You are on page 1of 12

Marine Biology 36, 61-72 (1976)

9 by Springer-Verlag 1976

Waste-Water Assay with Continuous Algal Cultures:


The Effect of Mercuric Acetate on the Growth of Some Marine
Dinoflagellates
H. Kayser

Biologische Anstalt Helgoland; List, Sylt, Germany (FRG)

Abstract

The e f f e c t of m e r c u r i c a c e t a t e was s t u d i e d in c u l t u r e e x p e r i m e n t s w i t h the dino-


f l a g e l l a t e s Scrippsiella faeroense (Paulsen) B a l e c h et Soares, Prorocentrum micans E h r e n -
berg and Gymnodiniu~splendens Lebour. I m p a i r m e n t of g r o w t h rates, in vivo c h l o r o p h y l l
f l u o r e s c e n c e , m a x i m u m cell d e n s i t i e s and m o r p h o l o g i c a l c h a n g e s s e r v e d as c r i t e r i a
for a s s e s s i n g s u b l e t h a l i n f l u e n c e s . T e s t s w e r e m a d e using the b a t c h - and c o n t i n u -
o u s - c u l t u r e t e c h n i q u e s . A d d i t i o n of Hg at c o n c e n t r a t i o n s of O.OO1 mg.1 -I and h i g h e r
r e s u l t e d in r e d u c t i o n of r e l a t i v e g r o w t h rates. In a few cases p o p u l a t i o n s r e c o v -
ered f r o m the i n i t i a l d e c l i n e and s h o w e d new growth. Cell counts c o r r e s p o n d e d v e r y
c l o s e l y to in ~ v o chlorophyll fluorescence measurements. Morphological variations
w e r e o b s e r v e d in S. faeroense, w h i c h r e s p o n d e d (even in s u b l e t h a l c o n c e n t r a t i o n s ) by
b u r s t i n g it's thecae, r e l e a s i n g n a k e d m o t i l e cells and f o r m i n g v e g e t a t i v e r e s t i n g
stages. The p r o b l e m s of o p t i m a l a l g a l - b i o a s s a y m e t h o d s are d i s c u s s e d also, in the
l i g h t of r e s u l t s o b t a i n e d by o t h e r authors.

Introduction m e d i u m ~ The a d d i t i o n of the toxic a g e n t s


can take p l a c e c o n t i n u o u s l y t o g e t h e r
Rearing experiments using marine plank- w i t h the m e d i u m inflow. Thus, as o p p o s e d
ton algae are u s e f u l for the i n v e s t i g a - to the b a t c h - c u l t u r e method, the algae
tion of toxic w a s t e s and w a s t e p r o d u c t s . are e x p o s e d p e r m a n e n t l y to the i n f l u e n c e
E v e n in s u b l e t h a l c o n c e n t r a t i o n s , im- of fresh, running, w a s t e water. The ex-
p a i r m e n t of g r o w t h rates, cell d e n s i t i e s p e r i m e n t a l time is t h e o r e t i c a l l y u n l i m -
and m o r p h o l o g i c a l or p h y s i o l o g i c a l p e c u - ited, as long as the c o n t r o l c u l t u r e
l i a r i t i e s of the cells can b e c o m e evi- m a i n t a i n s it's e x p o n e n t i a l growth. T h e r e -
dent. In the p r e s e n t i n v e s t i g a t i o n an fore, the c o n t i n u o u s - c u l t u r e m e t h o d can
a t t e m p t was m a d e to d e v e l o p an algal be used for the e x a m i n a t i o n of l o n g - t e r m
b i o a s s a y by m e a n s of c u l t u r i n g s e n s i t i v e e f f e c t s of p o l l u t a n t s .
test forms of the N o r t h Sea in n o n - a x e - The d i n o f l a g e l l a t e s Scrippsiella faeroen-
nic m o n o c u l t u r e s . T w o c u l t i v a t i o n m e t h - se, Prorocentrummicans and Gymnodinium splen-
ods w e r e u s e d for the test: (I) b a t c h dens s e r v e d as test forms. T h e s e algae
cultures, (2) c o n t i n u o u s c u l t u r e s u t i l i z - are c o m m o n m e m b e r s of the N o r t h Sea
ing the t u r b i d o s t a t p r i n c i p l e . B a t c h p l a n k t o n . T h e y w e r e c h o s e n b e c a u s e they
c u l t u r e s c o n s i s t of c l o s e d s y s t e m s w i t h could be c u l t i v a t e d d u r i n g the e x p e r i -
l i m i t e d e x p e r i m e n t a l time and volume. ments in pure sea w a t e r e n r i c h e d only
The cell density, the n u t r i e n t content, w i t h n i t r a t e and p h o s p h a t e . The a d d i t i o n
and p o s s i b l y the c o n c e n t r a t i o n s of dis- of c h e l a t i n g a g e n t s and trace m e t a l s
s o l v e d t o x i c a n t s c h a n g e w i t h i n one ex- could t h e r e b y be avoided; this is a very
p e r i m e n t . T o x i c s u b s t a n c e s can be a d d e d i m p o r t a n t p r e c o n d i t i o n of a test on
only once at the b e g i n n i n g of the e x p e r i - h e a v y m e t a l s , w h i c h o t h e r w i s e w o u l d be
ments. T h e r e f o r e , the b a t c h c u l t u r e s c o m b i n e d in a c o m p l e x f o r m even at the
serve o n l y for f i r s t e s t i m a t i o n s of sub- s t a r t of the e x p e r i m e n t s .
lethal and l e t h a l - l i m i t c o n c e n t r a t i o n s . The test was used to i n v e s t i g a t e the
In c o n t i n u o u s c u l t u r e s the cell d e n s i t y e f f e c t of m e r c u r i c acetate. M e r c u r y was
of the c u l t u r e s y s t e m can be h e l d at a c h o s e n as an e x a m p l e of a toxic i n d u s t r i -
c o n s t a n t and r e l a t i v e l y low level by al w a s t e product. The e x p e r i m e n t s w e r e
m e a n s of the f l o w rate of the c u l t u r e r e s t r i c t e d to c u l t u r e effects. M u l t i p l i -
62 H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates

c a t i o n rates, m a x i m u m cell d e n s i t i e s , in cm. A 14h:1Oh l i g h t : d a r k p e r i o d was m a i n -


