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The effects of organic environmental chemicals on the growth of the alga Scenedesmus subspicatus : A contribution to environmental biology
Chemosphere, Vol.14, No.9, pD 1355-1369, Printed in Great Britain
1985
0 0 4 5 - 6 5 3 5 / 8 5 $3.00 + .00 ©1985 P e r g a m o n Press Ltd.
THE EFFECTS OF ORGANIC E N V I R O N M E N T A L C H E M I C A L S ON THE G R O W T H OF THE ALGA S c e n e d e s m u s
subspicatus:
A C O N T R I B U T I O N TO E N V I R O N M E N T A L B I O L O G Y
Harald Geyer,
Gesellschaft
Irene S c h e u n e r t and F r i e d h e l m Korte
fur S t r a h l e n Institut
und U m w e l t f o r s c h u n g mbH M ~ n c h e n
fur O k o l o g i s c h e Chemie
Ingolst~dter LandstraBe
1
D-8042 N e u h e r b e r g Federal R e p u b l i c of G e r m a n y
ABSTRACT The e f f e c t i v e c o n c e n t r a t i o n s of 15 chemicals, inhibiting the cell growth of the alga S c e n e d e s m u s s u b s p i c a t u s by I0 % and 50 % during 96 hours, have been investigated in a static test under c o n t r o l l e d laboratory conditions.
i. I N T R O D U C T I O N
T o d a y there
is a great need for m e t h o d s
living organisms. States, market similar
According
to predict
the effects of c h e m i c a l s on
to r e g u l a t i o n s of the E u r o p e a n C o m m u n i t y Member
e c o t o x i c o l o g i c a l testing of new s u b s t a n c e s which are introduced into the 1 is n e c e s s a r y . The OECD Member c o u n t r i e s have, in principle, agreed to a testing scheme.
Studies of the effects of c h e m i c a l s on r e p r e s e n t a t i v e form a great part of e c o t o x i c o l o g i c a l with
fish, Daphnia,
is also required
testing and research.
e a r t h w o r m s and terrestrial
important p r i m a r y p r o d u c e r s
are c o n s u m e d by other aquatic organisms,
testing
and oysters.
in m o n o s p e c i e s
using the green
plants,
In a d d i t i o n to tests
a growth test with algae
in legislation 2.
A l g a e are the most
snails, m u s s e l s
species of flora and fauna
Several
in the aquatic environment.
such as Daphnia,
Rotatoria,
Amphipods,
algae have been used for e c o t o x i c o l o g i c a l
cultures 3-9 or in mixed c u l t u r e s I0-II. A batch test,
freshwater
They
alga S c e n e d e s m u s
1355
spec.
as a test organism,
was
1356
recently proposed by the OECD 12 and the German Federal Environmental Agency (Umweltbundesamt), they are released
Berlin 13, for screening and evaluating
new chemicals before
into the market and thus into the environment.
The present paper deals with the effects of 15 organic chemicals on the growth of the freshwater controlled
unicellular green alga Scenedesmus
laboratory conditions.
subspicatus Chodat under
One objective of this study was the
examination of the feasibility of the algae growth
inhibition test guideline 13.
The study involves also a comparison of the toxicity of the chemicals
to this
alga species and gives a hazard ranking of the tested organic substances.
2. MATERIALS AND METHODS
2.1 Chemicals 2,6-Dichlorobenzonitrile
was a gift of Shell Research Ltd., Sittingbourne,
U.K.
The other organic chemicals were purchased commercially and were used without further purification. volatility
They are listed in Table 1 including their use, purity,
from aqueous solution,
coefficient.
Acetone,
water solubility and n-octanol/water
used as a solvent, was of analytical
(purity{99.5 %) and purchased
partition
reagent grade quality
from E. Merck, FRG. All substances used for the
preparation of the nutrient medium were also of analytical
reagent grade quali-
ty. The water used was double distilled and sterilised. 2.2 Test Organism The usually unicellular
freshwater green alga Scenedesmus
(86, 81 SAG), belonging
to the order of Chlorococcales
phyceae), Hygiene,
was obtained
from Dr. Renate K0hn,
subspicatus Chodat
(Chlorophyta, Chloro-
Institute for Water, Soil and Air
Federal Health Office, Berlin-Dahlem,
FRG.
2.3 Stock-Breedin 9 Conditions The stock cultures of Scenedesmus stored
in 100 ml Erlenmeyer
subspicatus
placed on a white surface protected lighting by PHILIPS
in 20 ml nutrient solution were
flasks stoppered with metal caps against daylight,
fluorescent lamps
(Type: TL-D,
at 21-25 Oc. For maintenance of the test strain, prepared continuously at 10 days' flasks containing sterilized
intervals.
(Kapsenberg-caps),
and exposed to constant
58 Watts,
25 white universal)
fresh stock cultures were
For this purpose the Erlenmeyer
20 ml of nutrient solution 3 and stoppered with metal caps were
in a steam sterilizer
for 30 min. After cooling,
flask was inoculated with 2 ml algae suspension
the content of each
from a 10-day stock culture
1357
2.4 D e s c r i p t i o n of the Test P r o c e d u r e
The
test was p e r f o r m e d
Environmental Agency
in a c c o r d a n c e with the test g u i d e l i n e of the Federal
(Umweltbundesamt)
Berlin,
FRG 13. The nutrient m e d i u m and
washed g l a s s w a r e were sterilized by a u t o c l a v i n g prior to use. E x p e r i m e n t s stock c u l t u r e s were photosynthetically photons
• m -2
and
incubated at 22 + 2 °C at c o n s t a n t c o n d i t i o n s with a effective
light
intensity of 120 p E • m -2
• s-i(7.2
• 1019
s-i).
2.4.1 P r e p a r a t i o n of Test S u b s t a n c e S o l u t i o n
A defined q u a n t i t y of the c h e m i c a l
to be tested was diluted
in a defined volume
of sterilized double d i s t i l l e d water. A c e t o n e was the solvent carrier c h e m i c a l s of low water rophenol
s o l u b i l i t y such as h e x a c h l o r o b e n z e n e
(PCP), lindane
(~-HCH),
for
(HCB), p e n t a c h l o -
atrazine and t r i s ( 2 , 3 - d i b r o m o p r o p y l ) - p h o s p h a t e .
The m a x i m u m c o n c e n t r a t i o n of acetone was 0.I ml per i000 ml nutrient solution. The same amount of acetone was added to the control
subcultures.
In general
the
test was carried out without a d j u s t m e n t of the pH value of the test substance solution.
