Vyncke, W.

Open Marine Archive

Reprinted from Fishing News International, July 1968. Publishedby Arthur J. Heighway Publications Ltd.. 110 Fleet Street, London, E.C.4, England 
An accelerated microdiffusion 
Instltuufv.. - '^ 
-1"^ ?^ ".' method for the 
determination of ammonia 
in cartilaginous fish 
By 
W. VYNCKE 
Ministry of 
Agriculture, 
Fisheries Research 
Station, 
Ostend, Belgium 
NE X T to trimethylamine, ammonia is the most typical spoilage product in fish, molluscs and crustaceans. 
I t is generally determined together with other 
volatile nitrogen bases, as total volatile basic nitrogen 
(TVB). 
The objective quality determinations of cartilaginous 
fish such as dogfish and skate, however, raises special 
problems. These fish contain large quantities of urea (1.5 
to 2 per cent.) present in all parts of the body (muscles, 
intestines, blood, slime, etc.)^ By the action of a bacterial 
enzyme, the urease, urea is relatively quickly degraded to 
ammonia and carbon dioxide. 
Apart from this decomposition, ammonia is also formed 
during spoilage by dissemination of amino acids, either free 
or split off from proteins, and allied compounds (e.g. 
creatine), by oxidation of amines and by decomposition 
of nucleic bases. Hence, the possibility should be consid-
ered that the determination of free ammonia gives a 
better estimation of the freshness than the TVB-test. 
Furthermore, this latter method is rather difficult to 
carry out owing to the hydrolysis of urea during analysis, 
which produces ammonia, interfering with the results. 
Broad applications 
For the direct determination of free ammonia, different 
volumetric and colorimetric methods are available. In 
biological tissues and fluids, however, many interfering 
substances are generally present, making the preliminary 
isolation of ammonia a necessity. This can be achieved by 
ion exchangers or distillation, but in most cases, preference 
is given to the microdiffusion technique. 
The microdiffusion method of Conway^ has found broad 
apphcations, not only for determining ammonia, but also 
for a wide variety of other compounds (amines, acids, 
aldehydes, etc.). This method makes use of so-called 
Conway units with a concentric inner and outer chamber. 
Although relatively simple and very sensitive, this method 
has different disadvantages: the cell is comparatively 
expensive; the removal of the heavy grease used to make 
the cell airtight presents a cleaning problem; the metho'" 
requires several hours; the Uquid in the inner chamber 
must be pipetted out when applying colorimetry. 
In order to avoid these difficulties, Seligson and 
Seligson^ proposed an accelerated microdiffusion method 
for determining the free ammonia level in blood. They 
use penicillin bottles as diffusion cells; the rubber stopper 
is bored to fit a footed ground glass rod. The sample to be 
analysed is pipetted into the cell and made alkaline. The 
extremity of the rod is moistened with sulphuric acid. 
The cells are then rotated in horizontal position for a 
short period of time. The solution covers the inner wall 
completely, increasing the diffusion area considerably and 
keeping the depth of the layer at a minimal level. More-
over, the solution is constantly agitated. 
All these factors considerably shorten the diffusion 
time. After diffusion, the ammonia is determined colori-
metrically with Nessler reagent. 
Vyncke and Merlevede* have adapted this method to 
Fig. 1. Microdiffusion cell (dimensions in mm). 
24/29 
ground 
extremity 
fe&&i'^«''d»2Ba_^ 
"MrtMinarBfc 
/ 
shell fish and demonstrated its usefulness for the objective 
quality assessment of these species. The method has now 
been improved and applied to elasmobranch fish. 
Reagents. All solutions should be prepared in ammonia-
free water. Demineralized water meets this requirement. 
Normal distilled water should be purified by passing over 
an ion exchange column (e.g. Amberlite Monobed I I I or 
similar resin). 
Nessler reagent. The following reagent was found to 
give maximum sensitivity, reliability and stability. 
Solution 1: dissolve 40 g red Hgl2 and 30 g potassium 
