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,4-dintitrotoluene (2,4-DNT) is an explosive frequently found in the soil of military installations. Because reptiles can be common on these sites, ecological risk assessments for compounds such as 2,4-DNT could be improved with toxicity data specific to reptiles. Western fence lizards, Sceloporus occidentalis, were used to develop a laboratory toxicity model for reptiles. A hierarchical approach was used; acute to subchronic studies were conducted to provide toxicity data relevant to short- and longterm exposures. First, a modified median lethal dose (LD50) study was conducted on male and female lizards using a stage-wise probit model. The LD50 was 577 mg/kg for female and 380 mg/kg for male lizards. Subsequently, a subacute experiment was conducted to further assess 2,4-DNT toxicity to male lizards and to define exposure levels for a longer term, subchronic study. The subchronic study was conducted for 60 consecutive days; male lizards were exposed to 0, 9, 15, 25, 42, 70 mg/kg/d. Dosedependent mortality was observed in the three highest dose groups (25, 42, and 70 mg/kg/d); all other animals survived the study duration. Benchmark dose model calculations based on mortality indicated a 5% effect level of 15.8 mg/kg/d. At study termination, a gross necropsy was performed, organ weights were taken, and blood was collected for clinical and hematological analysis. Body weight, kidney weight, food consumption, postdose observations, and blood chemistries all were found to be significantly different from controls at doses above 9 mg/kg/d. Also, preliminary results suggest behavioral observations, and reduced food consumption may be a sensitive indicator of toxicity. The present study indicates Sceloporus occidentalis is suitable for evaluating toxicity of compounds to reptilian species.

DISCUSSION

Sceloporus occidentalis are relatively sensitive to 2,4-DNT. Acute exposures to high levels of 2,4-DNT showed male lizards may be more sensitive than female lizards. This may be due to general differences between sexes or may be complicated by changes in energy allocation dependent on cycles and/or potential to shunt 2,4-DNT to internal egg masses. Nonetheless, comparison of the LD50 indicates male lizards are more sensitive and were used for evaluating toxic effects of 2,4-DNT following longer exposure durations. Importantly, result interpretations must consider the large confidence intervals for the LD50 estimates, which are likely the result of individual variability in sensitivity and study design.

Following subchronic exposure, lizard survival was impacted significantly in a dose-dependent manner. Impact on survival first was seen in the 70 mg/kg/d 2,4-DNT dose group, but lizard deaths in this group occurred over an extended period of time. Some lizards exposed to 70 mg/kg/d survived 25 d past the death of the first animal. In comparison, lizards exposed to 42 mg/kg/d survived only 17 d following the first death in this dose group. Lizards exposed to 25 mg/kg/d appear to have similar survival rates to lizards in the 42-mg/kg/d dose group. However, 2,4-DNT at 9 and 15 mg/kg/d did not impact lizard survival for the 60-d duration. Nevertheless, significant differences were detected among lizards exposed to 0, 9, or 15 mg/kg/d in weight, food consumption, organ weight data, and blood chemistry parameters.

Generally, adult male lizards have fluctuating body weights. Based on extensive laboratory observations, following lizard hibernation or postreproduction, lizards will binge eat until maximum body fat has been stored. Then food consumption is reduced markedly, thus creating the weight fluctuations shown in Figure 2. However, despite the fluctuations, weights showed significant dose-dependent differences throughout the study. Furthermore, reduced food consumption trends can be linked to significant weight changes. Reduced food consumption appears to be an early indicator of a deleterious response to 2,4-DNT exposure. Even lizards receiving the lowest dose (9 mg/kg/d) showed a trend of reduced food consumption (Fig. 3) and the change in weight of the controls was consistently greater than that measured in the lizards receiving 9 mg/ kg/d of 2,4-DNT. However, no parameters between control and lizards exposed to 9 mg/kg/d 2,4-DNT were significantly different for the 60-d study duration; reduced food consumption trends suggest effects may become apparent in longer exposure durations.

Kidneys are a target organ for 2,4-DNT toxicity. Following exposure to 2,4-DNT, renal effects have been reported in birds, mice, rats, dogs, and possibly humans [22-27], In the present study, lizards exposed to 15 mg/kg/d 2,4-DNT had significantly heavier kidneys than controls, suggesting effects from 2,4-DNT exposure on renal function. Additionally, a significant increase in plasma uric acid and phosphorus concentrations in the 15 mg/kg/d 2,4-DNT lizards is consistent with renal effects. In an effort to better understand baseline hematology and plasma chemistry in lizards, a study reported a mean value of uric acid in bearded dragons (Pogona; n = 21) at 5.2 mg/dl, standard deviation of 2.6 [28]. Additionally, through previous efforts baseline blood data were obtained for western fence lizards that showed mean plasma concentrations of uric acid and phosphorus to be 6.42 mg/dl SE ± 0.76 (n = 14) and 12.0 mg/dl (SE ± 0.45; n = 15), respectively. Control lizards in the present study had a mean plasma uric acid value of 5.1 mg/dl (SE ± 0.30) that is very similar to the baseline data; however, lizards exposed to 15 mg/kg/d 2,4DNT had a mean plasma uric acid of 12.9 mg/dl (SE ± 4.63). This was much greater than the controls and the baseline data obtained for both lizard species. Elevated plasma uric acid and phosphorus are associated with renal failure in reptiles [29,30]. Furthermore, reduced renal function usually leads to increased kidney size and decreased uric acid excretion, which results in excess uric acid in the blood [31]. In summary, pathology results for western fence lizards confirm these findings; renal lesions, tubular degeneration and necrosis, and gout was seen in all treatment groups and frequency increased with dose [21].

Following exposure to 2,4-DNT, postdose behavioral observations were recorded daily. Three behaviors were displayed more prominently than others; therefore, lizards were observed for arched posture, dark color change, and hanging. The intention of postdose observation data is to show relative stress, not necessarily toxicity. A more robust approach to quantifying stress would be to measure stress hormones and/ or corticosteroid levels. However, postdose observation data provides preliminary insight into stress related to toxic exposures. For instance, Greenberg [32] reports that body color can be something of a window on the internal state of the green anole, Anolis carolinensis. Darker color changes have been linked to increased stress hormones in the green anole [32,33].

Consequently, lizards exposed to higher concentrations of 2,4-DNT displayed darker coloration more frequently than lizards exposed to 0, 9, or 15 mg/kg/d. Although arched posture more commonly is linked to lizard interactions (male and female), data analysis suggests it may be a stress response in this study, as all lizards exposed to 25, 42, or 70 mg/kg/d of 2,4-DNT displayed arched posture significantly more than control lizards. Finally, lizards exposed to higher concentrations of 2,4-DNT more frequently were seen hanging from the mesh top of the cage when compared to control lizards. This parameter was most useful in making objective, nonbiased observations and correlated well with the more subjective observations.