In the case of
nitrophenol
there were
the chemicals.
2,4,6-trichlorophenol, p e n t a c h l o r o p h e n o l
(PCP)
and 4-
indications of a d i s t i n c t change of pH value caused by
In these cases
the pH of the stock solution was adjusted
to pH 7
using NaOH.
2.4.2 P r e - c u l t u r e
The p r e - c u l t u r e was started
3 days prior
to the beginning of the test. Less than
one part of an algae s u s p e n s i o n taken from the stock culture was mixed with one part by volume of the c o n c e n t r a t e d
n u t r i e n t m e d i u m and 8 parts by volume of
water. After d i l u t i o n up to 10 parts by volume, p r e - c u l t u r e was
in the order of 104 cells/ml.
volume of 50 ml were
incubated
the cell c o n c e n t r a t i o n Of the
The p r e - c u l t u r e s with a total
in 300-mi E r l e n m e y e r
flasks with K a p s e n b e r g caps
for 3 days under the same t e m p e r a t u r e and light c o n d i t i o n s
as those of the test
culture.
2.4.3 P e r f o r m a n c e of the Test
The e x p e r i m e n t s were p e r f o r m e d by p r e p a r i n g 300-ml E r l e n m e y e r contained
flasks as follows.
80 ml of a saturated
The
test chemical
s u b s e q u e n t d i l u t i o n s were prepared
two parallel d i l u t i o n series
in
first flask of each d i l u t i o n series solution.
Starting
from this flask,
using a c o n s t a n t d i l u t i o n ratio of 40 ml
p r e l i m i n a r y chemical d i l u t i o n plus 40 ml d o u b l e - d i s t i l l e d water. T h e n 5 ml of m e d i u m and 5 ml of the algae s u s p e n s i o n of the p r e l i m i n a r y culture were added to each flask of the d i l u t i o n series
(40 ml). The algal s u s p e n s i o n of known cell
1358
TABLE Use, Purity and Relevant Physico-Chemical
NO. CHEMICAL
I
Hexachlorobenzene
MAJOR USES
Fungicide; manufactures of PCP and aromatic fluorocarbons
2 3
Tris-(2,3-dibromopropyl)-
Flame retardant in polyurethane foam and
phosphate (Tris)
plastics
Benzene
Solvent; intermediate for many chemicals; gasoline additive
4
Trichloroethylene (Tri)
5
Styrene-7,8-oxide
Solvent; cleansing; metal de~reasing In polymer industry as a diluent for production of epoxy resins
6
1,1-Dichloroethylene (Vinylidenechloride)
Production of copolymers and modacrylic fibres
7
Thiourea
Production of flame retardant resins;
8
1,2,4-Trichlorobenzene
vulcanization accelerator Solvent; transformer o i l ;
heat transfer
medium ; d i e l e c t r i c f l u i d ; dye c a r r i e r ; intermediate (2,4-D)
9
2,4,6-Trichlorophenol
Wood preservative; bactericide; herbicide and d e f o l i a n t
10 4-Chloroaniline
Intermediate in manufacture of many
11 2,6-Dichlorobenzonitrile (Dichlobenil: DBN)
Herbicide
12 Pentachlorophenol (PCP)
Wood preservative; herbicide; defoliant;
chemicals (herbicides)
sodium salt: algicide; fungicide 13 Atrazine
Herbicide
14 Lindane (~-HCH)
Insecticide; scabicide; pediculicide
15 4-Nitrophenol
Intermediate in manufactures of many chemicals
a) V o l a t i l i t y from aqueous solution (hour/m depth); Data from I. Scheunert22 b) N.D.: Not determined
1359
Properties of the Tested Organic Chemicals
Purity
V o l a t i l i t y a)
Water S o l u b i l i t y I~-17
n-Octanol/Water Partition Coefficient~a,15,18-21
(%)
ti/2(20°C)
(mg/l)
log
98.0
41
0.005
5.55
6.3
3.21
97.0
N.D. b)
KOW
99.7
20
1,710
2.11
99.5
18
1,100
3.24; 3.30
97.0
N.D.
2,800
1.16
99.0
1.6
3,210
1.87
99.5
N.D.
90,000
-1.61; -i.17
98.0
6.5
ig; 30
4.02; 4.09
98.0
low v o l a t i l e
420
2.80; 2.97
3,633
2,620
2.05
1,011
21 ; 18
2.65
2,984
2O
3.69; 3.81
98.0 ='94 99.0 99.0
N.D.
30; 47
2.64; 2.75
99.8 97.0
5,484 N.D.
7.6 12,400
3.20; 3.30 1.85; 1.92
1360
density After
was
taken
mixing,
controls shaken
the
were
daylight
fluorescent distance
The
120 ~ E
• m -2
effective
light
LI-185-A
Power
Supply
After
0, 72 and
from each
times
test c u l t u r e
using
numbers.
chamber
method
for
was m e a s u r e d
by means
Inc.,
USA,
with
colour
flasks
were
25 w h i t e
light
protected
universal).
intensity
The
was
s -I. The p h o t o s y n t h e t i c a l l y of the Quantum~Radiometer~Photo-
the special
the cell
and
from
growth
Quantum
Sensor
layer
of cell
of a 10-mm
the c o n t r o l s
PMQ II. The
established
The d e t e r m i n a t i o n
was m e a s u r e d
extinction
units were
standard
of the cell
LI-190
SB and
curve
numbers
suspensions
at 578 nm using converted
relating
a
to cell
extinction
was p e r f o r m e d
units
by the
of T o x i c E f f e c t s
the toxic
effects
96 hours.
These
are
are
the ECI0
the c o n c e n t r a t i o n s
and EC50
values
of the c h e m i c a l
tested,
of cells
is reduced
by i0 % or 50 %, respectively,
of that
area A below
the growth
curves
to
the number The
of a ch e m i c a l
is c a l c u l a t e d
according
(i):
F
( N o , , F N 1) 2
2 NO
] [ t 1 4-
o 2
of c e l l s / m l
at the b e g i n n i n g
N 1 = number
of c e l l s / m l
at time
N 2 = number
of cells/ml
at time
t I = time of
first m e a s u r e m e n t
t 2 = time of second
inhibition
control
Four
caps were
from the side by two
I0 cm. The photon
N O = number
The
Erlenmeyer
58 Watt,
phase.
104 cells/ml.
with K a p s e n b e r g
illuminated
TL-D
was
growth
of Uterm~h123.
within
of controls.
