Uptake of chromate in sulfate deprived wheat plants

EnvironmentalPollution, Vo]. 97, No. 1 2, pp. 131 135, 1997

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© 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0269-7491/97517.00+0.00

ELSEVIER

UPTAKE OF CHROMATE IN SULFATE DEPRIVED WHEAT PLANTS I. D. Kleiman* and D. H. Cogliatti Departamento de Eeologla, Facultad de Agronom[a, UNCPBA, C.C.178, (7300) Azul, Argentina

(Received 9 December 1996; accepted 25 April 1997)

teria (Ohtake et al., 1990; Coleman and Paran, 1991; Losi et al., 1994a), soils (Losi et al., 1994b), algae (Brady et al., 1994) and fresh water plants (Lenzi et al., 1994) are being studied, because they are cheaper and effective• Terrestrial plants also show high capacity to take up heavy metals, and are able to grow in liquid culture with adequate nutrient supply. Therefore, the use of such plants to decontaminate waste waters can also be considered. To design a decontamination system using terrestrial plants, kinetic parameters of chromate uptake, toxicity problems, and interactions between chromate and other nutrients should be investigated• In barley plants, chromate influx shows Michaelis-Menten kinetics at an external concentration ranging between 0.52-8.32/zgmi -1, and it is competitively inhibited by sulfate (Shewry and Peterson, 1974; Skeffington et al., 1976)• This suggests that chromate enters root cells using the same transport system as sulfate. Authors also report a lower affinity of carriers for chromate than sulfate. When plants are sulfate-deprived for a certain period, their capacity to take up sulfate increases 10-fold by 'de-repression' of their transport system (Deane-Drummond, 1987; Clarkson and Saker, 1989). Lee (1982) found that sulfate-deficient plants increased their sulfate uptake together with chemical analogues such as selehate. Therefore, it is possible that sulfate-deprived plants could show an increased capacity for chromate uptake. The aim of this paper is to study (a) if chromate uptake is inhibited by the presence of sulfate in culture solution, and (b) if sulfate deprivation pretreatments can improve chromate uptake•

Abstract Terrestrial plants have been proposed for the removal of chromate from waste waters. Since chromate seems to be absorbed in roots by the same transport system as sulfate, sulfate-deprivation pretreatment and sulfate absence during chromate uptake were tested in wheat in order to increase chromium uptake efficiency. At 1 and 5~tg Cr042-ml- t the highest chromate uptake was observed when plants suffered 5 days of sulfate deprivation pretreatment and absence of sulfate during chromate uptake. However, only at the lower concentration chromate net uptake was rapidly replaced by net efflux presumably due to toxic effects• At 511g CrO42-m1-1, the uptake of chromate was also enhanced by sulfate-deprivation pretreatment alone or lack of sulfate-competition. We conclude that sulfate is a strong inhibitor of chromate uptake, and when plants are going to be used to remove chromate from waste waters, sulfate-chromate interaction should be considered. © 1997 Elsevier Science Ltd

Keywords: Bioremediation, chromate uptake, chromatesulfate, waste waters, terrestrial plants.

INTRODUCTION Chromates are highly toxic to animals and plants due to their mutagenic, clastogenic and carcinogenic effects. They cross biological membranes, and have strong oxidative power that produces a high concentration of reactive species of 02 inside cells (Taylor et al., 1979; Barcel6 et al., 1987; Vfizquez et al., 1987; Bassi et al., 1990; Witmer et al., 1994). Waste waters with high chromate concentration are generated by different industrial processes and are frequently discharged into the environment without prior treatment (Poschenrieder et al., 1986). Exposure to high amounts of this pollutant cause a serious health risk to all forms of life. The most commonly used method to remove chromate from liquid effluents is chemical reduction and precipitation (Patterson et al., 1994). This method needs large quantities of expensive chemicals with high energy costs. Therefore, bioremediation techniques using bac-

MATERIALS AND METHODS Culture conditions

Two experiments with wheat (Triticum aestivum cv. Klein Atalaya) were performed in culture solution in a growth chamber at 20 J: l°C. The photon flux density at the level of plant canopy was 180/zE m -2 s -1 during a 16 h photoperiod provided by 400 W HPLN fluorescent and 100W incandescent lamps. In both experiments,

