FISHERIES RESEARCH BOARD OF CANADA

Translation Series No. 2014

An apparatus for continuous respirationmeasurement in marine organisms -

by J. A. Oertzen and V. Motzfeld

original title: Eine Apparatur zur koritinuierlichen Respirâtionsmessunc

an, marinen Organismen

From: Marine Biology, 3 : 336-340,.1969

Translated by the Translation Bureau(DJ)Foreign Languages Division

Department of the Secretary of State of Canada

Fisheries Research Board of Canada

Marine Ecology Laboratory

Dartmouth, N. S. . .

1972

15 pages typescript

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J.A. V®n Oertgen & V. Motzfeld

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An apparatus for continuously mea.suring'respiration in rrarine organisms.

Ti tle i-n foreign lasguagc- ( transli.terate f oT.e.iglm )Eine Apparatur zur kontinuierlichen Respiration-smessung an marinen

Organismen

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' Marine Biology

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Marine Biology

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0020g German DJ 28.3 . 72

AN APPARATUS FOR CONTINUOUSLY MEASURING RESPIRATION IN MARINE ORGANISMS

by J.A. von Oertzen and V. Motzfeld

(Biology Department and Physics Department of Rostock University, German Denocratic Republic)

Translated from: Marine Biology, Vol. 3, pp. 336-340, 1969

Abs tract

. . r MeaSilreMeRtS N ■.- r.E.; made with a Clark electrode in a closed system under conditions of differnit, regulatable iv.-iter

. current velocities. Six sir ultaneous parallel rneze-oirements are possible with only one electrode. "Ilie volume of respirution chambers amounts to 80 ml for small iii luta anti $00 ail for larger ones. Technical details of the apparatus and nie tint procedures are desrtrihed. '.I'he accuracy anniunts to 0.5 p.i 0 .: for the 80 ml chambers, and ..2 pi O. for the 300 nil chambers. The .apparatus has been successfulf ■- used on variais marine evertebrates and algae. .

(p.336) Introduction

In recent years a number of pepers have been published on

possible ways of measuring the respiration of aquatic organisms.

Aside from the papers by WESEMEIER, 1960, STEEN & IVERSEN, 1965 and

DAVIES, 1966, in which refined manometric methods were used, the bulk

UNEDITED TRANSLATION

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2.

of the publications dealt wi.with studies in which the principle of

electrochemical oxygen measurement using membrane-sheathed electrodes

was applied. BIELAWSKI, 1961; LENFANT, 1961; COURTNEY & NEWELL, 1965;

SCHRAMM, 1966; ERIKSEN & FELDMETH, 1967; PLATZER, 1967.

w. .

M+rane-sheathed electrodes are used in open or closed systems.

There are various technical versions of both systems. In the case of

the apparatus described in this paper we decided to employ the clo'sed

system. In doing so, however, we have not adopted the method used by

COURTNEY & NEWELL (1965) (measuring oxygen consumption in a closed

vessel), instead we have based our apparatus on the method of the closed

circulation system. (LENFANT, 1961; SCHRAMM, 1966; ERIKSEN & FELDMETH,

1967). For ecological experiments this method has the important

advantage that measurements can be carried out in flowing media.

addition, the method selected had to satisfy the following conditions

for the tests which we intended to perform:

1. A large volume of water was required in order to prevent the oxygen

concentration dropping below the ecological limit and to avoid any

influence from metabolic products during a measurement period.

2. Regulatable flow velocity of the medium.

3. Ability to provide the respiration chambers with various substrat es..

4. Opportunity to observe the organisms at any time during the test.

5. Maximum accuracy and minimum I9,'Pa^9 delay in the instrumentation.

6. Continuous,recording of measurements.

7. Statistically adequate number of synchronous parallel measurements.

3.

While we were in the course of constructing our apparatus the

paper by SCHRAMM (1966) appeared.. The measuring device employed by him

meets many of the stated requirements. But it would probably be an.

excessively complicated technical matter to conduct synchronous series

of measurements with SCHRAMM's apparatus. One would have to use at

least 6 electrodes. With our apparatus, as described in this paper,

parallel series of measurements can be made with only one electrode.

