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Author(s): Clive A. Marks, Lee Allen & Heli Lindeberg 
Title: Non-Lethal Dose-Response Models Replace Lethal Bioassays for Predicting the 
Hazard of Para-Aminopropiophenone to Australian Wildlife 
Year: 2023 
Version: Published version 
Copyright:   The Author(s) 2023 
Rights: CC BY 4.0 
Rights url: http://creativecommons.org/licenses/by/4.0/ 
 
Please cite the original version: 
Marks, C.A.; Allen, L.; Lindeberg, H. Non-Lethal Dose-Response Models Replace Lethal Bioassays 
for Predicting the Hazard of Para-Aminopropiophenone to Australian Wildlife. Animals 2023, 13, 
472. https://doi.org/10.3390/ani13030472 
 
Citation: Marks, C.A.; Allen, L.;
Lindeberg, H. Non-Lethal
Dose-Response Models Replace Lethal
Bioassays for Predicting the Hazard of
Para-Aminopropiophenone to
Australian Wildlife. Animals 2023, 13,
472. https://doi.org/10.3390/
ani13030472
Academic Editor: Marc Girondot
Received: 28 November 2022
Revised: 23 January 2023
Accepted: 23 January 2023
Published: 29 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
animals
Article
Non-Lethal Dose-Response Models Replace Lethal Bioassays
for Predicting the Hazard of Para-Aminopropiophenone to
Australian Wildlife
Clive A. Marks 1,2,*, Lee Allen 3 and Heli Lindeberg 4
1 Nocturnal Wildlife Research Pty Ltd., P.O. Box 2126, Melbourne, VIC 3145, Australia
2 Manaaki Whenua Landcare Research, Lincoln P.O. Box 69040, New Zealand
3 Queensland Department of Agriculture Fisheries and Forestry, Toowoomba, QLD 4350, Australia
4 Natural Resources Institute Finland (Luke), Production Systems, Halolantie 31 A, FI-71750 Maaninka, Finland
* Correspondence: cliveamarks@NocturnalWR.com.au
Simple Summary: Para-aminopropiophenone (PAPP) is registered as bait poison for the humane
control of red foxes and wild dogs in Australia. To classify the relative hazard of poisons, regulatory
bodies have historically demanded LD50 values (the dose lethal to 50% of an animal population) to
define the comparative sensitivity of pest and wildlife species. Instead, we developed a replacement
assay that used non-lethal dose-response methods to assess the sensitivity of 12 wildlife species
and laboratory rats to PAPP that did not require death to be used as an experimental outcome. By
establishing the relationship between non-lethal doses of PAPP and the formation of methaemoglobin
(MetHb), we found that we could accurately predict doses likely to be lethal. Our estimates very
closely approximated existing LD50 values determined for PAPP. We argue that laboratory-based
lethal-dose bioassays are unsuited to assessing the comparative hazard of toxicants to wildlife species.
In contrast, non-lethal assays that use biologically relevant measures can provide much more robust
and meaningful indications of relative hazard even in species of high conservation value, where
lethal experimentation can rarely be justified.
Abstract: Para-aminopropiophenone (PAPP) is a potent methaemoglobin (MetHb) forming agent
used for the lethal control of exotic carnivores and mustelids. To assess the sensitivity of Australian
wildlife to PAPP we developed an in vivo assay that did not use death as an endpoint. Sub-lethal dose-
response data were modelled to predict PAPP doses required to achieve an endpoint set at 80% MetHb
(MetHb80). The comparative sensitivity of non-target mammals referenced to this endpoint was found
to be highly variable, with southern brown bandicoots (Isoodon obesulus) the most sensitive species
(MetHb80 = 6.3 mg kg−1) and bush rats (Rattus fuscipes) the most tolerant (MetHb80 = 1035 mg kg−1).
Published LD50 estimates were highly correlated with PAPP doses modelled to achieve the MetHb80
endpoint (r2 = 0.99, p < 0.001). Most dose-response data for native mammals were collected in the field
or in semi-natural enclosures, permitting PAPP and placebo dosed animals to be fitted with tracking
transmitters and transponders and released at their point of capture. A protracted morbidity and
mortality was observed only in Australian ravens (Corvus coronoides). The combination of sub-lethal
dose-response assay and survival data collected in the field provided more relevant information
about the actual hazard of pest control agents to non-target wildlife species than laboratory-based
lethal-dose bioassays. We discuss the need to replace lethal-dose data with biologically meaningful
insights able to define a continuum of toxicological hazards that better serve the needs of conservation
and veterinary scientists and wildlife managers.
Keywords: para-aminopropiophenone; PAPP; methaemoglobin; MetHb; animal welfare; lethal-dose
bioassay; LD50; non-lethal assay; replacement; 3Rs
Animals 2023, 13, 472. https://doi.org/10.3390/ani13030472 https://www.mdpi.com/journal/animals
Animals 2023, 13, 472 2 of 17
1. Introduction
Since European settlement of Australia, an extensive fauna of exotic and feral species
became established throughout the continent [1]. Poison baiting is now used on a landscape-
scale to mitigate the impact of introduced pests within agricultural and natural environ-
ments [2,3]. Selective lethal agents are sought to mitigate the impacts of introduced preda-
tors such as feral cats (Felis catus) and red foxes (Vulpes vulpes) [4] whilst producing minimal
adverse impacts upon non-target native wildlife.
Lethal dose bioassays have remained the principal method used to assess the compar-
ative sensitivity of target and non-target species to prospective pest control agents [5–11].