vivo c h l o r o p h y l l f l u o r e s c e n c e and m o r p h o - tained. Cell counts and m i c r o p h o t o s w e r e
l o g i c a l a l t e r a t i o n s s e r v e d as c r i t e r i a m a d e w i t h an i n v e r t e d m i c r o s c o p e . In ad-
of toxic i m p a i r m e n t . C h e m i c a l a n a l y s e s dition, a C o u l t e r C o u n t e r was used. The
on the l o c a t i o n of the m e r c u r i c a c e t a t e in vivo f l u o r e s c e n c e of the algal c u l t u r e s
and it's a c c u m u l a t i o n by the cells dur- was d e t e r m i n e d by a T u r n e r F l u o r o m e t e r .
ing the e x p e r i m e n t a l time w i l l be con- S a m p l e s w e r e t a k e n d a i l y and at the same
s i d e r e d in later e x p e r i m e n t s . N e a r l y the time (10.00 hrs). To a c h i e v e a h o m o g e -
same test m e t h o d has b e e n used in e a r l i - neous d i s t r i b u t i o n of the algae in the
er i n v e s t i g a t i o n s of i n d u s t r i a l w a s t e continuous-culture experiments, a slight
p r o d u c t s of a t i t a n i u m d i o x i d e f a c t o r y t u r b u l e n c e of the c u l t u r e m e d i u m was ob-
(Kayser, 1969, 1970) and of "red mud" -- t a i n e d by a e r a t i o n (40 ml a i r . m i n - 1 ) .
a waste material from bauxite processing
(Kayser, 1973).
Results

Materials and Methods Scrippsiella faeroense

The d i n o f l a g e l l a t e s Scrippsiella faeroense Batch Cultures


(Paulsen) B a l e c h et S o a r e s I , Prorocentrum
micans E h r e n b e r g and Gymnodinium splendens The g r o w t h curves of 6 a l g a l c u l t u r e s
L e b o u r w e r e i s o l a t e d n e a r H e l g o l a n d and are p r e s e n t e d in Fig. I as cell n u m b e r
c u l t i v a t e d in n o n - a x e n i c m o n o e u l t u r e s . v e r s u s time. C o m p a r i s o n w i t h the c o n t r o l
The c u l t u r e m e d i u m c o n s i s t e d of s e a w a t e r c u l t u r e shows that the a d d i t i o n of O.OO1
from H e l g o l a n d (32 ~ 1.5~S, s t e r i l e - f i l - mg H g . l - 1 1 ~ s u l t e d in a s l i g h t l y de-
t e r e d t h r o u g h M i l l i p o r e f i l t e r of 0.22 c r e a s e d m a x i m u m cell density. A f t e r ad-
~m pore d i a m e t e r ) , e n r i c h e d w i t h O.1 g d i t i o n of O.01 mg H g . l -I, the c u l t u r e
N a N O 3 and 0.02 g N a H 2 P O 4 . 1 - 1 . The algae s t a g n a t e d on the f i r s t day, s h o w e d a re-
w e r e k e p t in series of 2-1 g l a s s b o t t l e s d u c e d m u l t i p l i c a t i o n rate, and r e a c h e d
(Jenaer Glas, D u r a n 50) and in an 8-1 a d i m i n i s h e d m a x i m u m cell d e n s i t y of
f e r m e n t e r . In the t u r b i d o s t a t e x p e r i - only 45% that of the control. A d d i t i o n
m e n t s the c o n t i n u o u s flow of the c u l t u r e of 0.05 mg Hg.l -I c a u s e d an i m m e d i a t e
m e d i u m was s e c u r e d by two 1 3 - c a n a l - p e r i - d e c r e a s e in cell d e n s i t y to 17% of the
s t a l t i c - m i c r o p u m p s w i t h T y g o n tubes. The i n i t i a l v a l u e w i t h i n the first few days,
p u m p s r a i s e d the c u l t u r e m e d i u m c o n t i n - f o l l o w e d by a s t a g n a t i o n phase. T h e n re-
u o u s l y from 5-1 stock b o t t l e s into the c o v e r y took place, so that at the end of
2-1 c u l t u r e v e s s e l s in an a m o u n t c o r r e - the e x p e r i m e n t n e a r l y the same d e n s i t y
s p o n d i n g to the m u l t i p l i c a t i o n rate of had b e e n r e a c h e d as the control. In O.1
the c o n t r o l cultures. S i m u l t a n e o u s l y , mg H g . l -I no r e c o v e r y stage was visible.
the pumps w i t h d r e w e x a c t l y the same In T a b l e I, the l i m i t i n g c o n c e n t r a t i o n s
q u a n t i t y of the a l g a l c u l t u r e f r o m the are s u m m a r i z e d for c o m p a r i s o n w i t h the
c u l t u r e vessels. M e r c u r y was added as r e s u l t s of the f o l l o w i n g e x p e r i m e n t s .
Hg(CH3COO) 2 f r o m a d i s t i l l e d - w a t e r stock- The in vivo c h l o r o p h y l l f l u o r e s c e n c e m e a -
s o l u t i o n [O.159 g Hg(CH3COO) 2 in 100 ml s u r e m e n t s of the c u l t u r e s are shown in
H20 , 20oc]. In the b a t c S - c u l t u r e series, Fig. 2 as f l u o r o m e t r i c units v e r s u s time.
the m e r c u r y was a d d e d once at the b e g i n n - The r e s u l t s agree very w e l l w i t h the cell
ing of the e x p e r i m e n t s , w h e r e a s in the counts in Fig. I. T h e r e was no e v i d e n c e
2-1 t u r b i d o s t a t series m e r c u r y was a d d e d that in ~ v o c h l o r o p h y l l f l u o r e s c e n c e was
t o g e t h e r w i t h the i n f l o w of the c u l t u r e m o r e a f f e c t e d than was cell number.
medium. O n l y in the 8-1 f e r m e n t e r e x p e r i - In a d d i t i o n to the r e d u c t i o n of
m e n t s was the m e r c u r y a d d e d d i r e c t l y g r o w t h c a p a c i t y a further, very c o n s p i c u -
from the stock s o l u t i o n in a m o u n t s corre- ous, r e a c t i o n was o b s e r v e d . W i t h the ad-
s p o n d i n g to the m e d i u m inflow, d i t i o n of m e r c u r y c o n c e n t r a t i o n s of O.O1
A l l c u l t u r e s w e r e set up in c o n s t a n t - mg Hg.l -I and higher, the m a j o r i t y of
t e m p e r a t u r e rooms at 18~ • IC ~ The the cells b e g a n w i t h i n as little as I h
b o t t l e s w e r e i l l u m i n a t e d by l a t e r a l l y - to sink and settle on the b o t t o m of the
p o s i t i o n e d d a y l i g h t f l u o r e s c e n t lamps c u l t u r e vessels. One hour later m i c r o -
(Osram - L 40 W/15) w i t h a light i n t e n s i - scopic e x a m i n a t i o n r e v e a l e d that in 0.O1
ty of a b o u t 6,000 lux. The d i s t a n c e f r o m mg H g . l -I a few cells, and in h i g h e r con-
the light source to the b o t t l e s was 10 c e n t r a t i o n s n e a r l y all cells, b u r s t
their thecae, d i s p l a y i n g a split b e t w e e n
the e p i t h e c a and the c i n g u l u m . E p i t h e c a
In a prior publication by the author (Kayser, and h y p o t h e c a w e r e gaping. A few m i n u t e s
1973), this species was named erroneously Peri- b e f o r e b u r s t i n g the p r o t o p l a s t w i t h d r e w
dinium trochoideum (Stein) Lemmermann (Balech s o m e w h a t from the apex of the epitheca.
and Soares, 1966) S u b s e q u e n t l y , e i t h e r (I) the p r o t o p l a s t
H. Kayser: E f f e c t of M e r c u r i c A c e t a t e on G r o w t h of M a r i n e D i n o f l a g e l l a t e s 63

