Acute toxicity of several oil dispersants towards the green algae Chlamydomonas and Dunaliella

Chemosphere No. 3, PP 247 - 255, 1978.

Pergamon Press.

Printed in Great Britain.

ACUTE T O X I C I T Y OF S E V E R A L OIL D I S P E R S A N T S TOWARDS THE GREEN ALGAE C H L A M Y D O M O N A S AND D U N A L I E L L A

Mikal Heldala{

Svein Norlanda!

T o r l e i v Lien a)

and Gjert 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 1 March 1978; received in UK for ~51ication 13 March 1978) Introduction

W i t h the increased oil d r i l l i n g a c t i v i t i e s

in the N o r t h Sea

and m o s t p r o b a b l y also further north in N o r w e g i a n waters,

the

p o s s i b l e needs for c h e m i c a l oil spill c l e a n i n g increase.

For

r e a l i s t i c c o n s e q u e n c e a n a l y s i s of the a p p l i c a t i o n of such chemicals,

t o x i c i t y data p e r t i n e n t to the actual c h e m i c a l s must be

available.

M u c h i n f o r m a t i o n has b e e n g a t h e r e d about the toxicity

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

towards animals,

and as an example we shall

m e n t i o n that a series of such c o m p o u n d s has b e e n a p p r o v e d

for use

at open sea by B r i t i s h a u t h o r i t i e s after testing for acute toxicity w i t h b r o w n shrimp~

U n f o r t u n a t e l y no similar data on effects

on p h y t o p l a n c t o n has b e e n a v a i l a b l e , a n d

c o n s i d e r i n g their key

role as p r i m a r y p r o d u c e r s such lack of t o x i c i t y data makes consequence a n a l y s e s of c h e m i c a l c l e a n up o p e r a t i o n s s e v e r e l y incomplete. We have t h e r e f o r e tested several d i s p e r s a n t s w i t h the unic e l l u l a r green algae C h l a m y d o m o n a s r e i n h a r d t i and D u n a l i e l l a marina.

Methods

Chlamydomonas reinhardti

(Cambridge C u l t u r e C o l l e c t i o n strain

no 11/32 b) was c u l t i v a t e d and s y n c h r o n i z e d as d e s c r i b e d in the 2 p r e c e e d i n g paper. Dunaliella marina

(G~ttingen Culture Collection,

247

synonymous

P48

No. 3

with was

D, biocula~a,

Cambridge

Chlamydomonas. which

was

supplemented

electronically

testing

a plate

(details

test,

are g i v e n

exponentially

using

for cell

Sweden).

the

transferred last well

charged. containing contained

1/3%

a control The p l a t e s

lids and

illuminated

After

48 h toxic

which

gave

sible. dead.

as w e l l s

Occasionally

Usually tures,

found b e t w e e n In the tube

w i t h NaCI, mits were

parallel

test w i t h

asynchronous estimated

to that w i t h

row,

to

suspension

Each plate of test

sub-

plastic

light at 2 k lux.

at higher

appeared

intermediate

completely

green

concen-

and almost

degrees

"all dead

also

the same dark

cells

discrepancies

50 ~i

At d i l u t i o n s

had

whereas

intermediate

invi-

to be

shades

of effect.

bleached cells",

microcul-

can e a s i l y

than one d i l u t i o n

step

series. Dunaliella

cultures

were

differently.

Chlamydomonas.

subfirstly

transfer

transparent

was b l e a c h e d

dessignated

Flow

from it and dis-

visible.

the a f f e c t e d

larger

was

mixing

instead

their

one or two w e l l s w i t h

and s e l d o m

of test

g r e e n algal

of w e l l s

giving

plates,

After

fluorescent

clearly

of the wells

in the f o l l o w i n g

be d e t e c t e d

thorough

removed

with

by Rasmus-

of a series

to each well.

of the control

dilution

of tests,

of the type used

dilutions

and so on.

the c o n t e n t s

representing

the h i g h e s t

plates

growth medium

covered

were

2.

50 ~i of test s u b s t a n c e

After

added

the m i c r o s c o p e

could be found,

were

effects

the c o n t e n t s Under

and then

from above with

no e f f e c t

appearance trations

were

no.

Chlamydomonas

Titertek/Linbro

50 ~i were

row with

stance.

types

like one d e v e l o p e d

150 ~i of a s l i g h t l y

NaHCO 3 were

done

p a p e r 2) and a tube test w i t h

of double

of the series.

and mixing,

three

titration

to the next well

Finally

were

in r e f e r e n c e

synchronous

To each well

50 ~i of g r o w t h medium,

for

medium

cultures.

(e.g.