The median lethal dose (LD50), based upon the method first described by Trevan (1927),
provides the principal comparative metric of species sensitivity. However, a requirement
for lethal outcomes means that high conservation value species will rarely be used given
their genetic value [12] and critical role in species recovery programs [13]. This constrains
the scope of toxicological risk assessments and fails to define non-target risk in those
species of greatest concern. While variants of the classical lethal bioassay methods can be
refined to substantially reduce the scale of animal use, many are not accepted by regulatory
agencies [14], frequently due to their poor precision [15]. Accordingly, viable non-lethal
assays able to generate data comparable to lethal dose bioassays are needed in order to
better define the hazard of prospective pest control agents especially in species with the
most precarious conservation status.
In 1981 the Organization for Economic Cooperation and Development (OECD) in-
corporated the use of the LD50 assay in its guidelines for the testing and regulation of
chemicals [16], although death as an experimental endpoint was gradually prohibited or
restricted in many government jurisdictions [17]. More recently the European Union and
OECD guidelines have emphasized the use of alternatives to lethal-dose bioassays [18].
As a consequence, many toxicological risk assessment methods have shifted towards the
adoption of predictive models based upon underlying mechanisms of action and biochem-
ical pathways involved in different gradients of exposure [19]. Pathway-based models
shown to be fit for purpose can be used in tandem with sub-lethal dose-response studies [20]
that seek to measure perturbations in relevant physiological or toxicological pathways as
alternative endpoints [21]. We sought non-lethal in vivo approaches to assess the sensitivity
of a range of Australian wildlife species to para-aminopropiophenone (PAPP), an agent
that showed potential as a new predator control agent for wild dogs and exotic foxes.
Orally ingested PAPP is rapidly N-hydroxylated to the active species, para-hydroxy-
laminopropiophenone (PHAPP) [22] which is a potent methaemoglobin (MetHb) forming
agent [23]. Methaemoglobin, arising through the oxidation of haemoglobin in the ferrous
valance (HbFe2+) to the ferric state (HbFe3+), is unable to transport oxygen (O2) as oxy-
haemoglobin (HbO), or carbon dioxide (CO2) as carbaminohaemoglobin. Because O2 is
required as the final electron acceptor in the electron transport chain to drive aerobic ATP
synthase [24], hypoxia arising from severe methaemoglobinaemia has profound conse-
quence for brain function, as the brain is reliant upon aerobic metabolism to maintain
sufficient ATP turnover to sustain consciousness and vital cell functions [25]. Lethal-dose
bioassays conducted in the early 1940s [26] implied that carnivores and mustelids had a
far higher acute sensitivity to PAPP in comparison to most other animal groups [27]. Oral
doses of PAPP given to red foxes under laboratory and pen conditions resulted in a toxico-
sis that appeared far more humane than that achieved using fluoroacetic acid (1080) [28],
which remains the principle pest control agent used for carnivore control in Australia [29].
However, as no PAPP toxicity data for Australian wildlife species were available, we devel-
oped a sub-lethal assay to establish the relationship between oral PAPP doses and MetHb
formation. This permitted the relative sensitivity of a wide range of high conservation
value species to be predicted, removing the need to use death as an experimental endpoint.
Animals 2023, 13, 472 3 of 17
2. Materials and Methods
2.1. Selection of Target and Non-Target Populations
The procedures and methods used to obtain, capture and handle wildlife species are
described in the supplementary information in more detail (Supplementary). Exotic red
foxes and wild dog/dingo hybrids (Canis lupus familiaris) were the principal Australian
target species and used to validate our alternative experimental endpoints. Non-target
mammals and birds present in south-eastern Australia were prioritized if their diets and
foraging strategies put them at risk of consuming meat baits used for the routine control
of foxes and wild dogs [30]. This included several carnivorous and omnivorous species
listed under the Australian Environment Protection and Biodiversity Conservation Act 1999 in-
cluding the spotted-tailed quoll (Dasyurus maculatus), eastern quoll (Dasyurus viverrinus),
Tasmanian devil (Sarcophilus harrisii) and southern brown-bandicoot (Isoodon obesulus).
Laboratory rats (Rattus norvegicus) were obtained from scientific suppliers. Five other
indigenous marsupials, 2 endemic rodents and 2 bird species were also seen as priority
species (see Table 1).
2.2. Dosage Methods
Prior to PAPP or placebo dosing animals were lightly sedated with 4 mg kg−1 intra-
muscular (IM) Zoletil (Virbac: Melbourne, Australia). Body weight for animals >5 kg
was determined using a digital balance accurate to ±1.0 g while smaller animals were
weighed on a digital laboratory balance accurate to ±0.001 g. A stock solution of PAPP
dissolved in dimethyl sulfoxide (DMSO) or polyethylene glycol (PEG) permitted maximum
PAPP dose concentrations of 375 mg mL−1 or 50 mg mL−1, respectively. Oral doses were
delivered when the anaesthetic was abating and the gag reflex was present. Doses of
PAPP (mg kg−1) were increased by multiples of 1.5 aiming to produce maximum MetHb
concentrations between 70 and 75% in wildlife species. Initially, dogs and foxes were dosed
with a wide range of PAPP concentrations using both the DMSO and PEG stock solution in
separate trials in order to assess the utility of each formulation and to validate an alternative
endpoint for latter trials with wildlife. Dosages using the PEG carrier were achieved by
gavage using a dog urine catheter. PAPP + DMSO formulations were delivered to the
back of the throat using an ejector system [28]. In small mammals, dosing was achieved
by slowly introducing the dose to the back of the tongue using a pipette. Animals were
held upright for a further two minutes and their necks massaged to ensure the dose had
been swallowed. Formulations of PAPP + DMSO were used exclusively in assessments in
non-target wildlife where doses of DMSO alone were used as a placebo.
