Abstract

Unhatched Alligator mississippiensis embryos were collected from six Florida, preserved in formalin and/or frozen and shipped to John Carroll University. The embryos were digested by a cold acid technique and analyzed by flame atomic absorption spectrophotometry for the following metals: As, Cd, Cu, Fe, Ni, Pb and Zn. The purpose of this study was to determine differences in metal levels in the alligator embryos between nest sites and to investigate the possibility of metal pollution as a cause for differences in relative hatching percentages between the sites. Analysis of the data indicated some differences between the nest sites but not proof of metal pollution problems.

Introduction

There is extremely little data concerning heavy metal loadings in reptiles. There does exist some information for whole eggs of the crocodile, Crocodylus acutus, which might provide a reference point for our purposes. Reported data included trace metal concentrations of the following metals in mg of element per kg (ppm) fresh weight (FW): As (0.2 FW), Cd (0.05 FW), Cu (15.0 FW), Pb (0.5 FW) and Zn (11.0 FW) (1).

The present study is preliminary work for further studies in heavy metal levels in American alligator embryos.

The female alligator lays bird-like eggs with a hard, calcified shell impervious to water penetration. An embryo encased in this calcified shell can only receive materials that are already inside the egg provided for by the mother (2). The specimens used in this study were embryos only. We did not receive yolk or other materials that were inside the egg.

Materials and Methods

Unhatched A. mississippiensis embryos were collected by the Florida Fish and Game Department from lakes Griffin (G), Apopka (A), Woodriff (W), Orange (0), Okeechobee (OK) and Jessup(J) which are scattered throughout Florida, then given to Dr. Cardeilhac, who forwarded the specimens to John Carroll University. The 151 samples had been preserved in formalin and/or frozen. The specimens were weighed (wet weight) and digested with a 1:1:1:1 mixture of deionized-distilled water, sulfuric, nitric, and perchloric acids at room temperature. Analysis for the metals arsenic (AS), cadmium (Cd), copper (Cu), iron (Fe), nickel (Ni), lead (Pb) and zinc (Zn) was accomplished by flame atomic absorption spectrophotometry using both air-acetylene and nitrous oxide-acetylene techniques (3).

Results

Arsenic, Cadmium and Lead

There were no detectable amounts (NDA) of arsenic, cadmium or lead in embryos from any of the six Florida lakes (Table 1).

Table 1.Average metal concentrations with standard deviations* (ppm) in alligator embryos from six lakes in Florida

*Aberrant data was not used in calculating average metal concentrations for each site.

Copper

Mean copper values in embryos from the different lakes G, A, W, 0, and OK ranged from 0.29 ± 0.21 ppm to 0.48 ± 0.71 ppm in which there were small differences between the sites. Embryos from Jessup Lake were relatively higher in copper levels (0.53 ± 1.18 ppm) (Table 1).

When dividing the samples into different weight groups there was the least amount of variance in calculated copper levels amongst those weighing over 1.0 g. These values ranged from 0.62 ± 0.18 ppm to 0.91 ± 1.28 ppm. Embryos weighing less than 0.1 g from orange Lake had the greatest copper concentration and variance (11.24 + 23.69 ppm) but one embryo in this group had 53.57 ppm of copper while the others had 2.65 ppm or less. Variance, as did copper concentration, tended to decrease as the sample.

Figure 1

Iron

There were small differences between mean iron concentrations in embryos from lakes G, A, W and 0 but were higher with an increased variability in embryos from lakes OK and J, both of whose embryos weigh less than 0.5 g. Values for lakes G, A, W and 0 varied from 8.76 ± 4.09 ppm to 9.97 ± 1.73 ppm, and values for lakes OK and J were 13.80 ±15.29 ppm to 16.42 ± 19.34, respectively (Table 1).

There was a tendency for variance to decrease with increased sample weight. Values ranged from 8.35 ± 2.69 ppm to 12.19 ± 1.04 ppm for samples weighing over 1.0 g. samples weighing less than 0.1 g had iron levels from 77.44 ± 146.28 ppm to 592.76 ± 973.59 ppm (Table 2).

Nickel

Mean nickel concentrations in embryos from lakes G, A, W, and 0 were similar but those of lakes OK and J were higher with increased variability. Embryos from lakes G, A, W, and 0 contained 1.18 ± 0.97 ppm to 1.67 ± 1.02 ppm of nickel while those of lakes OK and J contained 12.76 ± 11.58 ppm and 6.90 ± 10.05 ppm of nickel, respectively (Table 1).

Variance tended to decrease with an increase in sample weight. Samples weighing over 1.0 g had mean nickel levels of 0.62 ± 0.18 ppm to 0.91 ± 1.28 ppm while those weighing under 0.1 g varied from 1.14 ± 2.54 ppm to 33.41 ± 43.21 ppm (Table 2).