A =
300-mi
• m -2
a previously
2.4.4 E v a l u a t i o n
equation
Type:
approximately flasks
• 1019 p h o t o n s
96 hours,
to cell
for which
The
the e x p o n e n t i a l
185 PSA.
numbers,
obtained
was
continuously
intensity
spectrophotometer
The basis
density
per day.
(PHILIPS,
of L I C O R
during
Erlenmeyer
the two lamps
s -I = 7.2
meter
cell
300-mi
and were
lamps
between
the p r e - c u l t u r e
initial
used.
two to three
against
ZEISS
from
area A c and
A T , expressed
t2
of the test
(4th day)
after
JA
after
beginning beginning
is c a l c u l a t e d
the area ~ e l o w
as percent
(I)
t I (3rd day)
measurement
of cell growth
]
(t 2 _ tl )
the curve
of the control
c
the test the test
(3rd day) (4th day)
as the d i f f e r e n c e
(eq.
between
inhibited
the growth
2):
(2) x IOO
A
of
for the t o x i c a l l y
area A c
Ac - AT JA -
of
1361
The c o n c e n t r a t i o n - effect r e l a t i o n s h i p was plotted on s e m i l o g a r i t h m i c
paper.
The ECI0 and EC50 values were d e t e r m i n e d graphically.
3. RESULTS
In T a b l e
2 the results of the growth
(96-h ECI0 and 96-h EC50)
inhibition tests of S c e n e d e s m u s
are presented.
benzene no effects could be o b s e r v e d
subspicatus
In case of h e x a c h l o r o b e n z e n e
in saturated
and
aqueous solutions. The table
reveals that a wide range of toxicities was found among the tested chemicals: The 96-h ECI0 and 96-h EC50 values ranged over 5 orders of m a g n i t u d e 0.03 mg/l
and } 1360 mg/l, or 0.09 mg/l
to } 1360 mg/l,
(between
respectively).
4 . C O M P A R I S O N OF RESULTS W I T H L I T E R A T U R E V A L U E S
In l i t e r a t u r e m a n y data on the effects of c h e m i c a l s on algae growth are reported 3,4,7,24,25.
However,
it is d i f f i c u l t
d i r e c t l y with those from the literature,
to compare the above results
because d i f f e r e n t algae strains,
e v a l u a t i o n methods and test c o n d i t i o n s have been used. N e v e r t h e l e s s , cases
the literature data of toxic effects on algae
(EC50)
in some
are in good a g r e e m e n t
with our EC50 values.
For the alga S c e n e d e s m u s quadricauda,
A d e m a and Vink 26 reported a 50% reduction
of alga growth with p e n t a c h l o r o p h e n o l
at 0.08
of 50 % growth
inhibition for the alga S c e n e d e s m u s
per litre. L i n d a n e
insensitive
acutus was 0.055 mg a t r a z i n e
inhibited algae growth of S c e n e d e s m u s
3 days at a c o n c e n t r a t i o n of 5 mg/l various algae,
mg/l. B 6 h m 27 found that the level
including S c e n e d e s m u s
acutus by 51.4 % after
(Krishnakumari) 28. C u l l i m o r e 29 reported sp. and C h l o r e l l a
to 2 , 6 - d i c h l o r o b e n z o n i t r i l e
(dichlobenil)
sp., are,
that
in vitro,
in c o n c e n t r a t i o n s
up to
1 mg/l.
Most of the literature data on volatile c h e m i c a l s o b t a i n e d by means of growth inhibition tests on algae u n d e r e s t i m a t e difficulty
the toxicity.
in m a i n t a i n i n g c o n s t a n t c o n c e n t r a t i o n s .
This
is due to the
The 24-h EC50 of benzene to
an unknown alga species was 525 mg/l 30. The only recent data available t r i c h l o r o e t h y l e n e give EC50 values between 8 - 63 mg/130'31. investigating Selenastrum capricornutum 1,2,4-trichlorobenzene.
Galassi
for
and Vighi 32
found a 96-h EC50 value of 1.4 mg/l
for
1362
Table 2 Results of the Algal Growth Inhibition Test and Hazard Ranking Alga Scenedesmus subspicatus
No.
Chemical
96-h ECI0 a) (mg/l)
96-h EC50 D) (mg/l)
> 0 . 0 1 c)
for the
Hazard ~gnking Class
1
Hexachlorobenzene
2
Tr is- (2,3-dibromopropyl) -phosphate
3
Benzene
4
Tr ichloroethylene
5
Styrene-7,8-oxide
6
I, l-Dichloroethylene
7a 7b 7c
Thiourea
0.35} 0.3 0.42 0.6
8
i, 2,4-Tr ichlorobenzene
3.0
8.4
II (medium)
9
2,4,6-Tr ichlorophenol
I.I
5.6
II (medium)
i0
4-Chloroaniline
0.4
2.4
II
(medium)
ii
2,6-Dichlorobenzonitrile
0.3
2.7
II
(medium)
12
Pentachlorophenol (PCP)
0.03
0.09
III (high)
13
Atrazine
0.04
0.ii
III (high)
14
Lindane
15
4-N itrophenol
0.17 >1360 c)
(~-HCH)
> 0.01
uncertain
3.1
II (medium)
} 1360 c)
I
300
450
I
(low)
12
32
I
(low)
240
410
I
(low)
5.i} 4 6.8 10
II
(medium)
0.5
2.5
8.0
32.0
(low)
II (medium) I
(low)
a) 96-h ECI0: Effective 10 % in 96 h
concentration
inhibiting
the algal growth by
b) 96-h EC50: Effective 50 % in 96 h
concentration
inhibiting
the algal growth by
c) Water
solubility
concentrations
of the chemical
needed
limited
to determine
d) See text for explanation.
the examination
ECI0 and EC50 values.
of higher
1363
5. EVALUATION OF TEST RESULTS AND TOXICITY CLASSIFICATION RATING SCHEME The determined effective concentration Scenedesmus
levels which inhibit the 9rowth of
subspicatus by 50 % in 96 hrs
toxicity classes,
(96-h EC50)
ranging from low to high,
can be divided
into three
in accordance with the acute
toxicity for Daphnia33: CLASS I: Low hazard potential CLASS II: Medium
for the alga
(or uncertain)
(96-h E C 5 0 : i - I 0
hazard potential
scheme, tential
for the alga
the 15 chemicals classified
it can be seen that five chemicals
trichloroethylene,
styrene oxide,
(Class I) for Scenedesmus in tightly closed
inhibited.
