ORGANIC POLLUTANTS: AN ECOTOXICOLOGICAL PERSPECTIVE - CHAPTER 11 pptx

CHAPTER 11 The anticoagulant rodenticides 11.1 Background Warfarin, which was introduced to the market in the late 1940s, was the first of a series of anticoagulant rodenticides (ARs) related in structure to dicoumarol (Figure 11.1) (Meehan, 1986; Buckle and Smith, 1994). All of them are toxic because they act as anticoagulants – extending the clotting time of blood and so causing haemorrhaging. The anticoagulant properties of naturally occurring dicoumarol were discovered in the USA early in the twentieth century, when it was found to be the causal agent in cases of fatal haemorrhaging of cattle that had been fed spoiled clover. Subsequently, it was discovered that dicoumarol and rodenticides related to it have anticoagulant action because they act as vitamin K antagonists. For some years warfarin was by far the most widely used rodenticide of this type. In time, however, strains of rats resistant to warfarin began to appear, and the compound became ineffective in some areas where it had been regularly used. Resistance was overcome, at least in the short term, by a second generation of ARs sometimes called ‘superwarfarins’. Examples include brodifacoum, difenacoum, flocoumafen and bromodiolone. The superwarfarins are more hydrophobic, persistent and toxic than warfarin itself. They have usually been effective in overcoming resistance to warfarin and, consequently, have come into more wide-scale use in the last two decades. Although knowledge about their environmental fate and effects is at present very limited, enough is known to raise questions about the risks that might be associated © 2001 C. H. Walker The anticoagulant rodenticides 205 with their increasing use. The combination of persistence with very high vertebrate toxicity has set the alarm bells ringing. The ensuing account will be principally concerned with the second-generation rodenticides. 11.2 Chemical properties The formulae of some ARs are given in Figure 11.1, where it can be seen that they have some structural resemblance both to dicoumarol and to vitamin K in its quinone form. All possess quinone rings linked to unsubstituted phenyl rings. The phenyl rings of the rodenticides confer hydrophobicity, especially in the relatively large and Figure 11.1 Anticoagulant rodenticides. Brodifacoum O O OH Br Flocoumafen O O O OH CF 3 Dicoumarol O OO O OH OH CH 2 Warfarin O O CH 2 CO CH 3 Vitamin K (quinone form) O O R CH 3 R = phytyl (vitamin K i ) or a polyisoprenoid in the case of menaquinones © 2001 C. H. Walker 206 Major organic pollutants complex molecules of brodifacoum and flocoumafen. The chemical properties of some anticoagulant rodenticides are given in Table 11.1. All the compounds listed in Table 11.1 are solids. Flocoumafen and brodifacoum have particularly low vapour pressures. The hydrophobicity of brodifacoum and flocoumafen is reflected in their low water solubility. It should be remembered, however, that because they possess ionisable hydroxyl groups water solubility is pH dependent. With brodifacoum, for example, water solubilities are 3.8 × 10 –3 and 10.0 mg/L at pH values of 5.2 and 9.3 respectively. An increase in pH encourages some ionisation, and solubility increases accordingly. 11.3 Metabolism of anticoagulant rodenticides Metabolism has been studied in more detail for warfarin than for other related rodenticides. The metabolism of warfarin appears to be essentially similar to that of related compounds and will be taken as a model for the group. Two main types of primary metabolic attack have been recognised: (1) monooxygenase attack upon diverse positions on the molecule to yield hydroxy metabolites, and (2) conjugation of the hydroxyl group to yield, for example, glucuronides. More than one form of P450 is involved in warfarin metabolism. In a study of metabolism of [ 14 C]flocoumafen by the Japanese quail (Huckle et al., 1989), biotransformation was extensive and rapid, with eight metabolites detected in excreta. The elimination of radioactivity from the liver of Japanese quail was biphasic (Figure 11.2). After an initial period of rapid decline, there followed a period of slow exponential decline, indicating a half-life of more than 100 days. Slow elimination in the second phase has also been demonstrated in rodents and is attributed to very strong binding to one or more proteins of the hepatic endoplasmic reticulum. Other highly toxic ‘superwarfarins’ such as brodifacoum and difenacoum also show very slow ‘second-phase’ elimination from the liver, which is associated with strong protein