Apart from the fact that it is relatively uncomplicated, our apparatus

has other advantages to offer.

Design features

The apparatus (Fig. 1) consists of the electronic measuring

components and the testing stage proper. The electronic measuring

system comprises the measuring cell (platinum-silver electrode, Clark

principle), a measuring amplifier and a Zeiss universal potentiometric

recorder (the measuring amplifier and measuring cell were manufactured

by the firm of Metra/Radebeul).

The testing stage itself is located in a water-filled plastic

tank with a clear see-through front panel (60 x 50 x 20 cm). On the

bottom of the tank is located the heating system as well as the cooling

coil which can either be connected to a refrigeration unit or to the

water mains. A horizontal stirrer is used to achieve full circulation

in the tank. A constant temperature is one of the most important

conditions for the electrochemical method of measuring oxygen and this

is attained by allowing the cooling system to run all the time while

4.

simultaneously applying the minimum possible amount of heat (low

inertia heating elements controlled via contact thermometers and

thermionic.relays). The two . easy-to-remove roller pump systems are

mounted ou the back of the tank. Six respiration chambers, one control

chamber and the rotating-drum respirometer are mounted on an adustable

PVC grid. This arrangement permits the entire testing stage to be

removed from the tank in sections. The entire set-up is operated and

controlled from one switching unit.

Respiration chambers:

Various chambers have been made of Plexiglas for tests with marine

invertebrates (Fig. 2). For measuring the respiration of large bivalves

(species of the geiera Mytilus, Mya), chambers.of 300 ml volume are

used. For measurements on smaller creatures (for example, species of

the genera Macoma, Cardium), 80 ml chambers are used. The inlet-outlet

tube connections are located on one wall of the chamber. The covers,

also made of thick Plexiglas, are machined so that their inner surfaces

have a conical configuration to prevent air bubbles forming in the

water in the chamber. They screw in tight with a few turns and the

final seal is made by an 0-ring. A size,14 ground joint is provided

in the centre of the cover to accept a special thermometer. The chambers,

with the creatures in them,arenot fully closed, and measurements cannot

commence, until the thermometers have been inserted. The control chamber

is constructed in the same way.

(p. 33

0

—4

coil cooling

1—

C 0

0 0

o

Mwaa

967

/1.1.■

Figure 1: Overall diagram of the .apparatus. 1. measuring amplifier •

switching unit recorder cooling unit with roller pump respiration chaMbers rotating-drum réspirometer 02 electrode

contact thermometer

5.

2. 3. 4. 5. 6. 7. 8. 9.

Figure 2: Respiration chamber. 1. thermometer (ground fit) 2. cover 3. chamber housing 4. inlet/outlet connections.

6.

Roller pumps:

The roller pump operates on the principle of the circulation pumps

in heart-lung machines and artificial kidneys (Fig. 3).

A regulatable ac motor (approx. 20 kp/cm) drives an adjustable

roller system (30 to 80 rpm) acting on 4 tubes. Depending on the

speed of rotation and the roller pressure selected the flow rate can

be varied between 0 and 0.5 m/sec.. This is important when it comes

to studying the influence of the flow rate.

Rotating-drum respirometer:

The respirometer permits the p02 to be determined, using an electrode,

in 7 chambers with minimum time loss (measuring duration approx. 45 sec.).

The r:espirometer is also made of Plexiglas and consists of two parts

(Fig. 4). There are seven bores arranged in a circular pattern in the

housing. The diameter of these bores is matched to the radius of the

electrode. An inlet and outlet port leads to and from each of the

bores. The ports are so arranged that the water impinges directly

against the head of the electrode. The measuring cell is removable

and it is mounted in a PVC cylinder in the revolving drum of the

respirometer. The seal is made by a cap nut and 0.1 mm thick rubber

gasket. The rotating drum is pressed firmly face to face against the

floor of the housing by a cap nut and spring. The bore which accepts

the measuring head of the electrode is made in such a way that, firstly,

the membrane-sheathed.cathode surface is situated only 50 Pm above the

floor of the housing and, secondly, the bores align precisely during

the measuring phase. The amount of water entrained at the measuring

(.p.338'

7.

lead from onechamber system.to the other'when the drum is rotated is

so small (2. pl) that,.in View of the large chamber volume, it does not

affect the'measurements. This was proved in tests with gas-equilibrated

water. The'respirometer is connected to the respiration chambers and the

• roller punps by a tube system (rubber tubing, 5 mm dia.).