2.3. Blood Sampling and MetHb Determination
Methaemoglobin, as a percentage of total haemoglobin, was determined using a CO-
oximeter (OSM 3 Hemoximeter: Radiometer, Copenhagen, Denmark). Quolls, Tasmanian
devils, potoroos, bandicoots and pademelons were sequentially blood sampled with a
heparinised capillary tube after shaving the hair on the ear to expose a vein that was then
swabbed with 70% ethanol before venepuncture with a 23G needle, taking <0.1 mL of blood
on each occasion. For larger animals such as foxes and dogs, a dose of 10–15 mg kg−1 IM
Zoletil was administered, before blood was taken from the cephalic vein or jugular using a
23G needle and heparinised syringe. Blood was collected from small mammals (rats and
dunnarts) using the lateral saphenous vein collection method [31]. Birds were sampled
from the jugular or brachial vein. In animals dosed with PAPP, blood samples were taken
every 20 min until an asymptote of MetHb was detected as the peak MetHb concentration.
Thereafter, a sample was taken at 10 min intervals to confirm a decline in MetHb by two
sequential readings before the protocol ceased. At the end of the trials, euthanasia of foxes
and wild dogs was achieved via an intra-cardiac injection of 10 mL sodium pentobarbital
(Lethabarb: Virbac, Sydney, Australia or Mebunat Vet 60: Orion Pharma, Finland) while
the animal was under deep anaesthesia induced by an additional IM dose of 15 mg kg−1
of Zoletil.
Animals 2023, 13, 472 4 of 17
Table 1. Oral PAPP dose in mg kg−1 formulated with DMSO estimated to produce 50% (MetHb50) and 80% (MetHb80) MetHb based upon fitted models with upper
and lower prediction estimates (p < 0.05).
Common Name Specific Name Model * n m r2 F p MetHb50 MetHb80
Dose p < 0.05 Dose p < 0.05
mg kg−1 mg kg−1 mg kg−1 mg kg−1
Australian raven Corvus coronoides 1 7 1 0.97 220 <0.0001 33.82 35.1, 33.2 - -
Brushtail possum Trichosurusvulpecula 2 9 0 0.94 101 <0.0001 574.8 604, 590.1 614.9 760.0, 604.0
Bush rat Rattus fuscipes 3 7 0 0.99 433 <0.0001 293 205.5, 144.7 1035 1217.0, 634.0
Eastern quoll Dasyurusviverrinus 3 9 0 0.91 110 <0.0001 78.3 170.5, 25.8 277 504.5, 107.7
Fat-tailed dunnart Sminthopsiscrassicaudata 3 6 1 0.99 299 <0.0001 40 61.9, 22.7 101 140.5, 67.9
Laboratory rat Rattus norvegicus 3 8 0 0.97 234 <0.0001 46.5 76.5, 25.5 182.7 257.0, 106.0
Long-nosed
potoroo Potorous tridactylus 2 6 0 0.95 85.1 <0.001 87.0 121.8, 54.0 164.3 201.9, 127.4
Red fox Vulpes vulpes 4 9 3 0.99 371 <0.0001 4.0 5.3, 3.2 15.4 38.0, 11.48
Silver gull Chroicocephalusnovaehollandiae 4 7 0 0.97 67 <0.001 >1000 - >1000 -
Brown-bandicoot Isoodon obesulus 3 5 1 1 397 <0.001 2.96 3.3, 2.6 6.3 6.9, 5.8
Spotted-tailed
quoll
Dasyurus
maculatus 2 9 0 0.97 346 <0.0001 12.9 18.9, 9.7 27.1 29.3, 20.1
Swamp rat Rattus lutreolus 2 8 0 0.96 190 <0.0001 14.5 18.8, 10.4 26.1 29.9, 22.4
Tasmanian devil Sarcophilus harrisii 4 8 0 0.99 960 <0.0001 27.2 32.4, 22.9 120.3 156.0, 71.5
Tasmanian
pademelon
Thylogale
billardierii 3 7 0 0.91 80 <0.0003 88.5 191.0, 127.9 334 396.0, 103.0
Wild dog Canis lupusfamiliaris 4 9 2 0.99 268 <0.0001 1.94 2.7, 1.5 8.5 19.8, 4.4
Total 114
* (1) Iny = a + be−x , (2) Iny = a + bx3, (3) y = axb, (4) y = a + b−a
1+( cx )
.
Animals 2023, 13, 472 5 of 17
2.4. Recovery, Release and Monitoring
While anaesthetized, mammals were implanted with a 1.4 mm× 8 mm RFDI microchip
(Trovan ID-100: Microchips Australia, Keysborough, Australia) using the appropriate
cannula. Those animals >100 g in body weight were fitted with a single stage 173 MHz
transmitter (Supplementary). At the end of the procedure they were placed within a
darkened and insulated recovery box or covered cage filled with insulating material for
thermal support. Ambulatory animals were provided with water ad libitum and observed
until fully coordinated and then released at their point of capture. Over the following
4 weeks those exposed to oral doses of PAPP or a placebo dose of DMSO were monitored
regularly to determine their survival using a 3-element Yagi antenna and 173 MHz tracking
receiver (Titley Electronics: Brendale, Queensland, Australia). Animals that died during
the dose-response experiment were excluded from the survival study. Similarly, if animals
were euthanised due to injuries sustained during captivity they were also excluded. If
the fate of the animal was unknown (if it could not be located) no assumption was made
concerning its survival and it too was excluded.
2.5. Statistical Analysis
An experimental endpoint (85% MetHb) had been validated after preliminary trials in
red foxes [28] and feral cats (Marks, unpublished data) [32], where the Animal Ethics Com-
mittee had permitted death as an endpoint in order to validate the utility of non-lethal end-
points using MetHb thresholds. Published fox data for PAPP + DMSO (Marks et al. 2004)
were re-analysed in this paper alongside new data developed for PAPP + PEG formulations.
