J. mar. biol. Ass. U.K.

(1971) 51, 105-109 105

Printed in Great Britain

A SIMPLE METHOD TO DETERMINE THE O:N RATIO

OF SMALL MARINE ANIMALS*
By N. B. SNOWf AND P. J. LEB. WILLIAMS
Department of Oceanography,
The University, Southampton

(Text-fig. 1)

A simple method is described for determining the O: N ratio of small marine inverte-
brates. Oxygen consumption is followed using an oxygen electrode and the water sample
is subsequently analysed for a-amino nitrogen using a ninhydrin method. It is possible
to distinguish between ammo-nitrogen and ammonia-nitrogen in the water sample by
removing the ammonia under alkaline conditions. Results with the palaemonid prawn
Palaemonetes varians (Leach) indicate that ammonia is the main nitrogenous excretory
product and that the O:N ratio is 6-1 in winter. The ratio increases to 34-2 during early
summer, indicating a shift from protein catabolism to utilization of carbohydrate, fat, or
both of these energy sources.

INTRODUCTION
The atomic ratio of oxygen-consumed to ammonia-nitrogen excreted by marine
organisms (O:N ratio) is more conveniently and precisely determined with current
analytical procedures than the respiratory quotient (R.Q.). When an organism is oxidizing
protein exclusively the O: N ratio will be low, less than 7; it will be high when either fat or
carbohydrate is oxidized.
The usefulness of the ratio in nutritional studies of marine organisms was realized after
Harris (1959) first derived a figure of 7-7 for mixed zooplankton in Long Island Sound.
Corner, Cowey & Marshall (1965) determined a mean value of 13-5 for Calanus helgolandi-
cus and C.finmarchicus, and recently Conover & Corner (1968) have found that this ratio
varies on a seasonal basis for several species of zooplankton. This subject has been
reviewed by Corner & Cowey (1968), together with other biochemical aspects of marine
zooplankton.
Most aquatic invertebrates are usually assumed to be ammonotelic; although earlier
investigations on larger aquatic invertebrates indicated that considerable quantities of
amino acids were excreted. At the time of these earlier studies, however, reliable methods
for estimating ammonia were not available. Corner et al. (1965) concluded that ammonia
was the main nitrogenous excretory product of C.finmarchicus and C. helgolandicus using
three methods (two of which were specific for ammonia) for determining excreted
nitrogen. Corner & Newell (1967), using differential chemical methods, confirmed that
C. helgolandicus was primarily ammonotelic, excreting only small and variable amounts of
other nitrogenous substances.
* This work was carried out whilst one of us (N.B. S.) was in receipt of a Research Studentship from
the Natural Environment Research Council.
f Present address: Department of Zoology, University of Manitoba, Winnipeg, Manitoba.
106 N. B. SNOW AND P. J. LEB. W I L L I A M S
Recently, however, it has been reported that large amounts of amino acids are released
by zooplankton (Johannes & Webb, 1965; Webb & Johannes, 1967). Johannes & Satomi
(1967), using Palaemonetespugio as their experimental animal, reported a high release rate
of organic carbon. This may be amino-acid excretion. As biochemical studies were in
progress at this laboratory on the nutrition of a related species P. varians Leach, the
following simple method was devised primarily to determine ammonia and any a-amino
nitrogen excretion simply and separately. Ammonia excretion and respirometry data were
then used to determine to O:N ratio during summer and winter.
Methods for measuring ammonia in sea water are frequently based on the indolphenol
blue reaction. These are, however, exacting and have much more than the sensitivity
required for the samples obtained with present type of work. Such samples could be
diluted, of course, but a more satisfactory solution is to turn to a less sensitive method
for determining ammonia. The ninhydrin reaction has a suitable degree of sensitivity
and may be used to determine ammonia and a-amino nitrogen in sea water. The
method will not distinguish between ammonia and the a-amino nitrogen of amino acids.
The amino acids are generally stable under mild alkaline conditions, when ammonia
can be volatilized off. Thus, if the evaporated sample is subsequently re-analysed with
ninhydrin, then the residual ninhydrin reaction material may be attributed to amino
acids and other non-volatile a-amino nitrogen containing compounds. Ammonia is
thus determined by difference.

