Effect of atrazine herbicide on growth, photosynthesis, protein synthesis, and fatty acid composition in the unicellular green alga Chlorella kessleri

ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

29,349-358

(1994)

Effect of Atrazine Herbicide on Growth, Photosynthesis, Protein Synthesis, and Fatty Acid Composition in the Unicellular Green Alga Chlorella kessleri MOSTAFA

M. EL-SHEEKH,*

HOSSAM

M. KOTKAT,~

AND OLA H. E. HAMMOUDA$

Sublethal atrazine concentrations induced a general inhibition on growth. photosynthesis. and dark respiration in the green alga C%/orc~//a kedc~ri. 14C-protein hydrolysate indicated a maximum incorporation level by 5 & atrazine after 48 hr. Progressive reduction in protein synthesis was associated with increasing herbicide concentration at all experimental periods. The herbicide had preferential effect on the associated fatty acid composition of total and phospholipids. After 24 and 48 hr the herbicide stimulated fatty acids synthesis at concentrations where photosynthesis was inhibited. Meanwhile. stearic and miristic acids disappeared at I5 PM after 24 hr and the total polyunsaturated fatty acids were not affected after 48 hr. Fatty acid synthesis was sensitive to treatment at 72 hr by 5 and IO qt,f atrazine. whereas the total saturated fatty acids were completely inhibited. (2’ 1994 Academic PWSS.inc

INTRODUCTION

The genus Chlorella is very common in all kinds of freshwater bodies. It plays an important role in primary production and contributes to the purification of eutrophic waters. The presence or absence of certain species can be used for the evaluation of the water quality. In addition, the application of environmental pollutants can greatly affect the distribution of this genus. Extensive use of herbicides and pesticides for improving crop yields has given rise to the problem of their effect on soil and aquatic microorganisms. Triazine herbicides are toxic to algae by disrupting photosynthesis (Stratton, 1987). Atrazine is a common contaminant of surface water ofthe agricultural regions (Hallberg ef al., 1984). Atrazine effectively inhibits growth and photosynthesis in most plants, including freshwater algae (Mayasich et al., 1986; Abou-Wally et al., 199 la,b). De Noyelles et al. (1982) reported detectable effects of atrazine on algal productivity and species composition (Stadnyk et al., 197 1). Goldsborough and Robinson ( 1983) studied the effect of simazine and terbutryn on the productivity of freshwater march periphyton using chlorophyll (a) measurements and 14C assimilation. Green algae are very susceptible to atrazine (Hollister and Walsh, 1973; Walsh, 1972). The aim of this work is to assess the possible alterations in some physiological processes in the green alga Chlorella kessleri in response to atrazine herbicide. MATERIALS

AND

METHODS

Organism and Culture Conditions The unicellular green alga C. kessleri (Algal Culture Collection, Gottingen University, Germany) was cultured in an inorganic medium described by Kuhl(l962). The cultures 349

0 147-65 I3194

$6.00

Copyright Q 1994 by Academic Press. Inc. All ngba of rcproducl~on in any form merved.

350

EL-SHEEKH,

KOTKAT.

