Murdoch University
Animal Ethics Committee
STANDARD OPERATING PROCEDURE
SOP (0100 - 03): Wildlife (Marine) Remote biopsy sampling of cetaceans
0100 -03
Wildlife (Marine)
Remote biopsy sampling of cetaceans
Last approved or reviewed by AEC December 2013
1. Purpose.
To obtain small tissue samples (skin/blubber) from free-ranging cetaceans using a
remote biopsy system. Once obtained, samples are stored and later used for biological
and ecological studies including, but not limited to: species identification,
phylogeography, gene flow and population structure assessments (genetic analyses);
food web and dietary studies (stable isotope and fatty acid signature analyses); and
environmental and anthropogenic pollutant studies (contaminant analyses +/- biomarker
analyses).
2. Equipment.
 Specific
The PAXARMS biopsy system (0.22 calibre rifle with Pro-Point red-dot sight) uses blank
charges to project a lightweight, polycarbonate biopsy dart at wild cetaceans. This
particular biopsy technique (PAXARMS: http://www.paxarms.co.nz/) is now wellestablished as an international standard (e.g. Krützen et. al., 2002; attached).
 Description of equipment
The PAXARMS biopsy system uses a modified 0.22 calibre rifle with a detachable barrel, a
pressure (distance-) adjustable valve system (Fig. 1a) and a biopsy dart (Fig. 1b).
Figure 1. PAXARMS biopsy system. (a) Assembled 0.22 calibre rifle with Pro-Point reddot sight. (b) Biopsy dart and biopsy tip.
1a)
1b)
Murdoch University Standard Operating Procedure 0100 – 03 Wildlife (Marine) – Remote biopsy sampling of
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PAXARMS biopsy darts have a hollow polycarbonate body and a steel biopsy tip that is
bevelled inwards (Fig. 2a). For sample retention, three evenly distributed small triangular
shaped barbs are located 2 mm from the leading edge of the tip. The tip is welded into a
metal flange that acts as a stop and screws into the body of the dart (Fig. 2b). The body
is a thin-walled moulded tube made out of bright red polycarbonate. An internal wall just
below the thread for the metal flange prevents flooding of the body (Fig. 2a). An internal
thread at the tail end of the body is used to screw in a small piece of polycarbonate that
acts as a safety partition in case the tail flies off on impact (Fig. 2a). The polycarbonate
tail piece is also screwed in the tail end on top of the partition, with an o-ring to form a
watertight seal. A barb-resetting tool (Fig. 2c) is used to reset the barbs after each
successful hit. Its tip fits the inner diameter of the cutting tip and has an internal bevel at
one end and a handle on the other. The tool is inserted into the unscrewed steel tip from
the backside and pushed forward to reset the barbs.
Figure 2. Biopsy dart (extracted from Krützen, 2002). (A) Assembled biopsy dart
showing inserts of biopsy tip, safety partition, and plastic tail. (B) Detailed drawing of
biopsy tip. (C) Barb resetting tool.
0. D. = outer diameter.
The dart is positively buoyant and floats in an upright position. The total weight of an
assembled dart is approximately 21.5 g. Darts are fired using blank charges (PAXARMS).
A valve fitted to the chamber on one side of the rifle acts as a control for the pressure
propelling the dart. To facilitate aiming, the rifle is fitted with a Pro-Point red-dot laser
sight (Tasco). This makes aiming faster and more reliable compared to open sights.

