CA2088903A1

CA2088903A1 - Method for identifying marked fish

Info

Publication number: CA2088903A1
Application number: CA002088903A
Authority: CA
Inventors: Robert Brown; Shu-Heng Wang
Original assignee: BROWN MEDDINGS ELEMENTAL RESEARCH Inc
Current assignee: BROWN MEDDINGS ELEMENTAL RESEARCH Inc
Priority date: 1993-02-04
Filing date: 1993-02-04
Publication date: 1994-08-05

Abstract

METHOD FOR IDENTIFYING MARKED FISH
Abstract of the Disclosure A method is disclosed for marking fish with bone-seeking marker elements and for identifying fish from the distribution of marker elements in the scales and bones of the fish. The method may be used to assist in identifying the sources and patterns of migration of wild stock fish by providing information about the order and relative concentrations of naturally occurring bone- seeking elements to which the fish have been exposed from the spatial distribution of such elements in the fish's scales and bones. Fish may be marked by exposing the fish to a marker element such as Strontium or one of the Lanthanide series of rare earth elements. The method for identifying marked fish involves taking a sample of bony tissue, for example a scale, from a fish, exposing a surface of the sample, and sampling the constituent elements of the sample along a path on the surface which traverses successive growth rings in the sample. The sampling may be done by laser ablation and Inductively Coupled Plasma Mass Spectrometry (ICPMS), a technique which is extremely sensitive and provides fine spatial resolution. Because the method is sensitive to the distribution of elements in the sample as well as the amounts of the elements in the sample it is possible to detect a marked fish even if the fish has been exposed to a high background level of the same marker element which was used to mark the fish. The invention also provides a method for marking fish by successive exposures to different marker elements or combina-tions of marker elements. This allows a large number of unique markings for fish using relatively few marker elements.

Description

METHOD FOR IDENTIFYING MARKED FilSH

FIEID OF THE INVENTION

This invention relates to a method for identifying fish of the class Osteichthyes which have been exposed to a marker chemical. The method is also useful for monitoring the distribution and concentration of certain chemicals in the aquatic environment.

BACKGROUND OF THE INVENTION

In recent years there have been marked declines in fish stocks in various areas of the world. The decline in fish stocks has reinforced the need for better regulatTon of fisheries and for more effective programs to replenlsh fish stocks.

It is critically important to government agencies who are charged wtth regulating fisheries to obtain accurate data regarding the migrations of fish. This information is very important for determining how many flsh can be taken from a fishing ground at a particular time without harmlng the ability of the fish population to replenish itself.

The management of fish hatcheries is another important aspect of flsheries management. Many hatche!ies have now been built for the purpose of hatching and releasing juvenile fish into the wild. Millions of dollars are spent every year operating fish hatcheries in North America. It is Impossible to accurately assess the effectiveness of such hatcheries wlthout a means of monltoring the fate of luvenlle flsh after they are released from the hatcheries.
3û
Excluding man-made and natural spawning channels there are approximately S5 fish hatcheries in the Province of British Columbia, Canada alone. Each hatchery releases on the order of 5 million hatchlings ` 2088903 each year. A total of nearly 1.5 billion juvenile salmonids are released each year from hatcheries in British Columbia, Alaska, Washington, Oregon, Idaho and California.

The difficulty in monitoring the progress of fish after they are released from a hatchery is compounded by the life cycle of the fish.
Juvenile salmonids are hatched in fresh water. After they are released from the hatchery as juvenile fish they typically migrate to the sea where they may spend between eighteen months and four years. During their time at 10 sea, the fish may migrate for thousands of miles before returning to spawn at the same hatchery where they were born. Many of the fish are eaten by predators or harvested by fishermen. Determining whether a particular fish whlch has been caught at sea originates from a particular hatchery has obvlous dlfflculties.
Prlor art techniques for monltoring the progress of fish released from a hatchery rely upon physlcally marklng the fish In some way. Many traditional ways to mark fish involve mutllatlng the fish In some way. Such methods Include attachlng cllps to the fishes' fins, freeze branding the fish, 20 or implanting coded wire tags Into the fish. Implanting coded wire tags is now the most wldely used method.

In the coded wire tag ("CWr') method coded markings are applled to wlre tags by a laser marklng device for later Identificatlon. Such 25 wlre tags must be tiny because they must fit Inside the Juvenile flsh when the fish are released. The slze of Juvenile fish when they are tagged varies from specles to species. In some species, for example, salmon pinks the ~uvenile fish may be as small as approxlmately 1 cm long and weigh less than 1 g when they are tagged. Typical wire tags are approximately 1 mm 30 long and weigh several milllgrams. The wire tags are inserted Into the cartilage in a specific location in the head of the juvenile fish. The fish are generally anaesthetized before the tags are implanted.

There are several serious problems with the coded wire tag 5 method. Firstly, it is expensive to implement. In 1992, each wire tag cost approximately Cdn. $.12 to manufacture. Furthermore, implanting the coded wire tags is labour intensive. Each juvenile fish must be individually handled to implant the wire tag.