AND

HAMMOUDA

were illuminated continuously with fluorescent tubes maintaining the desired light intensity (140 W mm2). The cultures were incubated at 27°C and aerated with a mixture of 95% sterile air and 5% CO2 (v/v). The initial inoculum was 5 ml of algae cells from the exponential growth phase of optical density 1.5. Growth Parameter Measurements Growth of the cultures was monitored either by measuring the optical density of the cell suspension spectrophotometrically at 560 nm (Wetherell, 196 1) or as the increase in dry weight production as reported by Leganes et al. ( 1987). Chlorophyll concentration was estimated by the method described by MacKinney ( 194 1). Isotope Labeling To determine the rate of protein synthesis during atrazine treatment, one milliliter portions of the culture were removed at the designated intervals, pulse labeled with 0.5 Mbq of 14C uniformly labeled protein hydrolysate for 1 hr, and immediately precipitated with 10% trichloroacetic acid. The precipitates were collected by centrifugation and washed with cold 5% trichloroacetic acid and ethanol. The radioactivity was measured in a liquid scintillation spectrometer. Analysis of Lipids Lipids were extracted from IO-ml aliquots of culture by the method of Bligh and Dyer (1959). The polar lipids were separated by thin-layer chromatography on precoated silica gel plates (572 1, Merck, Darmstadt, Germany) with a mixture of petroleum ether:diethyl ether:acetic acid, 90: 15: 1 (v/v) (Malins and Mangold, 1960) as the mobile phase. After development, the plates were dried in a stream of CO2 and the lipids were identified using 8-anilinonaphthalin sulfate (ANS) fluorescence elution. The separated lipids were taken into ampoules containing 5% HCI in dry methanol and transesterified at 80°C within 2-3 hr under N?. The methylesters of the fatty acids were extracted from the esterification mixture after dilution with an equal volume of distilled water by n-hexane. Gas Chromatography Methylesters were separated using a Hewlett-Packard 5890 series II equipped with a capillary column coated with SP 2330 of 0.25 pm thickness (0.25 mm i.d. X 30 mi CPS-Li Quadrex, New Haven, CT). High purity nitrogen was applied at a flow rate of 230 kPa, hydrogen 100 kPa, and oxygen 280 kPa. The dual column system was programmed from 160 to 200°C to give partial separation of Cl 8:3 at the rate of 2.5”C min-‘. The detector and injector temperature was 220°C. Identification of the peaks was made using linoleonic standard and by plotting log relative elution temperature versus the number of carbon atoms (Schmidt and Wynne, 1967). To calculate the percentage composition using Hewlett-Packard 3396 A integrator all peaks emerging between the lauric ( 12:0) and linolenic ( 18:3) were included in calculations. Total Phosphorus Determination Phosphorus content from the total lipid extract was determined according to the method of Rouser et al. (1970) spectrophotometrically at 790 nm. The amount of phospholipids was derived from lipid phosphate as pmol - g-’ dry weight.

ATRAZINE

HERBICIDE

RESULTS

EFFECT IN GREEN

351

ALGA

AND DISCUSSION

Addition of atrazine to the cultures of C. kessleri did not inhibit the growth at concentration up to 3 @4. Higher concentrations of atrazine were, however, clearly inhibitory. Growth of the alga was monitored spectrophotometrically by measuring the optical density as well as the increase in dry weight. Abou-Wally et al. (199 la) have demonstrated that dry weight and optical density can be used as growth indicators for green and blue-green algae. The addition of sublethal concentrations of atrazine herbicide, up to 15 PM to the cultures, exhibited an immediate response on growth of alga cells (Figs. 1 and 2). Hersh and Crumpton (1987) suggested that the growth rates and other effects caused by toxins must be determined when nutrients and toxin concentrations are less affected by cell number. Treatment with atrazine at 5, 10, and 15 PLM inhibited growth as measured by optical density and dry weight production. The percentages of inhibition were 12, 3, and 30% with 5 PM, whereas these values increased to 2 1, 12, and 38% at 10 PM and 49, 2 1, and 49% at 15 PM atrazine after 24,48, and 72 hr, respectively. Similar inhibition effect was observed also by measuring the dry weight production. The immediate growth response of atrazine concentrations is consistent with the reports by Walsh ( 1972), Butler et al. ( 1975), and Butler ( 1977) who exposed algal species to atrazine in the laboratory. The pattern of growth and dry weight was highly consistent where the rate of induction of growth inhibition increased by increasing atrazine concentration and reaching maximum reduction at 15 pLM. Reduction of the growth rate by low concentrations of atrazine was reported by Stratton ( 1984) and Mayasich et al. ( 1986). Meanwhile, the growth rates of different isolates were dissimilarly affected. No significant difference in dry weight was observed at low atrazine concentration (5 PM) after 48 hr, whereas growth was slightly reduced by 2.8%. Stockner and Antia (1974) indicated that although short-term studies may be technically convenient for laboratory and field manipulations, a suitable experimental period is needed for adequate adaptation of the test algae to the pollutant or

24

FIG.

40 Time (hr)

72

I. Effect of different concentrations of atrazine on dry weight production of Chlordla

kessleri.

