fig. 2 indicate that they are variable among radio-isotopes and may be retained for considerable periods, e.g., 57Co and 65Zn.
longer periods of exposure. Our initial data also indicate that the patterns of uptake and loss of radio-isotopes by the egg-case are not greatly affected by the activities of the viable embryo. These results suggest that the egg-case material may have potential as an accumulating monitor of these elements. 5.4. Comparison of radio-isotope CFs in embryos and juveniles of S. canicula In Fig. 4 is shown a comparison of the mean (+ SD) CFs for six radio-isotopes for comparison between embryos and juveniles (mean wet weight 6.08 g) that were also determined after 15 days of uptake under experimental conditions (Jeffree et al., in press). The CFs are very similar between embryos and juveniles for 54Mn, 57Co and 109Cd but higher in juveniles by a factor of 3 for 134Cs, a factor of about 10 for 65Zn, and a factor of about 30 for 241 Am. Cadmium-109, 57Co and 54Mn, that show comparable CFs for embryos and juveniles, have uptake patterns in juveniles that approximate to equilibrium after the 15 days of uptake. In contrast, the radio-isotopes (134Cs, 65 Zn and 241Am) that show enhancement in juveniles, relative to embryos, also showed patterns of uptake in juveniles indicating that equilibrium CFs had not been reached in 15 days (Jeffree et al., in press). The increased CFs of 65Zn in juveniles, compared to their mean CFs in embryos, are also consistent with its increasing CF in embryos as they increase in size (Fig. 1). This phenomenon of increasing CF with increasing size indicates an expanding exchangeable pool for this radioisotope, in this species. With regard to potential chemotoxic effects the enhanced CFs for juveniles would make them more exposed to contaminant impacts, compared to embryos. However, with regard to radiation exposure from gamma-emitting isotopes the absorptive capacity of the egg-case (Table 1) would result in the potential for radio1000 Embryo Juvenile 100
CF for radioisotopes
1987). This would be consistent with 109Cd bioaccumulation being correlated with general metabolic rate and accord with 109Cd more even distribution throughout the body (Fig. 3). The contrasting increases in 57Co and 65Zn with increasing embryo length may be associated with the qualitative changes in anatomy and associated physiology that are taking place in the embryo over this size range, possibly with scale development in the skin, where these radio-isotopes are mainly located (Fig. 3) (Ballard et al., 1993). Clearly, more investigation is required to understand these contrasting patterns of bioaccumulation in the embryo.
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10
1
0.1
5.3. Radio-isotope kinetics in the egg case 0.01
Our results have shown that the egg case has not approached equilibrium concentrations (Fig. 2) during 15 days of exposure to these radio-isotopes and therefore is likely to have a much greater capacity to absorb them over
54-Mn
57-Co
65-Zn
109-Cd
134-Cs
241-Am
Fig. 4. Comparison of mean (+ SD) whole body to water CFs for six radionuclides in dogfish embryos (this study) and juveniles (from Jeffree et al., in press).
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toxic effects being much greater for the encased embryo. If the contaminants depurated from the egg case (Table 5) are also subsequently available for absorption by the embryo then their radiotoxic and chemotoxic effects may be more comparable. Acknowledgements The IAEA is grateful for the support provided to its Marine Environment Laboratory by the Government of the Principality of Monaco. The Oceanographic Museum, Monaco, is thanked for the provision of dogfish embryos used in this experiment. References Ballard, W.W., Mellinger, J., Lechenault, H., 1993. A series of normal stages for development of Scyliorhinus canicula, the lessed spotted dogfish (Chondrichthyes: Scyliorhinidae). J. Exp. Zool. 267, 318–336. Barker, M.J., Schluessel, V., 2005. Managing global shark fisheries: suggestions for prioritizing management strategies. Aquatic Conserv.: Mar. Freshw. Ecosyst. 15, 325–347.
Camhi, M., Fowler, S.L., Musick, J.A., Brautigam, A., Fordham, S.V., 1998. Sharks and their Relatives – Ecology and Conservation. IUCN/ SSC Shark Specialist Group. IUCN, Gland, Switzerland and Cambridge, UK. iv+39 pp. Diez, J.M., Davenport, J., 1987. Embryonic respiration in the dogfish (Scyliorhinus canicula L.). J. Mar. Biol. Assoc. UK 67, 249–261. Foulley, M., Mellinger, J., 1980. La diffusion de l’eau tritiee, de l’uree-14C and d’autres substances a travers la coque de l’oeuf de Roussette Scyliorhinus canicula. CR Acad. Sci. D, Paris t 290, 427–430. Irwin, S., Davenport, J., 2002. Iron status of adult dogfish (Scyliorhinus canicula) tissue and sources of iron during embryonic development. J. Mar. Biol. Assoc. UK 82, 4141/1-5. Jeffree, R.A., Warnau, M., Teyssie, J.-L., in press. Experimental comparison of the bioaccumulation of radio-isotopes from seawater by the spotted dogfish Scyliorhinus canicula (Chondrichthys) and the turbot Psetta maxima (Actinopterygii: Teleostei). Sci. Total Environ. Knupp, C., Chew, M., Squire, J., 1998. Collagen packing in the dogfish egg case wall. J. Struct. Biol. 122, 101–110. Luong, T.-T., Boutillon, M.-M., Garrone, R., Knight, D.P., 1998. Characterisation of Selachian egg case collagen. Biochem. Biophys. Res. Commun. 250, 657–663. Mellinger, J., Wrisez, F., Alluchon-Gerard, M.-J., 1986. Developmental biology of an oviparous shark, Scyliorhinus canicula. In: Uyeno, T., Arai, R., Taniuchi, T., Matsuura, K. (Eds.), Proc. Second Int. Conf. On Indo-Pacific Fishes, Tokyo, pp. 310–332.