Series

as follows:

to the first well were

micro

and c u l t u r i n g

given

with

test u s i n g

is e s s e n t i a l l y

flat b o t t o m e d

stance was made

performed

Dunaliella

test

typing

Laboratories,

added

was

as used

2,5%.

distributions

in the p r e c e e d i n g

grown

The p l a t e 3

to give

to the m e t h o d s

a tube

strain LB 19/4)

the C h l a m y d o m o n a s

and cell v o l u m e

according

Toxicity namely

w i t h NaCI

Collection

type of a p p a r a t u s

As m e d i u m was used

Cell c o u n t i n g

sen

Culture

g r o w n at 27 ° and in the same

Since

the m e d i u m was used,

Otherwise

and

supplemented

the t o x i c i t y

the test was

Dunaliella

grows

li-

analogous.

appreciably

No. 3

249

Table

1.

sants

alone

Results

(Experimental

Dispersant

Finasol

W

from

and water details

plate

test

extracts are

type

given

with

of

Chlamydomonas

mixtures

in

the

Dispersant

of

oil

and

and

disper-

dispersant.

text).

alone

Water extracts of mixtures of oil and dispersant

ii0

350

120

500

55

125

"

K

"

CC

"

OSR-2

150.000

800

"

OSR-5

195

175

(Norske

Fina

A/S)

"

OSR-7

230

250

iCorexit

7664

83.000

> 1000

"

8666

50.000

> 1000

"

9600

138.000

> 1000

"

9527

195

180

100.000

> 1000

i00.000

> i000

50.000

> i000

38.000

> 1000

BP

1100 "

X

"

WD

Shell

Dispersant

"

Oil

Dasic

Slickgone

Gamlen

LT

(nr.

Chlorothene Cold

Cleaner

Norclean Regal

A

C

X-12 Emulsol Servo

LT

Herder

CD

1000

Pero-Kleen

> 1000

LT2

100.000

> i000

154)

63.000

> 1250

7.500

> 1000

430

590

60

70

i00.000

> 1000

170

> 1000

> 250.000

> 1250

> 250.000

> 1250

> 250.000

> 1250

> 250.000

> 1250

Synperonic

26.000

> 1250

Sabreslick

> 250.000

635

BMD

(SM53)

868

360

250

No. 3

slower

than C h l a m y d o m o n a s

obtaining

significant

The t o x i c i t y wing

a 24 h testing

p e r i o d was n e c e s s a r y

for

results.

limits

were

estimated

according

to the follo-

espression:

Toxicity

effect

Bt - Btox

= I-

t

Bt B t is the b i o m a s s

of the control

and Bto x t of the

both at the end of the experiment. into a c c o u n t periment, degree

the g r o w t h

and h e n c e

the t o x i c i t y

of the c o n t r o l

the test period. number

of the culture

of u n d e r e s t i m a t i o n

biomass

culture

test culture,

does

not take

from the start of the ex-

in the p r e s e n t

increased

was m e a s u r e d

average

formula

is u n d e r e s t i m a t e d .

is small

Biomass

and w e i g h t e d

This

about

However,

case

25-fold

as the p r o d u c t

the

since

the

during of cell

cell volume.

Results

Our

investigation

concentration-series

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

i.

Dispersants

method of

developed

alone.

and

Table

i.

great

range

experiments

from

tested with

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

LC 50 values,

as well

These values

from a p a r a l l e l

plate

The most

99.5%

The a v e r a g e

toxic

test.

limit varies

system

higher

over

a

250 000 ppm

were

further

to e s t a b l i s h

giving

2 together of LC v a l u e s

using

synchronous

toxicity

in

99.5%

with

dead

results

extended

from

to 700 p p m for Cold Cleaner.

of the two methods, in the

than

compounds

in Table

The range

results

are listed

CC to more

Chlamydomonas

are g i v e n

plate

d e a d cells

test gave

tested by the plate

as those c o n c e n t r a t i o n s

1 ppm for Shell Oil H e r d e r Comparison

ving

were

for each c o m p o u n d

55 ppm for F i n a s o l

of m i x t u r e s

themselves.

It c a n be seen that the t o x i c i t y

compounds.

about

compounds

extracts

as test organism.

for several

cells.

water

such m i x t u r e s

All

with Chlamydomonas

3-5 p a r a l l e l

in such a w a y that we tested

of dispersants,

for

the c o n c e n t r a t i o n s system,

6 compounds

shows and

that

lower

githe

for

3.

No. 3

251

Table

2.

