The effect of oil industry “high density brines” on duckweed Lemna minor L.

~

Chemosphere,Vol.37, No. 13, pp, 2703-2715.1998

Pergamon

© 1998ElsevierScienceLtd.All rightsreserved 0045-6535/98/$ - see frontmatter

PII: S0045-6535(98)00156-8

THE EFFECT OF OIL INDUSTRY "HIGH DENSITY BRINES" ON DUCKWEED Lemna minor L.

Mirta Tkalec, ~.eljka Vidakovig-Cifrek* and Ivan Regula

Department of Botany, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia (Receivedin Germany13February1998;accepted23 April1998)

Abstract Duckweed Lemnn minor L. is a suitable plant model for toxicity evaluation of many substances due to its small size, rapid growth and ease of culture. Saturated water solutions of calcium chloride and calcium bromide and their 1:1 mixture are commonly used as "high density brines" for pressure control in oil wells. These solutions were added in Hoagland's nutrient medium in amounts appropriate to achieve 0.5%, 1.0%, 1.5% and 2.0% (v/v) dilutions and after two weeks of exposure the effect of tested chemicals on growth was estimated by counting fronds, measuring fresh and dry weights and determining total surface area of plants. Chlorophyll and carotenoid content in Lemna rather was also measured. Additionally, anthocyanin content in Spirodela polyrrhiza (L.) Schleiden was determined. During 14 days of exposure tested chemicals in lower concentrations (0.5%, 1.0% and 1.5% v/v) promoted the growth of Lenma minor, but they inhibited it in the highest (2.0% v/v). With increased concentration of tested solutions the concentrations of chlorophyll a and chlorophyll b were correspondingly higher in comparison with the control. Total carotenoid content and chl a/chl b ratio were also increased. The highest anthocyanin content in lower epidermis of Spirodela

polyrrhiza was noticed after the treatment with media containing 2.0% (v/v) CaC12 and 1:1 mixture of CaC12 and CaBr2, but lower concentrations of all three tested solutions also resulted in anthocyanin content increase. © 1998 ElsevierScienceLtd. All rightsreserved

Key words: Lemna minorL., calcium chloride, calcium bromide, high density brines, toxicity evaluation

2703

2704 Introduction Calcium chloride and calcium bromide aqueous solutions (densities 1300 g/1 and 1610 g/l, respectively) as well as their 1:1 mixture are industrial chemicals commonly used as high density brines, called also "heavy brines", for pressure control in oil wells. Accidental spills of these solutions can pollute ground waters and agricultural areas. Aquatic maerophytes are important primary producers and food source in water ecosystems. They are also among the first organisms reached by pollutants released in the water. The most of them are highly sensitive to wide range of pollutants so they could be used as the test organisms for assessment of toxicity in aquatic environments [ 1]. Duckweeds are small, vascular floating macrophytes that grow rapidly and reproduce vegetatively. Easiness of culture and possibility of manipulation in aseptic laboratory conditions make them suitable organisms for toxicity testing. They are very sensitive to many substances [1] and are already used as convenient test organisms for toxicity evaluation of a nurnber of pollutants including industrial and waste water effluents [2,3], herbicides [4,5], heavy metals [6,7], surfactants [8] and other common chemicals [9]. Among all species in family Lemnaceae, Lemna minorL, is the most commonly used for toxicity testing. The typical test end points are changes in the growth rate (expressed as frond production, fresh and dry weight and frond area) and changes in pigment content [10]. The purpose of our study was to investigate, under controlled laboratory conditions, the influence of CaC12, CaBr 2 and their 1:1 mixture on growth and photosynthetic pigments of Letrma ndnor L. In addition, the anthocyanin content in Spirodela polyrrhiza (L.) Schleiden was measured.

Materials and methods Lernna minor L. and Spirodela polyrrhiza (L.) Schleiden, both members of family Letnnaceae, were collected from Botanical Garden of Faculty of Science, University of Zagreb. Plants were sterilized with 50% (v/v) ethanol solution and 0.1% (w/v) mercuric chloride water solution [11]. They were then maintained as stock cultures under the axenic conditions. Every 14 days plants were transferred on the fresh Pirson-Seidel's medium [12]. The stock and experimental cultures were grown under 16 hours of light (40 W fluorescent light, 80 ~tEm'2s-1) at 24+_~2°C. All the experiments were carried out under axenic conditions and all media were sterilized. Saturated water solutions of CaC12 and CaBr 2 (concentrations 481.3 g/l and 1065.9 g/l, respectively) and their 1:1 mixture are industrial chemicals commonly used as high density brines in oil industry. They were used as stock solutions in our experiment. The