iodide in 100 ml water. Solution 2: dissolve 95 g sodium 
hydroxide in 1.000 ml water. Mix 1 and 2 and let stand 
for three days. Decant the clear supernatant into a brown 
flask and store in the dark. Dilute 1:25 before use. 
Apparatus. Microdiffusion cells. The penicillin bottles 
proposed by Seligson and Seligson^ were replaced after 
multiple modifications by flasks of 60 ml with conical 
sockets (24/29) fitted with polyethylene stoppers and 
footed ground glass rods (Fig. 1). The rod is fixed to the 
stopper with a household type clear adhesive, which also 
makes it airtight. 
The 24/29 standard socket greatly facilitates the 
insertion and the removal of the rod without touching 
the wall. This opening is maximal when taking the small 
volume of the flask into consideration. 
Microdiffusion rotator. The "Multi-Purpose Rotator" 
model 150 V (Scientific Industries, New York) was tried 
in the first experiments, but the device with clips for 
fixing the cells appeared not to be satisfactory. The 
Fig. 2. Microdiffusion rotator with cells in vertical position. 
rotation head was, therefore, replaced by a laboratory-
made model consisting of three plexiglass discs of 27 cm 
diameter (Fig. 2). The first disc serves as a ground plate 
for the bottles. The second is bored to fit 12 bottles. Both 
are fixed to the spindle at a distance of 3.5 cm from each 
other. The third plate is loose and has 12 holes of 1 cm 
diameter fitting the outer extremities of the glass rods 
and rests on the polyethylene stoppers. By means of a 
butterfly-nut turning on the spindle, the upper disc is 
screwed tightly against the rubber stoppers, thus holding 
the bottles in position and perfectly sealing them at the 
same time. The rotator can be turned in both vertical 
and horizontal position, allowing easy handling of the 
bottles. 
Procedure. Ten g. of previously chopped fish is homo-
genized for two minutes with 400 ml of demineralized 
water; 1 ml of the supernatant is pipetted into a micro-
diffusion flask and 1 ml of a saturated potassium carbonate 
solution is added. The extremity of the footed glass rod 
is dipped into 1 N sulphuric acid and the excess shaken off. 
The stopper with rod is immediately replaced on the 
bottle. 
All cells are now placed vertically into the rotator, 
turned in horizontal position and rotated for 30 min. at 
50 rpm. 
The rods, which now carry ammonium sulphate, are 
removed and dipped into 5 ml Nessler reagent 1:25. The 
absorbance of the yellow-orange solution is measured at 
400 m|j. after five minutes; the concentration of ammonia 
is read from a standard curve determined with solutions 
of 2.5, 5 and 7.5 ng NH3-N. 
All microdiffusion analyses should be performed in 
strictly uniform circumstances. Volume of cells, diffusion 
area and rotation speed can easily be kept constant and 
are therefore of less importance in practice. Marked 
variations in room temperature on the other hand should 
be avoided because rate of diffusion is affected by tem-
perature.^ 
The experimental conditions of the method were fixed 
in previous experiments.* The rotation time, however, 
was increased to 30 minutes since this time need not be 
very strictly adhered to. Moreover, small fluctuations in 
rotation speed are of no importance in this case. ' 
Interfering substances. With ammonia, different other 
compounds also produce colours with Nessler reagent. In 
fish, trimethylamine and (in teleost fish) dimethylamine 
are especially concerned; they are also liberated by 
alkalies, they diffuse and are bound as sulphate on the 
rod. Analyses carried out with 10 jxg N of both amines, 
corresponding to 40 mg N per cent, fish, gave no absorb-
ance, indicating that trimethylamine and dimethylamine 
do not interfere with the determination of ammonia. 
The possible hydrolysis of urea was also investigated. 
Experiments with 500 \ig of urea produced no ammonia, 
which is probably due to the short diffusion (and alkalini-
sation) time. The method thus allows the determination 
of ammonia in fish containing high amounts of urea. This 
should be emphasized since other methods (e.g. distillation) 