To ensure the survival of all wildlife species used, a maximum response of 75% MetHb was
sought, from which the alternative endpoint of 80% MetHb (MetHb80) was predicted. Peak
MetHb concentrations resulting from doses of PAPP were fitted to a statistical model that
best described the dose-response relationship with the highest level of significance. Linear
and non-linear dose-response models were tested for their strength of fit indicated by R2
values, significance and the Akaike Information Criteria [33]. Models were chosen with the
assistance of Statistical Package for the Social Sciences (SPSS) (Tablecurve: SPSS Version 14,
Chicago, USA). Fitted curves were calculated with 95% confidence and prediction inter-
vals [34] and used to estimate doses of PAPP mg kg−1 that would result in 50% (MetHb50)
and 80% MetHb (MetHb80). Validation of the predictive value of the models was sought
by regressing the MetHb50 and MetHb80 predictions against PAPP LD50 values for the
laboratory rat [35], feral cat (Marks, unpublished data) [36], fox [26], dog [37] and brushtail
possum [38]. A Fisher exact test was used to test for significant differences in the survival
of all animals in groups that received a PAPP dose or a placebo.
3. Results
3.1. Dose-Response Models for Red Foxes and Wild Dogs
Logistic dose-response models for PAPP + DMSO formulations were a strong fit
for fox (R2 = 0.99) and wild dog (R2 = 0.99) MetHb responses, predicting mean PAPP
doses of 15.4 mg kg−1 and 8.4 mg kg−1, respectively, to attain the MetHb80 endpoint
(Figures 1 and 2). Using PAPP + PEG formulations, marginally lower mean PAPP doses in
foxes (13.3 mg kg−1, R2 = 0.98) and wild dogs (6.85 mg kg−1, R2 = 0.98) were predicted to
attain the MetHb80 endpoint (Figures 3 and 4).
Animals 2023, 13, 472 6 of 17
Animals 2023, 13, x FOR PEER REVIEW 5 of 19 
 
 
Figure 1. (a) Methaemoglobin (MetHb %) response relative to oral dose of PAPP + DMSO in foxes 
and (b) the fitted logistics dose-response model for peak MetHb % with 95% confidence (dashed 
line) and prediction interval (solid line) and whether death (d) resulted. 
Figure 1. (a) Methaemoglobin (MetHb %) response relative to oral dose of PAPP + DMSO in foxes
and (b) the fitted logistics dose-response model for peak MetHb % with 95% confidence (dashed line)
and prediction interval (solid line) and whether death (d) resulted.
Animals 2023, 13, 472 7 of 17
Animals 2023, 13, x FOR PEER REVIEW 6 of 19 
 
 
Figure 2. (a) Methaemoglobin (MetHb %) response relative to oral dose of PAPP in DMSO in wild 
dogs and (b) the fitted logistics dose-response model for peak MetHb % with 95% confidence 
(dashed line) and prediction interval (solid line) and whether death (d) resulted. 
Figure 2. (a) Methaemoglobin (MetHb %) response relative to oral dose of PAPP in DMSO in wild
dogs and (b) the fitted logistics dose-response model for peak MetHb % with 95% confidence (dashed
line) and prediction interval (solid line) and whether death (d) resulted.
Animals 2023, 13, 472 8 of 17
Animals 2023, 13, x FOR PEER REVIEW 7 of 19 
 
 
Figure 3. (a) Methaemoglobin (MetHb %) response relative to oral dose of PAPP in PEG in foxes 
and (b) the fitted logistics dose-response model for peak MetHb % with 95% confidence (dashed 
line) and prediction interval (solid line) and when animals were euthanised (e) when they passed 
the 80% MetHb threshold. 
Figure 3. (a) Methaemoglobin (MetHb %) response relative to oral dose of PAPP in PEG in foxes and
(b) the fitted logistics dose-response model for peak MetHb % with 95% confidence (dashed line)
and prediction interval (solid line) and when animals were euthanised (e) when they passed the 80%
MetHb threshold.
Animals 2023, 13, 472 9 of 17Animals 2023, 13, x FOR PEER REVIEW 8 of 19  
 
Figure 4. (a) Methaemoglobin (MetHb %) response relative to oral dose of PAPP in PEG in wild 
dogs and (b) the fitted logistics dose-response model for peak MetHb % with 95% confidence 
(dashed line) and prediction interval (solid line) and whether death (d) resulted. 
All foxes died if they exceeded the MetHb80 threshold in the PAPP + DMSO group 
(peak MetHb % = 83.2, 85.7 and 86.5). Overall, 4 of 6 wild dogs (peak MetHb = 85.4, 85.5, 
85.6 and 86.7%) died in the PAPP + PEG group, with two others vomiting after oral dosing 
and surviving (peak MetHb = 83.1 and 84.2%), causing data from these individuals to be 
discarded. Because DMSO + PAPP was not associated with vomiting in foxes or dogs, and 
was capable of higher PAPP concentrations and low dose volumes, it was used consist-
ently thereafter in all dose-response trials with wildlife species. 
3.2. Comparative Species Sensitivity to PAPP 
Four different models were fitted to non-target data to take account of differences in 
the dose-response relationship that varied between being highly linear, sigmoidal or ex-
ponential in nature. A single fat-tailed dunnart and southern brown-bandicoot received 
PAPP doses that inadvertently caused MetHb levels to rise to 82% and 82.2%, respectively, 
resulting in their death. These were the only two unintentional deaths arising from the 
acute dose-response experiments in wildlife species. The southern brown bandicoot 
(MetHb80 = 6.3 mg kg−1) was the only mammal found to be relatively more susceptible to 
PAPP than wild dogs or foxes. Bush rats (MetHb80 = 1035 mg kg−1) were the most tolerant 
mammals, yet large variability in tolerance between rodents was evident, with swamp 
Figure 4. (a) Methaemoglobin ( et b %) response relative to oral dose of PAPP in PEG in wild dogs
and (b) the fitt d logistics dose-response model for peak MetHb % with 95% confidence (dashed line)
and prediction int rval (solid line) and whether death (d) resulted.
