Median lethal dose.
Both genders of S. macroura tested in the current study were significantly less sensitive to fipronil than the only other mammals tested, M. musculus (L. 1758; 94 mg kg-1) and Rattus norvegicus (Birkenhout 1769; 97 mg kg-1) (Food and Agriculture Organisation of the United Nations 1997) in the literature to date. This result directly contrasts with a 10 – 14 fold difference in acute oral toxicity for both dunnart species (S. crassicaudata = 129 mg kg-1 CI = 74.2 – 159.0; S. macroura = 97 mg kg-1 CI = 88.3 – 120.0) to the organophosphorous pesticide, fenitrothion, when compared to M. musculus (1100 – 1400 mg kg-1), using the same technique for the resolution of median lethal dose estimates (Story et al. 2011). Whilst the two chemicals mentioned above exert their influence on different physiological pathways, the significant differences in patterns of acute oral toxicity compound the lack of acute oral vertebrate toxicological data thereby reducing the predictive value of pesticide risk assessments for endemic Australian vertebrates.
Current risk assessment frameworks for pesticides generally use, in part, the lowest median lethal dose for mammals to assess hazard of a chemical (Newman 2015). Increasingly, median lethal dose estimates, either LD50 or LC50 data, obtained from chemical exposure studies can be incorporated into species sensitivity distributions (SSDs) to comparatively assess toxicity and derive hazard threshold values (Posthuma et al. 2002). However, the generation of a distribution using three data points, while possible with the assistance of extrapolation factors (as outlined in (Posthuma et al. 2002)), is less likely to provide a robust representation of the desired risk thresholds (e.g. HD05) rendering the estimation of safe residue levels problematic. Recent research has highlighted a similar problem in relation to the avian acute oral toxicity profile of fipronil. While previous risk assessments for this pesticide have cited a primarily bimodal toxicological profile with a highly sensitive species at one end (the northern bobwhite, Colinus virginianus L. 1758; LD50 = 11.3 mg kg-1) and an extremely insensitive species at the other (the mallard, Anas platyrhynchos L. 1758; LD50 = 2150 mg kg-1), Kitulagodage et al. demonstrated that, by testing other species, fipronil’s acute oral toxicity fits a distribution similar to that of other pesticides, and, moreover, is grouped along avian orders (Kitulagodage 2011; Kitulagodage et al. 2011b).
The advantages of using the UDP protocol for the derivation of median lethal doses over the traditional LD50 assessment techniques are well established (Newman 2013; Story et al. 2011). Specifically, a reduction in the number of individuals required to resolve an estimate of median lethal dose is desirable from an animal ethics perspective, particularly if the use of other chemical impact metrics (e.g. quantitative structure-activity relationships, QSARs) to assess the potential sensitivity of untested species to a pesticide are precluded due to a lack of data (Story et al. 2011). Additionally, the UDP method has been shown to produce a median lethal dose (LD50) estimate similar to that achieved from conventional toxicity testing with the LD50 values derived from this method being directly comparable to other acute toxicity testing classification systems, thus allowing a comparison of pesticide sensitivity of Australian marsupial fauna with non-native eutherian mammals (Story et al. 2011).
The assessment of agricultural and veterinary chemicals for registration in Australia is a process that is evolving over time as both the amount of data submitted to support registrations increases and assessment methodologies and detection levels improve (Hyman 1997). If the use of SSDs to assess protection thresholds in relation to Australian endemic species is to continue, then further sensitivity research will be required to circumvent the need to extrapolate from a narrow range of organisms tested under standard laboratory conditions to free-living populations or ecosystems. The results of the present study show the limitations of this approach and highlights the importance of evaluating the effects of pesticides on non-target species that are likely to be exposed, particularly when these species are phylogenetically distinct from those used in studies of pesticide sensitivity originating in North America or the European Union.
Fiprole (fipronil and metabolite) residues in tissues and body mass.
The use of the UDP methodology to quantify a median lethal dose unavoidably results in very small experimental groups, sometimes n = 1, thereby resulting in secondary data sets, such as residue loads from tissue samples, that are unable to be subjected to appropriate statistical analyses. Despite this limitation, the current study quantified fiprole residue levels in kidney, liver, plasma, brain and caudal and subcutaneous adipose tissue samples taken from individual dunnarts at either the time of death or at the end of the 14 day post-dose observation period. Obviously, these results need to be viewed with a great deal of circumspection. However, we report these results from the current study as a precursory dataset to maximise the amount of information derived and to better inform a subsequent study into the comparative metabolic fate of fipronil in two similar-sized, but systematically divergent species, M. musculus (eutherian) and S. macroura (metatherian) accepting the abovementioned limitations.
