A comparison of results from two test methods for assessing the toxicity of aminotriazole to Selenastrum capricornutum

Chemosphere, Vol.13, No.8, Printed in G r e a t B r i t a i n

pm

965

-

971,

1984

0045-6535/84 $3.00 + .00 01984 Pergamon P r e s s Ltd.

A COMPARISON OF RESULTS

FROM TWO TEST METHODS FOR ASSESSING THE TOXICITY OF AMINOTRIAZOLE TO Selenastrum capricornutum. N Adams and A J Dobbs Building Research Establishment Princes Risborough Laboratory Princes Risborough, Aylesbury Buckinghamshire, UK

ABSTRACT Two different

test methods

for assessing the toxicity of aminotriazole Growth medium composition is demonstrated effect on the toxicity of aminotriazole.

co~r~cornutum are compared.

to S e l e n a s t ~ to have a significant

INTRODUCTION In algal toxicity

studies there is a lack of information

dure on the toxic limits determined. procedures

on the influence of changes

This paper compares

with the same alga and toxicant.

A draft OECD guideline I on algal growth inhibition has recently been produced. derable

freedom in selection of test parameters

defined medium is recommended.

Bolds basal medium

(BBM) 2.

One experiment

the OECD guideline.

guideline but with phosphorus

EXPERIMENTAL

Laboratory

(termed the PRL method)

was carried out following

which uses

the PRL method and one

was performed

following

the OECD

from the medium.

METHODS AND MATERIALS

All glassware used in the experiments thoroughly

although a

of this OECD draft a slightly different

In addition a third experiment omitted

It allows consi-

including growth media composition,

Prior to the publication

procedure was devised at the Princes Risborough

following

in proce-

the results of experiments using two

was soaked in 5 per cent Decon 90 overnight,

in deionised water with a final rinse in particle-free

deionised water

rinsed (filtered

through 0.45 ~m pore size filters). Stock cultures of

Selenastr~ ccc3~cornutu~ CCAP 278/4 were maintained on agar slopes of modi-

fied BBM.

This species was selected because

it is in general use and maintains

throughout

its growth cycle:

suited to cell counting by electronic

it is therefore

counters.

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unicellularity particle

966

Growth medium and culture flask preparation In the modified

BBM employed in the PRL method, ammonium molybdate

instead of molybdenum trioxide,

as it is more soluble.

The OECD medium was used as recommended

in the 1983 draft test guideline except that zinc sulphate In all experiments,

(0.87 g 1 -I) was used

took the place of zinc chloride.

the medium was made up in particle-free

deionised water, mixed thoroughly,

divided into 90 ml aliquots and added to 250 ml narrow necked conical flasks. stoppered with

'Steristoppers'

for 20 minutes.

The flasks were

(Exogen Ltd) and sterilised by autoclaving at 120°C and ]5 psi

They were then stored at 4°C until required.

pared for each treatment level, and control.

Three replicate

flasks were pre-

The BBM had a pH of 6 at the start of the experi-

ment, as had the OECD medium, although the draft test guideline states that after equilibration with air the medium pH is 8. Amlnotriazole

solutions

Aminotriazole

(iH-l,2,4-triazol-3-amine)

at the site of carotenoid synthesis 3.

is a freely-soluble herbicide whose primary action is

Stock solutions were made up in sterile particle-free

deionised water immediately before the chemical was to be added to the culture flasks for the PRL method.

For the OECD method the chemical was made up the day before and stored in the dark

in a refrigerator until required.

The concentrations

tested were 0.2, 0.5, 1.0, 2.O, 5.0 and

iO.O ppm with the PRL method and i.O, 5.0, 10.O and 50.0 ppm with the OECD method.

Replicates

at iO0 and 500 ppm were also tested with the OECD method, but cell breakdown occurred making cell counting unreliable so data were not collected for these concentrations. Inoculum preparation and experimental procedures Algal cells were transferred using aseptic technique, slopes, to flasks containing either particle-free growth medium. about 4000 lux.

from stock cultures grown on BBbl agar

sterile BBM or particle-free

The flasks were incubated at 22°C and 175 rpm under constant

sterile OECD

illumination of

Two days before cells were required for the test, these flasks were sub-

cultured to give actively growing samples in the appropriate medium from which the inoculum could be prepared.