Zinc

There were no detectable amounts of zinc in about half of the embryos regardless of site of origin. The other half of the embryos containing zinc were calculated to have up to about 4000 ppm and even up to about 22000 ppm of zinc in one case. Embryos detected to have zinc were grouped together according to nest site and weight group, and it was found that there was generally a high amount of zinc but with a great degree of variance. Separating the data into the different weight groups revealed no regular pattern.

Relative Hatching % and Sample Sizes

Lake Griffin had the highest hatching percentage of the alligator nest sites, then Lake Okeechobee, then Jessup Lake, while Lake Apopka had the lowest hatching percentage. Data was insufficient to calculate relative hatching percentages for lakes Woodriff and Orange.

The sample sizes in the various weight groups for the various lakes were somewhat diverse. Embryos from lakes Griffin, Apopka and Woodriff tended to have died later in development than those from lakes Okeechobee and Jessup whose embryos died earlier in development. Embryos from Orange Lake tended to have died either very early in development (10 days or less) or much later in development (25 days or more) (Table 4).

Table 4: The number of samples (n) from each Florida lake in the different weight groups (A. B. C. D. E)

Key:
x= embryo weight
A- O.1g > x
B= 0. 5g > x > 0. 1g
C= 1.Og > x > 0.5g D= x > 1.0g
E= all weights

Discussion

Arsenic, cadmium and lead do not seem to be a pollution threat to the alligator embryos at these nesting sites. Copper was found in low amounts in most embryos so is not considered to be a pollution problem. Copper might be a problem, though, for embryos nesting at Jessup Lake. Iron and nickel might be a pollution problem to embryos from lakes Okeechobee and Jessup, but are not considered to be threatening to embryos from the other four lakes. It is unknown whether or not zinc was a general pollution problem due to the inconsistency of results, but the high values could be attributed to possible contamination (e.g. via containers, dissecting equipment or formalin used for embryo preservation) of the embryos upon sample collection.

Results from whole eggs of the crocodile, C. acutus, for the metals AS, Cd, Cu and Pb indicated higher levels than those obtained from the alligator embryos in this study. Zinc levels reported for crocodile eggs were within the same range of values obtained in the alligator embryos from this study. Differences in these results may be due to the difference in species or may be due to differences in the sample method used (i.e. whole egg vs. embryo only).

Metal pollution indications for lakes Okeechobee and Jessup may not be substantiated, however, since embryos from these lakes died very early in their development (10 days or less). Since they died so young it is unlikely that it can be determined whether or not it was metal poisoning or other factors such as a change in environmental conditions or genetic defects, which caused their demise. There may, however, be a solution to this problem: the use of whole eggs for heavy metal analysis in future studies, separating the embryo from the other contents of the egg (i.e. yolk and albumin) and analyzing both the embryo and the extra-embryonic material for metals.

Removing an embryo from the egg could lead to contamination and/or loss of extra-embryonic material. Loss of this extra-embryonic material would result in a lower-than-actual metal level reading. A young embryo has much more yolk and albumin to lose than an older embryo since an embryo utilizes this material for food and water. The variability in the amount of this material being lost by a young embryo would then result in a variable reading of metal levels. Therefore, calculated average metal levels for very young embryos with much material in the yolk sac can be expected to be varied from embryo to embryo of a particular nesting site.

Another problem arising from the use of very young embryos is sample weight. As the sample mass decreases, the greater the chance that even a small change in readings by the flame spectrophotometer will result in a higher-than-actual calculated metal level. The formula used to calculate metal concentration in ppm of a particular sample is total sample volume times the reading divided by the sample weight. It is easy to ascertain that even a small change in readings as low as 0.025 can change the calculated metal concentration astronomically as the sample weight decreases. The greater the sample weight, the better. As a general rule for heavy metal analysis, samples are recommended to weigh no less than 1.0g. The use of whole eggs would alleviate the sample weight problem since a whole egg has a mass well over 1.0g.

Taking these factors into consideration, copper, iron and nickel in embryos from Jessup Lake and iron and nickel in embryos from Lake Okeechobee may indeed be a metal pollution problem, but the low weight of these samples may have resulted in higher-than-actual calculated results.

References

1.  Eisler, R. 1981. Trace Metal Concentrations in Marine Organisms. Pergamon Press Inc. New York. pp. 624-625.

2.  Smith, HM and Brodie, ED. 1982. A Guide to Field Identification: Reptiles of North America. Western Publishing Inc.Wisconsin.

3.  LaCognata, S. and Skoch, EJ. 1987. A Comparison of Techniques for the Extraction of Heavy metals in Tissues. Proceedings of the 18th Annual Conference and workshop of IAAAM.