100000] ~
10 0 0 0 - I ~ ~ ~

000< T/I
100 o---_____

0~0~ O

\ = Control
10 \ o = 0.001 mg H g / I
\ ~ = o.o~ ....
~ 9 = 0.05 ....
= +,-_
. ~ +~ o : 0.1 "
+ = 1.0

1 . . . . , ,. . . . . , . . . . , , . , , ,
0 5 10 15 20 Days 25

Fig. i. Scrippsiella faeroense. B a t c h culture. Cell d e n s i t y after a d d i t i o n of m e r c u r i c acetate in


amounts of O.OO1 to i mg Hg.l -I at b e g i n n i n g of e x p e r i m e n t s in 2-1 bottles. M e a n v a l u e s and stan-
d a r d d e v i a t i o n s of 5 r e p l i c a t e s are given for 0.05 mg Hg.l -I

1 000 J /; ., ~ ~ , ~ ~~,~.--

100- " = Control


o = 0.001 m g H g t [
z~= 0.01
E / *, = 0 . 0 5 "
+ o= 0.1 -
+= 1.0

,T

10- ~

0 5 10 15 20 Days 25
Fig. 2. Scrippsiella faeroense. B a t c h culture. In vivo c h l o r o p h y l l f l u o r e s c e n c e after a d d i t i o n of
m e r c u r i c acetate in amounts of o.oo1 to i mg Hg.l -I at b e g i n n i n g of e x p e r i m e n t s in 2-i bottles.
M e a n values and s t a n d a r d d e v i a t i o n s of 5 r e p l i c a t e s are given for 0.05 mg Hg.l -I
@4 H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates

Table i. Scippsiella faeroense, Prorocentrum micans and Gymnodinium splendens.


Limiting concentrations (mg Hg.l-l) of Hg(CH3COO) 2 at which toxic impairment
occurs in batch and continuous cultures

Sequence of G. splendens S. faeroense P. micans


decreasing Continuous Batch Continuous Batch Continuous
toxic effects

(a) Break-down of 1 1 1 1
culture within 0.5
experimental period -O. 05
(b) Decreasing cell 0.5
density without o.1 o.1
recovery effect 0.01
(c) Decreasing cell o.1 o.1
density with 0.05 -0.05 -o. o 5
recovery effect
(d) Reduced multipli- 0.O1 O.O1 O.O1
cation rate, diminished -O 9 O O 1 -O 9OO 1
maximum cell density

left the theca via the split and swam s u r v i v e d the initial toxic decrease of
away, or (2) it r e m a i n e d in the theca the culture.
and, in the c o u r s e of a few weeks, as-
sumed a s p h e r i c a l t h i c k - w a l l e d shape. Case 2. The p r o t o p l a s t s r e m a i n e d in the
b u r s t thecae, e n l a r g e d s o m e w h a t in the
Case i. I m m e d i a t e l y after b u r s t i n g the course of the next few days, and formed
theca, the protoplast, w i t h it's apical a solid, smooth m e m b r a n e (Fig. 3 E). The
end foremost, s q u e e z e d t h r o u g h the lat- contents of the cells were s t a i n e d
eral split by means of a lateral flexure, s t r o n g l y by Lugol solution. Some e x a m p l e s
For a short time a l o n g i t u d i n a l f l a g e l - of this type r e m a i n e d u n a l t e r e d for more
lum was v i s i b l e at the a n t a p i c a l end of than 3 weeks in 0.05 mg Hg.l -I (Fi~. 3 F).
the e m e r g i n g cell; then the cell swam In the c u l t u r e to w h i c h I mg H g . l - " had
away by l o n g i t u d i n a l rotation. It was been added, all cells b u r s t laterally,
oblong, w i t h slower m o v e m e n t s than that but the p r o t o p l a s t s did not leave the
of a normal t h e c a t e d cell. This entire thecae, p r e s u m a b l y b e c a u s e of the imme-
p r o c e s s lasted a p p r o x i m a t e l y I min. By diate acute toxic e f f e c t of the mercury.
the f o l l o w i n g day m o s t of the o b l o n g All cells died in this stage w i t h i n 2
cells had d r o p p e d to the b o t t o m and lay weeks.
m o t i o n l e s s . Fig. 3 A s h o w s such cells, In the c o n t r o l c u l t u r e s c o m p a r a b l e
after the a d d i t i o n of 0.05 mg Hg.l -I, p r o c e s s e s w e r e not o b s e r v e d in the ex-
b e s i d e empty thecae. D i v i s i o n s (?) could p o n e n t i a l phase of the culture. Only
be o b s e r v e d o c c a s i o n a l l y (Fig. 3 B). At w h e n the c u l t u r e e n t e r e d the s t a t i o n a r y
times the o b l o n g cells could not come phase at d e n s i t i e s of 10,OOO to 20,000
off from the thecae entirely. In such c e l l s . m l -I were both l a t e r a l l y - b u r s t
cases they formed p o s t e r i o r l y one or a thecae and oblong naked m o t i l e cells ob-
chain of empty, smooth, o b l o n g p e l l i c u - served. In a d d i t i o n w e a k l y s t a i n e d m i c r o -
lae (Fig. 3 C). Such p e l l i c u l a e w e r e cells (ca. 20 ~m length), s t r o n g l y
also f o r m e d w h e n o b l o n g cells, a l r e a d y s t a i n e d m a c r o c e l l s (ca. 50 ~m length)
r e s t i n g on the bottom, r e s u m e d s w i m m i n g and cysts w e l l a r m o u r e d w i t h c a l c i t e
(Fig. 3 D). F a i r l y c o m m o n at this stage spiculae o c c u r r e d (Fig. 3 G,H,I, r e s p e c -
of the e x p e r i m e n t were empty thecae and tively). These cysts had an o r a n g e - c o l -
p e l l i c u l a e or chains of empty p e l l i c u - oured spot at the core. It is p o s s i b l e
lae, a t t a c h e d to a m o t i o n l e s s o b l o n g that these stages c o n s t i t u t e parts of a
cell. In 0.05 mg Hg.l -I all the o b l o n g sexual life cycle of this species (see
cells had s e t t l e d after I day. In the "Discussion").
s u c c e e d i n g days they b e g a n to die. Only
very few survived, a p p a r e n t l y by f o r m i n g Continuous Cultures
solid m e m b r a n e s . The i n c r e a s e in cell
n u m b e r s that o c c u r r e d after 13 days in The g r o w t h curves from six 2-1 turbid-
0.05 mg Hg.l -I (Figs. I and 2) was ostats are shown in Fig. 4. A flow rate
caused by n o r m a l l y t h e c a t e d cells, w h i c h of 400 m l . l - l . d a y -I m a i n t a i n e d the con-
H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates 65