Figure 3: Roller punp. 1. housing , 2. tubes 3. PVC rollers 4. knurled pressure nut 5. linkages controlling roller pressure setting

Figure 4: Rotating-drum respirometer1. 02 electrode2. rotating drum3. housing

handlé (for rotating the drum manually) with lockingin engagement

cap nut

Functinn and calibration

.:o, .The roller pumps generate a smooth, even water:flôw;.in a

closed circuit. The water travels from the pump into thë*chamber where

it circulates, from there it moves to the respirométer where it flows

past the measuring cell and from there it returns to the pump.

The measurements are taken in the following way: - The cfiâmbérs

and the tube'system.are filled by a water jet pump with normally

saturated, sterile sea water at a certain temperature and free from air

bubbles. Once the.test temperature has been,reached, the test organisms

are placed in the six respiration chambers. The control chamber remains

unoccupied. The chambers are closed and the chamber system is lowered

into-the -tank for .the. final. temperature : to be set and for the animals

to adapt..-themaelves:.:.to_:^the:' condi.tions. At the same time, the pumps

and-the measuring..,amplifier are'switched on. After the organisms have,

become adapted::(the tim^ required.depends on.the test conditions)

the thermometers-'-are::.inserted and after a.few minutes the arbitrary

zero.measurement.'.can be made.. To do this the electrode is rotated

clock-wise,from chamber.-.to chamber at 45 sec. intervals. The p02 in

the various:.chambers.is recorded by the measuring amplifier and the

recording.device... The recording procedure, which also includes

measurement of the.p02 in the control chamber, can.either be carried out

in.approximately continuous manner or - and this is what.we did in most.

cases - it,can be adjusted for hourly intervals. In this latter case

the electrode remains switched to one chamber system for.one hour while

the time course of the test creature's oxygen consumption is continuously.

recorded. Switching of the respirometer to the next chamber system

can be automated.

Evaluation takes the form of reading off the difference between

the arbitrary zero and the measurement taken.after the lapse of a

certain period of time and correcting the reading by applying the control

chamber value.

Calibration of the measuring cell is effected under test

conditions at the beginning of a test and it is accomplished by the

Winkler method. When large-respiration chambers are.used one 50 ml.

flaEkper chamber is filled using a thin glass syphon (inserted through

ground joint for the thermometer). In the case of the small Chambers,.

the set-up is calibrated by applying the microWinkler method after

FOX and WINGFIELD, 1938.

Gradation of the oxygen content is achieved by feeding in pure

N2. Unlike in the Winkler method, the oxygen content is determined

photometrically not titrimetrically (SCHULTZE, 1965). Since a linear

relationship exists between p02 and diffusion current a straight

calibration line can'be drawn up for eaCh test temperature. The

gradient of the straight line can be used directly as a conversion

factoTo check this factor - it is sufficient, within a series of tests, "

- 5tocatlit,one calibration measurement before each test is commenced:

LAccuracYand Applicability of the Method

.The measuring accuracy of our apparatus depends on the -:telat . _

temperature fluctuation, the read-out accuracy of the indicating instru- •

ments, the drift.of the electrode and amplifier and the accuraCy of

the calibration. The latter is limited by the Winkler method (1 0.62%

standàr“eviation according tp GRASSHOFF, 1962). °

Temperature fluctuation aMounts to ± 0.020C and,

tests showed, it can be ignored for the purposes of them

procedure. The read-out accuracy of the recorder is 0.05 ;iA per

Calibration unit on the scale and permits good averaging, although as

a result of the high amplification and the pressure fluctuations which

occur the recording trace is not a line but a band approx. 5 mm wide

(p • 339 )

11.

(Fig. 5). The intrinsic.oxygen consumption of the electrode is

0.0224 1.11/0 th,.the drift_of the measuring amplifier.is approx. 0.1 pA/h

consequentlyloth - values can_be ignored for short-term'measurements.