Studies investigating the biotransformation of fipronil in rats (Food and Agriculture Organisation of the United Nations 1997) have quantified 3 primary metabolites after hepatic transformation of the parent compound fipronil (Fig 1.). Of these metabolites, the fip-sulfone and fip-desulfinyl have been shown to be of toxicological concern in previous studies. The oxidative fip-sulfone metabolite has a six-fold higher binding affinity for the postsynaptic GABA receptor (Hainzl et al. 1998) and metabolism of the parent compound to this derivative has been shown to add synergistically to the overall toxicity of a fipronil-based formulation in pesticide-exposed birds (Kitulagodage et al. 2011b). Moreover, avian studies have demonstrated that inclusion of fip-sulfone residues in a regression analysis of post-exposure body mass loss provided a much better fit than regressions comparing loss of body mass with the parent compound, fipronil, alone in brain, liver and adipose tissues (Kitulagodage et al. 2011b). The overlap between symptoms of intoxication, the time course of fip-sulfone residues in brain, liver and adipose tissue, lack of post-dose feeding activity and subsequent weight loss in dosed birds provided insight into an observed increased selective toxicity to the three galliform species tested (Kitulagodage 2011; Kitulagodage et al. 2011b).
In the current study, fipronil and fip-sulfone residues were more prominent at the higher doses administered (e. g. 990 and 2000 mg kg-1) with the residue load occurring in subcutaneous and caudally stored fat, liver and brain, in descending order of magnitude. Slightly higher levels of fipronil were present in male (versus female) brains at the time of analysis, but very little difference existed between either fipronil or fip-sulfone levels in either subcutaneous or tail fat and plasma. Dunnarts not surviving the administered dose showed higher fipronil and fip-sulfone levels across adipose tissues, liver and brain. However, as was the case with dunnarts surviving a given dose, very little, if any, plasma-bound residue bringing into question whether the use of fipronil residue in plasma is suitable as a biomarker of pesticide exposure in wildlife monitoring studies. While the detection of fip-sulfone in the liver and adipose tissues of males and females across the various administered doses indicates the metabolism of fipronil to the fip-sulfone metabolite, the levels detected, in addition to low levels of this metabolite finding its way to brain tissue and an absence of weight loss in dunnarts surviving the administered dose, is contrary to the findings in the abovementioned avian studies. Further research into the metabolic fate of this pesticide in marsupials is required to better elucidate the role of the fip-sulfone metabolite in determining the overall toxicity of fipronil-based pesticide formulations, as seems to be the case in more sensitive avian orders.
Fip-desulfinyl is generally considered to be a photolytic breakdown product and not a metabolite as such. In the current study, analysis detected generally low levels of this compound (range = 0 – 46.07 ng g-1 with one male dunnart (dose = 99 mg kg-1) returning an outlier value of 281.89 ng g-1 in adipose tissue) and due to its toxicological significance, we have reported these results. Fip-desulfinyl is considered of high toxicity with an acute oral LD50 of 15 (males) – 18 (females) mg kg-1 for M. musculus (Food and Agriculture Organisation of the United Nations 1997). When administered orally to mice, the fip-desulfinyl metabolite has been shown to decrease body weight at doses of 30 and 60 ppm, whereas a lower dose of 3 ppm was seen to increased motor activity, irritability and aggression with convulsions also observed (Food and Agriculture Organisation of the United Nations 1997). Although present in small quantities, presumably as a result of photolytic breakdown of the dosing formulation immediately after preparation, it’s acute toxicity would necessitate its inclusion in residue analysis for any future field based trial investigating in situ wildlife impacts. Higher levels of the fip-sulfide metabolite (range = 0 – 85.78 ng g-1 with the same male dunnart as above (dose = 99 mg kg-1) returning an outlier value of 6345.34 ng g-1 in adipose tissue) were also found in adipose tissues of pesticide-exposed dunnarts. The higher LD50 values for this compound reported for mice (69 (males) and 100 (females) mg kg-1 (Food and Agriculture Organisation of the United Nations 1997)) indicates a moderate toxicity for this species, with similar toxicological signs as those reported for the other breakdown products (fip-sulfone and fip-desulfinyl) as well as the parent (fipronil).
The Australian arid zone is characterised by low productivity and highly variable rainfall (Stafford-Smith and Morton 1990). Species inhabiting these environments have evolved a range of adaptations which assist them in coping with the inconsistent, and often sparsely distributed resources - such as the ability for rapid, long-range movement enabling animals to access areas of recent rainfall and capitalize on the increase food resources (Dickman et al. 1995; Letnic and Dickman 2005). The Dasyuridae caudally store fat to provide an energy reserve that can be utilised during times of resource limitation (Morton and Dickman 2008a; Morton and Dickman 2008b). The ability for lipophilic xenobiotic compounds, such as agricultural pesticides and their toxic metabolites, to be stored along with these fat reserves has the potential to ensure that pesticide residues remain biologically available by being constantly metabolized as dunnarts utilise caudally stored fat to maintain the energetic resources necessary for sustaining daily life during times of drought. Conventional toxicity testing used for chemical risk assessments generally defines exposure times for the determination of median lethal dose values to quantify mortality (Newman 2015). The tendency for toxic substances to be stored in adipose tissue and later metabolized when animals are facing resource limitations, extends the exposure period for chemicals significantly beyond, for example, either the 48 hr acute oral toxicity test limit or the 30 d reproductive test limit more commonly used in pesticide risk assessments (Buttemer et al. 2008; Story et al. 2016).