Fig. 3. Scrippsiella faeroense. Morphological variations after addition of mercuric acetate. (A)
Naked oblong cells and empty thecae at bottom of the culture vessel, I day after addition of 0.05
mg Hg.l-l; (B) division stage of an oblong cell; (C) oblong cell which has not come off from the
hypotheca and has formed posteriorly a chain of empty, smooth, oblong pelliculae; (D) oblong cell
with empty, hyaline pelliculae; (E) oval protoplasts, remaining in the burst thecae; (F) oval and
oblong resting stages with a solid, smooth membrane, 3 weeks after addition of 0.05 mg Hg.l-1; (G)
control culture -- microcells in culture with a density of li,OOO cells.ml-l; (H) macrocell; (I)
cyst, partly armoured with calcite spiculae from 24th experimental day. (All cells coloured by Lu-
gol-fixation)
66 H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates

10 000
~ ~,Q:e . .
<---~2~2 z, ~.~.olO..o~O. "
~A 0--0~0~0
10o0 1 000

9 %,
A~x~a2(O\o/O~ o ~o. o_ ~
/'~ ~ 0
x9 /0~ D_D_O__O~[]
\ --
I O0 [] o I O0 ~'z~

/ AIAIA~A--A~A~ A ~A~A ~
~ ~. ~ /[3/D'O~[3 O--O\O/
o = 0.001 mg Hg/l O
10 + z~ = 0.01 _o 10 \ 9: Control
\ 9 = 0.05 \- - O = 0,001 mg Hg/I
\ z~: 0.01 ....
X [] = 0.1 o +~ +~ 9 = 0.05 . . . .
--~ + : 1.0
§ D = 0.1 ....
(D " - ' §+ - - = 1.0 ,,
] ' I . . . . I ' ,' r ' ""t 1 . . . . I . . \. . I . . . . I
0 5 10 Days 15 0 5 10 Doys 15

Fig. 4. Scrippsiella faeroense. Continuous cul- Fig. 5. Scrippsiella faeroense. Continuous cul-
ture. Ceil density during addition of mercuric ture. In vivo chlorophyll fluorescence during
acetate in amounts of O.OO1 to i mg Hg.1-1 in addition of mercuric acetate in amounts of
2-1 turbidostats O.O01 to i mg Hg.1 -I in 2-1 turbidostats

trol c u l t u r e at a n e a r l y c o n s t a n t d e n s i - found after the first day of the e x p e r i -


ty. As in b a t c h c u l t u r e s the f i r s t ef- ment.
fect on m u l t i p l i c a t i o n rate was o b s e r v e d
a f t e r a d d i t i o n s of O.OO1 and O.O1 mg Hg. Prorocentrum micans
1 -I (Table I). A f t e r 5 days the c u l t u r e s
b e g a n to d e c r e a s e in the c o u r s e of the Batch Cultures
f o l l o w i n g 10 days to 43 and 5.6%, r e s p e c -
tively, of the i n i t i a l density. A d d i t i o n The first v i s i b l e e f f e c t on m u l t i p l i c a -
of 0.05 and O.1 mg H g . 1 -I c a u s e d imme- tion rate o c c u r r e d after a d d i t i o n of
d i a t e r a p i d d e c r e a s e in cell number. Af- O.01 mg Hg.l -I, w h i c h c a u s e d s t a g n a t i o n
ter 4 days r e c o v e r y occurred, and sub- of the c u l t u r e d e n s i t y for the first 3
s e q u e n t l y the cell d e n s i t y r e m a i n e d near- days (Fig. 6, T a b l e I). A f t e r this ini-
ly c o n s t a n t at 4.3 and 2.1%, r e s p e c t i v e - tial lag phase, c u l t u r e d e n s i t y p a r a l -
ly, of the i n S t i a l density. A f t e r 4 days leled c o n t r o l d e n s i t y v e r y closely. Ad-
in I mg Hg.l-', the c u l t u r e d e c r e a s e d d i t i o n of O.1 m g H g . 1 -I d e c r e a s e d cul-
w i t h o u t r e c o v e r y to I c e l l . m l -I . The in ture d e n s i t y r a p i d l y f r o m the first day.
vivo c h l o r o p h y l l m e a s u r e m e n t s of the No r e c o v e r y occurred. In I mg Hg.l -I,
c u l t u r e s are p r e s e n t e d in Fig. 5. The the c u l t u r e d e n s i t y d e c r e a s e d to zero at
c o u r s e s of the curves f o l l o w v e r y close- the end of the f i r s t day. Fig. 7 shows
ly those of the cell counts. the c o r r e s p o n d i n g in ~ v o c h l o r o p h y l l
f l u o r e s c e n c e m e a s u r e m e n t s , w h i c h corre-
M i c r o s c o p i c o b s e r v a t i o n s h o w e d that s p o n d c l o s e l y w i t h the cell counts. A f t e r
the cells in Hg c o n c e n t r a t i o n s of O.O1 5 days, in vivo f l u o r e s c e n c e in the O.1
m g . l -I and h i g h e r s e t t l e d w i t h i n the mg H g . l - l - c u l t u r e s v a r i e d b e t w e e n I and
first day d e s p i t e a e r a t i o n t u r b u l e n c e . 10 f l u o r o m e t r i c units. T h e s e m e a s u r e -
As in b a t c h cultures, empty, l a t e r a l l y ments lie near the lower limits of p r e c i -
split t h e c a e and m o t i l e n a k e d cells ap- sion of the T u r n e r F l u o r o m e t e r . The re-
peared. Oval, t h i c k - w a l l e d r e s t i n g sults of 5 e x p e r i m e n t s are t h e r e f o r e
stages w h i c h had r e m a i n e d in the b u r s t p r e s e n t e d s e p a r a t e l y for this c o n c e n t r a -
thecae w e r e also observed. The n o n - t h e - tion (Fig. 7).
c a t e d m o t i l e cells died in the c o u r s e of
I week. O n l y n o r m a l l y t h e c a t e d m o t i l e Continuous Cultures
cells s u r v i v e d d u r i n g the i n i t i a l de-
c r e a s e in cell n u m b e r of the cultures. Figs. 8 and 9 show the r e s u l t s of a
In I mg H g . l -I no m o t i l e cells w e r e series of e x p e r i m e n t s run in six 2-1-
H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates 67