All foxes died if they exceeded the MetHb80 threshold in the PAPP + DMSO group
(peak MetHb % = 83.2, 85.7 and 86.5). Overall, 4 of 6 wild dogs (peak MetHb = 85.4, 85.5,
85.6 and 86.7%) died in the PAPP + PEG group, with two others vomiting after oral dosing
and surviving (peak MetHb = 83.1 and 84.2%), causing data from these individuals to be
discarded. Because DMSO + PAPP was not associated with vomiting in foxes or dogs, and
was capable of higher PAPP concentr tions and low dose volumes, was used consistently
there fter in all dose-response trials with wildlife species.
3.2. Comparative Species Sensitivity to PAPP
Four different models were fitted to non-target data to take account of differences
in the dose-response relationship that varied between being highly linear, sigmoidal or
exponential in nature. A single fat-tailed dunnart and southern brown-bandicoot received
PAPP doses that in dvertently caused MetHb l vels to rise to 82% and 82.2%, respec-
tively, resulting in their death. These were the only two unintentional deaths arising f om
the acute dose-response experiments in wildlife species. The southern brown bandicoot
Animals 2023, 13, 472 10 of 17
(MetHb80 = 6.3 mg kg−1) was the only mammal found to be relatively more susceptible to
PAPP than wild dogs or foxes. Bush rats (MetHb80 = 1035 mg kg−1) were the most tolerant
mammals, yet large variability in tolerance between rodents was evident, with swamp rats
(MetHb80 = 26.1 mg kg−1) and laboratory rats (MetHb80 = 182.7 mg kg−1) substantially
more sensitive. Similar variation was seen within the dasyurids, with fat-tailed dunnarts
(MetHb80 = 101 mg kg−1), eastern quolls (MetHb80 = 277 mg kg−1) and Tasmanian devils
(MetHb80 = 120.3 mg kg−1) being comparatively more tolerant compared to spotted-tailed
quolls (MetHb80 = 27.1 mg kg−1). Blood sampling was not attempted in brown antechinus
in the field due to their small body mass (<20 g) obviating repeated blood sampling, yet
this dasyurid recovered from PAPP doses as high as 571 mg kg−1. Both the long-nosed
potoroo (MetHb80 = 164 mg kg−1) and pademelon (MetHb80 = 334 mg kg−1) responses
indicated a much greater tolerance in macropodids compared to foxes and wild dogs.
Silver gulls (MetHb80 > 1000 mg kg−1) appeared to be the most tolerant species overall,
as despite a maximum practical dose of 857 mg kg−1, the MetHb response did not exceed
7.9%, meaning that no precise estimate of the MetHb80 was obtained (Table 1).
Overall, both red foxes and wild dogs were found to be highly sensitive to PAPP
relative to most other non-target mammals assessed. Mean doses of PAPP to achieve the
MetHb80 endpoint in all other species were 34-fold of those required to attain the same end-
point in wild dogs (±21.5-fold, p < 0.05) and 20-fold higher for foxes (±11.9-fold, p < 0.05).
Bush rats required doses 122-fold and 67-fold that of wild dogs and foxes, respectively,
to attain the MetHb80 threshold. However, southern-brown bandicoots were outliers and
susceptible to doses 0.7-fold and 0.4-fold that of wild dogs and foxes (Table 2).
Table 2. Comparative species sensitivity referenced to the multiple of red fox and wild dog sensitivity
based on the dose of PAPP mg kg−1 required to cause 80% MetHb in each with upper and lower
confidence estimates (p < 0.05).
Common Name × Dog p < 0.05 × Fox p < 0.05
Australian raven 17.4 * 18.1, 17.1 * 8.5 8.8, 8.3
Brushtail possum 72.3 89.4, 71.1 39.9 49.4, 39.2
Bush rat 121.8 143.2, 74.6 67.2 79.0, 41.2
Eastern quoll 32.6 59.4, 12.7 18.0 32.8, 7.0
Fat-tailed dunnart 11.9 16.5, 8.0 6.6 9.1, 4.4
Laboratory rat 21.5 30.2, 12.5 11.9 16.7, 6.9
Long-nosed potoroo 19.3 23.8, 15.0 10.7 13.1, 8.3
Red fox 1.8 4.5, 1.4 - -
Silver gull >118 - >64.9 -
Southern brown-bandicoot 0.7 0.8, 0.7 0.4 0.4, 0.4
Spotted-tailed quoll 3.2 3.4, 2.4 1.8 1.9, 1.3
Swamp rat 3.1 3.5, 2.6 1.7 1.9, 1.5
Tasmanian devil 14.2 18.4, 8.4 7.8 10.1, 4.6
Tasmanian pademelon 39.3 46.6, 12.1 21.7 25.7, 6.7
Wild dog - - 0.6 1.3, 0.3
Mean 34.1 20.1
p < 0.05 21.5 11.9
* Based upon MetHb50 comparison.
