- Top of page
Anuran embryos and larvae generally occupy thermally heterogeneous environments, and thermal regimes can strongly influence anuran development (Duellman & Trueb 1986; Ultsch, Bradford & Freda 1999). Temperature or temperature variability can alter the potency of some pesticides to tadpoles (Boone & Bridges 1999; Broomhall 2002). Less expected is the possibility that, because different life-history stages may respond differently to a given stress (Bridges 2000), temperature changes during early life-history stages may have substantial influences on later development.
The eggs of many anuran species develop over relatively short time periods, approximately 1–10 days (Duellman & Trueb 1986). Despite the shortness of the embryonic period, embryonic conditions can modify development. For example, Rana temporaria larvae that had received enhanced levels of UV-B as embryos subsequently metamorphosed later, and at a smaller size, than larvae that had not (Pahkala, Laurila & Merilä 2001). Egg temperature can also influence locomotor performance of ectotherms (Elphick & Shine 1998). Given these data, embryonic thermal history may change the way in which later pesticide exposure influences tadpole growth and behaviour. However, this possibility has not attracted any study to date.
Endosulfan is an organochlorine cyclodiene insecticide that is used extensively throughout the world. In 2001, endosulfan was among the 10 pesticide residues most frequently found on fruits and vegetables from the 15 member states of the European Union (European Commission 2003), and in the USA more than 625 000 kg of endosulfan active constituent is used annually (United States Environmental Protection Agency 2002). In Australia, endosulfan is sprayed on cotton crops, stone fruits, vegetables, tobacco, cereals, legumes, oilseeds, citrus, tropical fruits, ornamentals and others (NRA 1998). Cotton is cultivated in very large continuous areas and approximately 700 tonnes of technical endosulfan (95% active ingredient) are sprayed across cotton crops during the early part of the spring season every year (NRA 1998). Endosulfan sulphate, the predominant oxidation product, is frequently transported into riverine environments during storm events (Leys et al. 1998; Raupach & Briggs 1998; Leonard et al. 1999).
Many Australian frog species breed in spring (Tyler 1994) at a time when rivers and waterbodies exhibit strong short-term fluctuations both in levels of agrochemical residues (Cooper 1996; Muschal & Cooper 1998) and thermal regimes. Furthermore, toxic endosulfan degradates may remain in soil and sediments throughout the year (Nowak & Julli 1991), suggesting that even after periodic direct exposure tadpoles may be continuously exposed to sublethal levels of endosulfan via suspended particulate matter or ingestion of sediment.
The effects of endosulfan on Australia anurans remain unstudied despite their substantial phylogenetic distance from species tested in other parts of the world (Mann & Bidwell 1999), and a high risk of endosulfan contamination in spring breeders. This study tested the hypothesis that egg-rearing temperature might modify the effects of endosulfan on tadpoles of a frog species Limnodynastes peronii (Duméril and Bibron) from south-eastern Australia. The experiment was designed to mimic a complication that occurs in the real world variations in egg-rearing temperature. The study also attempted to ascertain if exposure to biologically relevant concentrations of endosulfan (in the commercial pesticide Thiodan®, AgrEvo, Sydney, Australia) affected survivorship or fitness-related traits in tadpoles. Sublethal response to toxicants is an important component of pesticide exposure in a broader ecological sense (Buckler et al. 1995). For example, glaucous gulls Larus hyperboreus with high blood concentrations of organochlorines exhibit increased absenteeism from the nest (Bustnes et al. 2001), perhaps leading to decreased reproductive success. Although sublethal reactions are rarely considered in amphibian research, recent papers highlight the importance of testing endpoints such as growth rates, swim speed and vulnerability to predation (Semlitsch, Foglia & Mueller 1995; Bridges 1997, 1999a,b).