2705 solutions were of technical grade, but amounts of heavy metals (Cd, Cr, Ni, V, Fe, Co) were not detectable. Detection limits for those metals are (rag/l): Cd<0.0005, Cr<0.07, Ni<0.008, V<0.1, Fe<0.005 and Co<0.006. The result of chemical analyse is shown in table 1. Stock solutions were added into the nutrient medium in amounts to achieve the following volume concentrations: 0.5%, 1.0%, 1.5% and 2.0% (v/v). These tested concentrations were chosen on the basis of preliminary experiments showing that 2.0% (v/v) concentrations already caused strong inhibition of plant growth and abscission of daughter fronds. However, salts which have toxic effects in higher doses can cause stimulation of growth in lower doses [13], so we also investigated the 0.5%, 1.0% and 1.5% (v/v) concentrations. Table 1. The chemical analyses of CaCI2 and Cal3r2 solutions (densities 1300 g/1 and 1610 g/l, respectively) commonly used as "high density brines". The results are expressed as rag/1.

Ca2+

Mg2+

Zn

C1

CaCI2

170000

6

1.8

301350

CaBr2

218000

195

6.1

Br

867500

Experimental cultures were started by picking out healty colonies with 2-3 fronds from stock cultures and transferring each of them into the 100 ml Erlenmeyer flasks containing 60 ml of modified Hoagiand's medium [14] supplemented with the solutions to be tested. The pH value of the medium was adjusted to 5.0 with 0.1 M KOH. Each treatment and the control were prepared in eight replicates. The frond mtrnber was cotmted during 2-weeks test period on days 0, 3, 5, 8, 10, 12 and 14. Fresh weights were determined at the beginning and at the end of:these period. Growth was estimated as a change in the frond number and fresh weight according to following formulas [15]: no. of flonds at day n - no. of fronds at day 0 no. offi-onds at day 0

flesh weight at day 14 - flesh weight at day 0 flesh weight at day 0

n = 3,5,8,12,14

(1)

~2)

After determination of fresh weight, plants from four replicates were dried at 80°C to constant weight. Dry to flesh weight ratios as percentage of control samples were

2706 calculated. The plants from the other four replicates were first used for determination of plants total surface area using image processing software "Adobe Photoshop" and afterwards photosynthetic pigments content was measured. The chlorophylls and carotenoids were extracted in 80% acetone and their content was determined speetrophotometrieally according to Arnon [16]. The experiment was repeated three times. The results are expressed as mgpigments gFW"1. The relative anthocyanin content was determined spectrophotometrically at 537 tim [ 17] and expressed as absorbauce per 0.1 g of fresh weight (A537x0. l g-1 FW). All result are given as mean + standard error of means. Treatments were compared to control using Student's t-test. Significance was inferred at the P<0.05 level.

Results Calcium bromide solution and 1:1 mixture of CaCl2 and CaBr 2 in 1.5% and 2.0% (v/v) concentrations caused the appearance of colonies with large number of overlapping fronds. This effect was also noticed after the treatment with CaCl 2 solution, but in 2.0% (v/v) concentration only. Results concerning the relative frond number during 14 days are shown in table 2. On the medium with 1.0% (v/v) of all three tested solutions after 12 and 14 days of growth, a significant increase (P<0.05) in the frond number in comparison with the control was noticed. Tested solutions of CaCl 2 and CaBr 2 in concentration 1.5% (v/v) also caused the significant increase in frond number (])<0.05) but only on day 14, while the medium containing 1:1 mixture of both tested solutions in the same concentration (1.5% v/v), caused it already on day 10. On the other hand, all three tested solutions present in media in 2.0% (v/v) concentration caused a significant decrease (P<0.05) in the frond number in comparison with the control already on day 5 and all subsequent days. In general, by increasing three lower concentrations of tested solutions (0.5%, 1.0% and 1.5%) the corresponding increase in frond number after 14 days of cultivation was noticed. On the contrary, the media with 2.0% (v/v) of tested solutions caused the significant reduction of growth (Tab. 2). Also, the highest concentration (2.0% v/v) of all tested solutions caused the significant decrease (P<0.01) in frond weight in comparison with the control, while solutions in lower concentrations increased frond weight, but significantly, P<0.05, only in 1.5% (v/v) concentration (Fig. 1).

2707

Table 2. Frond number of duckweeds exposed to different concentrations of tested solutions CaC12, CaBr2 and their 1:1 mixture during 14 days in cultures.