Therefore,
(96-h EC50:
(benzene,
and 4-nitrophenol) subspicatus.
that, like in the experiments reported performed
for the alga
mg/l)
CLASS III: High hazard potential When considering
(96-h EC50: > i0 mg/l)
l,l-dichloroethylene, possess low hazard po-
However,
in literature,
it should be considered
the tests could not be
flasks, since in this case algal growth
is strongly
the maintenance of the initial chemical concentration
the algal suspensions during 96 hours is not possible,
resulting
in
in EC50 values
which probably are higher than the real ones. Seven chemicals robenzene, lindane)
(thiourea,
4-chloroaniline,
1,2,4-trichlo-
2,6-dichlorobenzonitrile
are ranked in class II which means medium hazard potential.
hazard potential chlorophenol ricide,
tris(2,3-dibromopropyl)-phosphate,
2,4,6-trichlorophenol, (class III)
for the alga Scenedesmus
(PCP) which is used as a herbicide,
and
The highest
subspicatus showed penta-
algicide,
fungicide and bacte-
and atrazine which is used as a herbicide.
6. DISCUSSION The physico-chemical aqueous solution,
properties of the chemicals tested such as volatility
water solubility and n-octanol/water
covered a wide range of values
from
partition coefficient
(see Table i).
Most of the tested substances are important pesticides or industrial chemicals or intermediates
for other compounds
in large quantities. PCP, [ - H C H
(see Table i), which are produced and used
Some of them are dispersed
and trichloroethylene.
in the environment,
such as HCB,
HCB is also a by-product of the chlorination
of aliphatic hydrocarbons and other
industrial chlorination processes 34 as well
1364
as in waste combustion
35
ment and to humans. Tris
and is known as a chemical hazardous (2,3-dibromopropyl)phosphate
and styrene-7,8-oxide
shown mutagenic activity by the Ames test 36. For benzene• evidence
for carcinogenicity
in humans 37. There
for the carcinogenicity of HCB in experimental Wistar
rats HCB produced
produced hepatomas, limited evidence chloroethylene,
liver cell tumors.
styrene oxide,
mers 37'38. Therefore, are of increasing
have
there is sufficient
is also sufficient evidence
animals 37. In Swiss mice and
In Syrian golden hamsters, HCB
liver hemangioentheliomas
for carcinogenicity
to the environ-
and thyroid adenomas 38. There
in animals of l,l-dichloroethylene,
lindane and other hexachlorocyclohexane
is
tri-
iso-
studies on the fate and toxic effects of these chemicals
importance.
AS a consequence of the toxicity studies reported here, the following
facts can
be stated: i. The alga Scenedesmus subspicatus
is an organism which is sensitive to the
effects of the tested chemicals and, therefore,
can be considered
as a good
model for toxicity testing. 2. The guideline
for the algal growth
inhibition test of the German Federal
Environmental Agency can be sucessfully applied as a reliable screening test for obtaining
information on the concentrations of various chemicals
to be hazardous 3. In our opinion, benzene,
likely
to this alga. the test is not suitable
trichloroethylene
for volatile substances
and dichloroethylene.
such as
In these cases the real ECI0
and EC50 values probably are lower than those given in Table 2. 4. Since "open"
systems
(Erlenmeyer
flasks with Kapsenberg caps)
were deli-
berately employed to facilitate gas exchange and hence volatilisation, since adsorption on algae occurs 39,40,
the chemical concentrations
and
reported
apply only to the beginning of the test. 5. For chemicals
such as 2,4,6-trichlorophenol,
pentachlorophenol
and 4-nitro-
phenol there exists a dependency of the toxic effects upon the pH value. At pH values near the pKa value,
these chemicals are non-ionized
and more
toxic because weak acids penetrate biological membranes more easily in the non-ionized
form. The phenolic compounds are more toxic at pH values below 7
(see references 41 and 42). Changes of pH-values during the test may vary, depending on experimental
conditions.
This may provide an explanation
variations in ECI0 und EC50 values determined different laboratories. 6. Extrapolation of the results obtained using Scenedesmus freshwater
subspicatus
ecosystems
of natural populations
for these chemicals
for the
in
in the algae growth inhibiton test
to other algae species and to natural
is limited and problematic 43. Predicting is difficult
the response
because laboratory culture conditions
often differ greatly from natural conditions.
1365
7. In nature complex situations are met, where several toxicants may be present simultaneously
and where interactions
bioconcentration variations
and detoxication may occur.
in temperature,
possible to simulate 8. Nevertheless,
in processes such as degradation, In addition,
there exist natural
illumination and nutrient levels which are im-
in the laboratory.
the results show the differences
in the effects of individual
chemicals and the sensitivity of the alga Scenedesmus to these chemicals. They also give valuable
information on how to study these chemicals more
thoroughly under more complicated
test conditions.
7. CONCLUSIONS Since,
in aquatic ecosystems,
tions,
it would be logical
parameter
into account.
algae are exposed to chemicals
in ecotoxicity assessment
in dynamic condi-
to take this important
It has been shown by Jouany et al. 44 that the acute
toxicity of inorganic chemicals
such as Cu 2+, Cd 2+ and Pb 2+, which are accumu-
lated to a high extent by Chlorella,
is much higher under dynamic conditions
than under static conditions. We suggest that this would be similar organic chemicals. high volatility,
Especially,
in testing
for chemicals with low water solubility and/or
the static tests yield toxicity data which are lower than the
real ones. Thus it may be assumed that the static test gives optimistic and understates
the toxicity to algae. Therefore,
dynamic test as done by Jouany et al. 44. However, algae are difficult
to carry out and, therefore,
results
it is recommended to use a dynamic tests with unicellular it would be reasonable to
choose a pseudodynamic method of exposure 44 for a representative assessment of toxicity of chemicals, solubility,
at least of those with high volatility and/or low water
to algae.
ACKNOWLEDGEMENT
The authors would like to thank Dr. Renate Kdhn of the Institute for Water, Soil and Air Hygiene, Berlin, FRG,
for special
information regarding the algae growth
test and for providing us with algae cultures. We are grateful to Shell Research Ltd. Sittingbourne, Dr. H.Arent,
U.K.,
for providing
the 2,6-dichlorobenzonitrile,
Dr. R. Fischer and Dr. A. C. M. Willems
for valuable
and to
informations.
The experimental part of this study was funded by the Federal Environmental Agency, Berlin, FRG, Contract No. 106 04 011/02, by a grant of the Federal Ministry of the Interior, and of the Commission of the European Communities, Contract No. ENV-653-D(B).