3.3. Comparison with Published LD50 Data
Log MetHb80 estimates based upon dose response data obtained in wild dogs, foxes,
feral cats, brushtail possums and brown rats regressed strongly and significantly with
log LD50 data published for these five species (r2 = 0.99, p = 0.001). Regression of LD50
values with the doses of PAPP modelled to cause a 50% elevation in MetHb (MetHb50)
produced a weaker correlation (r2 = 0.93, p = 0.024) with 95% confidence intervals and
prediction boundaries indicating a less reliable predictive capacity for the MetHb50 estimate
(Figure 5a,b).
Animals 2023, 13, 472 11 of 17Animals 2023, 13, x FOR PEER REVIEW 12 of 19 
 
 
 
Figure 5. Comparison of log oral PAPP LD50 for (1) feral cats, (2) dogs, (3) foxes, (4) laboratory rats 
and (5) brushtail possums against (a) predicted log 80% MetHb level (Log MetHb80) and (b) log 50% 
MetHb level (Log MetHb50) with 95% confidence. 
3.4 10-Day Survival Assessment 
Beyond 10 days after the release of instrumented animals the detachment of trans-
mitters and/or their failure due to damage saw a rapid decline in the sample size, making 
14-day and 30-day survival monitoring impractical. Radio-tracking and recapture data 
revealed that 10 days subsequent to release, 77 of 79 PAPP-dosed and 58 of 59 placebo-
dosed animals were known to be alive. Overall, survival data for mammals indicated that 
there was no significant difference in mortality between the groups (p = 0.64). However, 
an Australian raven dosed with 380.7 mg kg−1 PAPP died within 24 h after a peak of 67.3% 
MetHb had been detected in the dose-response trials prior to release. Another raven that 
received 253.8 mg kg−1 (peak MetHb = 68.9%) was unable to fly for 24 h, appearing to 
recover but died within the 10-day monitoring period. All ravens dosed below 169.2 mg 
kg−1 PAPP survived the 10-day monitoring period. At a dose between 33.42 and 75.2 mg 
kg−1 there appeared to be no noticeable morbidity other than a temporary reduction in 
physical activity within 2 h after PAPP dosing. Morbidity in ravens appeared to be dose 
dependent where mild cyanosis was associated with lethargy increasing in severity from 
75.2 mg kg−1 PAPP (Table 3). 
(a)
(b)
Figure 5. Comparison of log oral PAPP LD50 for (1) feral cats, (2) dogs, (3) foxes, (4) laboratory rats
(5) brushtail possums against (a) predicted log 80% MetHb level (Log MetHb80) and (b) log 50%
MetHb level (Log MetHb50) with 95% confidence.
3.4. 10-Day Survival Assessment
Beyond 10 days after the release of instrumented ani als the detachment of transmit-
ters and/or their failure due to dam ge saw a rapid declin in th s mple size, making
14-day and 30-day survival monitoring impractical. Radio-tracking and recapture data
revealed that 10 days subsequent to release, 77 of 79 PAPP-dosed and 58 of 59 placebo-
dosed animals were known to be alive. Overall, survival data for mammals indicated that
th re w s no significant differ nce i mortality betw en the groups (p = 0.64). However, an
Australian raven dos d wi h 380.7 mg kg−1 PAPP died within 24 h after a peak of 67.3%
MetHb had been detected in the dose-response trials prior to release. Another raven that
received 253.8 mg kg−1 (peak MetHb = 68.9%) was unable to fly for 24 h, appearing to
recover but died within the 10-day monitoring period. All ravens dosed below 169.2 mg
kg−1 PAPP surv ved the 10-day monitoring pe iod. At a dose b tween 33.42 and 75.2 mg
kg−1 there appeared to be no noticeable morbidity other than a temporary reduction in
physical activity within 2 h after PAPP dosing. Morbidity in ravens appeared to be dose
dependent where mild cyanosis was associated with lethargy increasing in severity from
75.2 mg kg−1 PAPP (Table 3).
Animals 2023, 13, 472 12 of 17
Table 3. Summary of fate of animals known to be alive (KTBA) for a 10-day period after release as
determined from recapture, direct observation within captive colony or radio-tracking data for all
animals dosed with PAPP dissolved in DSMO (PAPP) or those given a placebo consisting of DMSO
alone (placebo).
Species
PAPP Placebo
Monitored KTBA Died Monitored KTBA Died
Antechinus 5 5 0 - - -
Brushtail possum 10 10 0 6 6 0
Eastern quoll 9 9 0 9 9 0
Little Australian raven 6 4 2 6 6 0
Long-nosed potoroo 6 6 0 6 6 0
Tasmanian pademelon 8 8 0 8 8 0
Silver gull 5 5 0 7 6 1
Southern brown bandicoot 4 4 0 6 6 0
Spotted-tailed quoll 5 5 0 1 1 0
Swamp rat 10 10 0 5 5 0
Tasmanian devil 9 9 0 5 5 0
Total 77 75 2 59 58 1
4. Discussion
4.1. Non-Lethal Data to Model Lethal End-Points
Doses of PAPP predicted to achieve the MetHb80 endpoint correlated strongly with
known LD50 values generated by bioassay. In generating data in 12 non-target wildlife
species and the laboratory rat (n = 13), 2 of 96 individuals (2.1%) died as a direct result
of the dose-response assay procedures where the MetHb80 endpoint was unintentionally
exceeded. Given an improved understanding of mammalian dose-response relationships
after these trials, it is now possible to better anticipate lethal responses and more reliably
reduce their occurrence. Nonetheless, the low overall prevalence of mortality in wildlife
was highly favourable in comparison to that hypothetically required by three OECD-
approved LD50 methods modified to reduce animal use. Here, between 2 and 40 animals
would have been used in each comparison [14] that for 13 species would require between
26 and 520 individuals in order to rank the toxicity of PAPP into six different categories
based upon 10-fold dose increments [39]. At best, such a categorical classification provides
an extremely low resolution of comparative hazard for wildlife species, where in the case
of PAPP susceptibility most of the species we assessed would be grouped into only two
categories of sensitivity.
