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Calcium chloride and calcium bromide aqueous solutions of

technical and analytical grade in Lemna bioassay

Marija Vujevic a, Zeljka Vidakovic-Cifrek a,*, Mirta Tkalec a, Mihovil Tomic b,Ivan Regula a

a Department of Botany and Botanical Garden, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatiab CROSCO Co. (Integrated Drilling and Well Services, INA-Group), Ulica grada Vukovara 18, HR-10000 Zagreb, Croatia

Received 1 October 1999; accepted 25 February 2000

Abstract

Saturated water solutions of calcium chloride, calcium bromide and their 1:1 mixture are commonly used as ``high

density brines'' for pressure control in oil wells. To compare the eect of these chemicals of technical grade with the

eect of the chemicals of analytical grade the Lemna test was used. The multiplication rate, fresh weight, dry to fresh

weight ratio, area covered by plants and chlorophyll content were measured as toxicity parameters. The concentrations

of tested chemicals were 0.025, 0.05, 0.075 and 0.1 mol dm3. Generally, the chemicals of both technical and analytical

grade in concentrations of 0.025 mol dm3 stimulated the Lemna minor growth, while tested chemicals in concentrations

of 0.05 mol dm3 did not aect the growth signicantly. The exceptions were results obtained by measuring fresh

weight. Most of tested chemicals in concentrations of 0.075 mol dm3 and all chemicals in concentrations of 0.1 mol

dm3 reduced the growth. No major dierences between eects of tested chemicals of technical and analytical grade on

plant growth were observed, except that tested chemicals of analytical grade in concentrations of 0.1 mol dm 3 in-

creased dry to fresh weight ratio much stronger than chemicals of technical grade. All tested chemicals in all con-

centrations increased chlorophyll content. After treatment with chemicals of analytical grade much higher increase of

chlorophyll a concentration in comparison to increase of chlorophyll b was noticed, while chemicals of technical grade

caused more prominent increase of chlorophyll b. 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Lemna minor L.; Lemna test; High density brines; Calcium chloride; Calcium bromide

1. Introduction

Duckweed Lemna minor L. is a small oating fresh-

water monocotyledon belonging to family Lemnaceae. It

has been used as convenient bioassay organism in phy-

totoxicity evaluation due to its small size, rapid growth,

vegetative reproduction, ease of culture and sensitivity

to numerous pollutants (Wang, 1986; Lewis, 1995). Till

now Lemna bioassay has been used in detection of

phytotoxicity of heavy metals (Smith and Kwan, 1989;Huebert and Shay, 1991; Huebert et al., 1993; Sajwan

and Ornes, 1994), phenols (Cowgill et al., 1991), herbi-

cides (Krsnik-Rasol and Rendic, 1977; Lockhart et al.,

1989; Peterson et al., 1994; Fairchild et al., 1997), surf-

actants (Dirilgen and Ince, 1995) and some chemical

mixtures (Clement and Bouvet, 1993; Kanekar et al.,

1993).

Saturated calcium chloride and calcium bromide

aqueous solutions of technical grade and their 1:1 mix-

ture are commercially known as oil industry ``high

density brines'' and have regularly been used in explo-

ration and production of crude oil and natural gas

(Schmidt et al., 1983). During the special operations,

Chemosphere 41 (2000) 15351542

* Corresponding author. Tel.: +385-1-48-26-262; fax: +385-

1-48-26-262.E-mail address: [email protected] (Z. Vidakovic-Cifrek).

0045-6535/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 0 7 0 - 9

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covered by plants and (5) chlorophyll a and chlorophyll

b content and their ratio. All procedures were described

by Tkalec et al. (1998).

Results obtained by evaluation of growth parame-

ters were represented as mean values of eight replicates.

The control was represented as 100% and the resultsobtained with treated plants were represented as per-

centage of control. Chemicals that aected Lemna mi-

nor growth signicantly dierent (P ` 0X05) from each

other and control were marked with dierent letters

according to Duncans New Multiple Range Test

(Duncan, 1955).

Experiment for determination of chlorophyll a and

chlorophyll b contents was repeated three times. Results

were calculated as mean values and represented as per-

centage of control.

3. Results

3.1. The inuence of tested chemicals on frond number

Tested chemicals (CaCl2, CaBr2 and their 1:1 mix-

ture) of both technical and analytical grade, added into

the modied Hoaglands nutrient solution in concen-

trations of 0.025 mol dm3 signicantly stimulated

(P ` 0X05) the multiplication of plants (Fig. 1). On the

contrary, all tested chemicals present in concentrations

of 0.05 mol dm3 and CaBr2 and 1:1 mixture in con-

centrations of 0.075 mol dm3 did not signicantly

change the frond number in comparison with the con-

trol. All tested solutions in concentrations of 0.1 mol

dm3 and CaCl2 in 0.075 mol dm3 caused a signicantdecrease (P ` 0X05) of frond number.