352

EL-SHEEKH.

KOTKAT.

AND HAMMOUDA

2.50

E

2.00

0 s i;j

1.50

z .z : 0 z 2 ‘: 0

1 .oo 0.50

-

0.00 24

40 Time

72 (hr)

FIG. 2. Effect of different concentrations of atrazine on growth of C/dorr//a density of the cell suspension.

ke.ss/cvi.measured as optical

to an alternate nutrient. Data in this study agree with this assessment, as in the treated samples, high rate of increase in dry weight and growth was obvious by increasing treatment time from the first to the second day. However, this rate declined at the third day. Similarly, Laughlin et al. (198 1) found that low doses of jet fuel initially enhanced the growth of algae, followed by a decrease in growth. The photosynthetic activity measured as oxygen evolution (Fig. 3) and dark respiration (Fig. 4) in Chlorella

600

,

0

I

24

46 Time

72

FIG. 3. Effect of different concentrations of atrazine on photosynthesis in Ch/ordu (0) 5 /M,

(cl)

10 /a.

(m) 15 /hf.

0

(hr)

kmhi.

(0) Control,

ATRAZINE

HERBICIDE

EFFECT

IN

GREEN

353

ALGA

0 0

24

40 Time

(0)

FIG. 4. Effect of different concentrations 5 pA4. (0) IO /AM. (W) I5 pM.

of atrazine

on dark

72

0

(hr) respiration

in Chlorella

kessleri.

(0)

Control,

in response to atrazine is not only highly consistent with the growth pattern but also more sensitive. Inhibition was immediately observed after 24 hr of treatment. Stratton (1984) reported that no correlation between photosynthetic and growth sensitivity appears to exist. However, the obtained pattern was in agreement with Hersh (1986) who demonstrated the photosynthetic differential response of different algal species to atrazine treatment. Low atrazine concentration of 5 PM progressively inhibited oxygen evolution by 68, 56, and 30% after 24, 48, and 72 hr, respectively. A similar but higher inhibitory pattern was exhibited by 10 and 15 &4. Oxygen uptake was markedly reduced by treatment after 24 and 48 hr by 5 1 and 62%, respectively, whereas recovery was obvious after 72 hr and progressively increased by 17, 33, and 59% at herbicide concentrations 5, 10, and 15 PM, respectively. In this respect De Noyelles et al. (1982) demonstrated that 14C uptake and biomass declined with atrazine concentrations of 20 and 500 pg/liter in plankton communities and growth was inhibited immediately within 2 days of exposure to atrazine. STriazine herbicides are often the most algicidal of any of the herbicide groups when tested on freshwater algae (Shehata et al., 1984) and their toxicity to algae is due to disrupting photosynthesis (Stratton, 1987). Aliquots (1 ml) of C. kessleri cultures were labeled for 1 hr with uniformly labeled 14C protein hydrolysate at different atrazine concentrations (5, 10. 15 PM) for 24,48, and 72 hr. Cell extracts prepared from the labeled cells and the newly synthesized protein level were determined (Fig. 5). Generally, an obvious reduction in the synthesis relative to the control can be clearly seen after 24 hr of atrazine treatment. Protein synthesis was progressively reduced by 6.7, 37, and 70% at 5, 10, and 15 FM, respectively. Gruenhagen and Moreland ( 197 1) tested the effect of 22 herbicides, including atrazine, on ATP content, oxidative phosphorylation, and RNA and protein synthesis in soybean hypocotyl tissue. All of these compounds have been reported to reduce RNA and protein synthesis. They related this inhibition to the reduction of ATP.

354

EL-SHEEKH,

0

KOTKAT,

24

AND

40

HAMMOUDA

72

96

Time (hr) FIG. 5. Effect of sublethal atrazine on “C-labeled protein synthesis in C/i/ore//u kcsderi. The alga was treated with 0.0 (0). 5 pM (O), 10 pM (Cl), and I5 pM (m) atrazine for 24, 48, and 72 hr at 27°C. Values are averages of three replicates.