Death marks the very end of a continuum of pathophysiological changes caused by
a gradient of toxicant doses. Lethal dose bioassays measure only dichotomous outcomes
(death or survival) and not a spectrum of prepathological and pathological consequences
leading to death [40]. In contrast, pathway-based studies can be used to define thresholds of
severity or adverse effects [41,42] revealed by changes in continuous or discrete responses
related to dose. MetHb-forming compounds, where an increasing gradient of adverse
effects are directly related to dose, are well suited to pathway-based assessments.
In vertebrates, MetHb is normally present at low concentrations (<1%), often due
to autoxidation of haemoglobin in the presence of O2 [43]. Exposure to MetHb-forming
compounds [44] can rapidly increase the concentration of MetHb in blood, potentially
resulting in a dose-dependent hypoxia and inhibition of aerobic ATP production [45]
that cause a continuum of signs and symptoms associated with a worsening hypoxia. In
humans, 10% MetHb caused by PAPP administration produced measurable performance
deficits under dynamic loads [46], although concentrations <20% MetHb may otherwise
be asymptomatic. Beyond 30% MetHb, symptoms such as dizziness, fatigue, headache
and weakness [47] are frequently reported by patients and are well known indicators of
advancing hypoxia. Cardiac arrhythmias and other signs of pathological hypoxia become
evident at >55% MetHb [48]. Peak MetHb values >70% are generally associated with
Animals 2023, 13, 472 13 of 17
unconsciousness and carry a risk of death if sustained [49]. Studies in populations of red
foxes showed that MetHb fractions >80% were required before brain death resulted, while
below this threshold all foxes appeared to recover uneventfully (Marks et al. 2004) as was
the case with wild dogs and mammals in our current study. Concentrations of MetHb >87%
were reported to be reliably lethal in dogs [26,50], implying that the MetHb80 endpoint
lies below doses that may correspond with an LD99 estimate. Adopting a lower endpoint
(MetHb80) was appropriate for our comparative studies to assure that death was avoided in
wildlife species, in part because some data suggested that deaths in smaller mammals may
occur marginally closer to the MetHb80 threshold (at 82% MetHb in the only two cases that
resulted in a lethal endpoint). Hence, we attempted to constrain doses of PAPP in wildlife
species to maximum responses ranging between 70 and 75% MetHb that appeared to be
well tolerated, ideally permitting the projection of the MetHb80 value over a narrow range.
4.2. Assessing Survival in the Field
Australian ravens were the only species observed to experience a protracted morbidity
and delayed mortality. Although the aetiology was unqualified during this trial, more
recent insights into the impact of PAPP on avifauna was obtained in later studies that better
elucidated the nature of the protracted toxicosis in some birds [51].
Our protocol of releasing and remotely monitoring wildlife survival differed markedly
from conventional lethal-dose studies that are routinely conducted in controlled laboratory
environments. From its first description, the LD50 value was known to be dependent
upon the experimental conditions under which it was derived [52], where variations in
factors such as temperature, food and thermal support, that were known to influence
mortality or survival, could be controlled [53]. However, the maintenance of captive
wildlife species in controlled environments does not adequately reflect the heterogeneity
of stressors normally encounter within their habitats. No wildlife species have uniformly
available resources, as is often the case in the laboratory with the provision of food, water
and thermal support. Periods of negative energy balance are frequently encountered
by wildlife [54] that must also defend their body temperature by behavioural as well
as autonomic responses [55]. Therefore, captive trials cannot mimic the stressors and
environmental conditions encountered by wildlife after ingesting bait poisons in situ.
Instead, captivity introduces novel stressors that elicit a large array of hormonal and
physiological changes that may be highly species-specific [56], resulting in acute and
chronic stress affecting the health and welfare of species quite differently [57].
It has been observed that the captive animal is physiologically quite distinct from its
wild counterpart. Captivity alone may confound a wide range of experimental results,
making toxicity data taken from captive wildlife populations potentially unreliable indica-
tors of outcomes in free-ranging species [58]. This led us to collect dose-response data in
the field so that captured animals could be assessed using sub-lethal dose-response assays
and released as soon as practical, their longer-term survival monitored remotely under
environment conditions normally encountered within their own habitats.
4.3. Re-Thinking How the Hazard of Bait Toxicants Is Assessed in Non-Target Wildlife
Since first described in 1927 [52], lethal-dose bioassays became a cornerstone of efforts
to achieve standardization in drug and chemical risk evaluation [59]. Regulatory bodies
used LD50 assay data from laboratory animals to routinely classify the relative toxicity and
hazard of substances [14] as a proxy for the human lethal dose data [60]. While the use of
laboratory animals to model human sensitivity to toxicants met with variable success [61],
lethal dose bioassays were not specifically developed as methods to assess the comparative
hazard of toxicants to wildlife species or to predict their impact upon animal health and
biological fitness. In contrast, wildlife managers, conservation scientists and veterinary clin-
icians are concerned with qualifying and quantifying the impact of poisons upon the fitness
and conservation status of individuals and populations. Already well-known limitations in
the interpretation of lethal-dose bioassay data become far more problematic when they are
Animals 2023, 13, 472 14 of 17
used to investigate the comparative hazard of toxicants within complex communities of
wild vertebrates. Genotypic polymorphism is correlated with variation in enzyme systems
involved with the activation and biotransformation of xenobiotic compounds [62,63] where
the presence of intraspecific and interspecific variation in toxicokinetics and toxicodynamics
is unassailable [64]. Unlike selectively bred laboratory species [65] that are typically used
in lethal-dose bioassays, wildlife species are far more polymorphic, requiring toxicology
assay methods that are fit for purpose and able to routinely survey the relative hazard of
poisons to large numbers of wildlife species, enabling intra-specific variations in sensitivity
to be revealed. For example, the co-evolution of wildlife species with fluoroacetic acid
bearing plants in the west of Australia resulted in a large comparative difference in regional
tolerance to 1080 compared to conspecifics in the east of the country where such plants are
absent [66]. Moreover, the selection of greater resistance in rabbit (Oryctolagus cuniculus)
populations frequently exposed to 1080 was revealed by bioassays [67]. Each is an example
of when ongoing monitoring for changing sensitivity in both target and non-target species
is needed in order to determine the overall cost-benefit of poison baiting. Although cur-
rently unexplored, similar forms of heterogeneity in sensitivity to PAPP can be investigated
using non-lethal dose-response assays and survival studies conducted in the field.