- Top of page
Egg-rearing temperature affected feeding rate and predator avoidance by L. peronii tadpoles (when tested 28 days later). Endosulfan significantly decreased feeding, growth and predator avoidance in tadpoles, but did not alter behaviour. Moreover, the effects of egg-rearing temperature and endosulfan on tadpole length were interactive. Consequently, these data indicate that egg-rearing temperature may alter the later effects of endosulfan on growth, and that short-term exposure to endosulfan can influence tadpole viability either immediately or over an extended period.
The lowered feeding rate in tadpoles hatched from cool eggs may reflect a fixed lower metabolic rate associated with cool egg-rearing temperatures (because all tadpoles were maintained at the same temperature). While existing data on thermal acclimation in amphibians are extremely variable across species and studies, Rome, Stevens & John-Alder (1992) concluded that, in general, physiological functions are slower if acclimated to low temperatures than they would be if acclimated to high temperatures. Unfortunately, there are so few data on the effect of embryonic developmental temperatures on subsequent metabolism and performance in anurans, it is difficult to explore potential reasons for this result.
Regardless of egg-rearing temperature, exposure to both concentrations of endosulfan continued to reduce the proportion of tadpoles feeding even on the last day tested, nine days after tadpoles were no longer exposed to endosulfan. Thus, reductions in feeding may have potentially continued longer than the 9 days in clean water used in this experiment. Decreases in feeding rate can limit energy gain and consequently have repercussions on growth rate. For example, lower food availability reduced size at metamorphosis in tadpoles of the spadefoot toad Scaphiopus couchii (Newman 1994). The same trend was evident in the data reported here, with tadpoles exposed to even the lowest concentration of endosulfan shorter than control tadpoles. Larger size at metamorphosis and earlier metamorphosis can enhance survivorship to maturity, hasten first reproduction and enable larger size at first reproduction (Smith 1987; Berven 1990). Thus, pulsed contact with low concentrations of endosulfan may affect metamorphosis and subsequent survivorship of this study species.
Tadpole behaviour did not appear to change in response to predator cues, egg-rearing temperature or previous endosulfan exposure. However, as tadpoles were tested 29–33 days after cessation of endosulfan exposure, potential short-term alterations in behaviour would not have been detected. It is none the less interesting to note that this species does not appear to respond to predator cues, in contrast to many northern hemisphere species (Relyea & Werner 1999; Eklov 2000; Laurila 2000; Relyea 2000). As tadpoles had been reared in laboratory conditions and thus had never previously experienced predator cues it may be possible, albeit unlikely, that behavioural responses are partially learned in this study species. Learned predator avoidance has been shown in lizard species (Marcellini & Jenssen 1991). On the other hand, anti-predator behaviour between naive and experienced tadpoles of Bufo americanus (American toad) did not differ (Gallie, Mumme & Wissinger 2001), arguing that in some species predator responses may be genetically determined. Alternatively, the methodology used in this study would not have detected other forms of response, such as reductions in the distances travelled (Anholt, Werner & Skelly 2000), or long-term morphological changes, such as deeper tail fins (McCollum & Van Buskirk 1996; McCollum & Leimberger 1997; reviewed by Chivers & Smith 1998; Van Buskirk & Saxer 2001).
The temperature that an egg experienced affected the resulting tadpole’s susceptibility to a predator 28 days later, despite experiencing a common (and different) temperature in those intervening days. Furthermore, endosulfan exposure increased vulnerability to a common predator when tested 9 days after cessation of pesticide exposure. Tadpoles hatched from warm eggs appeared to be better able to avoid predators than those hatched from cool eggs, yet they were also more adversely affected by endosulfan (Fig. 3). Consequently, it appears that both egg-rearing temperature and exposure to endosulfan can potentially cause long-term or perhaps permanent alterations in a tadpole’s physiology. As no behavioural change in the tadpoles according to endosulfan or egg-rearing temperature was detected, the mechanism by which this effect was manifested may have been a physiological one, such as a change in burst speed or a change in body shape.