binding. Depending on the compound and the species,< 2 µg/g (2 ppm by weight) of the residue of superwarfarins can be strongly bound in the liver. Most other tissues show less capacity for protein binding. Some of the strong protein binding in liver is to the target site of the rodenticide, the reductase of vitamin K 2,3-epoxide (see section 11.5). There is also evidence that flocoumafen can bind strongly to cytochrome P4501A1. In the Japanese quail, the Table 11.1 Properties of anticoagulant rodenticides Water solubility Vapour pressure Compound (mg/L) Log K ow (mPa) Warfarin 17 1.5 × 10 –3 Flocoumafen 1.1 4.7 1.3 × 10 –7 Brodifacoum 0.24 (pH 7.4) 8.5 << 1 × 10 –3 Difenacoum 2.5 (pH 7.3) © 2001 C. H. Walker The anticoagulant rodenticides 207 binding of flocoumafen to hepatic microsomes was linked to strong inhibition of EROD-ase activity, both in vivo and in vitro (Fergusson, 1994). Strong binding to two sites in the liver could explain the biphasic elimination of flocoumafen by this species (Figure 11.2). The marked persistence of the readily biodegradable superwarfarins in liver is something of a paradox. Ready biodegradability does not guarantee a short half-life. Superwarfarins are much more biodegradable than refractory pollutants such as p,p′- DDE, dioxin and higher chlorinated PCBs. Yet they can still be highly persistent at low concentrations in certain tissues because tenacious binding ensures that they are virtually unavailable to the P450s that can degrade them. Such long-term storage brings the risk of long-term toxic effects in individuals and in food chains. 11.4 Environmental fate of anticoagulant rodenticides Warfarin and related rodenticides are usually incorporated into baits and placed in locations where they will be found by rats, mice and certain other vertebrate pests. There is a potential risk that baits will be eaten by pets, farm animals and other non- target species. To guard against this, baits are made inaccessible to vertebrates other than the target species. For example, baits may be put in short lengths of piping, wide enough for rats and mice to enter but narrow enough to exclude cats and dogs. In Western Europe, warfarin is used both inside and outside farm buildings. The use µg Flocoumafen equivalents /g Time (days) 10 1 10 -1 0 2010 30 40 50 60 70 80 90 100 110 120 Figure 11.2 Loss of flocoumafen residues from quail liver. Depletion of radioactivity from Japanese quail after a single oral dose (14 mg/kg) of [ 14 C]flocoumafen. Data are presented as microgram equivalents of [ 14 C]flocoumafen per gram of tissue and are mean values of two animals. Data collected at day 7 and at day 12 were from four animals and three animals respectively. After Huckle et al. (1989). © 2001 C. H. Walker 208 Major organic pollutants of more toxic rodenticides such as brodifacoum and flocoumafen is often restricted to the interior of buildings, to reduce the risk to non-target species. The main concern about these compounds is that they can be transferred via rodents to terrestrial predators and scavengers that feed upon them. Species at risk include members of the crow family, e.g. raven (Corvus corax), magpie (Pica pica), carrion crow (Corvus corone) and rook (Corvus frugilegus), owls such as barn owl (Tyto alba) and tawny owl (Strix aluca), and large predators which are also carrion feeders such as buzzard (B. buteo) and red kite (M. milvus). The long half-lives of residues of the superwarfarins in the livers of rodents give particular cause for concern. Owls and other predators may acquire these compounds from their live prey. Individual rats and mice live for several days after consuming lethal doses of rodenticide before they die from haemorrhaging. Furthermore, some resistant strains of rodents can tolerate relatively high levels of rodenticide and so act as more efficient vectors of the pesticide than susceptible strains. Also, rodents in the later stages of poisoning may be more vulnerable to predation than normal healthy individuals; in other words, there may be selective predation, working in favour of the most contaminated members of the prey population. Apart from predation, there is the obvious concern that scavengers will feed upon poisoned individuals if they die above ground. A number of reports have established the presence of rodenticides in predators and scavengers found dead in the field [see, for example, reports by the UK Wildlife Incident Investigation Scheme (WIIS)]. Brodifacoum, difenacoum, bromodiolone and flocoumafen have all been found, albeit at low levels in most cases (< 1 ppm in liver). Sometimes, more than