1366
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0 0 4 5 - 6 5 3 5 / 8 5 $3.00 + .00 ©1985 P e r g a m o n Press Ltd.
THE EFFECTS OF ORGANIC E N V I R O N M E N T A L C H E M I C A L S ON THE G R O W T H OF THE ALGA S c e n e d e s m u s
subspicatus:
A C O N T R I B U T I O N TO E N V I R O N M E N T A L B I O L O G Y
Harald Geyer,
Gesellschaft
Irene S c h e u n e r t and F r i e d h e l m Korte
fur S t r a h l e n Institut
und U m w e l t f o r s c h u n g mbH M ~ n c h e n
fur O k o l o g i s c h e Chemie
Ingolst~dter LandstraBe
1
D-8042 N e u h e r b e r g Federal R e p u b l i c of G e r m a n y
ABSTRACT The e f f e c t i v e c o n c e n t r a t i o n s of 15 chemicals, inhibiting the cell growth of the alga S c e n e d e s m u s s u b s p i c a t u s by I0 % and 50 % during 96 hours, have been investigated in a static test under c o n t r o l l e d laboratory conditions.
i. I N T R O D U C T I O N
T o d a y there
is a great need for m e t h o d s
living organisms. States, market similar
According
to predict
the effects of c h e m i c a l s on
to r e g u l a t i o n s of the E u r o p e a n C o m m u n i t y Member
e c o t o x i c o l o g i c a l testing of new s u b s t a n c e s which are introduced into the 1 is n e c e s s a r y . The OECD Member c o u n t r i e s have, in principle, agreed to a testing scheme.
Studies of the effects of c h e m i c a l s on r e p r e s e n t a t i v e form a great part of e c o t o x i c o l o g i c a l with
fish, Daphnia,
is also required
testing and research.
e a r t h w o r m s and terrestrial
important p r i m a r y p r o d u c e r s
are c o n s u m e d by other aquatic organisms,
testing
and oysters.
in m o n o s p e c i e s
using the green
plants,
In a d d i t i o n to tests
a growth test with algae
in legislation 2.
A l g a e are the most
snails, m u s s e l s
species of flora and fauna
Several
in the aquatic environment.
such as Daphnia,
Rotatoria,
Amphipods,
algae have been used for e c o t o x i c o l o g i c a l
cultures 3-9 or in mixed c u l t u r e s I0-II. A batch test,
freshwater
They
alga S c e n e d e s m u s
1355
spec.
as a test organism,
was
1356
recently proposed by the OECD 12 and the German Federal Environmental Agency (Umweltbundesamt), they are released
Berlin 13, for screening and evaluating
new chemicals before
into the market and thus into the environment.
The present paper deals with the effects of 15 organic chemicals on the growth of the freshwater controlled
unicellular green alga Scenedesmus
laboratory conditions.
subspicatus Chodat under
One objective of this study was the
examination of the feasibility of the algae growth
inhibition test guideline 13.
The study involves also a comparison of the toxicity of the chemicals
to this
alga species and gives a hazard ranking of the tested organic substances.
2. MATERIALS AND METHODS
2.1 Chemicals 2,6-Dichlorobenzonitrile
was a gift of Shell Research Ltd., Sittingbourne,
U.K.
The other organic chemicals were purchased commercially and were used without further purification. volatility
They are listed in Table 1 including their use, purity,
from aqueous solution,
coefficient.
Acetone,
water solubility and n-octanol/water
used as a solvent, was of analytical
(purity{99.5 %) and purchased
partition
reagent grade quality
from E. Merck, FRG. All substances used for the
preparation of the nutrient medium were also of analytical
reagent grade quali-
ty. The water used was double distilled and sterilised. 2.2 Test Organism The usually unicellular
freshwater green alga Scenedesmus
(86, 81 SAG), belonging
to the order of Chlorococcales
phyceae), Hygiene,
was obtained
from Dr. Renate K0hn,
subspicatus Chodat
(Chlorophyta, Chloro-
Institute for Water, Soil and Air
Federal Health Office, Berlin-Dahlem,
FRG.
2.3 Stock-Breedin 9 Conditions The stock cultures of Scenedesmus stored
in 100 ml Erlenmeyer
subspicatus
placed on a white surface protected lighting by PHILIPS
in 20 ml nutrient solution were
flasks stoppered with metal caps against daylight,
fluorescent lamps
(Type: TL-D,
at 21-25 Oc. For maintenance of the test strain, prepared continuously at 10 days' flasks containing sterilized
intervals.
(Kapsenberg-caps),
and exposed to constant
58 Watts,
25 white universal)
fresh stock cultures were
For this purpose the Erlenmeyer
20 ml of nutrient solution 3 and stoppered with metal caps were
in a steam sterilizer
for 30 min. After cooling,
flask was inoculated with 2 ml algae suspension
the content of each
from a 10-day stock culture
1357
2.4 D e s c r i p t i o n of the Test P r o c e d u r e
The
test was p e r f o r m e d
Environmental Agency
in a c c o r d a n c e with the test g u i d e l i n e of the Federal
(Umweltbundesamt)
Berlin,
FRG 13. The nutrient m e d i u m and
washed g l a s s w a r e were sterilized by a u t o c l a v i n g prior to use. E x p e r i m e n t s stock c u l t u r e s were photosynthetically photons
• m -2
and
incubated at 22 + 2 °C at c o n s t a n t c o n d i t i o n s with a effective
light
intensity of 120 p E • m -2
• s-i(7.2
• 1019
s-i).
2.4.1 P r e p a r a t i o n of Test S u b s t a n c e S o l u t i o n
A defined q u a n t i t y of the c h e m i c a l
to be tested was diluted
in a defined volume
of sterilized double d i s t i l l e d water. A c e t o n e was the solvent carrier c h e m i c a l s of low water rophenol
s o l u b i l i t y such as h e x a c h l o r o b e n z e n e
(PCP), lindane
(~-HCH),
for
(HCB), p e n t a c h l o -
atrazine and t r i s ( 2 , 3 - d i b r o m o p r o p y l ) - p h o s p h a t e .
The m a x i m u m c o n c e n t r a t i o n of acetone was 0.I ml per i000 ml nutrient solution. The same amount of acetone was added to the control
subcultures.
In general
the
test was carried out without a d j u s t m e n t of the pH value of the test substance solution.
In the case of
nitrophenol
there were
the chemicals.