Anax dragonfly larvae preferentially kill Pseudacris triseriata tadpoles with wider and deeper bodies, narrower tail muscles and smaller tail fin depth (Van Buskirk, Mccollum & Werner 1997). Although the results reported here indicate that relative tail and body lengths did not differ between treatments, measures of body width and depth were not taken. Tail length can alter sprint speed and vulnerability to predation (although the direction of the change appears to be different in different species and situations; cf. Van Buskirk & Mccollum 2000; Parichy & Kaplan 1995). Because tail length did not differ between treatments, a more likely possibility for the differences in predation rates is a change in burst speed (as mediated by changes in muscle phenotype) rather than sprint speed.
Larval developmental temperature strongly affected burst speed in tadpoles of Hyla regilla Pacific treefrog regardless of acclimation or testing temperature (Watkins 2000). Tail myofibrillar ATPase activity, which is associated with the maximum shortening velocity of a muscle, was higher in cool-reared larvae (Watkins 2000). It may therefore be possible that egg-rearing temperature can also alter muscle phenotype, although this remains to be tested.
Whether the mechanism to explain the observed differences in predation lies with body shape or burst speed, predation is a major force structuring tadpole communities (Anholt & Werner 1995). Indeed, predators greatly influence the density and distribution of other organisms (Ormerod 2002). Thus, an increased predation risk over and above alterations in size may have substantial impacts on future survival in the current study species.
Although amphibians can often operate at suboptimal temperatures (reviewed by Rome, Stevens & John-Alder 1992), additional stresses imposed by exposure to agrochemicals (especially if they differ according to the thermal history of the tadpole, as is the case here with length) may substantially alter anuran population dynamics. The implications of changes in temperature, perhaps associated with global climate change, are therefore serious and warrant further repetition and investigation. This work has been repeated in the treefrog Litoria peronii, which, although it has a similar name, is from an entirely different family group, the Hylidae. Although there were subtle behavioural differences between species, egg-rearing temperature and endosulfan altered predator avoidance (S. Broomhall, unpublished data). In this way, changes in temperature regimes due to climate warming may potentially combine with agricultural practices to affect anuran populations.
Many standard toxicity tests do not address the possibility that pesticides may compromise future components of fitness, nor how pesticide exposure may interact with other factors present at the time of exposure. For example, the effects of a stressor differed at different population densities in the marine copepod Tisbe battagliai (Sibly, Williams & Jones 2000). An endosulfan concentration of 20·3 mg l−1 inhibited cortisol secretion in cells of rainbow trout Oncorhynchus mykiss by 76–82%, yet cell viability only decreased by 5% (Leblond, Bisson & Hontela 2001). This confirms that deleterious effects may occur at levels many times lower than those commonly used as endpoints. Similarly, Buckler et al. (1995) found that feeding activity (prey strike rates) was the most sensitive indicator of acidity-induced stress (and also aluminium-induced stress at pH 5·5) in Atlantic salmon Salmo salar, while mortality was a much less sensitive endpoint. Consideration of correlates of fitness such as those reported here are an important step towards gaining an understanding of the mechanisms by which exposure to agricultural chemicals may impact upon frog populations in a broader ecological context.
Of particular concern is that tadpole fitness was decreased by short-term exposure to an endosulfan concentration listed as the ANZZEC trigger value to protect 99% of species. Trigger values are generally expected to provide a greater than threefold protection from acute toxicity (ANZECC & ARMCANZ 2000b). Nevertheless, the current data show that this trigger concentration of 0·03 µg l−1 significantly (and persistently) impaired feeding, and also resulted in an increased risk of predation and significantly shorter tadpoles. This result has implications for natural resource management decisions, as existing water quality prescriptions may not provide adequate protection for populations over the long-term. This study demonstrates that extremely low concentrations of endosulfan have the potential to affect natural frog communities adversely and illustrates how interacting stressors may have cumulative effects on tadpoles. Furthermore, this study indicates that minor, short-term shifts in temperature may alter the impacts of agrochemical contaminants on anuran populations over the long-term.