*Fax: 54 0281 33291, e-mail: [email protected] 131

132

L D. Kleiman, D. H. Cogliatti

seeds were germinated on moistened filter paper in the dark for 48 h, and then transferred to 40 litre plastic tanks containing a thoroughly aerated complete nutrient solution made with deionized water. The composition of the nutrient solution was: KNO3 5mM; Ca(NO3)2 1.5mM; NaNO3 2mM; MgSO4 1.5mM; KHEPO4 l m M ; KCI 50#M; Fe-Edta 20/zM; H3BO3 25/zM; MnSO4 2/zM; ZnSO4 0.5 #M; CuSO4 0.5/zM; (NH4)6 MO7024 4H20 0.016/zM; pH 6.04-0.1. In the solution lacking sulfate, MgC12 was added instead of MgSO4, and micronutrients containing sulfate were replaced by: Cu(NO3)2 0.5/zM; MnC12 2/zM; ZnCI2 0.5/zM. KCI was added at a concentration of 0.017raM. Solutions were changed weekly during the first 14 days, and then every 72 h. Measurements of chromate uptake Two different methods to measure chromate uptake were performed depending on the chromate concentration used in the experiment. We had to choose different methodologies to measure chromate uptake because: at low concentration (1/zg CrO4 2- ml-l), measurements by successive harvests require very sensitive methods to detect chromium in plants or great amounts of plant material; thus a depletion method was chosen. On the other hand, at higher external chromate concentration (5/zgml-l), it is very difficult to detect changes in the solution in short periods; thus successive harvests were chosen to quantify Cr contents in plants. For the depletion method, on day 13 after germination, plants were divided into three groups according to pretreatment: (a) plants with continuous sulfate supply (+ S), (b) plants sulfate-deprived for 2 days (-S2d) and (c) plants sulfate-deprived for 5 days (-S5d). The sulfate-deprivation periods for (b) and (c) above were started such that they both finished on the same day. Chromate uptake was measured over a period of 24 h under continuous light in the presence (+ S) or absence (-S) of sulfate using a factorial design of the pretreatments, with eight replicates per combination. Depletion was measured in plastic beakers containing 70 ml nutrient solution with 1 ttg CrO42- ml -~ as K2CrO4. Four plants were placed into each beaker and were considered as one experimental unit. Solution was renewed at the end of each depletion period. The first renewal occurred 30min after chromate was supplied. From then on, the depletion period was 1 h in treatments which suffered sulfate-deprivation during 2 or 5 days combined with the sulfate-absence treatment, and 2 h in the remaining treatments. Chromium in solution was measured at 0.5, 2.5, 4.5, 8.5, 10.5 and 24 h after chromate was supplied. For the successive harvest method used for wheat grown in 5/zg CrO42- m1-1, plants were grown in groups of four in plastic beakers of 3.2 litre with complete nutrient solution. Based on the results of the depletion experiment, we decided to apply only two pretreatments starting on the 17th day after germination. Experimental units were then either supplied continuously with sulfate (+ S) or sulfate-deprived for 5 days (-S).

Each pretreatment contained 35 replicates. After pretreatment, five replicates were harvested from each, and K2CrO4 was added to the remaining plants with sulfate (+ S) and without sulfate (-S). Solutions were renewed daily and five experimental units were harvested from each treatment at 2, 4 and 6 days from the beginning of chromate supply. Plant and solution analysis In both experiments, dry weight of shoots and roots were determined after oven-drying samples at 80°C for 48h. Plant material was wet-ashed with a mixture of acids (nitric:percloric 3:2 v/v) for chromium determination. The chromium determinations in plant digests and in nutrient solutions were performed using atomic absorption spectrophotometry (AAS)(GBC model 902). Chemicals used were of analytical grade. Statistical analysis Results were analysed statistically by ANOVA and mean were contrasted by LSD multiple range test.

RESULTS Growth There were no significant differences in root or shoot dry wt or in ratio of shoot:root detected between control and sulfate-deprived plants after 5 days of pretreatment (Table 1). Cr depletion experiment (Fig. 1) All treatments showed decreasing chromate uptake rates with time. During the first 30 min of chromate supply treatments and pretreatments lacking sulfate showed a higher uptake rate than control plants (+ S + S). Plants with 5 days of sulfate-deprivation pretreatment and sulfate absence during chromate uptake ( - S 5 d - S ) showed the highest uptake rate and maintained a higher rate than the controls (+ S+ S) up to 4.5h from the beginning of chromate supply. Plants with 2 days of sulfate-deprivation pretreatment and sulfate absence when chromate was supplied ( - S 2 d - S ) also showed a chromate uptake higher than controls up to 4.5 h, but lesser than ( - S 5 d - S ) plants. In general, the remaining treatments did not differ from the controls after the first 30 min of chromate supply. At 8.5 and 10.5 h of chromate supply, ( - S 5 d - S ) and ( - S 2 d - S ) plants showed Cr etflux. After 1 day in chromate, all treatments showed a low but positive chromate Table 1. Effect of 5 days of sulfate deprivation on dry wt and shoot]root ratio in wheat plants

Treatment +S -S

Shoot(mg)

Root (mg)

Shoot/root

508a 445a

145a 123a

3.5a 3.6a

Results followed by the same letter within each column are not statistically different (a = 0.05).