Tested solutions

Conc. % (v/v)

Frond number on the first daya

Relative frond number by growth periods (days) according to formula (1) (mean value + standard deviation) 3

5

2.0

0.50+ 0.38

1.88_+ 0.23

3.00_+ 0.46

2.0

0.44_+ 0.50

1.69+ 0.59

CaBr2

2.0

0.5+ 0.46

Mixture

2.0

0.56+ 0.50

Control

3.63

12

14

6.38-+ 1.09

10.13+ 2.12

14.00-+ 3.21

3.31+ 1.07

7.13+ 1.89

11.56_+ 18.19+ 3.71 5.46

1.75+ 0.27

2.56+ 0.62

6.31+ 0.70

10.13+ 0.92

0.75-+ 0.32

1.75_+ 0.46 1.40_+ 0.30

2.75+ 0.85 2.30_+ 0.44

6.63-+ 1.96 3.96+ 0.90

10.56-+ 17.25+ 3.34 5.11 4.75+ 5.63_+ 0.82 1.08

3.25

0.78+ 0.33

1.18-+ 0.49

2.34+ 0.67

4.19+ 0.92

6.26-+ 1.61"

7.24_+ 0.72*

CaBr 2

3.25

0.74+ 0.23

1.24+ 0.46

0.96+ 0.18"

4.44+ 1.18

6.51-+ 1.69"

9.50_+ 2.27*

Mixture

2.75

0.75+ 0.25

Control

3.0

1.17+ 0.47

1.18+ 0.20 1.71+ 0.39

1.00-+ 0.30* 1.93+ 0.37

4.29-+ 0.71 3.23+ 0.93

6.19+ 0.74* 3.90+ 0.86

7.60+ 0.56* 4.29_+ 0.97

3.0

0.42+ 0.15"

1.33+ 0.01"

1.54-+ 0.25*

3.17+ 0.62

4.46+ 1.02

6.33-+ 1.55"

CaBr2

3.0

0.48_+ 0.30*

1.09+ 0.58*

2.02_+ 0.59

4.00+ 1.59

5.33+ 1.89

8.23+ 2.60*

Mixture

3.0

0.54-+ 0.43*

Control

2.38

0.69_+ 0.34

1.33+ 0.25* 1.88+ 0.56

1.83_+ 0.25 3.06-+ 0.60

4.46-+ 0.92* 5.96+ 0.74

6.33+ 0.80* 8.27+ 1.15

8.33_+ 1.14" 11.33-+ 1.50

2.38

0.52_+ 0.34

1.10+ 0.48*

2 . 0 6 - + 3.231_+ 0.66* 1.58"

5.10+ 2.34*

6.73_+ 3.46*

2.13

0.38_+ 0.44

1.04+ 0.65*

1.54-+ 0.93*

4.13_+ 2.36*

5.90+ 3.77*

2.13+ 2.46+ 3.75-+ 0.52-+ 0.90_+ 0.21 0.32* 0.55* 0.39* 0.93* * Statistically different (1'<0.05) from control using two-tailed Student" s t-test a Mean of eight cultures.

5.44+ 0.98*

Control CaC12

CaC12

CaCl2

CaCI2 CaBr2 Mixture

0.5

1.0

1.5

2.0

2.25

8

10

2.88-+ 1.25"

16.00_+ 1.81

2708

300

2 5 0

•

•

•

200 "~

--o-- Control --t3-- CaCI2 CaBr2 --o-- Mixture

o

150

,-g

100

50

O

I

I

L

q

0,5

1,0

1,5

2,0

Concentration of tested solution in medium, % (v/v)

Fig. 1. Relative fresh weight of Lcmna minor fronds (according to the formula (2)) cultivated 14 days in the nutrient media supplemented with tested solutions of CaCI2, CaBr 2 and their mixture (1:1) in comparison with the control represented as 100%. All three tested solutions in 2.0% (v/v) concentration increased the dry to fresh weight ratio (Fig. 2).

250

•~ ~ 150

--o--Control

o

--o-- CaCI2

o ,.~ ~ 10o

0

--n--CaBr2 1 --O--MixtureJ

I

t

I

I

0,5 1,0 1,5 2,0 Concentration o f tested solutions in

medium, % (v/v) Fig. 2. The dry to fresh weight ratio of L. minor after 14 days of cultivation in the nutrient media supplemented with tested solutions of CaCl 2, CaBr 2 and their mixture (1 : 1) in comparison with the control represented as 100%.