However, Van Hoogstraten (1972) demonstrated the inhibition of protein synthesis by herbicide treatment even when ATP was not limited. In Chlorella, atrazine concentration (5 PM) induced a maximum level of protein synthesis after 48 hr. After 72 hr, the level of induction was moderately reduced to 4.8%, relative to control. Protein synthesis decreased by 10 and 15 pA4 treatment and remained reduced compared to the controls over all the different treatment periods. At 15 @4 the synthesis of proteins normally present in cells was drastically reduced, reaching a maximum reduction of 82.8% after 72 hr. It has been reported that eukaryotes and prokaryotes respond to environmental changes by altering their pattern of growth and protein synthesis (Carr, 1973; Tissieres ef al.. 1974). Similarly, alterations in protein synthesis by heat stress have been reported by Ashbumer ( 1982) and Borbely et al. ( 1985). The effect of atrazine herbicide on the algal total lipid fatty acids and other lipid fractions was investigated and the results are displayed in Tables 1 and 2. The changes observed in fatty acid composition were reflected by the change in index Z unsaturation/ Z saturation. The detailed findings are as follows: After 24 hr in the total lipid, the quantity of lauric (12:O) and miristic (14:O) acids considerably increased by 5 and 10 PM atrazine, whereas 15 PM decreased these fatty acids. The other saturated fatty acids, palmitic and stearic acid, decreased significantly with the increase in atrazine concentration. The amount of monounsaturated fatty acids generally decreased with increasing concentration of atrazine, except palmitoleic acid (16: 1) increased only at 5 PM. The higher unsaturated fatty acids frequently display high increase in total lipid by the action of atrazine, e.g., Cl6:3 acid from 5.4 to 10.82 and Cl8:3 from 14.92 to 20.44. This could be attributed to the interference of atrazine with nitrite reduction; as a result, there is an accumulation of nitrite and this acts as a secondary phytotoxic agent and is responsible for initial injury and final death of cells (Klepper, 1974, 1975). In phospholipids (Table 3) palmitic acid increased with increasing atrazine concentrations, while C 14:O and C 18:O disappeared. Generally,

ATRAZINE

HERBICIDE

EFFECT TABLE

Emcr

OF ATRAZINE

IN

GREEN

I

HERWIDE ON THE FAT-W ACID COMPOSITION ISOLATED FROM C/2lorella kessleri Fatty

355

ALGA

OF TOTAL

LIPIDS

acid (mol%)

Time (hr)

Atra (PM)

12:O

I4:O

16:O

16:l

16:2

16:3

IS:0

18:l

IS:2

IS:3

Un/S

UI

24

0 5 10 15

13.94 28.42 28.63 15.89

8.0 9.35 10.08 7.70

18.69 II.88 12.33 14.85

2.33 6.23 2.03 1.78

I .84 I .14 2.89 2.54

5.39 8.08 9.17 10.82

6.65 2.99 2.44 3.82

17.63 9.62 6.99 II.78

10.69 1.26 7.1 I 10.35

14.92 14.35 IS.32 20.44

0.9 I.12 I.15 0.85

95 63 104 123

48

0 5 10 15

0.31 3.39 3.65 4.1

I .48 2.51 2.22 2.91

IO.65 10.3 11.17 10.2

6.85 5.14 6.59 8.43

11.18 12.5 I I .39 II.04

8.61 I I.14 II.02 10.98

1.2 0.8 I.1

4.98 2.38 3.62 2.81

24.5 21.77 20.7 18.57

29.58 29.95 29.66 29.8

0.16 0.20 0.21 0.23

173 178 176 174

72

0 5 10 15

2.1 2.2 1.73 -

3.32 1.91 2.73 -

18.21 13.51 10.8 12.03

-

9.4 12.82 13.45 12.09

5.85 8.14 9.8 9.25

6.31 4.76 0.98

8.07 5.64 3.87 4.0

26.42 26.9 26.4 26.45

20.24 24.8 31.2 35.9

0.29 0.29 0.35 0.15

138 155 180 190

Nore. Un/S, polyne index = sum of unsaturated fatty acids/sum of I2:O. l4:O. 16:O. and 18:O; UI, index of unsaturation = sum of percentage of weight multiplied by the number of olefinic bonds for each fatty acid in the mixture.