Removal o f chromate from waste waters 300 °

Table 3. Effect of sulfate treatments on chromate uptake (/zg g - i dry wt day -1) in wheat plants grown in 5/~g CrO4 2- ml -I

250a

Uptake period (days) 200-

" +S+S A -S2d+S

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Treatment

= +S-S o -S2d-S

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=

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50-

0

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+ S+ S + S-S -S + S -S-S

0

5

10

15

20

25

Time (h)

Fig. 1. Effect of sulfate treatments on rate of chromate uptake in wheat plants grown in 1/zgCrO4 2- ml-t over 24.5 h. Results are mean of eight replicates and bars respresent SE. uptake, with the exception of ( - S 5 d - S ) plants, which still showed efflux (negative chromate uptake). Successive harvest experiment

After 2 days, plants with 5 days of sulfate-deprivation pretreatment and sulfate absence when chromate was supplied ( - S - S ) showed the greatest Cr concentration in the shoots, while plants with sulfate-deprivation pretreatment alone ( - S + S) and plants with lack of sulfate during chromate uptake ( + S - S ) did not statistically differ from the controls ( + S + S). In the same period, the lowest Cr concentration in roots was found in control plants ( + S + S) and the highest in ( + S - S ) and ( - S - S ) plants. Total Cr content was lower in control plants than the remaining treatments, among which ( + S - S ) and ( - S - S ) showed the highest Cr contents (Table 2). Chromate uptake during the first 2 days from the beginning of chromate supply showed the following ranking ( + S + S) < ( - S + S) < ( + S - S ) < ( - S - S ) . From then on, the uptake was similar in all treatments and showed a decrease with time (Table 3). Plants ( - S - S ) not only showed the highest uptake rate, but also the highest chromium net translocation. The remaining treatments were not statistically different from each other. The percentage of transport in all treatments was less than 5% of the chromium taken up by the roots. Table 2. Effect of sulfate treatments on chromium concentration and total chromium content in wheat plants after 48 h in 5/zg

0-6

ll78a 1990c 1607b 2271d

875a 1522b 1293b 1277b

775ab 1071b 509a 1062b

Treatment

Plants ( + S - S ) showed a lower percentage of transport than ( + S + S ) and ( - S - S ) , but did not differ from ( - S + S) (Table 4).

DISCUSSION Sulfate-deprivation during 5 days did not affect growth in our experiment (Table 1). The effect of sulfate-deprivation pretreatment on chromate uptake, should not, therefore, be attributed to changes in growth demand. Chromate uptake at 1 ~tg CrO4 2- ml-1 was increased during the first 30 min by both sulfate-deprivation pretreatment or absence of sulfate during chromate uptake (Fig. 1). These results are in agreement with DeaneDrummond (1987) and Clarkson and Saker (1989), who reported that sulfate-deprivation induces an increase in sulfate uptake associated with a new or modified sulfate transport system with a higher affinity for sulfate. As both sulfate and chromate appear to be transported by the same transport system (Skeffington et al., 1976), the higher initial uptake of chromate in sulfate-deprived pretreated plants ( - S 2 d and - S 5 d ) may be the result of the de-repression of that system. The fact that both anions are transported by the same system may also explain the increase of chromate uptake by the lack of competition when sulfate is not present. However, after 2.5 h from the beginning of chromate supply, the effect of the presence or absence of sulfate on chromate uptake depended on the pretreatment. When plants were continuously sulfate supplied during pretreatment, a similar chromate uptake in the presence or absence of sulfate was observed. These results could be explained if sulfate efflux in ( + S - S ) plants was high enough, during the 2 h without a change of solution, to build up an external concentration sufficient to inhibit Table 4. Effect of sulfate treatments on net translocation rate and transport percentage of chromium into shoots of wheat plants after 48 h in 5 / z g CrO42- ml- !

CrO4 2- ml- I

+ S+ S + S-S -S + S -S-S

0-4

Results followed by the same letter within each column are not statistically different (a = 0.05).

o-50

0-2

Chromium concentration Shoot (#gg I dry wt)

Root (#gg-l dry wt)

Cr content (/Lg)

24a 26a 28a 40b

2210a 3761c 2737b 3996c

355a 592c 460b 623c

Results followed by the same letter within each column are not statistically different (a = 0.05).

Treatment +S+S + S-S -S + S -S-S

Translocation (~gg-1 dry wt day- 1)

Transport (%)

48a 47a 55a 80b

4.1b 2.4a 3.4ab 3.6b

Results followed by the same letter within each column are not statistically different (~ = 0.05).