one type of rodenticide has been found in one individual. The toxicological significance of these residues will be discussed in the next section. One study conducted in Britain between 1983 and 1989 was of barn owls found dead in the field. Ten per cent of the sample of 145 birds contained AR residues in their livers, difenacoum and brodifacoum were prominent among them (Newton et al., 1990). In another study, barn owls that had been fed rats dosed with flocoumafen showed that a substantial proportion of the rodenticide ingested by owls was eliminated in pellets (Eadsforth et al., 1991). The authors suggest that the exposure of owls to rodenticides in the field may be monitored by analysis of pellets dropped at roosts or regular perching places. 11.5 The toxicity of anticoagulant rodenticides The mode of action of warfarin and related rodenticides is illustrated in Figure 11.3. Vitamin K in its reduced hydroquinone form is a cofactor for a carboxylase located in the rough endoplasmic reticulum of hepatocytes. It is converted into an epoxide when it participates in the conversion of glutamate residues of certain proteins to γ-carboxy- glutamate (Gla). The regeneration of the hydroquinone form of vitamin K from the © 2001 C. H. Walker The anticoagulant rodenticides 209 2,3 epoxide is dependent on the function of a reductase. ARs bind to the reductase and prevent the conversion of the epoxide to the quinone. They also inhibit the subsequent reduction of the quinone to the hydroquinone. Thus, ARs can prevent the cyclic regeneration of the hydroquinone form so that carboxylation of glutamate slows down or ceases. In the case of prothrombin and related clotting factors, interruption of the vitamin K cycle leads to the production of non-functional, undercarboxylated proteins, which are duly exported from hepatocytes into blood (Thijssen, 1995). They are non- functional because there is a requirement for the additional carboxyl residues in the clotting process. Ionised carboxyl groups can establish links with negatively charged sites on neighbouring phospholipid molecules of cell surfaces via calcium bridges, and these links are necessary for the formation of blood clots. Because clotting proteins turn over slowly (half-life of prothrombin in the rat is approximately 10 h), several days will elapse after inhibition of the vitamin K cycle before the level of functional clotting proteins is sufficiently low to allow severe haemorrhaging. Typically, rats and mice begin to die from haemorrhaging 5 days or more after exposure to ARs. Owing to the strong affinity of superwarfarins for the reductase, the available binding sites may be progressively occupied by ARs over a period of weeks or even months of continuing exposure to low levels of the compounds. Evidently all the superwarfarins Figure 11.3 Action of warfarin and related rodenticides on the vitamin K cycle. © 2001 C. H. Walker Quinone reductase O O R O SH – SHI O R OH O R OH OH R OH OH R O O 2 O 2 O 2 CO 2 Carboxylase Epoxidase Epoxide reductase Quinone reductase Inhibition by anticoagulant rodenticides NAD(P) + NAD(P)H NAD(P) NAD(P)H COOH HC – COOH GLA COOH HCH Glutamate residue Protein S – S S – S SH – SH 210 Major organic pollutants bind to the same site, and it is to be expected that they will have an additive toxic effect. What is unclear, and can make interpretation of residue data difficult, is what degree of occupancy of the reductase binding sites by ARs will lead to serious haemorrhaging. Some individuals appear perfectly healthy when carrying liver residues that are high enough to cause haemorrhaging in others. On account of the uncertainties associated with the interpretation of residue data, it is important to have other evidence to establish toxic effects in the field. In the investigation of deaths in the field, haemorrhaging is usually easy to identify in a post-mortem examination if carcasses are in a reasonable condition. If early toxic effects are to be identified in live vertebrates, however, a biomarker assay is needed (Fergusson, 1994). The detection of undercarboxylated Gla proteins in blood has already been used to monitor human exposure to warfarin and other ARs (Knapen et al., 1993). The development of such an assay, e.g. an ELISA (enzyme-linked immunosorbent assay), that could be used to assay blood samples from predators/ scavengers exposed to rodenticides in the field has obvious attractions. It should then be possible to establish when levels of exposure in the field are high enough to begin to inhibit the vitamin K cycle and increase the blood level of undercarboxylated clotting