Anesthetic Agents
Ms 222 / Tricaine (chemical's full name is tricaine methanesulfonate)
(Other generic names are 3 aminobenzoic acid ethyl ester; ethyl
m-aminobenzoate; tricaine). Paling
banyak digunakan didunia.
Dosis : 50 – 100 mg/L
Karena pHnya rendah dapat di netralkan dengan
5-6 mL 10% saturated NaHCO3 / L 100 mg/L larutan MS222.
Benzocaine
(Chemical name: Ethyl-p-aminobenzoate).
Secara kimiawi
mirip dengan MS222. Tidak larut dalam
air, tetapi ethanol (1g/L 96% ethanol, dalam dark bottle). Neutral solution.
Dosis : 2.5 mL
stock solution / 10 L aerated water within 2-5 min. immobilzed fish and
recovery time 5 – 15 min.
Chlorbutanol-
Chlorbutol- Chorethone- Acetochloroform
Tidak banyak
digunakan. Menyebabkan iritasi pada
kulit, mata dan large quantity may cause unconsciousness.
Larut dalam ethanol
(30 g in 100 mL 96% ethanol)
Dosis : 10 mL base
solution in 10 L water.
Methomidate
chloride
Less depression of
respiration than MS222 dan benzocaine.
Konsentrasi 5
mg/L. Larut dalam air
Sudah dicoba dengan
hasil pada berbagai species.
Quinaldine
Tidak mudah larut
dalam air. Bahaya bagi kulit, tetapi
Quinaldin-sulphate lebih aman.
Merupakan larutan
pH rendah, buffered with Sodium bicarbonate.
Jarang digunakan.
Propanidide
Larutan 5% dapat
larut dalam air. Untuk anestesi waktu
singkat dan lama. Tidak mempengaruhi
laju respirasi dan sirkulasi darah.
Clove oil (Chemical name: eugenol
(4-allyl-2-methoxy-phenol).
Dikenal sebagai
bahan yang aman untuk anestesi.
Tidak mempengaruhi
kemampuan renang ikan.
Konsentrasi : 20 –
40 mg/L (anestesi ringan) atau 100 – 120 mg/L (strong anestesi)
FIXATIVES
1. Neutral Formalin 10%
Formalin
37% or 40% 1 mL + Aquades 9 mL.
Tambahkan 3 mL borax / L 10% formalin.
2. Bouin’s solution
Picric
acid : C6H3N3O7 : 15 mL (larutan jenuh picric acid dalam aquades)
Formalin : HCHO 40% : 5 mL
Acetic
acid (pure): CH3COOH : 1
mL
3. 10%Formalin - TBS :
- Tris
(hydroxymethyl) aminomethane : NH2C (CH2OH)3:
1.21 g
- NaCl :
8.775 g
diluted
into 600 ml DW and stir.
Adjust
the pH to 7.5 by adding HCl while stiring, then add up to 1 L by adding
formalin (37%). To get neutral pH, use
buffered formalin.
Mix pure
formalin (37%) : TBS = 1 : 9 to make 1 L solution.
4.
ALCOHOL
It is used to fix and preserve fish specimens, especially if
skeletal structures such as otoliths are to be examined. Alcohol is an excellent preservative but is
not recommended for fixation of soft tissues.
SUBJECT: Anesthetizing fish
with MS222
DATE: 4/96
Has anyone used MS222 (3-aminobenzoic acid ethyl ester) to anesthetize fish?
I recall using a weak solution for a physio lab as a grad student, but I
cannot remember (or find) a concentration. Also, any special tips on
getting it into solution?
Thanks very much-
Kate Toner
toner@bucknell.edu
Kate - I have used MS222 in a 20% solution to anesthetize worms. It
does go into solution, but very slowly (in water) with a lot of
stirring. I found that it looses potency quickly - I could not use
the same container two consecutive days. We eventually stopped using
MS222, moving to glycerol (I think - it has been a long time). Hope
that helps!
Kate Flickinger
ISU
Lab Coordinator - Zoology and Genetics
I don't know what formal conc. to use but some years ago I peppered
ms222 on the surface of a small amount of water to anesthetize Xenopus,
stirred gently, waited, and if still active, repeated the protocol until
the tadpoles were inactive, studied them, then after a few minutes
transferred them to water without ms222.