After 2 days growth, the cell concentrations

and dilutions were prepared in particle-free In the PRL method,

growth medium.

in these flasks were measured

These dilutions were the inocula.

aliquots were added to the culture vessels by aseptic technique to give an

initial cell concentration of IOO0 cells ml -I. tion was approximately

After 24 hours incubation

(the cell concentra-

104 cells ml -I) the aminotriazole was added at the required concentra-

tion for each treatment level and the flasks were made up to i00 ml with sterile particle-free deionised water.

This is termed day O in this procedure.

The cultures were incubated for 6

days as described above and the cell count for each flask was recorded every 24 hours. In the draft OECD method, algae and chemical were added together on day O and the flasks counted every 24 hours for 4 days. level.

An extra count was carried out on day 7 to establish the plateau

The inoculum culture was added to each flask to give an initial cell concentration of

IO 000 cells ml -I and a volume of i00 ml.

The aminotriazole

solution was then added using a

micropipette. Cell concentrations were determined throughout the experiment using an Elzone 80 XY electronic

967

particle counter

(Particle Data

Ltd).

Known volumes of cell suspension were removed from the

culture flasks and added to 20 ml of particle-free electrolyte 2 ml samples of this mixture were counted.

(Isoton II, Coulter Electronics);

The figure obtained was then multiplied by the

dilution factor to give the cell concentrations of the cultures. RESULTS Mean cell concentrations and coefficients of variance are presented in Table i. concentrations)

Log (mean cell

for the two methods are plotted as a function of time in Figures 1 and 2.

Figure 3 gives the mean cell concentrations expressed as a percentage of the control values and highlights differences that are obscured by the log transformations used for Figures 1 and 2. Table I

Mean cell concentrations of S. capricornutum (a)

cultures, treated with aminotriazole

Results obtained by the PRL Method Age of cultures (days)

Aminotriazole (ppm)

1

2

3

4

0

+0.67 *(5.7)

7.08 (3.7)

26.3 (6.7)

76.5 (3.4)

0.2

0.75 (II.0)

7.11 (3.0)

26.4 (5.5)

82.0 (7.5)

0.5

0.72 (4.3)

6.24 (2.4)

23.1 (4.2)

76.5 (3.2)

1.0

0.74 (5.6)

4.08 (14.3)

14.2 (16.4)

55.5 (7.6)

2.0

0.64 (8.0)

1.59 (6.5)

4.39 (8.3)

14.5 (18.4)

5.0

0.49 (5.6)

0.97 (4.5)

1.71 (11.4)

4.69 (3.2)

i0.0

0.49 (5.4)

0.68 (3.7)

1.00 (4.1)

2.0 (8.8)

(b)

Results obtained by the OECD method

Aminotriazole (ppm)

Age of cultures I

2

(days) 3

4

0

0.61 (8.4)

4.37 (14.9)

17.5 (16.7)

30.1 (21.7)

1

0.58 (4.7)

4.16 (6.2)

19.0 (IO.O)

32.6 (5.3)

5

0.59 (2.3)

3.86 (12.7)

16.1 (IO.i)

27.2 (7.7)

iO

0.44 (6.7)

1.97 (4.4)

8.43 (3.8)

24.1 (5.9)

50

O.15 (3.7)

0.33 (16.1)

0.44 (7.4)

0.88 (14.2)

+ = mean cell concentration

(x 105 cells ml-l).

* = coefficient of variance

(%)

968

1°7I I

i i

106i

10 e

_

v~

!

ppm

E E

: : ~ ,~

105

05 10 A 20 =

:: :: ,%

~o~i

50 10.0 500

50 10"0

1 0 4 L ..... ~ 1

I 3

2

~ . _ 4

I 5

1

6

Figure 1 PRL method: the effect of aminotriazo]e on growth of S. capricorn~t~, Mean cell number of 3 replicate flasks is recorded at each treatment level,

(95 per cent) differences

causing a significant analysed i ii

difference

in three different

cell concentrations area under

growth

the growth

rates

4

5

~i

7

obtained

the highest

from the controls

from the controls

concentration

causing n~

(NOEC) and the lowest concentration

(LCSE).