Because of the high cost of implanting wire tags in juvenile fish It is not practical to mark more than three to four per cent of the millions of hatchlings whlch are released each year from each fish hatchery. To obtain statlstlcally slgnlflcant informatlon about the migration patterns, rate of predation, rate of harvest and rate of return of tagged fish it is therefore 15 necessary to actively seek tagged fish. Each tagged fish must therefore be marked so that when it Is later caught it can be identified as being tagged and the tag can be extracted. Salmonids are usually marked by cllpping off their adipose fins. The trauma of being handled, having wire tags in]ected Into their heads and having their adipose fins clipped off 20 causes a significant number of the tagged juvenile salmonids to die prematurely.

Collecting tags from mature tagged fish relies principally upon commercial and sports fishermen. The fishermen are asked to save the 25 heads of any fish which they catch which have misslng adipose fins and to dellver the heads to the approprlate agency along wlth detalls of where and when the flsh were caught.

After the heads of tagged fish are recovered it is time 30 consuming to extract the wire tags and read them to identify the fish.
Mature fish may weigh 25 kg. Their heads, which were less than 1 cm long i :. :~: . ,, ~ .., :

. .

when the wire tags were implanted, may be over 15 cm long. Locating a ~ -1 mm long wire inside the head of a large fish is difficult.
The tags are magnetic so that they may be located in an adult fish's head by means of a magnetometer. Each fish head is scanned with 5 a sensitive magnetometer to determine whether a tag is present in the fish's head. The head is then bisected and the two halves are scanned to deter-mine which half holds the wire tag. This process is repeated until the portion of fish head bearing the tag is small enough that the tag can be manually located. The entire process may take 5 to 10 minutes to 10 complete if no difficulty is encountered. There is a significant risk that the tag will be damaged by the cutting blade as the head is sectioned into smaller and smaller pieces. When the tag is located it is cut out of the fish head and read.

The uncertainties inherent in the coded wire tag method make the information provided by the method unreliable. There are uncertainties associated with: the proportion of fish marked relative to the total release;
the mortality rate of tagged fish relative to the mortality rate of un-tagged fish; the fact that a fairly high proportion of the tags recovered from adult 20 fish are unreadable: and the percentage of tagged fish which fishermen recognize, and collect and return the heads.
:
Another method for tagging fish for later identification, which has been suggested in research papers but has not been implemented on 25 a large scale, is to mark the fish with Strontium (Sr). When ]uvenile fish are exposed to Sr the Sr is incorporated into the scales and bones of the fish.
Sr Is a suitable element for this purpose because It Is chemically similar to Calcium whlch Is a prlmary constltuent of bony tlssue. The Sr, In the form of a non-toxlc salt may be fed to Juvenile flsh or may be Introduced dlrectly 30 into thewatersurrounding the fish.
..~

.,~ . . .

~ 2088903 A problem with the use of Sr as a tagging element for fish is that Sr occurs naturally in sea water. A Sr marker may therefore be masked by Sr absorbed from sea water. Furthermore, Sr marking does not provide distinct tags for identifying fish from different hatcheries.
Snyder et al. Use of Dissolved Sr in Scale Markina of Juvenile Salmonids: Effects of Concentration and Exposure Time, Can. J. Fish.
Aa,uat. Sci. Vol. 49, No. 4, 1992, p. 780 suggest that elements, such as samarium or rubidium, which do not occur naturally in significant concen-10 trations, may be used alone, or in hatchery-specific combinations for marking fish.

Chemical analysls and X-ray spectrometry have been used to detect chemlcal marker elements In flsh. Chemical analysis typically 15 Involves dissolvlng fish scales or otoliths in acid and then using standard chemical techniques to detect the presence and concentration of marker elements in the resulting solution. X-ray spectrometry typically involves bombarding a portion of a sample fish scale or otoiith with x-rays and monitoring the energy of transmitted or reflected x-rays. Both techniques 20 lack both sensitlvlty and spatlal resolution and involve tlme consuming tests.

SUMMARY OF THE INVENTION

The method of the invention makes it possible to detect 25 chemlcal marker elements in fish at extremely low concentrations and with high spatial resolution. The method is readily adapted to automation and provides for vaporlzlng portlons of the surface of flsh scales by laser ablationand analyzlng the gaseous product uslng a mass spectrometer. The high spatlal resolution enhances the ability of the method to detect the pres-30 ence of a marker element despite the presence of a significant back-ground level of the marker element.

.

. . .. : : . ~ . : . . :

The invention provides a method of identifying a fish marked by a rnarker element during one period of the fish's growth, comprising removir,g a scale or other bony tissue from the fish and sampling the constituent elements of the scale or bony tissue along a path traversing 5 successive growth rings of the scale or bony tissue.