2709 'i'able 3. Relative plant surface area after 14 days of cultivation in media supplemented with tested solutions. Relative surface area of plants 1 (mean value~tandard deviation)

Concentrations

% (v/v)

Control

CaCI2

CaBr2

1:1 mixture

0.5

22.73+1.29

39.62+12.39

32.47+_3.59

37.95+12.42

1.0

4.42+_.2.07

5.63_-£-0.42

12.64+_3.94

10.96+4.32

1.5

2.48+1.07

4.99+1.18

9.27+_3.12

8.86_+0.61

2.0

10.95+_2.12

9.44+4.38

4.81_+0.86

9.46_+0.86

1Total surface area on day 14 in mm2/frond number on day 1.

Table 4. Photosynthetic pigments content and the chl a/chl b ratio in L. m/nor after 14 days exposure to media supplemented with tested solutions. Values are arithmetic means of three culturesS:standard deviations. Tested solutions

Conc¢lltrations % (v/,0

Chlorophyll a

(mg/gFW)

Chlorophyll b (mg/gF3y¢)

Carotenoids (mg/gFW)

chl ~¢chl b

0.468_+0.063

0.220_+0.030

0.220_+0.028

2.13

0.723_+0.149

0.281_+0.033

0.302-+0.042

2.57

0.723_+0.059

0.249_+0.018

0.331-+0.020

2.90

Mixture

0.768_+0.018

0.273_+0.006

0.348-+0.016

2.82

Control

0.364_+0.040

0.155_+0.010

0.183-+0.028

2.22

0.482_+0.031

0.185_+0.091

0.229_+0.047

2.60

CaBr2

0.599_+0.098

0.238_+0.018

0.182-+0.015

2.52

Mixture

0.579_+0.054

0.212_+0.024

0.273-+0.014

2.73

Control

0.272_+0.026

0.117_+0.028

0.153-+0.013

2.32

0.461_+0.105

0.182_+0.037

0.251_+0.050

2.53

Control CaC12 CaBr2

CaC12

CaC12

0.5

1.0

1.5

CaBr2

0.699_+0.085

0.230_+0.014

0.315_+0.013

3.06

Mixture

0.572_+0.139

0.203_+0.058

0.279-+0.067

2.83

Control

0.451_+0.051

0.189_+0.022

0.221-+0.087

2.39

0.745_+0.047

0.254_+0.05,*

0.345-+0.019

2.93

CaBr2

0.791_+0.013

0.245_+0.036

0.371_+0.021

3.18

Mixture

0.840_+0.081

0.264_+0.048

0.370-+0.020

3.19

CaC12

2.0

2710 The most observable decrease in surface area covered by plants (61%) was noticed after the treatment with 2.0% (v/v) of CaBr 2 solution. Mixture of tested solutions (1:1) in concentration 1.5% (v/v) caused 31% decrease in surface area (Tab. 3). Tested solutions caused the change of photosynthetic pigments' content in L. tmnor. With increased concentrations of tested solutions the concentration of chlorophyll a and chlorophyll b was correspondingly higher in comparison with the control. Total carotenoid content and the ratio chl ~¢chl b were also increased (Tab. 4). Tested solutions in all concentrations caused the increase in anthocyanin content in lower epidermis of

Spirodelapol.yn'hiza(Fig. 3).

25O

- - o - Control - o - CaC12 + CaBr2 - - o - Mixture

15o

0,5

1,0

1,5

2,0

I

Concentration of tested solutions in medium, % (v/v)

Fig. 3. Anthocyanin content in Spirodelapolyrrhiza after 14 days of exposure to tested solutions of CaCI2, CaBr 2 and their mixture (1:1) added in medium.

Discussion After the treatment with four concentrations of CaC12 and CaBr 2 water solutions and their 1:1 mixture, the appearance of overlapping fronds was noticed as a consequence of daughter-frond abscission prevention. This effect had already been observed but it was