MATERIALS AND METHODS

Measurement of oxygen consumption. A lead-silver oxygen electrode and meter (electrode:
Model 15A; meter: A15A, E.I.L., Richmond, Surrey) were used in conjunction with the
respirometer shown in Fig. 1, to determine the rate of oxygen consumption by P. varians.
The apparatus had a volume of 640 ml. Before an experiment the apparatus was partially
filled with diluted sea water (sea water from Southampton estuary, salinity approximately
32 %, diluted with an equal volume of distilled water), and the animals, usuallyfive,added.
The experimental animals were collected on the day prior to the experiment and held in
dishes containing 11. of the diluted sea water, during which time they were allowed to
feed on marsh-pool detritus, their normal food in the wild. These animals did not,
however, feed during the course of the experiment. The top was placed on and the
apparatus flushed with the diluted sea water until all residual bubbles of air were excluded.
The experiments were carried out in a constant temperature room, at environmental pool
temperature. Oxygen consumption was followed over a 3 h period. The meter recorded %
saturation and was previously calibrated against the Winkler method (Welsh & Smith,
i960). The measured uptake of oxygen was corrected for consumption by the electrode
and then converted to /ig oxygen consumed/mg animal wet weight.
At the end of each run the water contained in the respirometer was run off and its
ammonia content determined as described below.
Ninhydrin method. The method used was based on that described by Matheson &
Tattrie (1964). The reagents consist of a o-2M citrate buffer (pH 5-0) and a mixed
ninhydrin reagent, prepared by mixing 5 vol. of a 2-methoxyethanol solution containing
2
% (v/v) of aqueous o-oi M-KCN, with 1 vol. of 5 % (w/v) ninhydrin in 2-methoxy-
 DETERMINATION OF O:N RATIO 107

ethanol. The mixed reagent must be prepared at least one day before use, otherwise the
blanks will be high: Matheson and Tattrie recommend preparing it a week prior to use.
The assay consists of heating 2 ml. of the sample (containing 1-10 fig amino or ammonia
nitrogen) with 1 ml. of citrate buffer and 2 ml. of the mixed reagent at 100 °C in a water
bath for 7J min. The solution is then cooled and read in a 1 cm cuvette at 570 nm
against the appropriate blanks. Under these conditions 1 /igN will give an extinction of
approximately o-i. The method was standardized with a (NH4)2SO4 solution. It is
advisable to take reasonable precaution against NH 3 fumes and cigarette smoke.

Meter

Thermistor

Electrode

Stirrer

Fig. 1. The oxygen-electrode respirometer (horizontal arrows indicate direction of flow during flushing
of the chamber and broken lines indicate the position of plastic mesh.

Determination of amino acids. A replicate of the sea-water sample (a 50-100 ml. sample
is convenient to work with but smaller volumes may be used) is brought to pH 9-5-10 and
evaporated at 90-100 °C under reduced pressure in a rotary evaporator to between one-
fifth to one-tenth its volume. It is then brought up to its original volume with ammonia-
free distilled water, the pH of the solution adjusted to approximately pH 7 and then the
ninhydrin determination repeated. In the present work the samples were evaporated
twice, but the second evaporation appears to be unnecessary. The ammonia in the sample
is volatilized off during this procedure and the residual ninhydrin reacting material will be
amino acids and other stable non-volatile a-amino nitrogen compounds.
Amino-acid mixture. Amino-acid mixture used in the present work consisted of approxi-
mately equal weights of the following amino acids: glycine, taurine, DL-aspartic acid,
DL-methionine, DL-woleucine, DL-citruline, L-arginine, L-cystine, L-tyrosine, DL-serine,
/?-alanine, DL-valine, DL-tryptophan, L-lysine and DL-threonine.
108 N. B. SNOW AND P. J. LEB. W I L L I A M S

R E S U L T S AND D I S C U S S I O N
The results in Table l illustrate that the evaporation procedure meets the necessary
requirements. No detectable ammonia remained after a single evaporation at pH 10. The
mixture of amino acids appeared to be stable during the evaporation procedure. It is
necessary to carry out the evaporation at pH 10, for at pH 8 ammonia is not lost.