the monosaturated fatty acids increased by higher concentrations of atrazine; however, diunsaturated and polyunsaturated acids increased uniformly by all concentrations. Atrazine causes a blockage of electron transport and leads to increased generation of HzOz and 05 radical, which induces membrane damage and increased its fluidity TABLE EFFECTOFATRAZIN

ONTHE FATTY ACIDCOMPOSITION OF POLAR LIPIDS ISOLATED FROM Cldordla ke.vsleri

HERBICIDE

Fatty Time (hr)

Atrazin Wf )

I4:O

2

I6:O 2.17 13.7 17.3 15.8 *

acid (mol%)

16:l

l6:2

16:3

IS:1

IS:2

IS:3

Un/S

UI

-

3.3 4.8 9.6

6.47 IO.56 14.5 8.8

33.35 12.5 4.2 4.5

17.7 27.7 18.3 26.6

2 I .06 32.2 40.9 34.6

3.6 6.3 4.8 5.3

134 I75 198 I81

24

0 5 IO I5

-

48

0 5 IO 15

0.54 0.33 0.47 0.67

9.97 10.2 9.26 17.1

5.3 5.1 I 6.1 I -

14.3 13.7 12.17 14.9

12.94 14.6 13.5 9.7

1.59 2.9 1.6 3.4

22.9 20.7 21.23 28.2

32.4 32.37 34.6 25.8

8.5 8.5 9.3 4.6

194 197 201 168

72

0 5 IO 15

0.48 0.4 0.79 -

12.4 10.3 9.3 24.9

4.1 4.0 5.77 -

I5 15.8 14.8 -

IO.1 13.1 13.2 12.6

2.6 1.8 1.32 -

27.6 23.9 22.3 23.4

28.15 30.5 32.3 38.9

6.8 8.3 8.9 3.0

179 192 195 178

356

EL-SHEEKH.

KOTKAT,

AND

TABLE Emcr

3

OFDIFFERENTCONCENTRATIONSOFATRAZINEHERBICIDEON AND INORGANIC PHOSPHORUS (P~)IN Chlorella Control Pi

5 PM PL

Pi

Time W 24 48 72

HAMMOUDA

0.042 0.55 0.149

I .05 13.7 3.717

0.075 0.225 0.057

PHOSPHOLIPIDS(PL) CELLS

lojiM PL

(pmol

kessleri

-g-l 1.87 5.63 I .42

Pi

15 &iM PL

Pi

PL

dry weight) 0.155 0.296 0.065

3.87 7.4 I I .03

0.435 0.23 0.068

I .09 5.86 I .70

(Allen ef al.. 1983; Mattoo et al., 1984). Increasing herbicide concentration after 48 hr progressively increased the quantity of total lipid fatty acids and palmitic and palmitoleic acids in Chlorella. Meanwhile, polyunsaturated acids were unaffected by the treatment. Phospholipid fatty acids were markedly increased compared to controls. This is in agreement with reports by Aslam and Huffaker ( 1973) who indicated that atrazine action increased metabolism of nitrogen substances which led to stimulated formation of saturated fatty acids. Polyunsaturated fatty acids decreased with increasing atrazine concentration as a result of inhibition of COZ fixation which helps in elongation of saturated fatty acids forming polyunsaturated fatty acids (Ashton et al., 1960). After treatment for 72 hr, the total lipid fatty acids, saturated and monosaturated, decreased in response to 5 and 15 PM. In contrast, the polyunsaturated fatty acids markedly increased by increasing atrazine concentrations. The phospholipid quantity of the 14:0 and 16:O acids increased by treatment. Meanwhile, the quantities of palmitoleic and oleic acids gradually decreased until completely disappearing by increasing atrazine concentration. The quantities of polyunsaturated fatty acids increased by treatment at 5 and 10 PM and decreased at 15 pA4 atrazine. This suggested that high concentration of atrazine may induce elongation and desaturation via activation of desaturase enzyme and consequently increased changes in the saturated and unsaturated fatty acids. The quantities of inorganic phosphorus of phospholipids increased by increasing atrazine concentration in the first 24 hr (Table 3), whereas a progressive decrease was obvious after 48 and 72 hr compared to control. This could be attributed to the blocked synthesis of glycerolipids and glycolipids by atrazine, as a result of accelerated vacculation of cells. The reduced air space system leads to cessation of lipid metabolism (Lichtenthaler, 1990). Microalgae are quite sensitive to herbicides because they share many characteristics with higher plants. The sensitivity of algae toward herbicides varies, however, depending on the species and the kind of herbicide. The results presented here agree with previous reports (De NoyelIes et al., 1982; Mayasich et al., 1986; Abou-Wally, 199 1a,b) pointing out relative inhibitory effect toward atrazine herbicide. Toxicity appeared at herbicide concentrations higher than 3 PM. A marked decrease in the growth, photosynthesis, and other metabolic processes was attained at the concentration 15 PM, indicating that this is a lethal concentration for this algae strain. Altered photosynthesis (Fedtke, 1972), membrane lipids, and pattern of protein synthesis are typical responses to