Hope this helps until a more precise answer is available.
cheers
rrs@bradley.bradley.edu
Robert Rhea Stephens
Biology Department
Bradley University
Peoria, IL 61625
I found the following info in A LABORATORY COMPANION FOR GENERAL AND
COMPARATIVE PHYSIOLOGY, 2nd ed, by Hoar and Hickman:
"There is a very wide latitude in the permissible dosage and recommended
amounts vary from 1:5,000 to 1:20,000. In practice, a solution of about
1:10,000 is usually found satisfactory; animals are placed in the solution
until the desired level of narcosis is attained and then removed to their
natural water for injection or operation. The sustaining dose for prolonged
anesthesia (added to the water flowing over the gills) should be much more
dilute (1:45,000). Solutions of MS-222 gradually lose their activity but a
10% solution will remain fully active if stored in a brown bottle for up to
three days. Solutions in sea water will become toxic if exposed to light."
Hope this helps. Good luck.
George
George Edick
RPI - Dept. Biology
Troy, NY 12180
edickg@rpi.edu
I leafed through my file on anesthetics and found a methods paper on the
use of MS222 (generally referred to as tricaine methanesulfonate and now
available from Ayerst under the tradename Finquel). It gives all the
information you need:
Ohr, E.A. 1976. Tricaine methanesulfonate--I. pH and its effects on
anesthetic potency. Comp. Biochem. Physiol. 54C:13-17.
The gist: for goldfish start with 0.01% TMS in distilled water. The
spontaneous pH of this solution will be about 3.7. Titrate this solution
with NaOH to a pH of 7-8. Neutralizing the solution induces a quicker,
longer-lasting and less stressful anesthesia. You should titrate only the
final solution, not stocks if you make them up, because the MS222 will tend
to precipitate out. If you intend to anesthetize saltwater fish, note
that the buffering effects of seawater are much greater, resulting in a
spontaneous pH of much closer to neutral when MS222 is added; thus less pH
adjustment will be needed.
Induction time at this concentration should be around 5 minutes for a 2-5 g
goldfish. Once the fish is returned to freshwater, recovery time should be
around 4 minutes.
One last note from me, not the paper: MS222 is carcinogenic, so wear gloves.
Sara Hiebert
Biology Department
Swarthmore College
Swarthmore, PA 19081
610-328-8053
Hello Kate,
We have at our facility an Aquaculture unit and therefore regularly have
to anaesthetise fish. We have stayed away from using MS-222 because of its
carcinogenic nature although it is widely used in industry. We have used
both 2-phenoxyethanol and tertiary amyl alcohol to anaethetise fish:
2-phenoxyethanol has a narrower range of tolerance than tertiary amyl
alcohol so we now use the latter almost exclusively. We use a 1% solution
and find it works beautifully for goldfish, brook trout, charr & salmon.
It can be obtained from any chemical company (e.g. Sigma); the only
downside is that it is becoming somewhat expensive. If you need further
information please contact me at: Y7GX@UNB.CA
Best of Luck!
Evelyn Stillwell
c/o
Biology Department
University of New Brunswick
Fredericton, N.B.
Canada E3B 6E1
A URL for msds sheets is gopher://atlas.chem.utah.edu/11%2FMSDS
Unfortunately, it does not list ms222. What is the chemical's full name?
Sincerely,
Roger Christianson 503-488-0223 (home)
Department of Biology 503-552-6747 (office)
Southern Oregon State College 503-552-6415 (fax)
1250 Siskiyou Boulevard rchristi@wpo.sosc.osshe.edu
Ashland, OR 97520
The chemical's full name is tricaine methanesulfonate.
Sara Hiebert
Biology Department
Swarthmore College
Swarthmore, PA 19081
610-328-8053
MS222 is a brand name. Other generic names are 3 aminobenzoic acid
ethyl ester; ethyl m-aminobenzoate; tricaine.
