Toxicity testing with synchronized cultures of the green alga Chlamydomonas

Chemosphere No. 3, PP 231 - 245, 1978.

Pergamon Press.

Printed in Great Britain.

T O X I C I T Y T E S T I N G W I T H S Y N C H R O N I Z E D C U L T U R E S OF THE G R E E N A L G A CHI2%MYDOMONAS

Svein Norlanda!

M i k a l Heldala!

T o r l e i v Lien a)

and G j e r t Knutsenb! a) I n s t i t u t e of G e n e r a l M i c r o b i o l o g y .

b) Botani-

cal Laboratory. U n i v e r s i t y of Bergen,

All~gt.70,

5014 N Bergen

Norway. (Received in The Netherlands l March 1978; received in UK ~rpublication 13 March 1978) Introduction

An i n c r e a s e d use is seen of b i o a s s a y s w i t h m i c r o a l g a e for the d e t e c t i o n of s u b s t a n c e s w h i c h are toxic to a l g a e qua p r i m a r y producers.

In b i o a s s a y the alga

senses q u a l i t a t i v e l y and q u a n t i -

t a t i v e l y the c o m p o s i t i o n of its m i l i e u through i n c o r ~ i n g c h e m i c a l and p h y s i c a l

signals.

These signals i n f l u e n c e one or m o r e of the

m e t a b o l i c p a t h w a y s in the organism,

and the more the a f f e c t e d

p a t h w a y s are i m p o r t a n t to the d r i v i n g of the o r g a n i s m through its life cycle or to the m a i n t a i n a n c e of the total life process,

the

e a s i e r can the effect be d e t e c t e d in the o r g a n i s m or in the culture as a whole.

By d e t e c t i n g effects on single m e t a b o l i c or

m o r p h o l o g i c a l processes,

e.g. active

uptake of compounds,

and d e r e p r e s s i o n of e n z y m e synthesis,

induction

cell m o t i l i t y or division,

the cell s y s t e m can act as a m u l t i t r a n s d u c e r

instead of being only

a general t o x i c i t y sensor. A practical rate and simple,

test must be rapid,

reproducible,

sensitive,

accu-

and it m u s t be based on t h o r o u g h l y tested cell

systems so that a c c u r a t e c o n c l u s i o n s can be

drawn from the results

In this paper we shall o u t l i n e the m a i n features of the s y n c h r o n o u s culture of C b l a m y d o m o n a s r e i n h a r d t i and as an example show its a p p l i c a t i o n on one spill c l e a n i n g chemical.

231

232

No. 3

Methods

Chlam~domonas reinhardti tions at Cambridge,

England)

nitrate as N - s o u r c e I.

(no i1/32 b from the Algal Collec-

were grown in mineral m e d i u m w i t h

The c u l t u r i n g tubes, w i t h 40 mm inner dia-

m e t e r and conical bottoms,

u s u a l l y c o n t a i n e d 250 ml of culture.

An a e r a t i o n tube e x t e n d e d to the b o t t o m through a p o l y p r o p y l e n e c o v e r i n g cap.

The c u l t u r i n g tubes were s t a n d i n g in a glass-

w a l l e d w a t e r bath w i t h r o o m for 12.

E x p e r i m e n t s had shown that

all p o s i t i o n s were equal w i t h respect to g r o w t h and d i v i s i o n 2. The cells were s y n c h r o n i z e d by r e p e t i t i v e light dark shifts

(12 h light and 4 h dark),

a e r a t i o n w i t h 2% CO 2 in f i l t e r e d air.

at 35 ~ 0.1°C and

I l l u m i n a t i o n was by 3

Philips Y e l l o w White de Luxe and 2 D a y l i g h t f l u o r e s c e n t tubes, g i v i n g 20 K l u x at the front of the c u l t u r i n g

tubes.

D e t e r m i n a t i o n of cell n u m b e r and volume. were m e a s u r e d w i t h a Coulter analyzing system

These p a r a m e t e r s

Z B - C h a n n e l y z e r C 1000 p a r t i c l e volume

(Coulter Electronics,

England)

c o n n e c t e d to a

C o m p u c o r p 445 c o m p u t e r via a M e t r i c D a t a c o l l e c t o r (Metric Scandinavia,

Norway).