The invention further provides a method for identifying a fish which has previously been exposed to an elevated concentration of a bone seeking marker element from a sample of bony tissue from the fish.
10 The sample having a first part formed during the exposure, and a second part formed after the exposure. The method comprises the following steps.
First, vaporlzing a portion of the sample at a first point on a path on the surface of the sample to form a first vapour. The path passing through the flrst and second parts. Second collecting and ionizing the first vapour.
15 Third, transferring the ionized first vapour to a mass spectrometer. Fourth, determining the degree to which the marker element is present in the first vapour. Then the first through fourth steps are repeated at spaced intervals along the path and the relative degrees to which the marker element is present in the first and second parts is compared.
Another aspect of the invention provides a method of marking a scaled fish for later Identlflcation. The method comprises the steps of:
exposlng the flsh to a first marker element for a first period during th~
formation of a first region of a scale of the fish; and exposing the fish to a 25 second marker element for a second period during the formation of a second region of the scale of the fish.

Another aspect of the Inventlon provldes a method for identifying a scaled fish which has been marked by exposing the fish to a 30 first marker element selected from a group of marker elements for a first period during the formation of a first region of a scale of the fish and , ,: : , ~? 2088903 exposing the fish to a second marker element selected from the group for a second period during the formation of a second region of the scale of the fish. The method comprises the steps of: taking a sample of bony tissue from the fish; focusing a laser beam on a point on the sample on a path 5 extending between a region of the sample formed during the first period and a region of the sample formed during the second period: scanning the laser beam along the path to vaporize the surface of the sample along the path to form a vapour, the vapour having an elemental composition representative of the elemental composition of the sample at the point 10 along the path at which the laser beam is focused; transferring the vapour to a mass spectrometer and monitoring said vapour for the presence of marker elements from the group of marker elements as said laser beam scans along said path; and identifying and recording the sequence of marker elements whlch are detected along the path.
Another aspect of the invention provides a method for identify-Tng a scaled fish which has previously been exposed to a marker element wherein a scale from said fish has a first part formed during the exposure and a second part not formed during the exposure. The concentration of 20 the marker element being greater in the first part than in the second part.
The method comprises the steps of: taking a scale from the fish; vaporizing a portion of the scale at a first point on a path extending between the first part and the second part to form a first vapour; collecting the first vapour;
transferrlng the collected first vapour to a mass spectrometer; measuring 25 the amount of the marker element in the first vapour; vaporizing a portion of the scale at a second point on the path to form a second vapour;
collectlng the second vapour; transferring the second vapour to the mass spectrometer; measurlng the amount of the marker element in the second vapour; and determinlng whether the amount of the marker element In 30 the second vapour is significantly different from the amount of marker element in the first vapour. One of the first and second points lies within the , ~ , . .. . . .

first part and one of the first and second points does not lie within the first part.

BRIEF DESCRIPTION OF THE DRAWINGS
In drawings which illustrate specific embodiments of the invention, but which should not be construed as restricting or limiting the scope of the invention in any way:

Figure I is a schematic view of apparatus for practising the method of the invention;
, .
Flgure 2 iS a micrograph of a typical scale from a salmon;

Figure 3 is a plot of the variation in the ratio of Sr to Ca with position in a scale from a 21/2 year old salmon which was exposed to an elevated concentration of Sr when it was approximately 1 l/2 years old;

Flgure 4 Is a plot of the amount of Sr detected in a salmon 20 scale versus position across the scale during a continuous scan along a straight line passing from one edge of the scale, through the focus to the opposite edge of the scale;

Figure 5 is a plot of the ratio of Sr to Ca versus position in a 25 scale from a salmon whlch was exposed to an elevated concentration of Sr In Its Juvenlle phase along a llne extendlng radially outward from the focus of the scale to the outer edge of the fresh water growth reglon of the scale as measured by the method of the invention; and Figure 6 is a plot of the ratio of Sr to Ca versus position in a scale from an untreated salmon along a line extending radially outward ,. ' ! ' , . :,, ' ' ':

from the focus of a scale to the outer edge of the fresh water growth region of the scale as measured by the method of the invention.

Di~TAllED DESCRIPTION OF THE INVEi~lON

The method of the invention involves marking fish with low levels of selected bone seeking marker elements such as the Lanthanide elements, Strontium or Yttrium and subsequently detecting quantities of the marker elements in the fish. Some bone seeking marker elements are 10 present in the environment due either to natural sources or to human activity. Therefore, the method can also be used for monitoring the geo-graphical distribution and concentration of such marker elements.

Preferably the marker elements are selected from a group of 15 elements, such as the Lanthanide series of elements or Yttrium, which are not naturally occurring in signiflcant quantities in either fresh water or sea water. The marker elements may be introduced in the food of juvenile fish but are preferably introduced in the form of non-toxic salts into the ambient water surroundlng the fish. The concentration of the marker elements in ;
20 the water is preferably in the range of 10 to 50 micrograms per litre of water when the fish is exposed to the marker elements over an extended period but may be significantly higher when the exposure is for a short period. -Typlcally Juvenile fish are marked, however fish can also be 25 marked at later stages of growth. As the fish grow, the marker elements are taken up by the fish and are deposited in the bony tissue of the fish Includlng the fish's scales, vertebrae and otollths. In a typlcal Juvenile fish,the total mass of marker element absorbed by the skeletal structure of the flsh may be no more than a few nanograms. The total relative concentra-30 tion of the marker elements in adult salmon weighing up to 2û kg is typically1 part in 1012 (one picogram per gram) or less.