2711 induced by sugars under the certain conditions [18]. On the other hand, some pollutants, for example landfill le~.chate, caused partial or total breakup of colonies [2]. Chemicals tested in our investigation consist of elements (except bromine) which plants can use as nutrients. Influence of heavy metals on L. minor and other plants is well known, but the effect of concentrations of nutrients other than optimal has been rarely studied. Lower concenlrations of tested chemicals (0.5%, 1.0% and 1.5% v/v) had a stimulative effect on L. minor growth. The most of heavy metals' salts and other salts also have the same effect when present in nutrient solution in small amounts [13]. Water solution of CaBr 2 in 1.0% (v/v) concentration and 1:1 mixture of both tested chemicals first caused the significant decrease (P<0.001) in frond number (on day 8 of growth period) and then the increase (on day 12 and 14, P<0.05 and P<0.01, respectively). The same effect had 1.5% (v/v) concentration of all tested solutions, but the significant decrease in frond number (P<0.01) was observed on day 3 and 5, and the increase (P<0.01) on day 14, with the exception of 1:1 mixture which caused increase on day 10 (P<0.05). This results could be explained by adjustment phase in plants: first, tested solution decreases frond number, but

then the plant adjusts to solution and

continues to grow. However, in the presence of high amounts of toxic substances the adjustment would fall and plants would not be able to overcome toxic effects. Subhadra et al. [6] observed a stimulation of Lemna growth caused by low concentrations of mercury. Mixture of CaC12 and CaBr2 (1:i) in concentration 1.5% (v/v) caused the greatest increase in frond number (P<0.05) and in 2.0% (v/v) the greatest decrease (P<0.05). Also, CaBr 2 solution in concentration 1.5% (v/v) caused the increase (P<0.01) and in 2.0% (v/v) the decrease in frond weight. Since the greatest inhibition of growth has been observed after cultivation on nutrient solutions containing bromides, it is possible that bromides are responsible for such effect. Because calcium is a signaling molecule and can also act as an enzymatic cofactor [19], its increased concentration could change the activity of some enzymes and increase the growth. The effect of CaC12 solution in 2.0% (v/v) concentration might be the result of a higher osmotic value of that solution, although, we did not notice plasmolised cells in thin sections of plant tissue. It is known that salinity has two possible effects: the nonspecific osmotic effect and the specific toxicity of certain ions [20], but it is difficult to distinguish these two effects in plants. Further studies are needed to fred out exactly if some of the tested ions (Ca2+, Br- or C1-) are responsible for toxic effects and if plant accumulate any of them. In our further research we also plan to investigate the effects of tested solutions on enzyme activities (e,g. peroxidase activity) and protein content, because some salts as well as heavy metals

2712 induce changes in peroxidase and other enzyme activities [6, 21, 22] and in protein pattern [23]. It appears that the frond number as a parameter of growth is better indicator of toxicity than frond weight, because by counting the fronds we noticed the plant response to 2.0% (v/v) concentration of tested solutions already on day 5 (Tab. 2). The other authors used the frond number as indicator of cadmium toxicity on Lanmacene [24, 25] but the frond weight has also been used as enough sensitive parameter in investigation of zinc and cooper toxic effect [7]. The dry to fresh weight ratio has been often used as parameter in toxicity bioassays for a number of substances [7, 8]. An increase in this ratio occurs when chloroplasts become loaded with starch grains under the different stress conditions [18]. In our investigation the increase of dry to fresh weight ratio after the treatment with 2.0% (v/v) solution of tested solutions could be explained by the accumulation of starch grains. Our future experiments will be aimed towards the conformation of starch grains accumulation. All tested solutions caused the increase in chlorophyll & chlorophyll b and total carotenoids content. As mentioned earlier, Ca2+ ion could activate some enzymes as a cofactor, perhaps also those included in biosynthesis of photosynthetic pigments, and that could explain the achieved results. However, it is known that heavy metals, like Hg, Pb and Cd, inhibit chlorophyll biosynthesis [26]. The increase in chl a/chl b ratio is in agreement with the results of Gill [27] who tested the effects of cadmium on photosynthetic pigments in tomato. However, he found out that cadmium added in nutrient medium decreases the chl b content in greater extent than chl a. In all tested concentrations an increase in anthocyanin content was: noticed. It is well-known that anthocyanin accumulation in vegetative tissues of plants is indicator of osmotic stress [28] and other environmental stresses [29].

The results of our study are in agreement with already existing data about the suitability of Lemaaceae for testing the toxicity of various pollutants [30, 31, 32, 2, 6, 7, 8, 9]. Water solutions of CaCI2, CaBr 2 and their 1: 1 mixture promoted the growth of L. minor in lower concentration but they inhibited it in the higher. They also caused increased photosynthetic pigments' content and chl a/chl b ratio in L. n ~ o r . In addition, they caused the increase of antocyanin content in Spirodela polyrrhizn.

2713

Acknowledgments This work was supported by the Ministry of Science and Technology of the Republic of Croatia. We would like to thank Mihovil Tomig, M. Sc., INA - oil industry Zagreb for sample supply, advice and helpful discussions in the course of this work.

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