D e t e r m i n a t i o n of dry weight,

400 i n t e r f a c e

Other a n a l y t i c a l methods protein,

were:

RNA and DNA 3'4

Results and d i s c u s s i o n

G e n e r a l l y about s y n c h r o n o u s c u l t u r e s

The usual

l a b o r a t o r y c u l t u r e s of m i c r o a l g a e

g r o w t h phase c o n t a i n all stages of development.

in the e x p o n e n t i a l In c o n t r a s t the

cells of a s y n c h r o n o u s culture are in phase w i t h r e s p e c t to their life cycles.

The life cycle is d e f i n e d as the c e l l u l a r d e v e l o p -

m e n t d u r i n g the time period b e t w e e n d i v i s i o n s

(generation time),

and w h e n cells go through their life c y c l e s cme p r o c e s s e s d e v e l o p gradually,

e.g. growth,

Within experimental

and other abruptly,

e.g.

limits s y n c h r o n o u s c u l t u r e s

DNA synthesis. scale up the events

taking place in the i n d i v i d u a l cell as it goes through its life cycle.

S a m p l i n g and a n a l y z i n g such cultures at the m a c r o level

thus give i n f o r m a t i o n p e r t i n e n t to the single cell at its different d e v e l o p m e n t a l

stages.

No. 3

233

S y n c h r o n o u s cultures of C h l a m ~ d o m o n a s r e i n h a r d t i

A number of algae have b e e n s y n c h r o n i z e d and r e f e r e n c e s

to

the more r e c e n ~ l i t e r a t u r e are given by Zeuthen and c o w o r k e r s 5, and by L o r e n z e n and Hesse 6 . Chlamydomonas

is a unicellular,

w h i c h has b o t h a v e g e t a t i v e

biflagellated,

anda sexual life cycle

green alga (figure i).

The d e v e l o p m e n t of the former cycle is in short a p a s s a g e from a biflagellated

zoospore to a cell w h i c h goes through two or more

s u c c e s s i v e nuclear and c y t o p l a s m i c divisions.

Figure

1.

S c h e m a t i c drawing of the v e g e t a t i v e

life cycle of C h l a m y d o m o n a s .

The number of the d i v i s i o n s d e p e n d s on g r o w t h conditions. The 4 or more

(multiples of 2) new zoospores are c o n t a i n e d w i t h i n

the o r i g i n a l cell wall,

thus m a k i n g a sporangium.

After c o m p l e t i o n

of the spores they are r e l e a s e d from the s p o r a n g i u m upon its enzymic dissolution.

This release is c a l l e d sporulation.

Synchroni-

zation is a c h i e v e d by the I n t e r m i t t a n t I l l u m i n a t i o n / C u l t u r e Dilu-

234

No. 3

tion p r i n c i p l e Chlorella.

i n t r o d u c e d by L o r e n z e n for the s y n c h r o n i z a t i o n of

The c u l t u r e s are i l l u m i n a t e d intermittantly,

p r e s e n t case w i t h a r e g i m e of 12 h light/4 h of dark.

in the

At the end

of each d a r k p e r i o d the culture is d i l u t e d w i t h fresh m e d i u m to a s t a n d a r d cell number.

W i t h this m e t h o d s y n c h r o n i z e d c u l t u r e s

of C h l a m y d o m o n a s h a v i n g h i g h p r o d u c t i v i t y and r e p r o d u c i b i l i t y can be o b t a i n e d and m a i n t a i n e d for weeks. The m o s t c o n s p i c u o u s

feature of a s y n c h r o n o u s c u l t u r e is the

s t e p w i s e i n c r e a s e in cell number.

F i g u r e 2 shows how the cell

n u m b e r v a r i e d d u r i n g three d i f f e r e n t cycles of a series.

' -II

i I I

2 4 -: n

' I ~,--A

i I

C-'

-I 20

......

For all

'

I

I

' i I I

I -i

,s -I

I

t ~. 12 -i ,

I I

u c o ~-

I I I I @

-

8 4

-I I -I -~ ..-~

..j,

_-

- -

0

I

I

I

0

I

4

I

8

;

4,

I

1

12

I

~

0

4,A

.A~......-A.---k

- -

e

I

l

4

i

i

8

, 'I

12

I

0

"-- ¢ "-- ± ±_.jIL

I

I

4

e

e

8

a

a

I

12

I

16

Hours

F i g u r e 2. Cell numbers t h r o u g h o u t three d i f f e r e n t cell cycles of a series and w i t h one cycle b e t w e e n each.

three the cell number was c o n s t a n t for ii h and w i t h a slight increase d u r i n g the f o l l o w i n g half hour.