Ed Gruberg
7.2 ANAESTHESIA
7.2.1. Introduction
Rendering fish quiet (sedation) or unconscious (anaesthesia) is crucial to
several aspects of fish tagging. Summary sheets at the end of this section are
intended to help operators choose and use anaesthetics: they are also readily
downloadable as OHP slides. More information about anaesthesia may also be
gained by interrogating the WELFARE database. Operators should be aware
that there are legislative implications of use of anaesthetics on fish that are
to be released to the wild because of the perceived risk of chemical residues
reaching humans through the food chain (see section 6.2.4 in legislation
section).
7.2.2. Anaesthesia
A variety of handling methods have been applied during the tagging process,
ranging from use of blindfolding in calming fish, to full anaesthesia involving
continuous irrigation of the gills with fresh or seawater containing diluted
anaesthetic agents.
Under anaesthesia, handling stress will be reduced and tagging can be
accomplished more rapidly without risk of the fish hurting themselves when
trying to escape. Although the use of anaesthetics in some cases may be
unwanted due to their detrimental effects on the physiology and behaviour of
the fish, considerations of animal welfare will in most cases prohibit tag
attachment to unsedated fish if surgery is involved.
7.2.3. Choice of anaesthetics
Different handling procedures demand different anaesthetic approaches. Light
anaesthesia (=sedation) is defined as ‘reduced activity and reactions to
external stimuli’, and is sufficient for procedures such as transport or
weighing of fish. Full anaesthesia can be defined as ‘loss of consciousness and
reduced sensing of pain, loss of muscular tonus and reflexes’ and is needed
when surgical procedures are applied (MacFarland 1959).
The behavioural changes occurring in fish passing through sedation to full
anaesthesia were classified by MacFarland (1959). There are 4 stages with
subclasses ranging from normal (stage 0), where the fish reacts to external
stimuli and where the muscular tonus and swimming ability is normal, to the
stage of total physiological collapse (stage IV), where gill movements have
stopped and which in a few minutes will lead to heart failure. In a tagging
context, the stages where the fish is in a state of light/deep anaesthesia
(stages II and III) are of greatest relevance, as the animal is then
insensitive to pain caused by the attachment of transmitters or data storage
tags.
Choice of sedatives/anaesthetics must be based on the species to be tagged,
the number and size of fish involved, and the duration of the operation in
question. Water temperature and chemistry have also to be taken into
consideration when choosing the method. Lastly, the work often has to be done
under primitive field conditions without accurate control of concentrations and
exposure times. An anaesthetic with a good safety margin between effective
anaesthesia and irrevocable collapse is essential in such circumstances.
7.2.4. Categories of methods
(a) Physical sedation methods
Physical sedation can be obtained by rapid lowering of temperature or by
electric shock. The former method is mainly applicable for transportation (c.f.
Ho & Vanstone, 1961). Coldwater adapted species, and marine fish require
lower temperatures for sedation than warm water species and freshwater fish
(Chung 1980). Water cooling can also be used in conjunction with other
anaesthetics (e.g. Benzocaine) but the dosage must then be reduced by about 30%
(cf. Ross and Ross 1983). Electroanaesthesia has a number of advantages such as
rapid immobilisation of fish, no need for chemicals, rapid regain of
consciousness and low costs (Madden and Houston 1976, Gunstrom and Bethers
1985, Tytler and Hawkins 1981; Cowx & Lamarque, 1990; Cowx, 1990). But
these are outweighed by the fact that the method cannot be used in saline
water, and the danger of using inappropriate voltage levels, which may give
severe physiological stress responses in experimental fish (Shreck et al.
1976) due to hypoxia. There are also significant risks to experimenters,
principally from electric shock. In the U.K. the National Rivers (NRA) issued a
safety Code of Practice in 1995.
(b) Chemical sedation and anaesthesia
Chemical sedation is distributed to fish in liquid dilutions of varying
strengths depending on the agent used. The sedative is inhaled by the fish and
diffuses across the gill epithelia. In minor quantities it can also diffuse
into the fish via the skin (Fereira et al. 1984) - this may be a
particularly significant route in scaleless fish with well-vascularised skins.
Since these chemicals are absorbed and excreted predominantly via the gills,
fish with a large surface of gill ephithelium for a given body weight (e.g.
salmonids) require lower doses of anaesthetics than fish (e.g. eels) with
relatively smaller epithelial surfaces (Ross & Ross 1983). Other factors
affecting the absorption and excretion of chemicals are the relationship
between the surface of the gill epithelium and the body volume, thickness of
epithelium, type of anaesthetic, dosage and temperature.