: . . .
.. . ...
. .

lO- 2088903 The foregoing provides an easy method to simultaneously mark a great number of fish. The elemental marks are inexpensively and easily added to all of a selected group of growing fish at a hatchery. Further-more, the procedure is much less traumatic for juvenile fish than the coded 5 wire tag method.

It is very difficult, however, to detect extremely low concentra-tions of Lanthanide elements by conventional methods such as chemical testing or ICP (inductively coupled plasmal atomic emission spectrometry.
10 Furthermore, it is very difficult to use chemical tests to distinguish between different Lanthanide elements because these elements have very similar chemical properties.

Inductlvely Coupled Plasma Mass Spectrometry (ICPMS) is one 15 of the most sensitive known methods for detecting and differentiating between such elements. A sample of bony tissue from a fish, such as a scale, is analyzed by standard ICPMS by dissolving the fish scale in acid, introducing the resultant solution into a hot plasma where it is vaporized ~ -and ionized and introducing the ions into a quadrupole mass spectrometer 20 where the amount of marker elements in the sample relative to the amount of a naturally occurring constituent of the sample, such as Calcium, can be detected.

Due to the "dilution effects" brought about by the very large 25 increase in body mass of the salmon following its release to the wild and due to the accumulation of rnarker element in the fish from environmental sources after Its release to the wlld, standard ICPMS Is not sensitive enough to rellably detect the extremely small concentratlons of marker elements in an adult fish whlch was exposed to a low concentration of a marker 30 element when it was a juvenile fish, as described above. Furthermore, techniques such as standard ICPMS which require that a sample be ., . ~ ~ , . ..... . . .

11~ 2088903 dissolved in a solution for analysis do not provide any information about the distribution of the element being screened for within the sample. As described below, this information can be a very valuable tool in distinguish-ing extremely low levels of a marker element from background, for 5 monitoring the geographical distribution of particular elements, and for identifying fish which were marked by exposure to may be created using a given number of marker elements for marking fish.

The method of the invention enhances the sensitivity of ICPMS
10 for marker elements by focusing on the portion of a fish's anatomy where the marker elements would be concentrated if the fish had been exposed to such marker elements. For example, a bone seektng marker element will be much more concentrated In the portions of a fish's scales which were formed whlle the fish was living in water containing the marker element (or 15 eating food containing the marker element~ than in portions of the fish's scales which were formed either before or after the fish's exposure to the marker element.

In salmonld fish, scales grow outward from a central focus. As 20 the flsh grows, the successive rings of scale tissue are laid down around the periphery of the scales. After a portion of a scale is formed there is relatlveiy little change in the composition of that portion of the scale.
Therefore, In the scales of a fish which has been exposed to a marker element during a time interval, each scale will have a ring shaped zone 25 centred on the focus of the scale in which the concentration of the marker element is markedly greater than it is in other parts of the scale. When searchlng for the presence of a marker element In scales from a salmon whlch may have been exposed to a marker element when it was a Juvenile the region of interest is typically the region between the focus and the 30 outer periphery of the region of the scale which grew while the fish was Iiving in fresh water (the "fresh water growth region").

The focus itself may itself have an elevated concentration of a marker element if the fish was exposed to the marker element before or at the time that the scales of the fish began to grow. This may be the case, for example, where a fish egg is exposed to a marker element which 5 is absorbed into the yolk sac which forms part of the egg. The marker element may then be taken up by the juvenile fish over the period that it consumes the food in its yolk sac. If the fish's scales begin to develop during this phase then the foci of the scales may contain an elevated concentration of the marker element.
'~
The concentration of a marker element in the portions of a salmon's scales and skeletal structure which grew while the salmon was - -exposed to the marker element may be as much as several parts per mllllon dependlng upon the partlcular marker element chosen. The 15 concentratlon of the marker element in other parts of the salmon's skeleton and scales may be 1 part in 109 for the Lanthanide elements and as high as 1 part in 10~ for Strontium. The concentratlon of the marker element in the salmon's soft tissues may be so low that It is Impossible to detect by current methods, again depending upon the particular elements in 20 questlon.

The method uses a focused laser beam to selectively vaporize a chosen reglon of the surface of a sample, such as a fish scale, located on a movable stage withln a gas tight cell. The vaporized material from 25 the sample is transferred in a stream of Argon to a high temperature plasma where the components are atomized and lonlzed. The elemental composltlon of the vaporlzed materlal Is then determlned by mass spectrometry. Because the method can detect the presence of and measure the concentratlons of marker chemlcals at dlscrete points on a 30 sample of bony tissue, the method can be used to locate and identify the region of increased concentration of a marker element in a portion of a fish scale which was formed when the fish was deliberately exposed to a marker chemical. Because the method is sensitive to the spatial distribution of a marker element in a sample the method is capable of identifying a deliberately created region of increased concentration of a marker 5 element in a sample from a fish which was later exposed to a high back-ground level of the marker element.