TABLE 1. EXAMINATION OF LOSS OF AMMONIA AND STABILITY OF

AMINO ACIDS DURING THE EVAPORATION PROCEDURE
The solutions were analysed for their a-amino acid nitrogen content by the ninhydrin method. The
limits given are the standard error of the mean value: the number of replicates used for ninhydrin analyses
follows in parentheses.
The composition of the amino acid mixture is given in ' Methods': the amount added in the above
experiments was equal to approximately 4 figti.
Extinction coefficient
at 570 nm
Blank 007 + 0-03 (n = 10)
Sea water+ NH 3 (5 fig N) 0-55 ±0-04 (n = 10)
Sea water+ NH 3 (5 fig N) evaporated at
pH 8 and made up to volume 0-54 + 0-04 (n = 10)
Sea water+ NH 3 (5 /tg N) evaporated at
pH 10 and made up to volume 0-07 + 0-03 (n = 10)
Sea water + amino acid mixture 0-46 ±0-03 (n = 8)
Sea water + amino acid mixture, evaporated
at pH 10 and made up to volume 0-46 + 0-03 (n = 8)

TABLE 2. THE O:N RATIO FOR PALAEMONETES VARIANS DURING WINTER

AND EARLY SUMMER
Oxygen consumption Ammonia excretion
(fig O2/mg wet wt/h) (fig N/mg wet wt/h) Molar O:N Ratio
Winter
Dec. 1968 034 0054 5-5
029 0-040 6-4
Jan. 1969 067 0-080 7-4
Feb. 1969 0-73 0-130 49
084 0-121 6-2
Mean 0-57 0-085 6-1
Early summer
May 1969 0-71 0-016 397
June 1969 0-82 0-025 28-8
July 1969 086 0-022 34-3
Mean o-8o 0-021 34-2

Experimental runs using the estuarine natant decapod P. varians (Leach) gave results
in which the values of the residue after evaporation were not significantly diiferent from
blank values. It was therefore concluded that ammonia was the primary form of ninhydrin
reacting nitrogen excreted by these animals. The sensitivity of this method is such that
any a-amino nitrogen excretion is likely to be less than 5 % of the total nitrogen excreted.
The failure of P. varians to excrete appreciable amounts of amino acids apparently
contrasts with the general findings of Webb & Johannes (1969). We can offer no explana-
tion for this. Webb & Johannes suggest that the reason Corner & Newell (1967) failed to
detect amino acids in the excretion products of Calanus, was that in the course of their
 D E T E R M I N A T I O N OF O:N R A T I O 109
experiment (lasting 24 h) it was possible for a bacterial flora to develop sufficiently to
remove the excreted amino acids. The incubation times in the present work were short,
3 h, and it is unlikely that a sufficiently large flora would develop.
The O:N ratio was determined for P. varians during Winter and early Summer
months and the results are summarized in Table 2. The ratio was found to have a low
value in winter (average 6-i) indicating that protein is probably catabolized during this
period. In early Summer, however, the ratio increases to 34-2, when either carbohydrate
or fat, or both, are being utilized.

REFERENCES
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zooplankton. Biol. Rev., Vol. 43, pp. 393-426.
CORNER, E. D. S. & NEWELL, B. S., 1967. On the nutrition and metabolism of zooplankton. IV.
The forms of nitrogen excreted by Calanus. J. mar. biol. Ass. U.K., Vol. 47, pp. 113-20.
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