proteins. Toxic effects could be identified at an early stage before the occurrence of deaths due to haemorrhaging. Because of the delay in the appearance of haemorrhaging after exposure to warfarin and related anticoagulant rodenticides, a suitable interval must elapse between exposure of experimental animals to the chemical and the assessment of mortality in toxicity testing. Typically this period is at least 5 days. Some values of acute oral LD 50 of rodenticides to vertebrates are given in Table 11.2. Looking at the data overall, brodifacoum and flocoumafen are more toxic than warfarin to mammals. Toxicity of the former two compounds to birds varies considerably between species. On the available evidence, the galliform birds chicken (Gallus domesticus) and Japanese quail (C. coturnix japonica) are much less sensitive to flocoumafen than are mammals. The chicken is less sensitive to brodifacoum than mammals. The mallard duck (Anas platyrhynchus), however, is just as sensitive as mammals to brodifacoum; studies with the barn owl (T. alba) indicate that it is of similar sensitivity to the rat or mouse. Newton et al. (1990) fed mice containing brodifacoum to the owls and estimated that birds lethally poisoned by the rodenticide (n = 4) had consumed 0.150–0.182 mg/kg of the compound. The birds died within 6–17 days of receiving a single dose of brodifacoum, and the concentration of rodenticide in the liver was 0.63–1.25 mg/kg. Owls were also dosed with difenacoum in this study, which was found to be less toxic than brodifacoum. A number of studies have shown that predatory and scavenging birds can acquire liver residues of second-generation ARs when these compounds are used in the field (Newton et al., 1990; and Annual Reports of WIIS), and that the levels are sometimes high enough to cause death by haemorrhaging (Merson et al., 1984). In a field trial with brodifacoum conducted in Virginia, USA, five screech owls (Otus asio) that were found dead 5–37 days after treatment contained 0.4–0.8 µg/g of the rodenticide in © 2001 C. H. Walker The anticoagulant rodenticides 211 liver (Hegdal and Colvin, 1988). These residues are of a similar magnitude to the levels found in poisoned barn owls in the study mentioned above. Field deaths of red kites and goshawks have also been attributed to secondary poisoning caused by bromodiolone (WIIS scheme). In another study, ravens died of brodifacoum poisoning during a rat control programme on Langara Island, British Columbia, Canada (Howald et al., 1999). They had acquired the rodenticide directly from bait and indirectly by predating or scavenging poisoned rats. Post-mortem examination established that the birds had died of severe haemorrhaging, and contained 0.98–2.52 mg/kg brodifacoum in the liver, mean value 1.35 mg/kg (n = 13). In New Zealand, primary and secondary brodifacoum poisoning of wekas (Gallirallus australis), flightless rails which are predators and scavengers, has been reported (Eason and Spurr, 1995). As discussed earlier (p. 210), a problem with these field incidents is that the low levels of rodenticides found in many of the poisoned birds are of similar magnitude to those in birds that survive exposure. A low residue level may signify everything or nothing. Additional evidence is needed to establish that the concentrations of rodenticide present in the livers of birds (or mammals) found in the field are sufficient to have caused death, e.g. the presence of haemorrhaging in the carcasses. 11.6 Ecological effects of anticoagulant rodenticides The demonstration that owls and other birds can acquire lethal doses of superwarfarins in the field following normal patterns of use has raised questions about the possibility of these compounds causing population declines. In one case reported in Malaysia, a Table 11.2 Acute oral LD 50 values for some anticoagulant rodenticides Acute oral LD 50 Compound Species (mg/kg) Warfarin Rat 1 Warfarin Pig 1 Brodifacoum Rat (male) 0.27 Bridifacoum Rabbit (male) 0.3 Brodifacoum Mouse (male) 0.4 Brodifacoum Chicken 4.5 Brodifacoum Mallard duck 0.31 Flocoumafen Rat 0.25 Flocoumafen Mouse 0.8 Flocoumafen Chicken > 100 Flocoumafen Japanese quail > 300 Flocoumafen Mallard duck ~ 24 Data from Tomlin (1997). © 2001 C. H. Walker 212 Major organic pollutants population decline in owls was related to the use of superwarfarins (see Newton et al., 1990). In New Zealand, the entire population of wekas on one island was wiped out by primary and secondary brodifacoum poisoning (Eason and Spurr, 1995). In Britain, however, a widespread decline in the barn owl population during the 1980s