All known anaesthetics have unwanted side effects. Most of them are
barbiturates, which lead to unconsciousness, inhibition of the sensing of pain
and loss of muscular tonus and reflexes. The most important complication
connected with all forms of chemical anaesthesia is hypoxia due to reduced
respiration and vascular activity. This leads to physiological changes in the
blood (e.g. lowered pH), hypotonia (= reduced blood pressure), raised blood
glucose, blood lactate and haematocrit (Tytler & Hawkins 1981). In addition
to physiological deterioration of blood parameters, hypoxia can cause brain
damage, which interferes with directional orientation (Taylor 1988), or alters
temperature preferences (Goddard et al. 1974).
Widely used anaesthetics of the barbiturate group are:
MS 222- Tricaine methane sulphonate
Chemical name: ethyl- amino- benzoatemethanesulphonate. MS 222 is probably
the most widely used fish anaesthetic world-wide, and there are numerous
studies on the physiological effects of this agent (e.g. review by Bell 1987).
It is a crystalline powder easily dissolved in fresh and seawater. The
recommended dosage for anaesthesia is 50- 100 mg/ l (Klonz 1964; Fereira et al.
1979). It should be observed that MS222 becomes toxic in seawater exposed to
sun (Bell 1987). MS222 gives an acid solution and a dosage of 75 mg l-1
can cause the pH to fall to 4.0 in soft water (Wedemeyer 1970). This effect
can, however, be mediated by adding 5- 6 ml saturated (10%) solution of NaHCO3
to 1 litre of 100 mg l-1 solution of MS222.
Benzocaine
Chemical name: Ethyl-p-aminobenzoate. This chemical is also very widely used
in fish anaesthesia. It is chemically close to MS- 222, both being derivatives
of p- aminobenzoic acid. Benzocaine is a white crystalline powder, which is
insoluble in water and has to be dissolved in ethanol in a ‘master solution’ of
1 g l-1 96% alcohol. The master solution should be stored in a dark
bottle, and has a life of up to a year. The recommended dosage is 2.5 ml of
this master solution to 10 l of aerated water. With this dosage the animals
should be immobilised in 2 - 5 min. and the recovery time will be 5 - 15 min.
Benzocaine gives a neutral solution (Egidius 1973). The time to obtain
anaesthesia was observed to take 1.5 min longer time for trout (Salmo trutta)
and 3 min longer for pike (Esox lucius) in 7° C water than at 12 ° C
(Dawson & Gilderhus 1979). According to Wedemeyer (1970) a comparison
between Benzocaine and MS-222 as anaesthetics for salmonids was slightly in
favour of Benzocaine as less metabolic change was observed. More recent studies
by Soivio et al. (1977) showed few differences between the two; both caused
hyperglycaemia. However, benzocaine caused somewhat lesser hyperglycaemia than
MS- 222. With the exception of occasional allergic reactions, health hazards to
humans are not normally recorded with the use of benzocaine ( MND 1986).
Chlorbutanol- Chlorbutol- Chorethone- Acetochloroform
Chemical name: Chlorbutanol. Although classified as a safe anaesthetic for
fish (Johansson 1978), it has not been widely used outside Scandinavia due to
health hazards to humans connected with its use. Inhalation of larger
quantities may cause unconsciousness, it can also irritate human skin and eyes.
Chlorbutanol (Cb) is a crystalline colourless powder that has to be dissolved
in ethanol. The usual base solution is 30 g to 100 ml 96% ethanol, and the dose
10 ml base- solution to 10 litres aerated water. Johansson (1978) states that
the time for falling into stupor and wakening is inversely dependent to the
water temperature, the higher the temperature the lesser the time needed for
sedation. The dosage varies somewhat with the size and species of fish but is
considered sufficient when the fish rolls on it side after 3-5 min.