Then the rate of sporula-

tion i n c r e a s e d a b r u p t l y and w i t h i n the two next hours all the cells had sporulated. mination,

Mean s p o r u l a t i o n times from the onset of illu-

u s u a l l y d e f i n i n g the start of a cycle, were 12.5 ± 0.4,

12.4 ~ 0.3 and 12.4 ± 0.4 h r e s p e c t i v e l y . is thus sharp and v e r y r e p r o d u c i b l e

The s p o r u l a t i o n s y n c h r o n y

from one cycle to another.

The

No. 3

235

r e l a t i v e p r o g e n y numbers were for the same cycles

16, 17 og 16.

The a v e r a g e spore number d e p e n d s s t r o n g l y on the t e m p e r a t u r e and the s t a r t i n g cell density,

for instance at 35 ° w i t h 5 • 104

cells/ml an average of 30 spores per s p o r a n g i u m is p r o d u c e d in contrast to the a b o v e m e n t i o n e d 16 w i t h 1.4 m i l l i o n cells per ml. Figure

3 shows time courses for dry weight,

w e i g h t e d average cell volume.

20

A

~

18

total p r o t e i n and

All three f u n c t i o n s were c o n t i n o u s

B

C

J ~o..i-,

o

12

o

;

o/~

.

•

>

~P~--J

/i

o

-~

2

~

0

~---o I

0

I 4

I

I 8

I

z 12

I

I 16 0

I 4

8

I 12

~r~

I

I

16 0

I 4

I

I 8

i

I

z

12

z 16

Hours

Time courses of dry w e i g h t (frame B) and total p r o t e i n

(frame A), average w e i g h t e d volume (frame C) of three cycles of a series

w i t h one week b e t w e e n each cycle.

d u r i n g the whole light period.

During the first part of the dark

the dry w e i g h t per culture volume d e c r e a s e d to reach a c o n s t a n t level,

the d e c r e a s e m o s t p r o b a b l y being due to the d i s s o l u t i o n

of the s p o r a n g i u m w a l l d u r i n g sporulation. No p r o t e i n a c c u m u l a t e d

in the dark.

The w e i g h t e d average

cell volume n a t u r a l l y d e c r e a s e d during sporulation,

r e a c h i n g the

236

No. 3

same v a l u e

as that of the

The v o l u m e data, were

while taken

from s t u d e n t

Three

turing

stock culture, one c y c l e

cultured

analyzed

number.

from v o l u m e

on three

and s i m p l i c i t y no p r e v i o u s

the f o l l o w i n g

Cells were

as d e s c r i b e d

taken

distributions

average

The data

laboratory

course,

of the cell experience

experiment

in the M e t h o d s

cell v o l u m e s

of the kind

sets of

sys-

in cul-

under

from an a s y n c h r o n o u s

for the three a b o v e m e n t i o n e d

The w e i g h t e d

cycle.

on two sets.

of a g r a d u a t e

with

a l g a e did

apart:

are b a s e d

is b a s e d

protocols

of s t u d e n t s

one week

of the p r o c e e d i n g

curves curve

the r e p r o d u c i b i l i t y pairs

and a n a l y z i n g

guidance,

cell

and p r o t e i n

the dry w e i g h t

illustrating tem.

zoospores

shown

section

parameters were

and and

computed

in figure

4.

1200 I

800 I.)

"6 600 400 ~'

,

it 0

~ 0

i 200

I

I 600

I

I I000

I

I 1400

I

I 1800

I

I 2200

I

I 2600

pm 3

Figure

4.

Volume

distributions

cell

cycle.

The

respectively,

after

in each d i s t r i b u t i o n full

scale"

mode

registered

times

at d i f f e r e n t

of s a m p l i n g

were

the s t a r t of illumination. differ

greatly

of the Channelyzer.

due

times

throughout

a

0, 4, 8 and 12 hours The total

to use of the

counts

"stop

at

No. 3

237

It should be noted that the volume d i s t r i b u t i o n of zoospores and s p o r a n g i a

(0 and 12 h, respectively)

were c o m p l e t e l y separated,

m e a n i n g that the s m a l l e s t cell before s p o r u l a t i o n was larger than the largest one after. Finally,

time courses of RNA and DNA

by the d i p h e n y l a m i n e

method as

(DNA being d e t e r m i n e d

well as by the f l u o r e s c e n c e m e t h o d

14 12 I0 ~"

/

0

®

/"

4

0

•

0 [

I

i

l

I

0

2

4

6

8

I

I

I0

12

l

14

I

16

Hours Figure

5.