do not always meet this requirement. 
Some precautions should be taken before carrying out 
free ammonia determinations on dogfish (Squalus acan-
thias L). In the shme and the blood on the skin of the fish, 
ammonia is easily formed, esf)ecially when the fish has 
been exposed to higher temperatures for some time, such 
as on deck before stowage in the hold or in the auction 
hall. In this case, ammonia can easily be detected by 
smell, but this does not always indicate the fish to be of 
definitely inferior quahty. In the muscles themselves, it is 
very possible that little or no free ammonia had yet been 
formed. Before sampUng, all fish must thus be thoroughly 
washed with ice cold water. 
In Belgium, as in several other countries, dogfish is 
usually beheaded and skinned shortly after auction to be 
sold as "sea-eel". During this operation, much blood is 
released at the surface of the skinned fish (the red colour 
being an index of freshness) and ammonia can be formed 
easily. This skinned fish should also be washed carefully 
before sampling. 
An average spoilage curve for dogfish is shown in 
Fig. 3. The fish were gutted and stored in ice at 1 deg. C 
immediately after capture. They were caught in the 
southern part of the North Sea during the months of 
October to January. Each dot on the figure represents an 
average of ca. 30 analyses. The range of the observations 
is also indicated. 
It can be noted that ammonia increéises very slowly 
during the first eight days of storage but rather quickly 
after that period, to reach values of 75 mg N per cent, 
after 14 days. The limit of edibility can be set tentatively 
at about 60 mg N per cent. 
Ammonia content correlated fairly well with organo-
leptic judgment which indicates that the method could be 
useful for the objective quality assessment of elasmobranch 
fish. Further experiments, however, are necessary to draw 
a definite conclusion in this matter. 
Finally, it should be emphasized that the method was 
shown to be very valuable for comparative experiments. 
It was already used with good results for the study of 
the influence of different temperatures on the shelf life of 
dogfish. 5 
Summary 
The microdiffusion method described makes use of 
small diffusion cells with conical polyethylene stoppers 
mi, 
.NH,-N« 
1 
• \ » ' : • 
H 
1 ' 
y-/ 
» II u n U «K 
Fig. 3. Average spoilage curve for dogfish ("Squaius acan-
tliias L.") Itept at 1 deg. C in ice. 
fitted with a glass rod and ground extremity, which is 
moistened with sulphuric acid. A fish extract is pipetted 
into the flask and made alkaline with potassium carbonate. 
By rotating the cells in a special microdiffusion rotator, 
diffusion time is shortened to 30 minutes. Ammonia is 
determined with Nessler reagent. 
There is no interference from urea, dimethylamine, or 
trimethylamine. The method is very simple and can be 
used for quaUty assessment of elasmobranch fish. An 
average spoilage curve for dogfish (Squalus acanthias L.) 
is given. 
References 
1. W. Simidu and K. Oisi — Bulletin of the Japanese Society of 
Scientific Fisheries, 16, 547, 1951. 
2. E. Conway — Microdiffusion Analysis and Volumetric Error, 
5th Ed., Crosby Lockwood & Son Ltd., London, 1962. 
3. D. Seligson and H. Seligson — A microdiffusion method for the 
determination of nitrogen liberated as ammonia — Journal of 
Laboratory and Clinical Medicine, 38, 324, 1951. 
4. W. Vyncke and E. Merlevede — Spoilage of fish and crustace-
ans ; Rapid determination of volatile ammonia by accelerated 
microdiffusion — Archives Beiges de Médecine Sociale, Hygiene, 
Médecine du Travail et Médecine Legale, 21 (3), 147, 1963. 
5. W. Vyncke — The influence of temperature on fish as measured 
by objective quality methods — Fishing News International, 6, 
39, 1967. 
Printed by Richardsons & Coppin Ltd., Lady smith Road, Grimsby, England 


An accelerated microdiffusion method for the determination of ammonia in cartilaginous fish

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An accelerated microdiffusion method for the determination of ammonia in cartilaginous fish

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