The elemental composition of different sections of a fish's scales and skeletal structure will vary depending upon the elements which were 10 present in the fish's environment when those portions of the fish's anatomy were formed. One way of using this information is to identify the origin of wild fish caught at sea. The rivers in which many wild fish hatch typically contaln concentrations of at least some bone seeking elements. The water In each rlver Is llkely to contaln a different mlxture of such elements. The 15 river of orlgln of a wlld flsh may therefore be determined by comparing the relatlve concentratlons of elements found in the fresh water growth reglon of the wild fish's scales or skeleton to the relative concentration of elements found in the various rivers in the region from where the fish could have come.
A second way to use this information is to monitor the concentration and distribution of certain elements in the environment. For example, If It is known that a particular fish has spent time in an area of Interest then Informatlon about the concentration of various bone seeking 25 elements in the area of interest may be obtained by measuring the concentrations of those elements in the portion of the fish's scales or skeleton whlch was formed when the flsh was llving In the area of Interest.

Flgure 1 is a schematlc dlagram of the apparatus whlch may 30 be used to practise the method of the invention. A fish scale 1 is placed in sample cell 3. Sample cel, 3 is mounted on computer controlled X-Y

positioner 5 and elevation stage 7. X-Y positioner 5 allows the position of sample 1 to be changed in increments of approximately 0.1 ,um. Elevation stage 7 allows adjustment of the vertical position of sample 1. The surface of sample 1 can be viewed through microscope 11 on television monitor 9.
Laser beam 12 from laser 13 is focused on the surface of sample 1 at focal point 14 by lens 15 and mirror 17. Laser 13 may be, for example a pulsed Nd YAG laser with a peak output power of up to 400 millijoules at a wavelength of 1 û64 nanometres. A laser of a different wave~
10 length may provide better coupling with fish scale samples without perforat-ing the samples. A laser with an output power of less than 400 millijoules is preferable. Optimally, the light output from laser 13 should have a power of below lûû mllliJoules per pulse to avold perforating the sample and to malntain hlgh spatial resolution.
When a pulse of llght from laser 13 hits the surface of sample 1 It Is absorbed in the sample. The energy deposited In sample 1 by laser beam 12 vaporizes a portlon of the surface of sample 1 In the vicinity of focal point 14. The vaporized portion of the sample expands into sample 20 cell 3.

The vaporized portion of sample 1 is carried from gas tight sample cell 3 by an inert gas such as Argon. The Argon gas is introduced into sample cell 3 through port 21, flows through cell 3 and escapes through 25 exit port 23. The Argon gas then carries the vaporized constituents of sample 1 through tube 27 to plasma torch 29. The volume of sample cell 3 and tube 27 should preferably be kept small to prevent excesslve dllutlon of the vaporlzed portion of the sample and to provide a short gas clearance time.

Plasma torch 29 preferably contains an inductively coupied plasma. The vaporized constituents of sample 1 are atomized and ionized within plasma torch 29. The temperature within the plasma of plasma torch 29 is preferably high enough so that elements with a first ionization energy 5 less than 10 eV are fully ionized in plasma torch 29. Good results can be obtained when the temperature within the plasma in plasma torch 29 is approximately 8000K.

The ions produced in plasma torch 29 are extracted from the 10 central channel of the plasma and delivered to mass spectrometer 30. The ions pass from the plasma through a one millimetre aperture in a water cooled cone (not shown) into an area of reduced pressure. The ions are transmltted through the reduced pressure stage, through a second cone, referred to as a sklmmer, and Into mass spectrometer 30. Mass spectrome-15 ter 30 comprises an ion lens, a mass filter, a detector, a multichannel ana-lyzer, and a computer for data analysis. Ions pass through the skimmer into an ion lens region (not shownJ, which operates at further reduced pressure.
The ion lens forms the ions into a focused and energy corrected ion beam.
The lon beam passes into a quadrupole mass filter (not shown) where the 20 lons are separated according to their mass to charge ratio. A detector (not shown) at the exit to the mass filter counts the number of ions which pass through the mass filter. The mass to charge ratio of the ions is measured by scanning the mass which is passed by the mass filter from llthium at m/e 6 through uranium at m/e 238. The mass is controlled by 25 varying the radio frequency voltage applied to rods within the quadrupole mass filter.

The plasma torch 29 and mass spectrometer 30 may be components of an Integrated inductively coupled mass spectrometer such 30 as a model PQ2PLUS mass spectrometer manufactured by Fisons instru-ments of Winsford, Cheshire, England.