could not be explained in terms of rodenticide use. Only a small proportion (2%) of the barn owls found dead during the period 1983–9 contained residues of brodifacoum + difenacoum of 0.1 ppm or above in the liver. Thus, no more than 2% of the dead owls contained residues of rodenticides high enough to suggest poisoning. However, it should also be pointed out that the use of superwarfarins was restricted at the time of the survey. The recommended use of brodifacoum, for example, was (and still is) restricted to the interior of buildings. Superwarfarins have mainly been used in areas where resistance has developed to warfarin. The critical question is whether increasing use of superwarfarins will bring a significant risk to owls and other species exposed to them. During the 1990s, cases have been reported of red kites and other birds of prey dying as a consequence of poisoning by superwarfarins (see reports of WIIS). This is a controversial issue because red kites have been reintroduced into England in recent years. The population is still small, but growing. It has been suggested that mortality due to rodenticide poisoning may prevent the re-establishment of the species in certain areas of the country where ARs are more widely used. In summary, there is as yet no clear evidence that superwarfarins have caused any widespread declines in predators/scavengers that feed upon rodents. However, the persistence and very high cumulative toxicity of these compounds suggest that they could pose a serious hazard to such species if they are more widely used. The situation should be kept under close review. Resistance to warfarin has developed in populations of rats after repeated exposure to the rodenticide (Thijssen, 1995). One resistant strain discovered in Wales was found to have a much reduced capacity to bind warfarin to liver microsomes in comparison with susceptible rats. Resistance was due to a gene which encoded for a form of vitamin K epoxide reductase that was far less sensitive to warfarin inhibition than the form found in susceptible rats. Another resistant strain, which arose in the area of Glasgow, was found to differ from the resistant Welsh strain. Resistance was again due to an altered form of vitamin K epoxide reductase. However, the strain from the Glasgow area contained a form of the enzyme which bound warfarin just as strongly as that from susceptible rats! The difference was that the binding was readily reversible (Thijssen, 1995). In addition to these strains, another from the Andover area may owe its resistance to enhanced detoxication by a P450-based monooxygenase. 11.7 Summary Warfarin and the second-generation superwarfarins are anticoagulant rodenticides that have a structural resemblance to dicoumarol and vitamin K. They act as vitamin © 2001 C. H. Walker The anticoagulant rodenticides 213 K antagonists, thereby retarding or stopping the carboxylation of clotting proteins in the hepatic endoplasmic reticulum. The build-up of non-functional, undercarboxylated clotting proteins in the blood leads eventually to death by haemorrhaging. Brodifacoum, difenacoum, flocoumafen and other superwarfarins bind strongly to proteins of the hepatic endoplasmic reticulum and consequently have long half-lives in vertebrates, often exceeding 100 days. Thus, they present a hazard to predators and scavengers which feed upon rodents that have been exposed to them. A number of species of predatory and scavenging birds have died as a consequence of secondary poisoning by superwarfarins in field incidents, and questions have been asked about the long-term risks associated with expanding use of these compounds. 11.8 Further reading There is a shortage of appropriate texts on the ARs. Buckle and Smith (1994) describe the use of ARs in rodent control. Thijssen (1995) gives a concise account of mode of action and resistance mechanisms. For effects on non-target species, reference should be made to the individual citations given in the foregoing text. © 2001 C. H. Walker . strains, another from the Andover area may owe its resistance to enhanced detoxication by a P450-based monooxygenase. 11. 7 Summary Warfarin and the second-generation superwarfarins are anticoagulant. Major organic pollutants complex molecules of brodifacoum and flocoumafen. The chemical properties of some anticoagulant rodenticides are given in Table 11. 1. All the compounds listed in Table 11. 1. that can degrade them. Such long-term storage brings the risk of long-term toxic effects in individuals and in food chains. 11. 4 Environmental fate of anticoagulant rodenticides Warfarin and related