2,4,6-trichlorophenol, p e n t a c h l o r o p h e n o l
(PCP)
and 4-
indications of a d i s t i n c t change of pH value caused by
In these cases
the pH of the stock solution was adjusted
to pH 7
using NaOH.
2.4.2 P r e - c u l t u r e
The p r e - c u l t u r e was started
3 days prior
to the beginning of the test. Less than
one part of an algae s u s p e n s i o n taken from the stock culture was mixed with one part by volume of the c o n c e n t r a t e d
n u t r i e n t m e d i u m and 8 parts by volume of
water. After d i l u t i o n up to 10 parts by volume, p r e - c u l t u r e was
in the order of 104 cells/ml.
volume of 50 ml were
incubated
the cell c o n c e n t r a t i o n Of the
The p r e - c u l t u r e s with a total
in 300-mi E r l e n m e y e r
flasks with K a p s e n b e r g caps
for 3 days under the same t e m p e r a t u r e and light c o n d i t i o n s
as those of the test
culture.
2.4.3 P e r f o r m a n c e of the Test
The e x p e r i m e n t s were p e r f o r m e d by p r e p a r i n g 300-ml E r l e n m e y e r contained
flasks as follows.
80 ml of a saturated
The
test chemical
s u b s e q u e n t d i l u t i o n s were prepared
two parallel d i l u t i o n series
in
first flask of each d i l u t i o n series solution.
Starting
from this flask,
using a c o n s t a n t d i l u t i o n ratio of 40 ml
p r e l i m i n a r y chemical d i l u t i o n plus 40 ml d o u b l e - d i s t i l l e d water. T h e n 5 ml of m e d i u m and 5 ml of the algae s u s p e n s i o n of the p r e l i m i n a r y culture were added to each flask of the d i l u t i o n series
(40 ml). The algal s u s p e n s i o n of known cell
1358
TABLE Use, Purity and Relevant Physico-Chemical
NO. CHEMICAL
I
Hexachlorobenzene
MAJOR USES
Fungicide; manufactures of PCP and aromatic fluorocarbons
2 3
Tris-(2,3-dibromopropyl)-
Flame retardant in polyurethane foam and
phosphate (Tris)
plastics
Benzene
Solvent; intermediate for many chemicals; gasoline additive
4
Trichloroethylene (Tri)
5
Styrene-7,8-oxide
Solvent; cleansing; metal de~reasing In polymer industry as a diluent for production of epoxy resins
6
1,1-Dichloroethylene (Vinylidenechloride)
Production of copolymers and modacrylic fibres
7
Thiourea
Production of flame retardant resins;
8
1,2,4-Trichlorobenzene
vulcanization accelerator Solvent; transformer o i l ;
heat transfer
medium ; d i e l e c t r i c f l u i d ; dye c a r r i e r ; intermediate (2,4-D)
9
2,4,6-Trichlorophenol
Wood preservative; bactericide; herbicide and d e f o l i a n t
10 4-Chloroaniline
Intermediate in manufacture of many
11 2,6-Dichlorobenzonitrile (Dichlobenil: DBN)
Herbicide
12 Pentachlorophenol (PCP)
Wood preservative; herbicide; defoliant;
chemicals (herbicides)
sodium salt: algicide; fungicide 13 Atrazine
Herbicide
14 Lindane (~-HCH)
Insecticide; scabicide; pediculicide
15 4-Nitrophenol
Intermediate in manufactures of many chemicals
a) V o l a t i l i t y from aqueous solution (hour/m depth); Data from I. Scheunert22 b) N.D.: Not determined
1359
Properties of the Tested Organic Chemicals
Purity
V o l a t i l i t y a)
Water S o l u b i l i t y I~-17
n-Octanol/Water Partition Coefficient~a,15,18-21
(%)
ti/2(20°C)
(mg/l)
log
98.0
41
0.005
5.55
6.3
3.21
97.0
N.D. b)
KOW
99.7
20
1,710
2.11
99.5
18
1,100
3.24; 3.30
97.0
N.D.
2,800
1.16
99.0
1.6
3,210
1.87
99.5
N.D.
90,000
-1.61; -i.17
98.0
6.5
ig; 30
4.02; 4.09
98.0
low v o l a t i l e
420
2.80; 2.97
3,633
2,620
2.05
1,011
21 ; 18
2.65
2,984
2O
3.69; 3.81
98.0 ='94 99.0 99.0
N.D.
30; 47
2.64; 2.75
99.8 97.0
5,484 N.D.
7.6 12,400
3.20; 3.30 1.85; 1.92
1360
density After
was
taken
mixing,
controls shaken
the
were
daylight
fluorescent distance
The
120 ~ E
• m -2
effective
light
LI-185-A
Power
Supply
After
0, 72 and
from each
times
test c u l t u r e
using
numbers.
chamber
method
for
was m e a s u r e d
by means
Inc.,
USA,
with
colour
flasks
were
25 w h i t e
light
protected
universal).
intensity
The
was
s -I. The p h o t o s y n t h e t i c a l l y of the Quantum~Radiometer~Photo-
the special
the cell
and
from
growth
Quantum
Sensor
layer
of cell
of a 10-mm
the c o n t r o l s
PMQ II. The
established
The d e t e r m i n a t i o n
was m e a s u r e d
extinction
units were
standard
of the cell
LI-190
SB and
curve
numbers
suspensions
at 578 nm using converted
relating
a
to cell
extinction
was p e r f o r m e d
units
by the
of T o x i c E f f e c t s
the toxic
effects
96 hours.
These
are
are
the ECI0
the c o n c e n t r a t i o n s
and EC50
values
of the c h e m i c a l
tested,
of cells
is reduced
by i0 % or 50 %, respectively,
of that
area A below
the growth
curves
to
the number The
of a ch e m i c a l
is c a l c u l a t e d
according
(i):
F
( N o , , F N 1) 2
2 NO
] [ t 1 4-
o 2
of c e l l s / m l
at the b e g i n n i n g
N 1 = number
of c e l l s / m l
at time
N 2 = number
of cells/ml
at time
t I = time of
first m e a s u r e m e n t
t 2 = time of second
inhibition
control
Four
caps were
from the side by two
I0 cm. The photon
N O = number
The
Erlenmeyer
58 Watt,
phase.
104 cells/ml.
with K a p s e n b e r g
illuminated
TL-D
was
growth
of Uterm~h123.
within
of controls.
A =
300-mi
• m -2
a previously
2.4.4 E v a l u a t i o n
equation
Type:
approximately flasks
• 1019 p h o t o n s
96 hours,
to cell
for which
The
the e x p o n e n t i a l
185 PSA.
numbers,
obtained
was
continuously
intensity
spectrophotometer
The basis
density
per day.