Toxicity

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

synchronous

cultures

Synchronous

and w i t h

as e v a l u a t e d

the p l a t e

cells

Plate

with

test.

test i

Conc. giving 99,5% d e a d cells

LC 50

Lowest conc. giving "all cells

dead"

i

Shell

Oil H e r d e r

Nokomis

III

1

195-390

10

Butyl J a y s o l v e

435

85

Finasol

CC

37

60

49

Finasol

K

37

38

98

Finasol

W

12

13

98

Finasol

OSR 7

440

630

195

Finasol

OSR 5

510

900

195

Corexit

9527

575

900

350

150

350

49-93

25

28

195

700

950

195-781

Norclean

A

X-12 Cold

Cleaner

2.

Mixtures

a)

Plate

We m i x e d

test w i t h w a t e r 5 ml E k o f i s k

shaken

shaking

several

about

section.

Two p a r a l l e l

and in the cases w h e r e

during

we used

94.5 ml C h l a m y d o m o n a s funnel.

an 8 h period,

before

then tested extractions

the w a t e r

were

done

2.5 ml d i s p e r s a n t

The m i x t u r e

and from the

last

in the M e t h o d s

for each c o m p o u n d

gave d i f f e r i n g

and tests were

medium

p h a s e was collected.

as d e s c r i b e d

the two e x t r a c t s

new e x t r a c t i o n s

OSR-2,

and C h l a m y d o m o n a s .

oil with

in a 250 ml s e p a r a t o r y

16 h e l a p s e d

e x t r a c t was

Finasol

extracts

crude

times

The w a t e r

in the test,

h

of oil and d i s p e r s a n t .

and 0.5 ml d i s p e r s a n t was

h

performed.

results With

and 92.5 ml m e d i u m

plus

OSR

Finasol

Finasol

~

OSR

OSR

Finasol

~.~• ~

7

5

2 x)

Dasic

~! ul

9527 x)

Corexit

Corexit

OSR

Finasol

~

O i l s o l v e x'

x)

Slickgone

Dispersol X-12

7

5

2 x)

L T 2 x)

O S D x)

9527 x"~

OSR OSR

Finasol Finasol

o~

%

1000 xj

Synpersonic

CD

~ ml ~ o ~ ~o ~

Servo

%

ocox) U ~ Q

S a b r e s l i c k x)

L.W. x)

~.~~

Emulsol

Cleaner

(SM 53)

Jaysolve

I I 0 0 W D x)

Cold

BMD

ppm

I I 0 0 X x)

1100

Butyl

BP

BP

BP

100-1000

U . K . 1.

Pero-Kleen

the

S a m l e n x)

X-12

in

Oil Herder

A

Shell

Norclean

Finasol

III

K

W

Finasol

Nokomis

CC

< 100 p p m

approved

Finasol

cals

LT

9600

Dasic

Slickgone

7664

O i l s o l v e x)

Corexit

ppm

8666

! D i s p e r s o l x)

Shell

Corexit

Corexit

1000-10000

LT2 x)

C

Corexit

Regal

> 10000

7644

ppm

E f f e c t s of m i x t u r e s of E k o f i s k c r u d e o i l w i t h d i s p e r s a n t s in i:i r a t i o on C h l a m y d o m o n a s a n d Dunaliella. T h e c o m p o u n d s a r e g r o u p e d a c c o r d i n g to l o w e s t c o n c e n t r a t i o n w h i c h k i l l e d a l l c e l l s of the t e s t p o p u l a t i o n . C o m p o u n d s m a r k e d x) a r e o n t h e W a r r e n S p r i n g L a b o r a t o r y list of chemi-

O~

3.

~!

>

o

Table

o

No. 3

253

5 ml oil,

the f i v e - f o l d

being

to the p r o c e d u r e

due

producer.

The results

compounds

we have only

below which

more

comparison with tions e m p l o y e d

increased fold

in d i s p e r s a n t

of a p p l i c a t i o n

are g i v e n

e x a c t results

their about

toxicity. 200-fold

effect.

are given,

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

alone

and testing

Exception

and F i n a s o l

by the

i, and for m o s t of the

concentrations,

had no acute

for e x t r a c t i o n

concentration

recommended

in T a b l e

registered

the c o m p o u n d s

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

not change

increase

1000 or 1250 ppm, For

the rest of

and they

that under

the condi-

the p r e s e n c e

are F i n a s o l which

show upon

of oil did

OSR-2 w h i c h

decreased

about

35-

in toxicity. The most

British

recent method

a i:i ratio. series

Our final

test was

of such m i x t u r e s

test was

well

as w i t h Dunaliella.

of all cells 1000-10 tested

000 p p m and over

in this way.