134

L D. Kleiman, D. H. Cogliatti

chromate uptake. On the other hand, when plants were sulfate-deprived for 2 or 5 days, a chromate uptake similar to that of control plants (+ S + S) was observed only in the presence of sulfate ( - S 2 d + S and - S 5 d + S plants). These results may be due to the presence of sulfate that down regulates its own uptake and consequently chromate uptake. A rapid repression of sulfate influx was reported by Clarkson and Saker (1989) in wheat plants returned to + S solution after a 5 day sulfate-deprivation pretreatment. The massive uptake of sulfate repressed sulfate influx by a rapid increase of cytoplasmic sulfate concentration (Clarkson and Saker, 1989; Bell et al., 1995). Finally, when plants were sulfate-deprived during pretreatment and treatment ( - S 2 d - S and - S 5 d - S plants), chromate uptake was effectively increased. The longer the sulfate-deprivation pretreatment, the higher the increase of chromate uptake. This was probably due to the de-repression effect of the pretreatment and treatment on the sulfate transport system, added to the lack of competition of sulfate on chromate uptake by this system. It is worthwhile here to consider the rapid decrease of chromate uptake observed in the ( - S 2 d - S ) and ( - S 5 d - S ) plants. After 8 h of chromate supply, chromate net uptake was changed into a net Cr efttux. Because chromate has a deleterious effect on biological membranes (Barcel6 et al., 1987; V~zquez et al., 1987; Bassi et al., 1990), it is possible that the high initial uptake built up an internal chromium concentration sufficient to increase permeability of root cell membranes, resulting in a net chromium efflux. All treatments showed a decrease in chromate uptake during the 24h time course experiment, but only ( - S 2 d - S ) and ( - S 5 d - S ) plants showed net efflux. Because plants were not expose to chromate prior to the experiment, the initial measurements of chromate uptake equalled chromate influx. After that, the internal Cr concentration started to build up progressively, and the greater the internal Cr concentration, the higher the Cr efflux and the lower the chromate uptake. When chromate was supplied at a higher concentration (5/zg CrO42- ml-l), a higher chromate uptake than that of control plants was observed in plants that received sulfate-deprivation pretreatment or absence of sulfate during chromate treatment (Table 3). At this chromate concentration, ( + S - S ) plants showed a higher chromate uptake than (+ S + S) plants during 4 days. These results may be due to the derepression of the sulfate transport system during the 48 h without sulfate added to the lack of competition. On the other hand, the inhibitory effect of sulfate efflux when plants + S were transferred to - S nutrient solutions, appeared to be less important in this experiment due to the high concentration of chromate used here. The greater chromate uptake of the ( - S + S) plants in comparison with controls (+ S + S) does not agree with the rapid repression of the sulfate transport system when the sulfate supply is restored (Clarkson and Saker, 1989; Bell et al., 1995). These results could be obtained

if sulfate uptake was decreased by the competitive inhibition performed by the high chromate concentration. The highest chromate uptake also occurred in this experiment in the combination of sulfate-deprivation pretreatment with absence of sulfate when chromate was supplied ( - S - S ) . This result could also be explained by a de-repression of the sulfate transport system added to the lack of competition. At 5/zg CrO42- ml -l, chromate net uptake decreased along the experimental period in all treatments; however, net effiux was not observed in any of them, including ( - S - S ) plants. It is possible that roots still affected by the toxic level of chromate remained able to take up chromate due to the higher external chromate concentration used here. Further experiments would be necessary to clarify this question. The combination ( - S - S ) not only showed higher chromate uptake, but also higher chromium translocation into the shoots, even when they showed the same percentage of transport as the control ( + S + S ) (Table 4). It has been reported (Bell et al., 1995) that in sulfate deprived plants, S-flux into the xylem is higher than in continuously sulfate-supplied plants. It is worth considering if chromate was released into the xylem instead of sulfate in sulfate-deprived plants, using the same transport mechanism.

CONCLUSIONS According to the results reported here, sulfate appears to be a strong inhibitor of chromate uptake. When the sulfate transport system is de-repressed by a previous sulfate-deprivation, it is possible to obtain a higher chromate uptake in the presence of sulfate only if chromate concentration was high (5/xg CrO4 2- ml-l). In high chromate concentration (5/zg CrO42-ml-I), the absence of sulfate when chromate was supplied (+ S - S plants) was enough to increase chromate uptake after 48 h. In both low and high chromate concentration, the highest uptake was obtained with a de-repressed transport system in the absence of sulfate-chromate competition ( - S - S plants). This high efficiency of removal of chromate from solution did not remain for an extended period, presumably due to toxic effects of chromate. Finally, when plants are to be used to remove chromate from waste waters, interaction with sulfate should be considered and the interaction with other nutrients need to be investigated.

ACKNOWLEDGEMENTS This research was supported by the Universidad Nacional del Centro de la Pvcia de Buenos Aires (UNCPBA). The authors express thanks to Dr J. Rogers for the correction of manuscript, and Jorge Cardozo for technical assistance.

Removal o f chromate f r o m waste waters

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