Safety operating procedure for PAXARMS rifle:
1. Prior to departure on marine vessel, biopsy heads are boiled for 10-20 mins and
flamed in ethanol.
2. Always wear clean surgical gloves when handling the dart and biopsy tip, for the
dual purpose of minimising infection of both the dolphin and the operator, and to
minimise cross-contamination by DNA/RNA from either the operator or other
dolphin samples. Change gloves between animals.
3. Using forceps, dunk heads in 95% Ethanol and then flame. Make sure to hold the
biopsy head down so no ethanol can run down the forceps. Repeat once and let
biopsy heads cool down.
4. Assemble biopsy darts according to manufacturer's instructions. Make sure that
biopsy heads and tails are not screwed in too tightly. Use only dart bodies without
Murdoch University Standard Operating Procedure 0100 – 03 Wildlife (Marine) – Remote biopsy sampling of
cetaceans Version 3 July 2009
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cracks.
5. Store assembled darts in clean, watertight container.
6. (Actual rifle use starts here) Insert dart into muzzle, pushing gently.
7. Attach muzzle to PAXARMS rifle.
8. Load blank cartridges into magazine, put magazine into rifle.
9. Put gun into receptacle in boat.
10. Use biopsy gun only at the bow of the boat, make sure crew stands behind person
operating the gun.
11. Prior to darting, cock the gun, and then make sure safety catch is on.
12. Always communicate with your crew to make sure that everyone on board knows
when a shot is imminent. If cetacean group is deemed dart-able, release safety
and attempt to dart cetaceans on lateral side below dorsal fin (see Krützen et al.
2002). Only take a shot when it is safe to do so. Make sure you follow the darting
protocol as set out in Krützen et al. 2002.
13. After the dart has been fired, put gun in safe place, retrieve biopsy dart.
14. Process sample.
15. If more than one animal is darted, start over at step 5.
16. Between darting, make sure that no cartridge is in the rifle.
17. At the end of the day, store biopsy gun in Pelican case, making sure no blank
cartridges are left in the gun. Store ammunition and cartridges separately.
18. Clean gun according to manufacturer's instruction at the end of the day.
3. Description of procedure.

General description
Following sighting of a cetacean of interest, and upon assessment that sampling is
appropriate (behaviour of cetaceans, conditions at sea); the individual is approached and
darted with the PAXARMS biopsy dart from the assembled PAXARMS biopsy system. The
biopsy dart is retrieved and the sample is extracted and stored in a small vial containing
either ethanol or salt-saturated 20% DMSO. The cetacean’s behavioural response is
monitored and notes are recorded on data sheets. Numerous studies report on responses
of cetaceans to biopsy darting and the exemplar is attached.

Method
Cetaceans are approached to distances of 5-25m on small (generally <7m) research
vessels for attempted biopsy sampling. Slow speeds are maintained so as not to
disturb/startle cetaceans. The animals are always aware of our presence (motor noise).
An assessment by professional and experienced personnel is made as to whether
sampling is appropriate and likely to be successful with minimal disturbance to animals.
The individual’s behaviour is observed and recorded, together with information on group
composition, age class and other information of interest. Based on our location,
environmental conditions, cetacean behaviour and the presence/absence of small calves,
we determine whether the situation is suitable for biopsy sampling. If conditions are
favourable, approaches are made to the cetaceans and darting is attempted.
Figure 3. a) The PAXARMS system in use, and b) dart bouncing off a dolphin.
Murdoch University Standard Operating Procedure 0100 – 03 Wildlife (Marine) – Remote biopsy sampling of
cetaceans Version 3 July 2009
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 Required conditions
Biopsy sampling only takes place in appropriate conditions (no rain, light winds and seas
less than 1.0m). Attempts to dart cetaceans are only made when they are between four
and 20 metres from the vessel. In most circumstances, cetaceans are darted only after
identification photographs have been taken. Only cetaceans in robust nutritional
condition (based on assessment of their dorsal epaxial muscle mass on either side of
their dorsal fin, as per Pugliares et al., 2007) will be biopsied; those in thin to emaciated
condition will not.
 Personnel
During darting attempts, the sampler stands in the bow of the boat. Another researcher
stands behind the sampler taking identification photos. A third person drives the boat
and a fourth person (optional) is responsible for retrieving darts and recording notes.
Immediately after successful darting, the dart is retrieved using a hand-net. All personal
confer on behavioural responses and appropriate data is recorded.

Biopsy samples and sites
Samples are generally taken from lateral to and below the dorsal fin, where the blubber
layer is thickest. The dart strikes the cetacean, penetrates to a stop (generally 4-5mm
into the blubber layer), then bounces off and floats on the water surface for collection.
Wounds resulting from darting heal quickly and without complication (Fig. 4). In Shark
Bay, the mean number of days until wounds healed was 47.5 ± 24.2 days (n = 25). This
is likely an overestimate as individuals could not be tracked on a daily basis. For four
animals that could be monitored daily, the mean number of days until the wound was
healed was 23.3 ± 5.6 days. For comparison: even large wounds inflicted by sharks heal
relatively quickly (Corkeron et al., 1987; Orams and Deakin, 1997).
6-June
11-June
16-June
27-June
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cetaceans Version 3 July 2009
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24-July
Figure 4. Wound healing on an adult bottlenose dolphin darted 5th June 1999.
Fifty days after darting the wound was healed completely and the scar barely visible.
The PAXARMS biopsy system used by members of MUCRU was co-developed by Dr.
Michael Krützen (a Murdoch Adjunct Appointee) and has been used in sites the world
over for more than a decade. Simon Allen (Murdoch University Research Fellow) has used
the system without incident (to animal or researcher) in hundreds of sampling events.