3.2. The inuence of tested chemicals on fresh weight

CaCl2, CaBr2 and their 1:1 mixture of both technical

and analytical grade in concentrations of 0.025 mol

dm3 did not change the relative growth of fresh weight

in comparison to control plants, except CaBr2 of ana-

lytical grade that stimulated the growth (P ` 0X05).

CaCl2 and CaBr2 of analytical grade in concentrations

of 0.05 mol dm3 and CaBr2 of technical grade in con-

centration 0.075 mol dm3

also had no signicant eecton fresh weight (Fig. 2). Other solutions in 0.05 and

0.075 mol dm3 and all solutions in 0.1 mol dm3

inhibited the growth of fresh weight (P ` 0X05).

3.3. The inuence of tested chemicals on dry to fresh

weight ratio

Tested chemicals of both technical and analytical

grade present in concentrations of 0.025 and 0.05 mol

Fig. 1. Relative growth of Lemna minor frond number after two week exposure to tested chemicals of analytical (left) and technical

grade (right). Dierent letters on the top of the column indicate signicant dierences between treatments at P ` 0X05 by Duncans

New Multiple Range Test.

M. Vujevic et al. / Chemosphere 41 (2000) 15351542 1537

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dm3 did not inuence dry to fresh weight ratio signi-

cantly (P ` 0X05). Tested chemicals in higher concen-

trations (0.075 and 0.1 mol dm3) increased observed

ratio (Fig. 3). The exceptions were CaCl2 of analytical

grade and CaBr2 of technical grade in concentrations of

0.075 mol dm3 and CaCl2 and CaBr2 of technical grade

in concentrations of 0.1 mol dm3. The most signicant

increase was obtained by 0.1 mol dm3 tested solutions

of analytical grade (P ` 0X05).

3.4. The inuence of tested chemicals on the relative area

covered by plants

Tested chemicals of both technical and analytical

grade in concentrations of 0.025 mol dm3 did not sig-

nicantly increase the relative area covered by plants in

comparison to control plants except 1:1 mixture

(P ` 0X05). The relative area covered by duckweed

treated with tested solutions in 0.05 and 0.075 mol dm3

was similar to the relative area covered by control plants

(Fig. 4). CaCl2, CaBr2 and their 1:1 mixture of both

technical and analytical grade added into the modied

Hoaglands nutrient solution in concentrations of 0.1

mol dm3 decreased the relative area covered by plants

(P ` 0X05).

3.5. The inuence of tested chemicals on photosynthetic

pigments content

The chlorophyll a and chlorophyll b content in-

creased correspondingly with the increase of tested

chemicals concentration of both technical and analyti-

cal grade (Figs. 5 and 6). In duckweed treated with

tested chemicals of analytical grade the increase of

chlorophyll a content was more prominent than the in-

crease of chlorophyll b content. The consequence was

increased chlorophyll a to chlorophyll b ratio (Fig. 5).

On the contrary, in duckweed grown on nutrient solu-

tion supplemented with tested chemicals of technical

grade, the chlorophyll b content increased to a greater

extent than chlorophyll a content, so the ratio was

slightly decreased in comparison to control (Fig. 6).

4. Discussion

Tested chemicals in concentrations of 0.025 mol

dm3 stimulated the Lemna minor growth similarly to

results of Tkalec et al. (1998) obtained with 0.5% (v/v)

dilution of high density brines (0.5% v/v CaCl2 solution

contains 0.0218 mol dm3 CaCl2, while 0.5% v/v CaBr2

Fig. 2. Relative growth of Lemna minor fresh weight after two week exposure to tested chemicals of analytical (left) and technical

grade (right). Dierent letters on the top of the column indicate signicant dierences between treatments at P ` 0X05 by Duncans

New Multiple Range Test.

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solution contains 0.0266 mol dm3 CaBr2). This stimu-

lative eect of tested salts in lower concentrations could

be explained either by establishing better growth con-

ditions by additional amounts of nutrients brought in

with the sample (Wundram et al., 1997) or by positive

eect of increased turgor (Oertli, 1975, cited by von Sury

and Fluckiger, 1983). We also noticed that the inhibition

caused by chemicals in concentrations of 0.1 mol dm3

corresponded to results of Tkalec et al. (1998) obtained

with high density brines in 2.0% (v/v) dilution (2.0% v/v

CaCl2 solution contains 0.0867 mol dm3 CaCl2, while

2.0% v/v CaBr2 solution contains 0.1066 mol dm3

CaBr2). Since the present results as well as results of

Tkalec et al. (1998) showed no signicant dierences

between eects of CaCl2, CaBr2 and their 1:1 mixture in

concentrations of 0.1 mol dm3, the inhibition could be

due to the osmotic eect.

Chemical analysis (Table 1) showed that there were

not signicant amounts of heavy metals in high density

brines solutions. But, it could be possible that other

impurities (of inorganic or organic nature) were present

in the samples. The eect of high density brines solutions

on duckweed growth showed no major dierences in

comparison to the eect of chemicals of analytical grade.