(PHILIPS,
of L I C O R
during
Erlenmeyer
the two lamps
s -I = 7.2
meter
cell
300-mi
and were
lamps
between
the p r e - c u l t u r e
initial
used.
two to three
against
ZEISS
from
area A c and
A T , expressed
t2
of the test
(4th day)
after
JA
after
beginning beginning
is c a l c u l a t e d
the area ~ e l o w
as percent
(I)
t I (3rd day)
measurement
of cell growth
]
(t 2 _ tl )
the curve
of the control
c
the test the test
(3rd day) (4th day)
as the d i f f e r e n c e
(eq.
between
inhibited
the growth
2):
(2) x IOO
A
of
for the t o x i c a l l y
area A c
Ac - AT JA -
of
1361
The c o n c e n t r a t i o n - effect r e l a t i o n s h i p was plotted on s e m i l o g a r i t h m i c
paper.
The ECI0 and EC50 values were d e t e r m i n e d graphically.
3. RESULTS
In T a b l e
2 the results of the growth
(96-h ECI0 and 96-h EC50)
inhibition tests of S c e n e d e s m u s
are presented.
benzene no effects could be o b s e r v e d
subspicatus
In case of h e x a c h l o r o b e n z e n e
in saturated
and
aqueous solutions. The table
reveals that a wide range of toxicities was found among the tested chemicals: The 96-h ECI0 and 96-h EC50 values ranged over 5 orders of m a g n i t u d e 0.03 mg/l
and } 1360 mg/l, or 0.09 mg/l
to } 1360 mg/l,
(between
respectively).
4 . C O M P A R I S O N OF RESULTS W I T H L I T E R A T U R E V A L U E S
In l i t e r a t u r e m a n y data on the effects of c h e m i c a l s on algae growth are reported 3,4,7,24,25.
However,
it is d i f f i c u l t
d i r e c t l y with those from the literature,
to compare the above results
because d i f f e r e n t algae strains,
e v a l u a t i o n methods and test c o n d i t i o n s have been used. N e v e r t h e l e s s , cases
the literature data of toxic effects on algae
(EC50)
in some
are in good a g r e e m e n t
with our EC50 values.
For the alga S c e n e d e s m u s quadricauda,
A d e m a and Vink 26 reported a 50% reduction
of alga growth with p e n t a c h l o r o p h e n o l
at 0.08
of 50 % growth
inhibition for the alga S c e n e d e s m u s
per litre. L i n d a n e
insensitive
acutus was 0.055 mg a t r a z i n e
inhibited algae growth of S c e n e d e s m u s
3 days at a c o n c e n t r a t i o n of 5 mg/l various algae,
mg/l. B 6 h m 27 found that the level
including S c e n e d e s m u s
acutus by 51.4 % after
(Krishnakumari) 28. C u l l i m o r e 29 reported sp. and C h l o r e l l a
to 2 , 6 - d i c h l o r o b e n z o n i t r i l e
(dichlobenil)
sp., are,
that
in vitro,
in c o n c e n t r a t i o n s
up to
1 mg/l.
Most of the literature data on volatile c h e m i c a l s o b t a i n e d by means of growth inhibition tests on algae u n d e r e s t i m a t e difficulty
the toxicity.
in m a i n t a i n i n g c o n s t a n t c o n c e n t r a t i o n s .
This
is due to the
The 24-h EC50 of benzene to
an unknown alga species was 525 mg/l 30. The only recent data available t r i c h l o r o e t h y l e n e give EC50 values between 8 - 63 mg/130'31. investigating Selenastrum capricornutum 1,2,4-trichlorobenzene.
Galassi
for
and Vighi 32
found a 96-h EC50 value of 1.4 mg/l
for
1362
Table 2 Results of the Algal Growth Inhibition Test and Hazard Ranking Alga Scenedesmus subspicatus
No.
Chemical
96-h ECI0 a) (mg/l)
96-h EC50 D) (mg/l)
> 0 . 0 1 c)
for the
Hazard ~gnking Class
1
Hexachlorobenzene
2
Tr is- (2,3-dibromopropyl) -phosphate
3
Benzene
4
Tr ichloroethylene
5
Styrene-7,8-oxide
6
I, l-Dichloroethylene
7a 7b 7c
Thiourea
0.35} 0.3 0.42 0.6
8
i, 2,4-Tr ichlorobenzene
3.0
8.4
II (medium)
9
2,4,6-Tr ichlorophenol
I.I
5.6
II (medium)
i0
4-Chloroaniline
0.4
2.4
II
(medium)
ii
2,6-Dichlorobenzonitrile
0.3
2.7
II
(medium)
12
Pentachlorophenol (PCP)
0.03
0.09
III (high)
13
Atrazine
0.04
0.ii
III (high)
14
Lindane
15
4-N itrophenol
0.17 >1360 c)
(~-HCH)
> 0.01
uncertain
3.1
II (medium)
} 1360 c)
I
300
450
I
(low)
12
32
I
(low)
240
410
I
(low)
5.i} 4 6.8 10
II
(medium)
0.5
2.5
8.0
32.0
(low)
II (medium) I
(low)
a) 96-h ECI0: Effective 10 % in 96 h
concentration
inhibiting
the algal growth by
b) 96-h EC50: Effective 50 % in 96 h
concentration
inhibiting
the algal growth by
c) Water
solubility
concentrations
of the chemical
needed
limited
to determine
d) See text for explanation.
the examination
ECI0 and EC50 values.
of higher
1363
5. EVALUATION OF TEST RESULTS AND TOXICITY CLASSIFICATION RATING SCHEME The determined effective concentration Scenedesmus
levels which inhibit the 9rowth of
subspicatus by 50 % in 96 hrs
toxicity classes,
(96-h EC50)
ranging from low to high,
can be divided
into three
in accordance with the acute
toxicity for Daphnia33: CLASS I: Low hazard potential CLASS II: Medium
for the alga
(or uncertain)
(96-h E C 5 0 : i - I 0
hazard potential
scheme, tential
for the alga
the 15 chemicals classified
it can be seen that five chemicals
trichloroethylene,
styrene oxide,
(Class I) for Scenedesmus in tightly closed
inhibited.