Time courses of RNA amine m e t h o d e acid

(m) and DNA.

DNA was a n a l y z e d w i t h the d i p h e n y l -

(o) and the f l u o r e s c e n c e m e t h o d w i t h d i a m i n o b e n z o e i c

(A).

w i t h d i a m i n o b e n z o e i c acid)

are d e p i c t e d in figure 5.

While RNA

a c c u m u l a t e d c o n t i n o u s l y d u r i n g the w h o l e light p e r i o d and w i t h a t e m p o r a r y d e c r e a s e in a c c u m u l a t i o n rate b e t w e e n 8 and 9 h, DNA s y n t h e s i s was stepwise.

No RNA or DNA a c c u m u l a t e d in the dark.

The a m o u n t of DNA was c o n s t a n t for the first 8 h of the cycle, w h e r e upon it i n c r e a s e d almost 16-fold d u r i n g the next 4 h.

D u r i n g that

2~

No. 3

time

an a v e r a g e

of 4 s u c c e s s i v e

also

occurred.

DNA s y n t h e s i s

about

25 % of the cell

cycle.

With

cultures

of the

synchronous

life cycle The

indeed high

to manage,

productivity

to the c u l t u r e s into

we have

here,

p o i n t of view,

is simple

and c y t o p l a s m i c

divisions

thus o c c u p i e d

explored

several

only

aspects

of C h l a m y d o m o n a s 7'8'3'4

few data p r e s e n t e d

a cell b i o l o g y

nuclear

and cell d i v i s i o n

are not d i s c u s s e d

the p r e s e n t

reproducible

of the cells

are m u c h

and w h i c h

show that

cell

and accurate.

negative

effects

easier measured

from

system Due

to the

on them by a d d i t i v e s

than w i t h cells

dividing

two. Furthermore

plasmic tested

effects

divisions than w i t h

at d i s t i n c t

or other cells

times

when

cells

time w h e n

is such

growth

cells

around

system,

toxicity added

specifically

since

they a p p e a r

The r e s o l u t i o n

the v o l u m e

analyzing

detected

part of the cycle

are used,

than

in

system

within

B a s e d on protein, is less

the s u i t a b i l i t y

we p r e s e n t

of the oil d i s p e r s a n t

The

suspension,

15 min

and in less dry w e i g h t

this.

of

150 m m lenght

and w i t h

illuminated

at 35 ° and

of the cells

as the process

we used a v e r a g e

of the

The c o m p o u n d

was

at 0 h of a s y n c h r o n o u s

in test tubes

tubes of 20 m m d i a m e t e r

extending

to the bottom,

20 Klux.

from the

start of the cell

for r e g i s t e r i n g

weighted

9527.

zoospores

aeration

of the Chlamy-

from the testing

Corexit

contained

and

Growth

and p o t e n t i a l

the results

were

chosen

cultures,

cultures.

6 h are used.

to 25 ml s u s p e n s i o n s

culture.

can be much more

that w i t h

the r e s o l u t i o n

and cyto-

applications

To i l l u s t r a t e domonas

nuclear-

can be s i g n i f i c a n t l y

from the e a r l i e r

and RNA m e a s u r e m e n t s

Practical

processes,

in a s y n c h r o n o u s

in the s y n c h r o n o u s

time of the s y s t e m w h i c h we employ,

on DNA synthesis,

cell

volumes

the effect, obtained

and

cycle was for this

from v o l u m e

distri-

butions. The v o l u m e were we

followed

found

that

distributions

over

of cells

the i l l u m i n a t e d

the cells were

either

in d i f f e r e n t

part of the killed

concentrations

life cycle,

or they were

and

only

slightly

No. 3

239

'i

/ ' ~ CONTROL

,!~

~ DeADCELLS - -

,, '~

V

II

"i

i

LIVING

~,.3

126.7 ~3

CELLS

i

/~

400

Ppm

1211,7 ~3

[~ I

5 0 0 ppm

22.7 =,~

,

114,2 ~ 5

I

Ll

600

ppm

25,2 ~ 3

, I

i i i

116,5.M] 7 0 0 ppm

i

l'

18,5 ~,M3

I q I i

'i I

1

'

800

ppm

'

18,5 ~.3

I

-

100 UM3

i i

SIZE

Figure

6.