There were few exceptions according to the Duncans

New Multiple Range Test. Some solutions of analytical

grade caused stronger stimulation of growth than solu-

tions of technical grade (P ` 0X05): for example, inu-

ence of mixture in concentrations of 0.025 and 0.075 mol

dm3 on frond number (Fig. 1) and inuence of 0.025

mol dm3 CaBr2 on fresh weight (Fig. 2). CaBr2 and 1:1

mixture of analytical grade in concentrations of 0.1 mol

dm3 inhibited the increase of frond number stronger

than the same substances of technical grade (Fig. 1). The

most obvious dierence between chemicals of technical

and analytical grade was noticed in dry to fresh weight

ratio, which was signicantly higher (P ` 0X05) aftertreatment with all three samples of analytical grade in

concentrations of 0.1 mol dm3. The reason for such a

result could be the starch accumulation in chloroplasts

of treated plants due to disturbed sugar metabolism

(Hillman, 1961; Severi, 1991). In our recent investiga-

tions (data not shown) we conrmed the starch accu-

mulation in chloroplasts of treated plants by microscopy

and centrifugation of isolated chloroplasts in sucrose

gradient.

A few signicantly dierent results obtained after

treatment with chemicals of analytical and technical

grade could be explained by possible interactions of

impurities with constituents of nutrient solution or with

Fig. 3. Dry to fresh weight ratio in Lemna minor plants grown on modied Hoaglands nutrient solution supplemented with tested

chemicals of analytical (left) and technical grade (right). Dierent letters on the top of the column indicate signicant dierences

between treatments at P ` 0X05 by Duncans New Multiple Range Test.


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Fig. 5. Chlorophyll a and chlorophyll b content and their ratio in Lemna minor grown on modied Hoaglands nutrient solution

supplemented with tested chemicals of analytical grade. Values are the average of three dierent experiments represented as percentage

of control.

Fig. 4. Relative area covered by Lemna minor plants grown on modied Hoaglands nutrient solution supplemented with tested

chemicals of analytical grade (left) and technical grade (right). Dierent letters on the top of the column indicate signicant dierences

between treatments at P ` 0X05 by Duncans New Multiple Range Test.


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ions of tested solutions. Interactions between the con-

stituents of the nutrient solution are well known (Wang,

1992). As a consequence of such interactions, certainsubstances could be more available to plants than others

and vice versa.

Chemicals of both analytical and technical grade in-

creased chlorophyll a and chlorophyll b content, but

some dierences between chemicals of analytical and

technical grade were noticed. Chemicals of analytical

grade caused much greater increase of chlorophyll a

content in comparison to increase of chlorophyll b

content, so the chlorophyll a to chlorophyll b ratio was

also increased (Fig. 5). On the other hand, chemicals of

technical grade caused greater increase of chlorophyll b

content in comparison to increase of chlorophyll acontent, so the ratio was slightly decreased (Fig. 6). It is

possible that the presence of impurities in chemicals of

technical grade promoted the formation of chlorophyll b

from the chlorophyll a, possibly by oxidising methyl

group on pyrol ring no. 2 into aldehyde group (Gil et al.,

1995).

Only minor growth dierences caused by chemicals

of analytical and technical grade support the hypothesis

that eect noticed after treatment with chemicals of

technical grade used as high density brines was due to

the major constituents of these solutions (Ca2, Cl and

Br ions) and was not the consequence of impurities

commonly present in chemicals of technical grade.

However, chemicals of analytical and technical grade

had dierent eect on chlorophyll pigments content in

duckweed plants.

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Marija Vujevic received her B.Sc. degree in 1998 from the

University of Zagreb, Croatia. She is M.Sc. student at theFaculty of Science, University of Zagreb. The subject of hercurrent research is in vitro propagation of rare and endangeredCroatian plant species.

Zeljka Vidakovic-Cifrek received her B.Sc. (1990), M.Sc. (1993)and Ph.D. (1999) degree from the University of Zagreb,Croatia. She is research assistant at the Department of Botany,Faculty of Science, University of Zagreb. Her research interestsfocus on inuence of various environmental factors on physi-ological processes in plants.

Mirta Tkalec received her B.Sc. degree in 1996 from the Uni-versity of Zagreb, Croatia. She is currently M.Sc. student at theFaculty of Science, University of Zagreb. The main topic of herresearch is evaluation of stress factors by Lemna test.

Mihovil Tomic received his B.Sc. degree from the University ofSarajevo, Bosnia and Herzegovina and M.Sc. degree from theUniversity of Zagreb, Croatia. He works in INA-Oil industryon analytical aspects in exploration and production of gas andoil as well as on pollution problems in oil industry.

Ivan Regula is full professor of Plant Physiology at the De-partment of Botany and Botanical Garden, Faculty of Science,University of Zagreb, Croatia. His scientic interests includestructure and function of indolic compounds in plants as well asinuence of xenobiotics on plant metabolism.


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