Chlorbutanol gives a light anaesthesia, but it is normally sufficient when the
fish only needs to be handled for a short time handling, such as in tagging
(Johansson 1978, Horsberg and Høy 1989). Chlorbutanol is considered a safe
anaesthetic for fish, although a study by Hansen and Jonsson 1988 showed an 87
% reduction in return rates of Atlantic salmon (Salmo salar) smolts
anaesthetised before release in comparison with untreated fish. Chlorbutanol
has also been tested on Atlantic halibut (Hippoglossus hippoglossus),
but with a dosage of 50 ml base solution dissolved in 10 l water. The smallest
fish are most rapidly sedated; they also have the shortest recovery time.
Methomidate chloride
Methomidate is a hypnotic (sleeping-agent) and not a barbiturate. It
therefore causes less depression of respiration than MS-222 or Benzocaine. This
may lead to fewer and less serious side-effects. Methomidate is water-soluble.
Mattson & Riple (1989) report an effective concentration of 5 mg l-1.
Methomidate was tested on rainbow trout in the early 1980s by Gilderhus &
Marking (1987), and showed in these tests to give a relatively long wake-up
time and also some mortality after treatment. However, during the late 1980s
this anaesthetic has been tested with good results for handling salmonids and
other fish in culture, such as cod and halibut at the Department of
Aquaculture, Institute of Marine Research, Norway, (Mattson & Riple 1989;
Huse, pers. Com.; Furevik, pers. com). From 1992 onwards methomidate has been
the only anaesthetic used at the Dept. of Aquaculture (Holme, pers. com.); the
only negative feature is the high cost of the product.
Quinaldine
Quinaldine is not easily soluble in water, and is also reported to be irritating
to human skin and mucus membranes. Quinaldine-sulphate does not have these
negative effects, but gives an acid solution, and must therefore be buffered
with sodium bicarbonate (Blasiola 1977). It has been used in acetone solution
for the capture of intertidal fish living in rock pools. Reports that it may be
carcinogenic currently restrict use.
Propanidide
In a 5% solution this chemical is water-soluble. Propanidide seems to
have few physiological side effects, and can be used both for short- and long-duration
anaesthesia. The main reported asset of this anaesthetic is that it does not
reduce the ventilatory rate of the fish (Ross & Ross 1987). The
blood-circulation can also remain unaffected as reported by Veenstra et al.
(1987) from studies of S. fontinalis embryos and 7 days old alevins of
amargosa pupfish (Cyprinodon nevadensis amargosae). It has also been
tested on carp (Jeney et al. 1986) rainbow trout and smolts of Atlantic
salmon and sea trout (Siwicki 1984) with good results.
Clove oil
Chemical name: eugenol (4-allyl-2-methoxy-phenol). Recent experiments
(Anderson et al. 1997) have shown that clove oil is just as effective an
anaesthetic for both juvenile and adult rainbow trout (Onchorhynchus mykiss)
as MS-222. Clove oil does not affect swimming performance and it also provides
swift induction and recovery from anaesthesia. It is regarded as a GRAS
(‘generally recognised as safe’) substance by the US Federal Drugs
Administration (FDA) and is suitable for use in field studies where immediate release
of the fish into the food chain is required. Anderson et al. (1997) have
shown that concentrations of 20-40 and 100-120 mg/l will induce light and heavy
anaesthesia, respectively. At a concentration of 120 mg/l induction times are
significantly faster than MS-222 for both juveniles and adults. At a
concentration of 40 mg/l there is no difference for juveniles but induction
times are significantly faster for adults. Recovery times for adult fish are
rather longer than MS-222 at the higher concentration but no different at the
lower concentration.
Download able information sheets that will assist in the choice of
anaesthetics for specific purposes have been prepared; they are displayed in
Appendix II (7.10) of this chapter and are also available on the CATAG web site
(http://www.hafro.is/catag).
7.3. EFFECTS OF CONVENTIONAL TAGS ON FISH
Consideration of conventional tagging (including procedures such as fin-clipping)
will be given here. Generally such tagging procedures are innocuous and there
is little or no stress to fish beyond that involved in capture and handling
(e.g. chinook salmon, Onchorhynchus tshawytscha, Sharpe et al.,
1998; see also Gjerde & Reftstie, 1988, Hansen, 1988). The main problem
associated with tags is that of pathological lesions caused by tagging or fin
clipping (Roberts et al., 1973a, b, c; Morgan & Roberts, 1976), or
indeed any breach of fish skin. Such lesions may be subject to secondary
infections and are likely to cause effects on growth rate and reproductive
performance. Uncontrolled infections may well be a source of mortality, but it
seems probable that this is very rare.