Volume

distributions

r e c o r d e d at 4 h of a s y n c h r o n o u s c y c l e

c u l t u r e s of d i f f e r e n t c o n c e n t r a t i o n s

of C o r e x i t

9527.

in

240

No. 3

affected vation

or not a f f e c t e d

that

a fraction

the rest b e c a m e

smaller

cells w h i c h w e r e persant. 15 min

Apart

after

at all.

than

affected

depended

the a d d i t i o n

cells.

from the obsernormally,

The

looked

the a f f e c t e d

within

cells

under

of

of the dis-

c o u l d be d e t e c t e d

very a b n o r m a l

while

fraction

on the c o n c e n t r a t i o n

which

of d i s p e r s a n t ,

and they

inferred

developed

the st a r t i n g

from shrinking,

their p i g m e n t a t i o n

This was

of the p o p u l a t i o n

lost

the m i c r o -

scope. This

pattern

from an e x p e r i m e n t from 400 to 800 tered

after

control

is i l l u s t r a t e d

in figure

with

concentrations

ppm.

different

The p r e s e n t e d

4 h of g r o w t h

in the p r e s e n c e

cell d i s t r i b u t i o n

starting

cells.

at 4 h is well

The average

~m 3 and that of the control As the c o n c e n t r a t i o n two cell p o p u l a t i o n s represents dent

living

than

became

apparent.

starting

as c o m p a r e d shows

The

about

The cells

from the

period,

all

zoospore

had an a d d i t i o n a l

the c o n t r o l of 49,3

cells.

This

towards

cells w h i c h

in size is evi-

smaller

increases

with

are smaller

volume

than 3 of 50,0 ~m

ones.

Figure

7

at the end of a dark

8 h of light plus

above.

50,0

from 400 ppm,

The peak w h i c h

of the culture

The

cells was

The one d e c r e a s i n g

latter had an average

4 h stage d e s c r i b e d

volume

increased

20 ~m 3 for the a f f e c t e d

the size d i s t r i b u t i o n s

period. dark

cells. with

regis-

127 ~m 3.

affected

is due to the a f f e c t e d

9527

were

from that of the

starting

of the peak

culture.

results

of the d i s p e r s a n t . separated

of the

slightly

shows

of C o r e x i t

distributions

at 4 h was

of C o r e x i t

and only

the control

concentration the

volume cells

from the slight d i s p l a c e m e n t

volumes

volume

6, w h i c h

4 h of

At the end of the dark

cells had s p o r u l a t e d g i v i n g an average 3 ~m and an a v e r a g e p r o d u c t i o n of p r o g e n y

of 20,5 per sporangium.

Three

features

i)

All

of this

the cells

the end of the dark butions The

when

final

figure

shall be noted:

at 4 h w h i c h period.

This

they are c o m p a r e d

number

of cells

had not shrunk is e v i d e n t

with

had

sporulated

at

from the size distri-

that of the c o n t r o l ' c e l l s .

in the c u l t u r e

decreased

with

increasing

No. )

241

~ >" Z W w

"

TROL

l~ ~

ppm

pm

ppm

~

=

49,3 ~M3

N

=

32,8 ' 106 CELLS/ML

X

=

20,5

N

"

28,5 ' 106 CELL$/ML

X

=

19.5

7

52,6 ~M3

N

24,5 ' 106 CELLS/ML

X

=

7

=

20,3

53.7 .M3

N =

21,9 ' 106 CELLS/ML

X

=

26

~

=

58,8 ~M3

N

=

x -

6,2 ' 106 CELLS/ML

30

800 ppm =

60.8 .e 3

N =

1,q , 106 CELLS/ML

X --

30

SIZE Figure

7.

Volume

distributions

Figure

6.

at

16 h o f

the

same

cultures

as d e s c r i b e d

in

242

No. 3

concentration. trations

The d i s t r i b u t i o n p a t t e r n s of the two h i g h e s t c o n c e n -

clearly

show

c e l l s and the o t h e r each culture

two peaks,

one c o r r e s p o n d i n g

to the n e w z o o s p o r e s .

to the n o n g r o w i n g

The total cell n u m b e r of

is t h e r e f o r e a s u m of the new s p o r e s and the a f f e c t e d

cells.

2) higher

The a v e r a g e n u m b e r of p r o g e n y per s p o r a n g i u m was in the 600 - 800 p p m c u l t u r e s

significantly

than in the c o n t r o l

and the

400 - 500 p p m c u l t u r e s .