100 ppm,

trations

above

one range more cating domonas

namely

i0 000 ppm.

that D u n a l i e l l a with respect

With

twenty-two

is s o m e w h a t

killed

and only

Of the r e m a i n i n g

more

when

all cells

1 at c o n c e n -

9 compounds

sensitive

ppm,

6 did so

7 were

than for C h l a m y d o m o n a s

effects

death

of the

extents,

whereas

as

in

100-1000

to equal

000 range,

to n e g a t i v e

culture

ranges g i v i n g

i00 ppm,

p p m range,

for D u n a l i e l l a

dilution

are p r e s e n t e d

of the c o m p o u n d s

3 in the I000-i0

toxic

which

affected

in the 100-1000

by in

oil and d i s p e r s a n t s .

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

under

10 000 ppm.

Eighteen

is s p e c i f i e d

performed with

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

b o t h algae were

at c o n c e n t r a t i o n s below

crude

The results,

under

of the culture,

compounds

grouped

therefore

done w i t h C h l a m y d o m o n a s

3, are g r o u p e d

which

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

of E k o f i s k

This

Table

of t e s t i n g

a u t h o r i t i e s 4 uses m i x t u r e s

indi-

than C h l a m y -

of the tested oil dis-

persants.

Discussion

Comparison dispersants the m a j o r i t y systems

of t o x i c i t y

with

data o b t a i n e d

the similar

of the c o m p o u n d s

are by and

large m o r e

data

w i t h our a l g a e and 1 shrimps shows that

for b r o w n

w h i c h have b e e n toxic

towards

tested with both

algae

than

towards

the animals. The p l a t e

test has

its m e r i t s

as a s c r e e n i n g

test,

its m a i n

254

No. 3

advantages

being

to p e r f o r m

so that h i g h l y

the c h e a p

that no e q u i p m e n t

is n e e d e d

Its r e p r o d u c i b i l i t y precision with

those

trend

wards

table

w i t h oil

the f r e s h w a t e r

according

to the

spondance

between indicates

structures

cells

points

the p l a s m a

aware

difficulties

screening

tests

concentration

The toxicity without

are

which

between

that

corre-

compounds

act on g e n e r a l

The s h r i n k i n g

site of action.

is a long w a y

from l a b o r a t o r y

spill

site.

tests

over

cleaning

value

at sea,

give

to-day. a ranking

chemicals.

between

toxicity

the ranking

the least h a r m f u l

a few

at each c o n c e n -

are not p o s s i b l e

and the effects

the

with performing

can at least

spill

However,

of s u b s t a n c e s

of the t r a n s f e r

in c h o o s i n g

of the a f f e c t e d

as the

knowledge

user

to-

Dunaliella,

response

in the l a b o r a t o r y a potential

Our c l a s s i f i -

to toxic

respect

are c o n n e c t e d

oil

test.

The close

investigations

potential

well

with

a small n u m b e r

such

tube

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

and say w i t h two p a r a l l e l S

so g r e a t

laboratory

the

is to test

and the m a r i n a

of a p o t e n t i a l

even w i t h

steps,

laborious, chemicals

ranking

membrane

that there

to e c o s y s t e m s

of the results.

of testing.

or processes.

practical

tration,

organisms

that the tested

towards

We are well conditions

a group

alga C h l a m y d o m o n a s

these

strongly

but m o r e

and

it c o m p a r e s

in a one to one ratio 4.

gives

it is simple

into a c c o u n t

with

oil c l e a n i n g

latest p r i n c i p l e

cellular

Taking

obtained

accurate,

in t e s t i n g

therefore

that

is unnecessary,

for the r e g i s t r a t i o n

the r e s u l t s

from the more

t h e m in m i x t u r e cation

needed,

labour

is satisfactory.

of the test

The next

equipment skilled

of

Even found

can a s s i s t

compound.

Aknowledgments

We wish

to thank The N o r w e g i a n

and the H u m a n i t i e s Council to M a r t a

and The N o r w e g i a n

for f i n a n c i a l Nesvik

cal a s s i s t a n c e

Research

support

and M a r i a n n e

Fisheries

of this work. Str#mme

and J o r u n n A k e r v o l l

Council

Research

We are also

for their

for typing

for Science

skilled

indepted

techni-

the m a n u s c r i p t .

so. 3

255

References i.

Jeffery,

P.G. and Nichols,

J.A. 1974:

(OP), Warrens Spring Laboratory, 2.

Heldal, M, Norland,

Report No LR 193

Stevenage,

Herts, England.

S., Lien, T. and Knutsen, G. 1978:

Chemosphere 3.

Rasmussen,

P. 1976:

Organiska MiljWgifter i Vatten.

Nordic Symposium on Water Research. NORDFORSK, 4.

Wilson,

Milj~v~rdssekretariatet,

K.W. 1976:

Agriculture,

Helsinki.

Personal communication.

Ministry of

Fisheries and Food, Fisheries Laboratory,

Burnham-on-Crouch,

Essex,

England.

Twelfth

p. 553.