Non-lethal and pathway-based assays that measure appropriately selected continuous
or discrete variables can also denote dose-dependent adverse effects thresholds, such
as the no observable effect level (NOEL) and the lowest observed adverse effect level
[LOAEL] [41,42]. The NOEL denotes doses that have no measurable impact on health and
welfare, while the LOAEL is associated with the lowest dose associated with the onset
of adverse effects. Each were employed to better define the relative hazard of PAPP to
New Zealand bird species, generated by a non-lethal dose-response assay [51]. Historically,
the emphasis on the statistical significance of death and survival as markers of the dose-
response relationship [68], is a legacy of a regulatory culture primarily concerned with the
classification of human risk, not one seeking to determine the comparative risk of toxicants
to wildlife populations. Determining only the probability of death from exposure is an
extremely course measure of hazard to wildlife species. Instead, methods most suited to
describing the hazard of bait toxicants to a wide range of wildlife species should seek to
establish thresholds of acceptable and unacceptable animal health and welfare.
5. Conclusions
Australian mammals were found to vary greatly in their comparative sensitivity
to PAPP (MetHb80 = 6.3–1035 mg kg−1). Modelling dose-dependent MetHb responses
permitted MetHb80 endpoints to be predicted, that were strongly correlated with known
LD50 values for five species. Survival data were collected in the field, avoiding the need
for captivity in a laboratory setting where animals would be subjected to a range of novel
stressors. Survival data collected over a 10-day period allowed protracted effects upon
Australian ravens to be identified. Overall, sub-lethal dose-response methods were a viable
alternative to lethal-dose bioassays for establishing the comparative hazard of PAPP to
Australian wildlife.
Lethal dose bioassays measure only dichotomous data—death and survival. In con-
trast, assays that monitor continuous variables linked to dose-dependent pathophysiolog-
ical changes may be correlated with changing animal welfare states. Non-lethal assays
potentially allow thresholds to be set to demarcate acceptable and unacceptable welfare
impacts associated with perturbations in pathophysiological markers at levels well below
those that cause mortality. Non-lethal assays ensure that sensitivity data can be collected in
a wide range of species, including high conservation value species, where lethal experi-
mentation can rarely be justified.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ani13030472/s1.
Animals 2023, 13, 472 15 of 17
Author Contributions: Conceptualization, C.A.M.; methodology, C.A.M. and H.L.; formal analysis,
C.A.M.; investigation, C.A.M., H.L. and L.A.; resources, C.A.M., H.L. and L.A.; data curation, C.A.M.;
writing—original draft preparation, C.A.M.; writing—review and editing, C.A.M., H.L. and L.A.;
visualization, C.A.M.; supervision, C.A.M., H.L. and L.A.; project administration, C.A.M. and H.L.;
funding acquisition, C.A.M. All authors have read and agreed to the published version of the
manuscript.
Funding: This work built upon preliminary IP funded and developed by Nocturnal Wildlife Research
Pty Ltd. All research described in this paper was funded by Australian Wool Innovation Pty Ltd. via
the Invasive Animals Cooperative Research Centre. Further analysis and publication was supported
by the Department of Conservation (New Zealand) Predator Free 2050 Tools to Market Programme
under contract (reference number: 3054988; contract report: LC4147) and a research programme grant
(2223-28-009 A) provided by the New Zealand Ministry of Business, Innovation and Employment
as part of a research contract with Manaaki Whenua Landcare Research. The APC was funded by
Manaaki Whenua Landcare Research.
Institutional Review Board Statement: All research procedures were carried out in accordance with
the 2004 Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were
approved by the Department of Primary Industries (Metropolitan) AEC as protocols 2648, 2649
and 2650. Procedures in foxes conducted in Finland were approved and overseen by the Kuopio
University Animal Ethics Committee as protocols 2003–84 and 2004–1. Scientific procedures were
conducted under Victorian field license SPFL129, Tasmanian wildlife permit TFA 05121 and New
South Wales National Parks and Wildlife Service permit 11743.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is available for fair use to support further scientific research upon
request to the corresponding author.
Acknowledgments: We thank Bob Seamark for his encouragement and for providing the headspace
from which this project emerged. Frank Gigliotti and Frank Busana of the Department of Primary
Industries (Victoria) made a significant contribution towards the technical development and labora-
tory management of the project. Anitta Helin and Elina Reinikainen provided an exceptional level of
technical support throughout the work conducted at the University of Kuopio (Finland). Particular
thanks are given to Paul Meek, Mick Statham, Al Glen, Terry Coates, Randy Rose, Peter Frappell,
Michael Johnston and Nick Mooney for their expertise in helping to obtain animals at various field
sites. The authors wish to thank Elaine Murphy and Brian Hopkins for their ongoing interest and
support of this work.
Conflicts of Interest: The authors declare no conflict of interest.
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