10000
~...~_~ "\ ..~..~_~../~

1 000

9 = Control
O 0.001 m g / l Hg
100
z~ 0.01
[] = 0.1

--- -- 1.0

~o', 'y\,

1
0 5 10 15 20 25 Ooys 30
Fig. 6. P r o r o c e n t r u m micans, Batch culture. Cell density after addition of mercuric acetate in
amounts of O.O01 to i mg Hg.l -I at beginning of experiments in 2-1 bottles. Mean values and standard
deviations of 5 replicates are given for O.1 mg Hg.! -I

/o.c-__ 2 /"
1000

- _ / AI~

100
~ ~ / 9 = Control
o = 0.001 m g / I Hg
B B
z~ = 0.01
o = 0.1
+ = 1.0

10 ~=Io8 o

(n + o B o o
[] o
E =I=o I
o
r_
o B Bo B
E
o noBR o o [] o
o

LL

I i o , [ ,000 I . . . . ~ , o , , i B , , o I
0 5 10 15 20 25 Days 30
Fig. 7. P r o r o c e n t r u m micans. Batch culture. In vivo chlorophyll fluorescence after addition of mer-
curic acetate in amounts of O.O01 to 1 mg Hg.l -I at beginning of experiments in 2-1 bottles, Sepa-
rate measurements of 5 parallel replicates are illustrated for O.i mg Hg.l -I
68 H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates

1 000-

10 O00 Betch-stQge I Continuous-stage


Botch-stage Continuous - stage
without I 241ml / l / day, Hg added
without 241mill/day, Hg added
addition of I
addition of
Hg Hg A - - - ~ - z ~ , ~z~
1 000 ~n-9~m~--_x/ A~.wv
100

/ \{ o %0
- \ / 9 : Control
.O
100 ~_u/ 0 = 0.001 mg Hg/I
v : 0.005 ,,
z~: 0.01 ,. 2
9
2 A: 0.05
~J
o ~= 0.1
10 . . . . I . . . . i . . . . , 10 . . . . I . . . . I ' ' ' ' t

5 10 Days 15 5 10 Doys 15

Fig. 8. P r o r o c e n t r u m micans. Continuous culture. Fig. 9. P r o r o c e n t r u m micans. Continuous culture.


Cell density during addition of mercuric ace- In vivo chlorophyll fluorescence during addi-
tate in amounts of O.OO1 to O.i mg Hg.l -I on tion of mercuric acetate in amounts of O.OO1 to
5th day of experiment in 2-1 turbidostats O.i mg Hg.l -I on 5th day of experiment in 2-1
turbidostats. Symbols as in Fig. 8

--->
i addition of Hg (rag/I)
1 000 0.01~]<-- 0.1~ / ~ 0.5 "~j-1.0 ->
[<-- addition of Hg ( m g / I ) - -
/
100 i0.01~1~ 0.1 ~" 0.5 ~T1.0 ~

100

10
10
• X~x
\
x~ x
\•
1 \ E L
I

O~
O
I
01 1 . . . . , I . . . . I . . . . .
o . . . . . . . . lb . . . . Days 2O 0 5 10 15 Days 20

Fig. iO. P r o r o c e n t r u m micans. Continuous cul- Fig. ii. P r o r o c e n t r u m micans. Continuous cul-
ture. Cell density in B-1 turbidostat (ferment- ture. In vivo chlorophyll fluorescence in 8-1
er). Mercuric acetate was added once daily di- turbidostat (fermenter). Mercuric acetate was
rectly to culture vessel in amounts of O.O1 to added once daily directly to culture vessel in
I mg Hg.1 -I in succession from 5th day of ex- amounts of O.O1 to i mg Hg.l -I in succession
p e r i m e n t on from 5th day of e x p e r i m e n t on
H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates 69

t u r b i d o s t a t s . D u r i n g the first 5 days Bo t c h - ! C o n t i n u o u s - s t a g e