The output from the detector is amplified and accumulated in a high capacity multichannel analyzer (not shown). The resulting data is subsequently processed by a microcomputer (not shown) to determine the elemental composition of the vaporized portion of sample 1. `
A profile showing the variation of the elemental composition of sample 1 with position can be developed by moving sample 1 using X-Y ~ `positioner S, and measuring the elemental composition of sample 1 as described above at a number of discrete points on the surface of sample 1. , Depending upon the Intensity of laser 13 and the characteris-tlcs of sample 1 It may be deslrable to expose each polnt of sample 1 to two or more pulses of laser 13 before moving sample 1 to a new location.
15 Using more than one pulse per locatlon increases the sensitivity of the method by vaporizing a larger amount of material at each sample point without sacrificing spatial resolution. The power of each pulse of the laser should be optlmized and controlled to provlde sufflcient ablation of the sample without causing penetration through the thin sample.
As an alternative to measuring the elemental composition of sample 1 at a number of discrete points, the variation of the amount of a selected element or elements along a path on sample 1 may be measured by operating laser 13 continuously and slmultaneously movlng sample 1 so 25 that focal point 14 moves along the desired path. As focal point 14 moves along the path, the portion of sample 1 whlch lles along the path Is at least partlally vaporlzed. The vaporlzed fractlon of sample 1 can then be carried to the mass spectrometer for slmultaneous analysls as descrlbed above.
To ensure that varlations in the elemental composition recorded by mass 30 spectrometer 30 can be correlated with position on sample 1, where data about the elemental composition of sample 1 is obtained during a continuous scan the volume of sample cell 3 and tube 27 should be small and should be designed so that the inert gas used to carry vaporized portions of sample 1 to the mass spectrometer flows through sample cell 3 and tube 27 with minimum turbulence.
Laser 13, X-Y positioner 5 and mass spectrometer 30 can be controlled by a. single computer system to facilitate automatic collection of data. An automatic sample changer may also be provided to allow numerous samples to be processed in sequence without manual interven-1 0 tion.

Figure 2 is a micrograph of a typical scale from a salmonid fish.Some other families of fish have similar scales. The focus of the scale 41 lies near the centre of the scale. Surrounding the focus are rings 4i3 which were 15 successively formed during the development of the fish. The central portion of the scale with the closely spaced rings is the portion of the scale which grew while the fish was in fresh water. The fresh water growth region is of variable radius depending upon the fish species and is characterizeci by relatively closely spaced rings 43. The outer reglon 44 of the scale which 20 Is characterized by more widely spaced rings 43 Is the portion of the scale which grew while the fish was growing in salt water. Rings 43 are more wldely spaced In the salt water growth region 44 than they are in the fresh water growth region because fish typically grow faster when they are living In the open ocean than when they are living in fresh water. The portion 45 25 of the scale in which rings 43 are indistinct is the portion of the scale which Is exposed to the water. The portion 47 of the scale in which rings 43 are dlstlnct Is thls the portlon of the scale that attaches to the flsh. Generally It Is preferable to take measurements In portlon 47 of the scale because the dlstlnct rlngs ser~e as clear polnts of reference whlch may be used to 30 Identify the areas of Interest in thescale.

- 18- 2088903 ::

As described above, if the fish were exposed to a suitable -~
bone-seeking marker element while the scale was growing, the marker element would be concentrated in a ring shaped region of the scale which was formed while the fish was exposed to the marker element. The prior art 5 experimental methods for detecting the presence of a marker element in a fish scale are hampered because they are incapable of focusing on the ~ ;
portion of the scale in which the marker element is most concentrated.
Furthermore, the prior art experimental methods are incapable of distin-guishing between a deliberately introduced marker element and back-10 ground levels of the same element.

Figure 3 is a plot showing the variation in the relative concen- `
tratlon of Strontlum and Calcium in a scale from a fish which was exposed to an increased concentration of Strontium when it was a Juvenile fish. In 15 Flgure 3 the lighter coloured regions correspond to higher relative concen-trations of Strontium.

It is convenient to measure the amount of the marker element relatlve to the amount of an element, such as Calclum, which is naturally 20 present throughout sample 1. This avoids the need to compensate for variatlons In the amount of sample 1 which is vaporized by laser 13 at each sampllng point and the amount of vaporized sample which Ts actually dellvered to mass spectrometer 30.

The region 51 of the plot corresponds to the portion of the scale near the focus which was formed before the fish was exposed to the Sr marker element. Region 53 of the plot corresponds to the portlon of the scale whlch was formed after the fish was exposed to the Sr marker element. In regions 51 and 53 there is a detectable concentration of Sr which arises as a consequence of the natural background level of Sr in the environment. Region 55 corresponds to the area which was scanned but .,"
, - 19- 2088903 -:

which lies outside of the scale. No Sr is detected in region 55. The boundary between region 53 and region 55 corresponds to the outline of the scale. Region 57 corresponds to the portion of the scale which grew while the fish was marked by exposure to an enhanced level of Sr. As 5 shown in Figure 3, region 57, is very clearly distinguishable frorn regions 51, and 53.