3)

Similarly

the a v e r a g e v o l u m e of the n e w z o o s p o r e s w a s also

l a r g e r at the h i g h e s t c o n c e n t r a t i o n s .

The e f f e c t s on p r o g e n y n u m b e r and size m i g h t be i n t e r p r e t e d as a s t i m u l a t i o n of c e l l u l a r p r o c e s s e s .

However,

p r o b a b l y due to the e s t a b l i s h i n g of m o r e

favourable growth condi-

tions

in the c u l t u r e s w i t h h i g h e s t kill.

t h e y are m o s t

The l a r g e r the p r o p o r t i o n

of the c u l t u r e w h i c h dies e a r l y in the

life cycle,

cal p r o p e r t i e s

l i v i n g cells

exist

rest of the cycle. number

for the r e m a i n i n g We h a v e

shown that l o w e r i n g

f r o m the one w h i c h we use as standard,

in p r o g e n y

the b e t t e r o p t i throughout

leads

to i n c r e a s e

size.

95 o

8O

5O J

~o J

,

,

400

Figure

8.

Probit

plot

for

Corexit

i

500

9527

i

i

600

of

700

lethality

800 ppm

versus

the

the s t a r t i n g cell

concentration.

No. 3

243

Toxicity

was

evaluated

4 h.

We c a l c u l a t e d

tion,

and

rithme

from size d i s t r i b u t i o n s

the p r o p o r t i o n

from p r o b i t

plots

of p e r c e n t

of the c o n c e n t r a t i o n ,

50 % lethality.

A representative from it a LC-50

the

line of b e s t

fit

(linear

plot

registered

versus

the loga-

the c o n c e n t r a t i o n

for C o r e x i t

9527

giving

is shown

of 575 ppm can be e s t i m a t e d

regression

at

at each c o n c e n t r a -

lethality

we d e t e r m i n e d

in figure

8, and

of dead cells

using

analysis).

Conclusion

i)

~domonas

is one of the best

physiology,

2)

The cells which

on these cells. and cell

effects

4)

number

on g r o w t h

and more

3)

important

accurate

The cells

The o r d e r e d nuclear

cycle,

can be studies

sequence

at more

of m a j o r

detected,

dividing

with

binary.

simple m e a n s

assuring

and

that an

16 hours.

b o t h using

processes

division,

continuous

as well

on g r o w t h

can for i n st a n c e

effects

on D N A - s y n t h e s i s , on sexual

on v e g e t a t i v e

division

time points

of o t h e r

in the cell

processes

asynchronous

of a

cultures.

be d i s t i n g u i s h e d or spore

reproduction

development.

like D N A - s y n t h e s i s ,

the a p p e a r a n c e

of the t a r g e t

than w i t h

Effects

effects

than

or less d i s t i n c t

less d i f f i c u l t

larly effects

should be e a s i e r

short,

its

one g e n e r a t i o n ,

cultures.

make the e l u c i d a t i o n

toxicant

less

cultured,

of a test

increases

during

in cells

is r e l a t i v e l y

and c y t o p l a s m i c

processes

than

autotrophically

test takes

as s e m i c o n t i n u o u s

5)

and d e v e l o p m e n t measured,

acute

effects

regarding

reproducibly

Since Chlam~domonasj

time

Chronic

and very

up to t h i r t y - f o l d

are g r o w i n g

algae

and u l t r a s t r u c t u r e .

for the r e p r o d u c i b i l i t y

the g e n e r a t i o n toxicity

studied

genetics

are h o m o g e n e o u s l y

is m o s t

based mass

biochemistry,

from

release.

can be s e p a r a t e d

Simifrom

244

No. 3

6)

The i m p o r t a n t fact about the mode of action of this c o m p o u n d (which also holds for several others tested)

that it affects

some cells of the p o p u l a t i o n and leaves the rest s e e m i n g l y unaffected,

could hardly have been r e v e a l e d w i t h o u t syn-

c h r o n o u s cultures. such c u l t u r e s

This again illustrates

for t o x i c o l o g i c a l

the p o t e n t i a l of

research.

Acknowledgement

This r e s e a r c h has p a r t l y b e e n supported by The N o r w e g i a n R e s e a r c h Council for Science and the H u m a n i t i e s and by The Norwegian Fisheries appreciated.

R e s e a r c h Council.

Their support is g r e a t l y

We also w i s h to thank Mrs J o r u n n A k e r v o l l

for typing

the manuscript.

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