How the animal is monitored
During and following sampling events, cetacean behaviour is monitored by experienced
observers. If either the sampled individual or other group members display behaviour
indicative of being disturbed (e.g. repeated rapid surfacing and dives in a direction away
from the boat), sampling efforts are terminated and the research team moves away.
Digital photographs of the sampling event are obtained whenever possible to allow for
individual identification of those cetaceans sampled. If a sampled cetacean is resighted
days, months and years following sampling, efforts are made to photograph the biopsy
wound to ensure normal healing.
Attempted darting generally results in mild, short-term ‘flinch’ or ‘buck’ reactions, with
most cetaceans then continuing their pre-biopsy behaviour and remaining in the vicinity
of the research boat (Krützen et al., 2002). Studies on bottlenose dolphins in multiple
geographic areas (by Krützen, Allen and others) have shown that dolphins tend to react
equally to the darting procedure regardless of being hit or missed - indicating the
reaction is primarily a result of the unexpected disturbance (i.e. a ‘startle response’
regardless of whether the dart strikes the animal or the water next to it). Additionally,
the PAXARMS system elicits the same reactions irrespective of sex, size, age or
population, making it a particularly useful system for darting small cetaceans in all
coastal habitats. Biopsied individuals in Shark Bay, Bunbury, and off the Pilbara, Western
Australia, as well as in various locations along the New South Wales coast, have not
altered their long-term behaviour since they are still easily approached for systematic
surveys and individual focal follows (Bejder and Allen, personal observations).

How long is the animal restrained for
As this is a remote biopsy technique, cetaceans are not handled or restrained in any way.

How long does the actual procedure take, number of attempts
Research vessels are generally in the vicinity of cetaceans for between 5 mins and 1 hour
for photo-identification and behavioural observations. Approaches for biopsy sampling
are generally limited to 15-20 mins. The ‘actual procedure’ is instantaneous and usually
occurs within the first 15 mins of approaching cetaceans. To avoid harassment of
cetaceans, darting attempts cease if animals display active avoidance of the research
vessel or we have not obtained a sample after 15-20 mins. If a dart strikes a dolphin but
no sample is obtained, one week must elapse before another attempt is made to dart the
same individual. If a dart completely misses a dolphin, we may try again the same day,
provided the dolphin shows no evidence of changed behaviour (i.e. no flinch or other
reaction), and continues to follow the boat.

Training required
This procedure must only be performed by personnel approved as competent or under
the direct supervision of a competent person. The only MUCRU members who are
sufficiently trained and competent in the use of this equipment/technique are Lars
Bejder, Simon Allen, Hugh Finn, Alexander Brown and Delphine Chabanne. Should a new
MUCRU member without experience be required to use it, they must first receive training
and guidance by one or more of the aforementioned 5 personnel. Should a new MUCRU
member with experience (i.e. training elsewhere) be required to use it, this action must
first be approved by one or more of the aforementioned 5 personnel after assessment of
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cetaceans Version 3 July 2009
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their level of competency has occurred, providing it is deemed adequate (if not, then
further training and guidance must take place, as per a new, inexperienced member).
A current firearms licence is required (held by Murdoch University). All individual MUCRU
personnel trained in use of the PAXARMS must be listed by name on this firearms licence.

General recommendation
Advantages of the PAXARMS system over others:
Among the systems available for taking skin/blubber biopsy samples from free-ranging
cetaceans (i.e. crossbows or pistols), the design of the PAXARMS biopsy system make it
the best available for darting cetaceans.
 Rifles are inherently more accurate than other systems. This is crucial when
darting small delphinids that are fast-moving and frequently found in tight groups.
 The PAXARMS system allows the fine adjustment of the velocity at which the dart
leaves the barrel. The system is thus readily adjusted to accommodate variation
in distance to target animals (i.e. cetaceans often approach boats).
 The PAXARMS system has been successfully employed to obtain biopsy samples
from numerous delphinids including: bottlenose dolphins (Tursiops spp.), common
dolphins (Delphinus delphis), Hector's dolphins (Cephalorhynchush hectori),
snubfin dolphins (Orcaella heinsohni) and humpback dolphins (Sousa chinensis).
 The large diameter of the stop helps the dart to bounce free of an animal without
the need for retrieval systems (which can lead to entanglement problems).
 The cost-effective modular assembly of the darts allows parts to be exchanged.