The method of the invention involves measuring the concentra-tion of a marker element in a fish scale (or other portion of the skeletal 1û structure of a fish) both inside and outside of the region where the marker element is most concentrated. As the precise location of the region of maximum marker element concentration in a particular scale may not be known in advance, it is preferable to measure the distribution of the selected marker element at several points along a path which intersects 15 the probable location of the area of maximum marker element concentra-tion. That is, it is preferable to measure the distribution of the marker element along a path which passes from region 51 through region 57 and Into region 53 (or vice versa). Where the sample Is a scale from a salmonid fish, a suitable path is a line extending radially from near the focus of the 20 scale to the outer edge of the freshwater growth area.
: :
In routine practice it may not necessary to measure the concentratlon of a marker element relative to a naturally occurring element. The method is stable enough that peaks in the concentration of 25 a marker element can readily be detected in many cases with reference only to the amount of marker element detected. Figure 4, is a plot of the amount of Sr detected versus posltlon In a scale from a salmon which had previously been exposed to an elevated level of Sr. The data plotted in Figure 4 was taken by scanning a pulsed laser beam at a constant linear 30 velocity along a straight line passing across the scale from one edge, through the focus to the opposite edge of the scale. As the laser beam ,:, was scanned across the scale a portion of the surface of the scale along the line was vaporized. During the scan, a continuous flow of inert gas was maintained across the surface of the scale. The inert gas carried the vaporized material from the scale to a mass spectrometer where the 5 amount of Sr in the vaporized material was measured as a function of time.
The twin peaks shown in Figure 4 correspond with areas where the scan line crossed the ring shaped region of the scale which grew when the fish was exposed to an enhanced level of Strontium.

The peaks of Sr concentration shown in Figure 4 are very distinct and well above the background level of Sr found in other parts of the scale. In some cases, the fish may be exposed to a very high background level of a marker element after it has been deliberately marked with the same marker element. In such cases, traditional tech-15 niques for detecting marker elements in a fish scale which do not have high spatial resolution cannot tell whether the scale is from a deliberately marked fish or not. With the method of the invention, in many cases, the distinct well defined peak in marker element concentration which arises from deliberate exposure to a marker element for a defined period can be 20 Identified and distinguished by its sharpness from even a significant back-ground level of marker element.

Figure 5 is a plot of the variation in the ratio of Sr(88) to Ca(48) wlth position along a straight line passing outward from the focus of a 25 salmon scale to the outer edge of the fresh water growth region of the scale. The salmon had been treated by feeding it a diet enriched in Sr(88) durlng Its Juvenlle phase. The peak of the graph corresponds to the portion of the scale whlch grew during the time the salmon was treated.

Figure 6 is a plot of the variation in the ratio of Sr(88) to Ca(48) with position along a straight line passing outward from the focus of a : :- ;

salmon scale to the outer edge of the fresh water growth region of the scale. The scale came from a salmon which had not been exposed to an elevated level of Sr(88) during its juvenile stage. The plot of Figure 6 lacks completely the prominent peak in the ratio of Sr(88)to Ca(48) which is shown in Figure 5.

The method can be easily adapted to identify fish which have been coded by exposure to various combinations of marker elements. For example, fish from a hatchery could be marked with a combination of two or more marker elements which is unique to that hatchery. Any fish which was caught and which had scales containing the particular combination of marker elements used by the hatchery could be positively identified as havlng coming from the hatchery. The method of the invention is sensitive enough to detect vdriations in the relative proportions of marker elements and not just the presence or absence of specific marker elements.
Therefore, different fish or groups of fish can uniquely marked by exposing each fish, or group of fish to the same combination of marker elements and varying the relative conc:entrations of the marker elements in each case.
' Because the coded markings in the fish are permanently incorporated in the fish's bones, the origin of a marked fish may be determined even after the fish has been caught and processed. This may assist in policing the illegal practice of open ocean drift net fishing.

Another method of marking Juvenile fish wlth coded markers for later identificatlon is to expose the Juvenile fish to a flrst marker element (or comblnatlon of marker elements~ durlng the formatlon of a flrst region of the flsh's scales and to later expose the same fish to a second marker element (or combination of marker elements~ during the formation of a second region of the fish's scales. The fish may be further exposed to other marker elements in subsequent periods. Because the scales of fish typically ~ ~ , .............. . .
~ ., . . .:
.. . . .

grow outward from a central focus, the first and second regions of the scales will form concentric rings. Because the marker elements are preferentially taken up in growing scale tissue the different marker elements (or combinations of marker elements) will be maximally concentrated in 5 concentric rings in the fish's scales.

This technique allows fish to be marked with a code according to the order of exposure of the fish to various different deliberately added marker elements. Using this technique makes a great number of unique 10 codes available for marking fish with a few marker elements. The number of unique codes can be increased even more by exposing the fish to different combinations of several marker elements during subsequent intervals. The number of unlque codes may be further increased by varying the relative concentrations of the marker elements to which the fish is 15 exposed. The fish can be identified from the order in which the rings of different marker elements appear in its scales, which can easily be deter-mined according to the method of the invention as described above. As noted above, the order of appearance and relative concentrations of naturally occurring elements whlch are found in a fish's scales may assist in 20 Identifying the sources and patterns of migration of wild stock fish.

As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practlce of this invention without departing from the spirit or scope thereof.
25 Accordingly, the scope of the invention is to be construed in accordance wlth the substance defined by the followlng clalms.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a fish marked by a marker element during one period of the fish's growth, comprising removing a scale or other bony tissue from said fish and sampling the constituent elements of said scale or bony tissue along a path traversing successive growth rings of said scale or bony tissue.