the algae w e r e c u l t i v a t e d as b a t c h cul- stage ! 350 ml/I/day, Hg a d d e d
tures. T h e n the c o n t i n u o u s m e d i u m flow
was s w i t c h e d - o n at a f l o w r a t e of 241 ml. I 000 ~
l - l . d a y -I . S i m u l t a n e o u s l y , m e r c u r y ace- ] . J ' J~.c-'~'--'--~
~\
tate was added c o n t i n u o u s l y t o g e t h e r I/ II\~\A
w i t h the m e d i u m inflow. The 0.001 to
O.O1 mg H g . l - l - c u l t u r e s v a r i e d little
f r o m the c o n s t a n t cell n u m b e r s of the
c o n t r o l culture. In 0.05 mg Hg.l-1, how-
100 il~ ~\~
"A~A~ a
ever, a d i s t i n c t d e c r e a s e in cell n u m b e r
o c c u r r e d d u r i n g the f i r s t 2 days. A f t e r
this i n i t i a l d e c r e a s e the c u l t u r e re- X ~A
c o v e r e d rapidly. The same r e s u l t s w e r e 9 = Control
o b t a i n e d w i t h the 0.1 mg H g . l - l - c u l t u r e . A = 0.01 m g / I Hg
The in ~ v o c h l o r o p h y l l f l u o r e s c e n c e i = 0.05
measurements displayed analogous results
(Fig. 9).
o
\
[]\
\, \i
m =
....
0.1
0.5
A second continuous-flow experiment,
to d e t e r m i n e the lethal H g - c o n c e n t r a t i o n s 1 . . . . , .\ , . . ,\ . . . . ,
u n d e r t u r b i d o s t a t i c c o n d i t i o n s , was con- 0 5 10 Days 15
d u c t e d in an 8-1 f e r m e n t e r (Figs. 10,11).
The e x p e r i m e n t v a r i e d f r o m the first in Fig. 12. Gymnodinium splendens. Continuous cul-
two details: (I) the f e r m e n t e r had only ture. Cell density during addition of mercuric
I c u l t u r e v e s s e l -- t h e r e f o r e d i f f e r e n t acetate in amounts of O.O1 to 0.5 mg Hg.1-1,
Hg c o n c e n t r a t i o n s c o u l d be i n v e s t i g a t e d beginning on 3rd day of experiment in 2-1
only in succession; (2) the m e r c u r y was turbidostats
a d d e d d i r e c t l y once d a i l y to the c u l t u r e
v e s s e l -- this s e m i c o n t i n u o u s a d d i t i o n
was d e s i g n e d to p r e v e n t p o s s i b l e in-
a c t i v a t i o n of the m e r c u r y in the m e d i u m a d d i t i o n of 0.01 mg H g . l -I d e c r e a s e d the
stock b o t t l e s b e f o r e it r e a c h e d the test c u l t u r e d e n s i t y to n e a r l y 2% of con-
c u l t u r e v e s s e l by the f l o w - t h r o u g h sys- trol d e n s i t y w i t h i n 2 weeks. In 0.05 and
tem. A f l o w rate of 325 m l . l - l . d a y -I O.1 mg Hg.l-1 cell d e n s i t y d r o p p e d to
m a i n t a i n e d the i n i t i a l d e n s i t y at a less than I c e l l . m l - 1 even after 11 and
c o n s t a n t level (Fig. 10). A d d i t i o n of 7 days, r e s p e c t i v e l y . In 0.5 m g Hg.l-1,
0.01 mg H g . l -I on the f i f t h day caused, all cells d i e d w i t h i n the f i r s t day of
w i t h i n 3 h, a s h o r t - t e r m d e c r e a s e in Hg addition. No r e c o v e r y was o b s e r v e d at
cell numbers, p r e s u m a b l y by s e t t l e m e n t any of the c o n c e n t r a t i o n s tested.
since on the f o l l o w i n g day the c u l t u r e
had r e c o v e r e d to p r e - m e r c u r y values. The
same o c c u r r e d in the in viva f l u o r e s - Discussion
cence m e a s u r e m e n t s (Fig. 11). S u b s e q u e n t
i n c r e a s e of Hg c o n c e n t r a t i o n to O.1 mg C o m p a r i s o n of the three a l g a l species
Hg.l -I c a u s e d a r e n e w e d d e c r e a s e in cell shows that Gymnodinium splendens is m o s t
n u m b e r s w i t h i n 2 h. In this case, how- s e n s i t i v e to m e r c u r i c a c e t a t e (Table I),
ever, cell n u m b e r s r e m a i n e d a r o u n d this f o l l o w e d by Scrippsiella faeroense and Proro-
level for the f o l l o w i n ~ 4 days. Upon ad- centrum micans. E f f e c t s of t o x i c i t y w e r e
d i t i o n of 0.5 mg H g . l -i the cell n u m b e r s m o s t o b v i o u s i m m e d i a t e l y after the ad-
and c o r r e s p o n d i n g in ~ v o fluorescence d i t i o n of m e r c u r y in b o t h b a t c h and con-
d e c r e a s e d r a p i d l y w i t h i n the next w e e k t i n u o u s - c u l t u r e e x p e r i m e n t s . The e x p e c t -
and w i t h no signs of r e c o v e r y . A d d i t i o n ed i n t e n s i f i e d t o x i c i t y under c o n t i n u o u s -
of I mg Hg.l -I c a u s e d total d e s t r u c t i o n f l o w c o n d i t i o n s did not occur. The Hg-
of the c u l t u r e w i t h i n I day. a d d i t i o n v i a the c o n t i n u o u s m e d i u m in-
flow, as c a r r i e d out in the 2-1 t u r b i d -
o s t a t series, had the same e f f e c t on the
Gymnodini um splendens algae as had the s e m i c o n t i n u o u s d i r e c t
input of Hg into the c u l t u r e v e s s e l dai-
Continuous Culture ly by hand, as c a r r i e d out in the 8-1
f e r m e n t e r e x p e r i m e n t . The r e c o v e r y o b -
The e x p e r i m e n t was c a r r i e d out in a s e r v e d in s. faeroense and P. micans m a y
series of five 2-1 t u r b i d o s t a t s (Fig.12). have r e s u l t e d f r o m v o l a t i l i z a t i o n or
A f t e r an i n i t i a l b a t c h stage of 3 days, f i x a t i o n of ionic Hg to w a l l s or to
a c o n t i n u o u s f l o w - t h r o u g h of 350 m l . l -I. p a r t i c u l a t e or d i s s o l v e d o r g a n i c cam-
day-1 m a i n t a i n e d the c o n t r o l c u l t u r e at pounds, in w h i c h form it is less toxic
n e a r l y c o n s t a n t cell density. C o n t i n u o u s to the algae. P e r h a p s these c o m p o u n d s
70 H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates

are p r o d u c e d by the algae themselves. cies very difficult, since only a few
C h e m i c a l a n a l y s e s of the l o c a t i o n of the forms can grow w i t h o u t a d d i t i o n of
Hg during the e x p e r i m e n t s may p r o v i d e n u t r i e n t s and other s u b s t a n c e s to their
p r e c i s e information. c u l t u r e medium. P a r t i c u l a r l y steno-
Davies (1974) r e p o r t e d a c o m p a r a b l e p l a s t i c and s e n s i t i v e forms may require
r e c o v e r y e f f e c t w i t h cultures of Isochry- special m e d i a for normal growth. Sensi-
sis galbana: a d d i t i o n of 10.5 ~g.l -I m e r - tivity of the test forms is n e v e r t h e l e s s
curic c h l o r i d e at the b e g i n n i n g of the a very i m p o r t a n t c r i t e r i o n for evaluat-
e x p e r i m e n t s p r e v e n t e d g r o w t h after 5 ing r e s u l t s from a w a s t e - w a t e r test.
days. On the 15th day r e c o v e r y occurred, T e s t o r g a n i s m s should be c h a r a c t e r i s t i c
f o l l o w e d by r e n e w e d e x p o n e n t i a l g r o w t h m e m b e r s of the p l a n k t o n c o m m u n i t i e s in
of the culture. W i t h the d i f f e r e n c e that the area. The test should not be re-
in the p r e s e n t study g r o w t h was h i n d e r e d s t r i c t e d to one species but should in-
even from the first day on (Figs. I and clude as m a n y forms as can be c u l t i v a t e d
2), Davies' e x p e r i m e n t c o r r e s p o n d e d to w i t h i n the assay conditions.
my 0.05 mg Hg.l -I e x p e r i m e n t on Scripp- M o r p h o l o g i c a l changes in test algae
siella faeroense. A c c o r d i n g to B e n - B a s s a t p r o v i d e very r e l i a b l e and d i s t i n c t cri-
et al. (1972) , chlamydomonas rheinhardii is teria for a bioassay. In scrippsiella
e x t r e m e l y t o l e r a n t to inorganic m e r c u r y faeroense, H g - a d d i t i o n causes the emer-
and c o n t i n u e s to g r o w in c o n c e n t r a t i o n s gence of naked m o t i l e cells, and the ob-
of I mg Hg.l -I a l t h o u g h w i t h a length- s e r v a t i o n of n o n - m o t i l e r e s t i n g stages
ened lag phase. At 2 mg Hg.l -I, however, even at low cell d e n s i t i e s in the expo-
g r o w t h ceases. In their experiments, the n e n t i a l phase of the cultures. In the
total m e r c u r y levels of the a e r a t e d cul- c o n t r o l cultures, c o m p a r a b l e p r o c e s s e s
tures i n i t i a l l y c o n t a i n i n g 0.2 mg Hg.l -I o c c u r r e d only after m a x i m u m cell densi-
d e c r e a s e d by about 40% over a p e r i o d of ties had been reached. This p h e n o m e n o n
8 days. The authors s u g g e s t e d that the was also o b s e r v e d in aged cultures by
m e r c u r y had b e c o m e v o l a t i l e through some B r a a r u d (1958) and Sousa e Silva
b i o l o g i c a l process, thus a l l o w i n g it to (1962). B o l t o v s k o y (1973) p o i n t e d out
leave the cultures. that the e m e r g e n c e of naked cells in
D u r i n g culture e x p e r i m e n t s inorganic d i n o f l a g e l l a t e s occurs under u n f a v o u r -
m e r c u r y c o m p o u n d s can be c h a n g e d into able e n v i r o n m e n t a l conditions, and Pro-
organic forms by m i c r o b i a l a c t i v i t y (Ma- fessor H.A. von S t o s c h (personal commu-
gos et al., 1964). This is i m p o r t a n t in nication) reports this even from d a m a g e d
v i e w of the fact that Harris et al. (1970) p l a n k t o n samples. This p h e n o m e n o n is,
and N u z z i (1972) r e p o r t e d that m o n o a l k y l - therefore, not a s p e c i f i c e f f e c t of m e r -
and a r y l - m e r c u r y salts are c o n s i d e r a b l y cury. C o m p a r i s o n with the u n a f f e c t e d
more toxic than their inorganic forms, control c u l t u r e s indicates, however,
Matida et al. (1971) showed also that that the a d d i t i o n of Hg induces the pro-
Scenede~mus dimorphus was not i n h i b i t e d by cess. Cu-ions p r o d u c e the same effect
50 ppb m e r c u r i c chloride, but that (Dr. S.M~ Saifullah, p e r s o n a l c o m m u n i c a -
g r o w t h was r e t a r d e d by 10 ppb m e t h y l mer- tion).
curic chloride. A c c o r d i n g to K n a u e r and The a d d i t i o n a l o c c u r r e n c e of m a c r o -
M a r t i n (1972), a s s i m i l a t i o n of m i x e d and m i c r o c e l l s and of c a l c i t i c resting
p h y t o p l a n k t o n c o m m u n i t i e s was slowly in- spores at m a x i m u m cell d e n s i t i e s in the
h i b i t e d by c o n c e n t r a t i o n s of over 0.5 control cultures suggests that some
ppb m e r c u r i c chloride, w h e r e a s as little stages of a sexual life cycle may be in-
as O.1 ppb m e t h y l m e r c u r y i n h i b i t e d J p r o - volved. A c c o r d i n g to P r o f e s s o r von
d u c t i o n to 30%. On the other hand, Han- Stosch (personal c o m m u n i c a t i o n ) , the
nan and P a t o u i l l e t (1972) and H a n n a n et m a c r o c e l l s could be p l a n o z y g o t e s , that
al. (1973) i n d i c a t e d that m e r c u r i c chlo- later change into h y p n o z y g o t e s (Fig. 3
ride is m o r e toxic than d i m e t h y l mercury. H). The m i c r o c e l l s c o u l d be gametes
Similar effects of d i p h e n y l m e r c u r y on (Fig. 3 G). However, only an exact anal-
f r e s h - w a t e r p h y t o p l a n k t o n was noted by ysis of the nuclei and l o n g - t e r m obser-
Harris et al. (1970). v a t i o n s of the living cells can p r o v i d e
Results of t o x i c i t y tests d e p e n d on v a l i d data (von Stosch, 1973).
the c o m p o s i t i o n of the test m e d i u m used. Nuzzi (1972) d e s c r i b e d p h a t h o l o g i c a l
T o x i c i t y varies i n v e r s e l y w i t h the con- changes in the size and form of Phaeodacty-
c e n t r a t i o n of n u t r i e n t s p r e s e n t (Hannan lum tricornut~ and Chlorella sp. after the
and P a t o u i l l e t , 1972). In a p r a c t i c a l a d d i t i o n of o r g a n o m e r c u r i a l compounds.
w a s t e - w a t e r t o l e r a n c e test it is best, Davies (1974) o b s e r v e d a c o n s i d e r a b l e
therefore, to use pure local sea water e f f e c t of m e r c u r i c c h l o r i d e upon the
from that area w h i c h is b e i n g c o n s i d e r e d m e a n cell v o l u m e s of Isochrysis galbana;
for w a s t e - w a t e r d i s c h a r g e p u r p o s e s (Kay- cell v o l u m e s a l m o s t d o u b l e d at the high-
ser, 1971; J e n s e n and Rystad, 1974). est s u b l e t h a l c o n c e n t r a t i o n s he e x a m i n e d
This makes the choice of the test spe- (10.5 ~g.l-1).
H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates 71

The measurements of t h e in vivo c h l o - Acknowledgements. I gratefully acknowledge