Therefore,
(96-h EC50:
(benzene,
and 4-nitrophenol) subspicatus.
that, like in the experiments reported performed
for the alga
mg/l)
CLASS III: High hazard potential When considering
(96-h EC50: > i0 mg/l)
l,l-dichloroethylene, possess low hazard po-
However,
in literature,
it should be considered
the tests could not be
flasks, since in this case algal growth
is strongly
the maintenance of the initial chemical concentration
the algal suspensions during 96 hours is not possible,
resulting
in
in EC50 values
which probably are higher than the real ones. Seven chemicals robenzene, lindane)
(thiourea,
4-chloroaniline,
1,2,4-trichlo-
2,6-dichlorobenzonitrile
are ranked in class II which means medium hazard potential.
hazard potential chlorophenol ricide,
tris(2,3-dibromopropyl)-phosphate,
2,4,6-trichlorophenol, (class III)
for the alga Scenedesmus
(PCP) which is used as a herbicide,
and
The highest
subspicatus showed penta-
algicide,
fungicide and bacte-
and atrazine which is used as a herbicide.
6. DISCUSSION The physico-chemical aqueous solution,
properties of the chemicals tested such as volatility
water solubility and n-octanol/water
covered a wide range of values
from
partition coefficient
(see Table i).
Most of the tested substances are important pesticides or industrial chemicals or intermediates
for other compounds
in large quantities. PCP, [ - H C H
(see Table i), which are produced and used
Some of them are dispersed
and trichloroethylene.
in the environment,
such as HCB,
HCB is also a by-product of the chlorination
of aliphatic hydrocarbons and other
industrial chlorination processes 34 as well
1364
as in waste combustion
35
ment and to humans. Tris
and is known as a chemical hazardous (2,3-dibromopropyl)phosphate
and styrene-7,8-oxide
shown mutagenic activity by the Ames test 36. For benzene• evidence
for carcinogenicity
in humans 37. There
for the carcinogenicity of HCB in experimental Wistar
rats HCB produced
produced hepatomas, limited evidence chloroethylene,
liver cell tumors.
styrene oxide,
mers 37'38. Therefore, are of increasing
have
there is sufficient
is also sufficient evidence
animals 37. In Swiss mice and
In Syrian golden hamsters, HCB
liver hemangioentheliomas
for carcinogenicity
to the environ-
and thyroid adenomas 38. There
in animals of l,l-dichloroethylene,
lindane and other hexachlorocyclohexane
is
tri-
iso-
studies on the fate and toxic effects of these chemicals
importance.
AS a consequence of the toxicity studies reported here, the following
facts can
be stated: i. The alga Scenedesmus subspicatus
is an organism which is sensitive to the
effects of the tested chemicals and, therefore,
can be considered
as a good
model for toxicity testing. 2. The guideline
for the algal growth
inhibition test of the German Federal
Environmental Agency can be sucessfully applied as a reliable screening test for obtaining
information on the concentrations of various chemicals
to be hazardous 3. In our opinion, benzene,
likely
to this alga. the test is not suitable
trichloroethylene
for volatile substances
and dichloroethylene.
such as
In these cases the real ECI0
and EC50 values probably are lower than those given in Table 2. 4. Since "open"
systems
(Erlenmeyer
flasks with Kapsenberg caps)
were deli-
berately employed to facilitate gas exchange and hence volatilisation, since adsorption on algae occurs 39,40,
the chemical concentrations
and
reported
apply only to the beginning of the test. 5. For chemicals
such as 2,4,6-trichlorophenol,
pentachlorophenol
and 4-nitro-
phenol there exists a dependency of the toxic effects upon the pH value. At pH values near the pKa value,
these chemicals are non-ionized
and more
toxic because weak acids penetrate biological membranes more easily in the non-ionized
form. The phenolic compounds are more toxic at pH values below 7
(see references 41 and 42). Changes of pH-values during the test may vary, depending on experimental
conditions.
This may provide an explanation
variations in ECI0 und EC50 values determined different laboratories. 6. Extrapolation of the results obtained using Scenedesmus freshwater
subspicatus
ecosystems
of natural populations
for these chemicals
for the
in
in the algae growth inhibiton test
to other algae species and to natural
is limited and problematic 43. Predicting is difficult
the response
because laboratory culture conditions
often differ greatly from natural conditions.
1365
7. In nature complex situations are met, where several toxicants may be present simultaneously
and where interactions
bioconcentration variations
and detoxication may occur.
in temperature,
possible to simulate 8. Nevertheless,
in processes such as degradation, In addition,
there exist natural
illumination and nutrient levels which are im-
in the laboratory.
the results show the differences
in the effects of individual
chemicals and the sensitivity of the alga Scenedesmus to these chemicals. They also give valuable
information on how to study these chemicals more
thoroughly under more complicated
test conditions.
7. CONCLUSIONS Since,
in aquatic ecosystems,
tions,
it would be logical
parameter
into account.
algae are exposed to chemicals
in ecotoxicity assessment
in dynamic condi-
to take this important
It has been shown by Jouany et al. 44 that the acute
toxicity of inorganic chemicals
such as Cu 2+, Cd 2+ and Pb 2+, which are accumu-
lated to a high extent by Chlorella,
is much higher under dynamic conditions
than under static conditions. We suggest that this would be similar organic chemicals. high volatility,
Especially,
in testing
for chemicals with low water solubility and/or
the static tests yield toxicity data which are lower than the
real ones. Thus it may be assumed that the static test gives optimistic and understates
the toxicity to algae. Therefore,
dynamic test as done by Jouany et al. 44. However, algae are difficult
to carry out and, therefore,
results
it is recommended to use a dynamic tests with unicellular it would be reasonable to
choose a pseudodynamic method of exposure 44 for a representative assessment of toxicity of chemicals, solubility,
at least of those with high volatility and/or low water
to algae.
ACKNOWLEDGEMENT
The authors would like to thank Dr. Renate Kdhn of the Institute for Water, Soil and Air Hygiene, Berlin, FRG,
for special
information regarding the algae growth
test and for providing us with algae cultures. We are grateful to Shell Research Ltd. Sittingbourne, Dr. H.Arent,
U.K.,
for providing
the 2,6-dichlorobenzonitrile,
Dr. R. Fischer and Dr. A. C. M. Willems
for valuable
and to
informations.
The experimental part of this study was funded by the Federal Environmental Agency, Berlin, FRG, Contract No. 106 04 011/02, by a grant of the Federal Ministry of the Interior, and of the Commission of the European Communities, Contract No. ENV-653-D(B).
1366
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in an alga.
Acute toxicity of some
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ion transport
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42. Kuivasniemi,
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(Received in Germany 16 July 1985)
in algae. Ecotox.