ATRAZINE

HERBICIDE

EFFECT

IN

GREEN

ALGA

357

photosystem II (PS II) herbicides and perhaps are linked to the metabolism of Dlprotein of PS II (Mattoo et al., 1984). Data in this study have successively proved the concept that in algae the toxicity of atrazine which is related to intracellular oxygen generation affects the extra- and intracellular membrane lipids directly, changing the quantitative value, presumably by its direct action on the enzymes influencing the fatty acid metabolism. As reflected in Fig. 3, photosynthesis is swiftly inhibited by toxic levels of atrazine. Thus, a harmful effect of the herbicide on green algae, which are the main components of phytoplankton in aquatic systems, is likely to occur at the onset of its field application. A better understanding of the environmental relevance of these findings requires further study of the effect of herbicides on natural populations of algae under field conditions. REFERENCES ABOU-WALLY, H.. ABOU-SETTA, M. M., NIGG, H. N., AND MALLORY, L. L. (1991a). Dose-response relationship of Anabaena jos-aqlrae and Selenasfrwn cupricorm~rum to atrazine and hexazinone using chlorophyll (a) content and “C-uptake. Aq~ut. Toxicol. 20, 195-204. ABOU-WALLY, H., ABOU-SET-TA,M. M.. NIGG, H. N., AND MALLORY, L. L. (1991b). Growth response of fresh water algae, Anubuenu Josuqltue and Selenusrrtrm cupricornalttm to atrazine and hexazinone herbicides. Bnll. Environ. Contam. Tosicol. 46, 223-229. ALLEN, M. M., TURNBURKE, A. C., LAGACE, E. A., AND STEINBACK, K. E. (1983). Effects of photosystem 11herbicide on photosynthetic membranes of the cyanobacterium Aphunocupsu 6308. Plunf Physiol. 71, 388-392.

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SCHMIDT, J. A., AND WYNNE, R. B. (1967). Relative elution temperature: A simple method for measuring peak retention in temperature programmed gas chromatography. J. Gas Chromatogr. 4, 325-328. SHEHATA, A. S., ALY, 0. A., AND FARAG, H. (1984). Effect of bladex, etazine, dicryl and baylucide on the growth of Scenedesmus. Environ. Inf. 10, 225-234. STADNYK, L., CAMPBELL, R. S.. AND JOHNSON,B. T. (197 I). Pesticide effect on growth and r4C assimilation in a fresh water alga. Bull. Environ. Conram. Tosicol. 6, 1-8. STOCKNER,G. M., AND ANTIA, N. G. (1976). Phytoplankton adaptation to environmental stresses from toxican and pollutants-A warning. J. Fish Res Board Can. 33, 2089-2096. STRATTON,G. W. ( 1984). Effectsof herbicides atrazine and its degradation products, alone and in combination. on phototrophic microorganisms. Arch. Environ. Con!om. Tosicol. 13, 35-42. STRATTON,G. W. (1987). The effects of pesticides and heavy metals towards phototrophic organisms. Rrr. Environ.

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