2. The method of claim 1 wherein said sampling is done by laser ablation and mass spectrometry.

3. A method for identifying a fish which has previously been exposed to an elevated concentration of a bone seeking marker element from a sample of bony tissue from said fish wherein said sample has a first part formed during said exposure, and a second part formed after said exposure, said method comprising the steps of:

(a) vaporizing a portion of said sample at a first point on a path on the surface of said sample, said path passing through said first and second parts, to form a first vapour;

(b) collecting and ionizing said first vapour;

(c) transferring said ionized first vapour to a mass spectrometer;

(d) determining the degree to which said marker element is present in said first vapour;

(e) repeating steps (a) through (d) at spaced intervals along said path; and (f) comparing the relative degrees to which said marker element is present in said first and second parts.

4. The method of Claim 3 wherein said fish was exposed to two or more marker elements during said exposure and step (d) further includes determining the relative concentrations of said two or more marker elements in said first vapour.

5. The method of Claim 3 wherein said sample has a third part formed before said exposure, said path passes through said third part, and step (f) comprises comparing the relative degrees to which said marker element is present in said first, second and third parts.

6. The method of Claim 3 wherein said sample is a fish scale.

7. The method of Claim 3 wherein said scale has a focus and a fresh water growth region and said path comprises a portion which extends radially between said focus and the outward edge of said fresh water growth region.

8. The method of Claim 7 wherein said path extends between the outward edge of said fresh water growth region on a first side of said focus through said focus to the outward edge of said fresh water growth region on a second side of said focus.

9. The method of Claim 7 wherein step (d) comprises measuring the ratio of the amount of said marker element in said first vapour to the amount of a naturally occurring constituent element of said scale in said first vapour.

10. The method of Claim 9 wherein said naturally occurring constituent element of said scale is Calcium.

11. The method of Claim 7 wherein said fish was exposed to two or more marker elements during said exposure and step (d) further includes determining whether the ratio of the amount of each of said two or more marker elements to the amount of said naturally occurring constituent element of said scale is more than a threshold amount.

12. The method of claim 3 wherein step (a) comprises focusing and firing a pulsed laser at said portion of said sample.

13. The method of claim 12 wherein step (a) comprises firing two or more pulses of said laser at said portion of said sample.

14. The method of Claim 3 wherein said marker elements are selected from Strontium, Yttrium, and the group of elements having atomic numbers between 57 and 71.

15. The method of Claim 6 wherein said marker elements are selected from Strontium, Yttrium and the group of elements having atomic numbers between 57 and 71.

16. A method of marking a scaled fish for later identification, said method comprising the steps of:

(a) exposing said fish to a first marker element for a first period during the formation of a first region of a scale of said fish; and (b) exposing said fish to a second marker element for a second period during the formation of a second region of said scale of said fish.

17. The method of Claim 16 wherein said marker elements are selected from Strontium, Yttrium and the group of elements having atomic numbers between 57 and 71.

18. The method of Claim 17 wherein said fish is exposed to two or more of said marker elements during said first period.

19. The method of Claim 17 wherein said fish is exposed to two or more of said marker elements during said second period.

20. A method for identifying a scaled fish which has been marked by exposing said fish to a first marker element selected from a group of marker elements for a first period during the formation of a first region of a scale of said fish and exposing said fish to a second marker element selected from said group for a second period during the for-mation of a second region of said scale of said fish said method comprising the steps of:

(a) taking a sample of bony tissue from said fish;

(b) focusing a laser beam on a point on said sample on a path extending between a region of said sample formed during said first period and a region of said sample formed during said second period;

(c) scanning said laser beam along said path to vaporize the surface of said sample along said path to form a vapour, said vapour having an elemental composition representative of the elemental composition of said sample at the point along said path at which said laser beam is focused;

(d) transferring said vapour to a mass spectrometer and monitor-ing said vapour for the presence of marker elements from said group of marker elements as said laser beam scans along said path;

(e) identifying and recording the sequence of marker elements which are detected along said path.

21. The method of Claim 20 wherein said path extends from a region of said sample formed before said first period through regions of said sample formed during said first and second periods to a region of said sample formed after said second period.

22. The method of claim 20 wherein said sample of bony tissue is a scale.

23. A method for identifying a scaled fish which has previously been exposed to a marker element wherein a scale from said fish has a first part formed during said exposure and a second part not formed during said exposure, the concentration of said marker element being greater in said first part than in said second part, said method comprising the steps of:

(a) taking a scale from said fish;

(b) vaporizing a portion of said scale at a first point on a path extending between said first part and said second part to form a first vapour;

(c) collecting said first vapour;

(d) transferring said first vapour to a mass spectrometer;

(e) measuring the amount of said marker element in said first vapour;

(f) vaporizing a portion of said scale at a second point on said path to form a second vapour;

(g) collecting said second vapour;

(h) transferring said second vapour to said mass spectrometer;

(i) measuring the amount of said marker element in said second vapour; and (j) determining whether the amount of said marker element in said second vapour is significantly different from the amount of marker element in said first vapour.

wherein one of said first and second points lies within said first part and one of said first and second points does not lie within said first part.