Under these'conditions the apparatus can detect changes in oxygen

of 0.5 or 2 }.11 0 2 , depending on the chamber volume (80 ml or 300 ml

respectively).

The choice of material is also of decisive importance for the

measuring accuracy of the apparatus. A wide range of Opinions can

be found in the literature on the subject of plastics used for 02

measurements (LENFANT, 1961; STEEN and IVERSEN, 1965; SCHRAMM, 1966).

The plastics used so far are not inert vis à vis oxygen and

other gases. The same holds true for a very large range of tubing

material. Since glass or stainless steel are not ideally suited for

technical and methodological reasons for the simple construction of

efficient apparatus, we have, despite the attendant disadvantage, made

our apparatus of Plexiglas.

.The solubility of oxygen in Plexiglas is taken into account by

recording the "oxygen loss" via the control chamber readings. The

control chamber values are then deducted from the respiration value.

A large number of preliminary tests showed ihat this "oxygen loss" was

the same. for all chambers. •

The poor thermaL conductivity of Plexiglas may be regarded as

an advantage.because once the test temperature has been reached the

• plasti c . acts as a very good heat barrier. The Measuring delay occasioned

12.

by the length of the-tubing system is slight and.amounts on average

to 20 sec. The entire system is cleaned between individual measure-

ments by means.-.of . a fine disinfettant (Fesiamon) and subsequent flushings

with Aqua destillata. After . a test series is concluded, the individual

parts are cleaneein warm, neutral detergent solution, placed for a

time in Aqua destillata and then stored dry.

For experiments lasting a long time it has proved of advantage,

to add a little streptomycin (30 to 50 mg/i, PARANJAPE, 1967) to the

test water to inhibit any bacterial growth that would falsify the

measurements.

The apparatus described in this paper is universally suited for

respiration or assimilation measurements on large and small aquatic

invertebrates, small aquatic vertebrates and aquatic plants. Only the

volume (and perhaps the shape) of the respiration chambers needs to

be adapted to the size of the test subjects. It should be no,ted that

for chamber volumes Over 300 ml a stirrer must be installed in the

chamber to guarantee full circulation. It is quite possible to use

other electrode systems. In this case the rotating drum of, the respiro-

meter need only be slightly redesigned.

In order to . test the verSatilityof the apparatus . we sucéessfully

carried out tests_with polychaetes (Harmothoe), bivalves (Cardium, adult

Mya), fishes-(Gobius), amphibians (Xenopus) and Rhodophycaeae (Delesseria).

-

13.

Figure 5: Original recording trace of the oxygen consumption ofCardium Lamarkii.(REEVE) - 84 mg dry weight at 15°C and15%. The respiration rhythm can be clearly seen.

1. each scale division = 0.5 p1 022. time in minutes

Summary

1. The respirometer described in this paper permits six parallel

measurements to be carried out with only one electrode.

2. The respiration measurements are carried.out in a closed system at

variable flow rates of the medium.

3.-Depending on the size of the animal, respiration chambers of different

volume are.used (80 or 300 ml). Further variations can be produced

withouttoo much expenditure of time and effort.

4. The sensitivity of the apparatus is dependent on the type of electrode

used and on the associated measuring amplifier. It is 0.5 N1 02

for the 80 ml'chambers and 2 pl 02 for the 300 ml chambers.

5. The wide versatility of the.apparatus has been proved on pôlychaetés,

bivalves and fish, also amphibians and algae.

14.

We should.like.to thank Prof..Dr, E.A. ARNDT for his critical

appraisal of.the manuscript. We.are particularly indebted to the

workshop of the-Physics Department (especially to foreman SASS) as

well to the.central workshop (especially foreman STAVE) for technical

support.

15.

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Studies ôri:.oxygen determination'^^n.s'éâ'water.. ^; :•;^.^-:-

. Continuous measurement of assimilation and respiration;.:of:;marine

algae using the electrochemical method of oxygen detërmina'tion.

.of Chironomidae ( Chironomus strenzkei) (FITTKAU).Studies of the temperature adaptation of the tropical species

4. Contributions on the:subject of temperature adaptation in the eel.

5. Studies of metabolic reduction.

It.