This was applied

to the results

ways:

measured

(b) cell concentration iii

3

Figure 2 OECD method: the effect of aminotriazole on growth of ~. ~a~P/a~P~xt~,. Mean ceil number of 3 replicate flasks is recorded at each treatment level.

The data were analysed using Dunnetts method 4 to give significant

2

Age of c[dlure{days}

Age of culture (days)

on each day of the culture period;

curve computed

from

(a) log

(cell concentration)

- time and

of log

(cell concentration)

against

- time plots; by linear regression

The NOEC and LCSE values obtained

for each of these alternatives

are presented

time.

in Table

2.

DISCUSSION Toxic values The NOEC values those obtained

in Table

2 sho~: that the two experimental

by the OECD method

can be at least an order of magnitude

The results based on cell counts or areas under of 0.2 or 0.5 ppm, whereas are obtained

methods

difference

higher

is 5.0 ppm in all cases.

in results

res~Jlts

in some cases.

curve by the PRL method

I.O ppm for the PRL method

but the order of magnitude

is maintained.

the growth

that for the draft OECD m e t h o d

from growth rate measurement

draft OECD method,

9ro~'edures give \,e~y d[f!erent

give

a NOEC

Higher NOEC

and 5 or IO ppm for the from the different

test

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Age of c u l t u r e s ( d a y s )

Figure 3

Percentage cell concentrations. At each culture age, cell concentrations for each treatment level are presented as a percentage of the control cell concentration.

The two essential differences between the methodologies

are

(i) the time of chemical addition

and algal inoculation and (ii) the composition of the growth medium. eliminate

It is not possible to

the first of these as the cause of the different toxic ]imits.

that it is an unlikely cause because in practice at the time of chemical addition,

However we believe

the experiments had the same cell concentration

and because the algal inoculum is prepared in the same medium

as that used for the toxicity test to minimise stress and there is no evidence of a lag phase induced by inoculation in either medium. The growth medium Bold's Basal Medium nutrients

(BBM) is on the whole richer than the OECD medium;

in both and their concentrations.

present in markedly different concentrations

(Table 3 lists the

The main nutrients, nitrogen and phosphorus are and ratios in the two cases.

Nitrogen,

as nitrate,

in BBM is present at about 10 times the concentration of that in the OECD medium where it is present as an ammonium salt.

The BBM medium contains nearly 150 times as much phosphorus

as the

OECD medium. The nitrogen:phosphorus

ratio is important in cell growth and is nearly 2:1 in BBM but is about

24:1 in the draft OECD medium.

The BBM does not meet the limits of nitrogen and phosphorus

required in the draft OECD guidelines which are nitrogen 714 x 10 -6 M and phosphorus

970

Table 2

Highest No Effect Concentrations (NOEC) and Lowest Concentrations causing a Significant Effect (LCSE) of aminotriazole treated cultures. Comparison of values (ppm) calculated using four different methods. Method of calculation (see text)

i

Cell concentration

Age of culture (days)

measured

daily

iia Area under growth curve (log cell concentration

against

time)

11b Area under growth curve (cell concentration iii Growth rate

(linear regression

cell concentration

Table 3

Compound

against time) of

against time)