Adipose fin clipping (commonly performed on Pacific salmon) may be
detrimental because there is some evidence that these fins are secondary sexual
characters, which perform an important function in mate selection.
Most tagging experiments are based on the assumption that the behaviour,
growth and survival of tagged fish is similar to that in untagged fish and that
data generated from these studies is unaffected by the type of tag used or the
tagging procedure implemented. Few studies have been carried out to assess the
impact of simple external tags on the behaviour of fish (e.g. Lewis &
Muntz, 1984; McFarlane & Beamish, 1990), probably because they are
difficult to design and carry out. Furthermore, tag effects are sometimes
examined under controlled laboratory experiments, which often provide conditions
different from the natural environment.
While many of the internal tags or marks may have minimal or negligible
effect on the behaviour of marked fishes (Buckley & Blankenship, 1990),
external tags may affect the behaviour of tagged fish. Small individuals may
have problems with relatively large tags and the application of the tag may
cause problems, such as wounds around the attachment. External tags may effect
feeding or evasive behaviour and the fish may therefore be more vulnerable to
predation. Especially in demersal fish, tags may become overgrown with algae
and/or mussels, becoming heavier and more cumbersome. An external tag that has
not been anchored firmly into the muscle may continue to irritate the fish,
preventing the wound from healing causing a chronic wound.
Growth of sablefish, Anoplopoma fimbria, was found to be affected by
the tag or tagging procedure in a comparison of wild, tagged fish with untagged
fish (McFarlane & Beamish, 1990). Thus, extrapolating growth information
from tagged fish resulted in altered estimates for mortality and mean age at
maturity for this species. On the other hand, no effect on growth was observed
in similar studies with Arctic char (Salvelinus alpinus) (Berg &
Berg 1990).
Carlin tagging and fin clippings are commonly used in studies on salmon or
trout migration, survival or growth. Saunders & Allen (1967) showed
negative effects of this tagging method on survival of Atlantic salmon, Salmo
salar, implying that mortality estimated from tagged salmon smolts would
result in an underestimation of the survival rates to adults. This was
confirmed in later studies on the same species by Isaksson & Bergman (1978)
and Hansen (1988). The increased mortality was attributed to handling,
anaesthesia and marking of fish. Carlin tagging was found to have a higher
impact on survival than fin clipping, although the latter was not without
impact, probably due to stress from handling and anaesthesia. In a laboratory
study on snapper (Pagrus auratus), no effect of dart tags on survival or
growth was observed on three length sizes of fish during a one-year period
(Quartararo & Kearney 1996).
All tagging or marking of fish involves treatment, which disturbs the fish
and may stress or harm the fish. Careful handling procedures throughout the capture
and marking process are of highest importance. Physiological research has shown
fish to be stressed for a prolonged period after handling; for example, levels
of lactic acid may be elevated for more than 24 hours after stressing the fish
at certain temperatures (Wendt 1965, 1967; Wendt & Saunders, 1973).
Histopathological studies on the effects of Disc-dangler tags on Atlantic
salmon (Morgan & Roberts, 1976) revealed that external tags of these types
can leave severe traumatic wounds which may lead to secondary infection.
Similar observations were made by Vogelbein & Overstreet (1987), who
reported histopathological problems with internal anchor tags used on spot, Leiostomus
xanthurus. The incomplete healing of the integument during the life of the
fish may affect the normal behaviour of the fish and result in biased estimates
of biological parameters.
A possible (and virtually unstudied) effect of all types of external tagging
(whether conventional or with electronic tags) is that tags may become fouled,
causing enhanced drag, so disadvantaging the fish. Anecdotal evidence has been
collected during CATAG of the existence of such fouling (e.g. by barnacles and
seaweed) but more investigation is needed. In particular, it would be desirable
if systematic fouling trials could be conducted on tags and tag materials - it
is quite possible that fouling could be a source of unremarked mortality of
tagged fish.