rophyll fluorescence corresponded closely the Deutsche Forschungsgemeinschaft which sup-
tO the c e l l c o u n t s in the e x p e r i m e n t s ported this investigation by a grant. I express
d e s c r i b e d here. T h i s is m a i n l y b e c a u s e my thanks to Dr. G. Drebes for the cultures of
the s a m p l e s w e r e t a k e n s i m u l t a n e o u s l y . Scrippsiella faeroense and Gymnodinium splendens
Blasco (1973) s h o w e d t h a t in the d a r k and to Mrs. M. Boy and Miss J. Willf~hr for tech-
the fluorescence of a l g a e is s i g n i f i c a n t - nical assistance. I am much obliged to Dr. K.-R.
ly e n h a n c e d . In h i s e x p e r i m e n t s the ra- Sperling for critical reading of the manuscript
t i o of in vivo f l u o r e s c e n c e : c h l o r o p h y l l and to Dr. J. Markham and Mr. G. Baber for aid
a was not constant for different species in correcting the English text.
and, for the s a m e s p e c i e s , w a s n e g a t i v e l y
c o r r e l a t e d w i t h the c o n c e n t r a t i o n of
chlorophyll p e r cell. In vivo f l u o r e s - Literature Cited
c e n c e d e p e n d s a l s o o n the p h y s i o l o g i c a l
characteristics of the c e l l s . M e a s u r e - Balech, E. y L. de O. Soares: Dos dinoflagelados
m e n t s of p h o t o s y n t h e s i s , however, seem de la Bahia de Guanabara y proximidados (Bra-
to c o n s t i t u t e a very much more sensitive sil). Neotropica 12, 103-109 (1966)
method for the examination of t o x i c s u b - Ben-Bassat, D., G. Shelef, N. Gruner and H.J.
stances. Harris et al. (1970), f o r e x a m - Shuval: Growth of Chlamydomonas in a medium
ple, s h o w e d a r e d u c e d a s s i m i l a t i o n rate containing mercury. Nature, Lond. 204, 43-44
in the d i a t o m Nitzschia delicatissima in as (1972)
l i t t l e as O.] p p b of o r g a n o m e r c u r i a l Blasco, D.: Estudio de los variaciones de la re-
fungicides. The same was reported by lacion fluorescencia in vivo/chlorofila a, y
Knauer and Martin (1972) f o r m i x e d p o p u - su aplication en oceanografica. Influencia de
l a t i o n s of p h y t o p l a n k t o n with methyl la limitatiSn de diferentes nutrientes, efec-
mercury. to del dia y noche y dependencia de especie
So far, a l g a l b i o a s s a y s in t h e l a b o - estudiada. Investigaci6n pesq. 37, 533-556
ratory have been made almost exclusively (1973)
on m o n o c u l t u r e s in t h e e x p o n e n t i a l Boltovskoy, A.:Formacion del arqueopilo en tecas
growth phase and under optimal culture de dinoflagelados. Revta esp. Micropaleont. 5,
conditions. These correspond o n l y to a 81-98 (1973)
s m a l l e x t e n t to c o n d i t i o n s in t h e n a t u - Braarud, T.: Observations on Peridinium trochoi-
ral environment. Interspecific competi- deum (Stein) Lemm. in culture. Nytt Mag. Bot.
tion, n u t r i e n t limitation, non-optimal 6, 39-42 (1958)
light and temperature conditions, and Davies, A.G.: The growth kinetics of Isochrysis
g r a z i n g r a t e s of p l a n k t o n p r e d a t o r s lim- galbana in cultures containing sublethal con-
it a l g a l p o p u l a t i o n s in situ and, t h e r e - centrations of mercuric chloride. J. mar.
fore, m a y i n c r e a s e t h e i r s e n s i t i v i t y to biol. Ass. U.K. 54, 157-169 (1974)
additional s t r e s s e s by t o x i c a n t s . The Fisher, N.S., E.J. Carpenter, C.C. Remsen and
objective i m i t a t i o n of n a t u r a l s t r e s s C.F. Wurster: Effects of PCB on interspecific
f a c t o r s in a l a b o r a t o r y t e s t is v e r y competition in natural and gnotobiotic phyto-
difficult. Interspecific competition plankton communities in continuous and batch
c o u l d be i m i t a t e d b y m u l t i s p e c i e s cul- cultures. Microb. Ecol. 1, 39-50 (1974)
t u r e s . F i s h e r et al. (1974) s h o w e d in Hannan, P.J. and C. Patouillet: Effect of mercu-
two-species c u l t u r e s of Dunaliella tertio- ry on algal growth rates. Biotechnol. Bio-
lecta a n d Thalassiosira pseudonana a n d in engng 14, 93-101 (1972)
natural plankton communities t h a t 0.01 -, P.E. Wilkniss, C. Patouillet and R.A. Carr:
p p b of p o l y c h l o r i n a t e d biphenyl (PCB) Measurement of mercury sorption by algae.
d i d n o t e f f e c t a l g a l g r o w t h in p u r e N.R.L. Rep. 7628, 1-28 (1973)
cultures but caused substantial disrupt- Harris, R.C., D.B. White and R.B. McFahrlane:
i o n in c o n t i n u o u s - c u l t u r e communities. Mercury compounds reduce photosynthesis by
Nutrient-limited conditions in c o n t i n u - plankton. Science, N.Y. 170, 736-737 (1970)
ous-test cultures could be achieved by Jensen, A. and B. Rystad: Heavy metal tolerance
using chemostats. This method, however, of marine phytoplankton. I. The tolerance of
involves relatively high and unnatural three algal species to zinc in coastal water.
cell densities. In the p r e s e n t w o r k t h e J. exp. Biol. Ecol. 15, 145-157 (1974)
turbidostat technique was preferred be- Kayser, H.: Zfichtungsexperimente an zwei marinen
c a u s e it a l l o w s o b s e r v a t i o n of l o w c e l l Flagellaten (Dinophyta) und ihre Anwendung im
densities, b y m e a n s of t h e c u l t u r e f l o w - toxikologischen Abwassertest. Helgol6nder
through system, and avoids nutrient lim- wiss. Meeresunters. 19, 21-44 (1969)
i t a t i o n . In f u t u r e w o r k it is i n t e n d e d -, Experimental-ecological investigations on
to s u p p l e m e n t the present experiments Phaeocystis poucheti (Haptophyceae) : cultiva-
by continuous multispecies cultures tion and waste water test. Helgol6nder wiss.
using the chemostat technique, and by Meeresunters. 20, 195-212 (1970)
c h e m i c a l a n a l y s e s of the l o c a t i o n s of -, Produktivit6tsmessungen an Phytoplanktonorga-
the added toxicants. nismen aus KOstengew6ssern als Standardmetho-
72 H. Kayser: Effect of Mercuric Acetate on Growth of Marine Dinoflagellates

de f~r einen Abwassertest. Thalassia jugosl. Sousa e Silva, E.: Some observations on marine
7, 139-150 (1971) dinoflagellate cultures III. Goniaulax spini-
-, 0bet den Einflu~ yon Rotschlamm auf die Kul- fera (Clap. and Lach.) Dies., Goniaulax tama-
tur einiger mariner Planktonalgen. Helgol~n- rensis Leb. and Peridinium trochoideum (Stein)
der wiss. Meeresunters. 25, 357-383 (1973) Lemm. Notas Estud. Inst. Biol. mar., Lisb. 26,
Knauer, G.A. and J.H. Martin: Mercury in a ma- 1-24 (1962)
rine pelagic food chain. Limnol. Oceanogr. 17, Stosch, H.A. yon: Observations on vegetative re-
868-878 (1972) production and sexual life cycles of two
Magos, L., A.A. Tuffery and T.W. Clarkson: Vola- freshwater dinoflagellates, G y m n o d i n i u m p s e u -
tilization of mercury by bacteria. Br. J. ind. dopalustre Schiller and Wo!oszynskia apicula ~
Med. 21, 294-298 (1964) ta sp. nov. Br. phycol. J. 8, JO5-134 (1973)
Matida, Y., H. Kumada, S. Kimura, Y. Saiga, T.
Nose, M. Yokote and H. Kawatsu: Toxicity of
mercury compounds to aquatic organisms and Dr. H. Kayser
accumulation of the compounds by the organ- Biologische Anstalt Helgoland
isms. Bull. Freshwat. Fish. Res. Lab., Tokyo Litoralstation List
21, 197-227 (1971) Hafenstrasse 3
Nuzzi, R.: Toxicity of mercury to phytoplankton. 2282 List, Sylt
Nature, Lond. 237, 38-40 (1972) Germany (FRG)

Date of final manuscript acceptance: February 13, 1976. Communicated by O. Kinne, Hamburg