Emergency intervention
Biopsy of cetaceans is not without risk to researchers or animals, since a firearm is in use
on a moving vessel. However, our experienced personnel have not encountered any
human or animal welfare concerns in many hundreds of biopsy sampling events.
Experienced personnel conduct fieldwork in a measured and professional fashion so as to
reduce the chance of mishap. Sampling is only ever attempted in appropriate conditions.
The greatest response recorded thus far during our combined biopsy experiences is:
multiple leaps because of a dart not bouncing off the dolphin, but remaining attached to
the skin. In the rare instances where this has occurred, the dart dislodges shortly
thereafter. On another occasion in which a dart remained attached, the dolphin carried
on engaged in its pre-biopsy behaviour. This dart dislodged of its own accord within 24
hours. These animals have been part of long-term research programs and are under
observation from year to year with no long-term effects detectable.
Should an animal ever react in such a way that indicates distress in future, the research
vessel will endeavour to stay alongside and monitor the animal(s). A researcher will only
enter the water should the animal be deemed to be at immediate risk of further injury. In
such a case, a researcher will attempt to physically support the animal and provide
whatever intervention is required e.g. removal of dart, buoyancy support if shock is being
experienced. The animal will then not be released until such time as they are deemed
able to support themselves, or the major health or welfare issue has been controlled.
It is not likely that a veterinarian will be immediately available. Given the nature of
marine fieldwork on cetaceans and their size, handling and transport of an animal is
unlikely to be feasible. It should be noted here, however, that such an event occurring
due to use of this biopsy system is extremely unlikely, and has not occurred in any
previous biopsy attempts.
Any injured, stranded, deceased or entangled cetaceans encountered should be reported
to Department of Parks and Wildlife (DPaW; formerly DEC) via the Wildcare 24 hour
Helpline 9474 9055 or directly to Douglas Coughran (Senior Wildlife Officer, DPaW) on
(08) 9219 9849 (office), 0419 947 708 (mobile), or by email at
Douglas.Coughran@DPaW.wa.gov.au.
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4. Substances administered.
No substances are administered
5. Related Policy and attachments.
This SOP must only be used in conjunction with the following:

An Animal Ethics Permit with remote biopsy of cetaceans listed as an approved
procedure. Verification of currently approved AEC permits is available from the
Research Ethics & Integrity Office.

Personnel are registered with Murdoch University for Animal Competency and
Experience and are listed on the approved Animal Ethics Permits.

Current and valid permits from relevant authorities for WA and Commonwealth
waters.

The PAXARMS system is registered under Murdoch University’s firearms license
and a current copy available.

A Licence to use animals for scientific purposes from Department of Agriculture
and Food WA Animal Welfare Act 2002 (for Murdoch University and any other
institution involved in collaboration). Laminated copies are available from the
Research Ethics & Integrity Office and should be carried on board whenever
biopsy is being undertaken.
6. References.
Corkeron, P.J., Morris, R.J., Bryden, M.M., 1987. A note on healing on large wounds in
bottlenose dolphins, Tursiops truncatus. Aquatic Mammals 13, 96-98.
Krützen, M., Barré, L.M., Möller, L.M., Heithaus, M.R., Simms, C., Sherwin, W.B., 2002. A
biopsy system for small cetaceans: darting success and wound healing in Tursiops
spp. Marine Mammal Science 18, 863–878.
NHMRC: Australian code of practice for the care and use of animals for scientific purposes
7th Edition 2004
Orams, M.B., Deakin, R.B., 1997. Report on the healing of a large wound in a Bottlenose
Dolphin Tursiops truncatus. In: M. Hindell, C.M. Kemper (Eds.), Marine Mammal
Research in the Southern Hemisphere Volume I: Status, Ecology and Medicine.
Beatty and Sons, Chipping Norton, pp. 170-173.
Pugliares, K.R., Bogomolni, A., Touhey, K.M., Herzig, S.M., Harry, C.T., Moore, M.J.,
2007. Marine mammal necropsy: an introductory guide for stranding responders
and field biologists. Woods Hole Oceanographic Institution Technical Report
WHOI-2007-06, p. 55.
7. Availability of SOP.
Restricted Access: to personnel of and projects undertaken by the Murdoch University
Cetacean Research Unit (MUCRU).
Murdoch University Standard Operating Procedure 0100 – 03 Wildlife (Marine) – Remote biopsy sampling of
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