Concentrations

of Nutrients

Bolds basal medium Moles ~-I

NaNO 3

PRL method

OECD method

NOEC

LCSE

NOEC

LCSE

1

2.O

5 .O

5 .O

IO .O

2

0.2

0.5

5.0

I0.0

3

0.2

0.5

5.0

I0.O

4

0.5

i.O

5.0

IO.0

1 to 3

0.2

0.5

5.0

i0.O

1 to 4

0.5

i.O

5.0

IO.0

1 to

3

0.2

0.5

5.0

10.O

1 to 4

0.2

0.5

5.0

IO.0

at 3 days

1.0

2.0

5.0

10.O

at 4 days

I.O

2.0

iO.O

50.0

in Bolds Basal Medium and OECD Medium

Compound

OECD medium Moles

280

f-i

2942

x 10 -6

NH4CI

CaCI2.2H20

170

x 10 -6

MgC]2.6H20

MgSO4.7H20

304

x 10 -6

CaCI2.2H20

K2HPO 4

431

x 10 -6

MgSO4.7H20

60.9

x 10 -6

KH2PO 4

11.8

x 10 -6

59.0 122

x 10 -6 x 10 -6 x 10 -6

1290

x 10 -6

KH2PO 4

NaCI

428

x 10 -6

FeCI3.6H20

0.30 x 10 -6

Na2EDTA.2H20

171

x 10 -6

Na2EDTA.2H20

0.30 x 10 -6

KOH

553

Fe2SO 4 N3BO 3

17.9

H3BO 3

2.99 x 10 -6

MnCI2.4H20

2.10 x 10 -6

x 10 -6

ZnCI 2

x 10 -6

COC12.6H20

MnCI2.4H20

7.28 x 10 -6

CaCI2.2H20

(NH4)6Mo7024.4H20

0.70 x 10 -6

Na2MoO4.2H20

CuSO4.3H20

6.29 x 10 -6

NaHCO 3

ZnSO4.7H20

Co(NO3)2.6H20 H2SO 4

185

x 10 -6 x 10 -6

30.7

1.68 x 10 -6 -6 x IO

2000

22.0

x 10 -9

6.30 x 10 -9 58.7

x 10 -12

28.9

x 10 -9

595

x 10 -6

971

22.6 x iO

-6

M, although in all other respects micronutrients,

pH, chelators

and hardness

it is

within the range specified. The controls

indicate that the composition

of the growth medium has a significant

rate of growth and the plateau cell concentration growth rate observed

in BBM could be advanced

of the alga to toxicant view that organisms Figure

in this medium;

Indeed,

the faster

for the greater susceptibility

an explanation which contrasts with the generally held

are more susceptible

1 does show, however,

of S. capricor~utum.

as one explanation

effect on the

to toxicants

as environmental

that a high growth rate is maintained

stresses

are increased.

on the richer

(BBM) growth

medium for up to 24 hours after addition of the aminotriazole. Phosphate

free test was carried out exposing selenastr~ capricor~ut~ to aminotriazole

An experiment

medium with phosphate being accidentally

omitted.

using the OECD

Despite this omission the procedure

achieved

all the criteria required in the draft OECD guideline. The medium fitted the requirements

set, and the cell concentrations

increased by a factor of at least 16 in the three days pH was 6.0 throughout. significant

Aminotriazole

in the control

cultures

(the increase was 19.2 times),

was tested at concentrations

and the

up to IO ppm and displayed no

inhibitory effects.

CONCLUSION The results of this investigation medium composition,

capricornut~n.

show that changes in test methodology,

have a significant

effect on the toxicity of aminotriazole

It appears that reductions

the alga

more tolerant of this toxicant;

magnitude

in the different media studied.

of growth medium composition

in the basic nutrients

in algal growth studies.

undoubtedly results.

important

In this respect

in the recommended nutrients

in affecting

In general

insufficient

toxic limits.

Changes

in concentrations

it is important

is known about the influence

There is however every reason to will be generally

of nitrogen

to note that the concentrations

medium of the OECD draft guideline

it is not possible

limits with changes

an order of

and phosphorus

toxic limits but trace nutrients may also influence

to predict the magnitude

in media composition.

of some trace nutrient~

in other components.

or direction of changes in algal toxic

Until this is resolved,

from tests conducted with growth media of different

are the

are so low that larger amounts of these

will probably be added to the medium as impurities

At present

to Sele~strum

the toxic limits change by approximately

on the experimental

growth

nitrogen and phosphorus make

expect that the variation of toxic limits with growth media composition observed

principally

interpretation

composition will be difficult

of toxic limit: and comparison

of results will be impossible. REFERENCES i

OECD. Final Draft Update of Test Guideline Environment Directorate, Paris. 1983.

2

George E A (Ed). 'List of Strains' 36 Storey's Way, Cambridge, 1976.

3

Cremlyn R.

4

Zar J H.

(Received

Pesticides, Biostatistical

in

UK

20 M a y

Preparation Analysis. 1984)

201,

'Alga, Growth Inhibition Test'.

OECD

The Culture Centre of Algae, Protozoa, and Mode of Action. Prentice-Hall,

1974.

John Wiley and Sons.

1979.