Effects of sodium dodecyl sulphate on the aquatic macrophytes Azolla ...

Plant Biosystems, Vol. 142, No. 3, November 2008, pp. 665–668

CELL AND DEVELOPMENTAL PLANT BIOLOGY

Effects of sodium dodecyl sulphate on the aquatic macrophytes Azolla and Lemna

C. FORNI1, F. GIORDANI1, M. PINTORE2, & L. CAMPANELLA2 1

Dipartimento di Biologia, Universita` degli Studi di Roma ‘‘Tor Vergata’’, Roma, Italy and 2Dipartimento di Chimica, Universita` degli Studi di Roma ‘‘La Sapienza’’, Roma, Italy

Abstract In this work, we determined the tolerance to sodium dodecyl sulphate (SDS) of Azolla filiculoides and of Lemna minor. The presence of the detergent in the media affected growth more than the chlorophyll content. On the basis of the effect indices, Lemna is more tolerant to SDS than Azolla. The fern had a better capacity, compared with duckweed, to remove and accumulate the pollutant.

Abbreviations: SDS, sodium dodecyl sulphate; SE, standard error; EI, effect index; TI, tolerance index; EC50, half maximal effective concentration Keywords: Azolla, Lemna, chlorophyll, growth, SDS

Introduction Surfactants are widely used in household and industrial products, and the detergents, utilised for personal and domestic uses, share formulations designed to combine good foaming properties together with low skin irritancy (Shore & Berger 1976). After use, the residual surfactants are mainly discharged into sewage treatment plants or directly to surface waters, and then dispersed in the environment. Sodium dodecyl sulphate (SDS) is one of the most widely used surfactants, and even though it can be degraded by microbes, this anionic molecule, or its degradation products, can be toxic for aquatic organisms. The toxic effects on algal growth have been studied, (Nyber 1988), but there are few data concerning the effects on higher plants (Dirilgen & Ince 1995). To understand how SDS may affect aquatic plants, we assayed the effects of this detergent on two species of floating macrophytes: Lemna minor L., used as test organism in aquatic pollution and phytoremediation studies (Forni et al. 2001b, 2006), and the fern Azolla filiculoides Lam., shown in previous studies to be tolerant to and capable of

accumulating different pollutants (Forni et al. 2001a,b, 2002, 2006). In this study, we determined the tolerance of these species towards SDS by means of EC50 and effect indices (EI), and their capacity to absorb the detergent.

Materials and methods A. filiculoides and L. minor, originally from the Botanical Garden of the Federico II University of Naples (Italy), were maintained under environmental conditions in 1/10 Hoagland medium, pH 6.8 (Van Hove & Diaria 1983); the medium was N-free for Azolla and added with KNO3 for Lemna (Forni et al. 2001b). To test the effect of SDS on plants, 5 g/l of Azolla and 4 g/l of Lemna, corresponding to 80% of surface covered, were inoculated in plastic bowls containing 1000 ml of medium, added or not with 2.5, 10, 25, 50, 100 ppm SDS (Sigma). Detergent concentrations were nominal (i.e. not measured analytically). Test media were not renewed during the experiment.

Correspondence: C. Forni, Dipartimento di Biologia, Universita` degli Studi di Roma ‘‘Tor Vergata’’, Via della Ricerca Scientifica, 00133 Roma, Italy. Tel: þ39 06 72594345. Fax: þ39 06 2023500. Email: forni@uniroma2.it ISSN 1126-3504 print/ISSN 1724-5575 online ª 2008 Societa` Botanica Italiana DOI: 10.1080/11263500802411460

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Plants were kept indoors under environmental conditions. Based on the preliminary experiments, we applied short-term exposures of 1 week to detect the tolerance of these macrophytes to SDS. A randomised block design was applied; each treatment was performed in triplicate and the experiments were repeated three times. The data are expressed as mean values+SE. Plant growth and chlorophyll determination To determine the effect of SDS on growth, the plants were collected after 7 days, wiped off on a sheet of filter paper and weighed (fresh weight). To detect the amount of chlorophyll, the plants were sampled at the end of the experiments, weighed and frozen at 7208C until use. Chlorophyll was extracted from homogenised samples in 80% cold acetone, and the extracts were kept for 60 min in the dark. The solutions were centrifuged at 3000g for 20 min. The absorbance of supernatants was measured by a spectrophotometer (UVIKON 860Spectra). Chlorophyll quantification was made according to Lorenzen and Jeffrey (1980). Chlorophyll concentration is expressed in mg/g fresh weight. EC50 and EI To test the tolerance to SDS EC50 and EI were determined. EC50 is the concentration that results in 50% of the response, using growth as the parameter, EI was calculated as EI ¼ 17TI, where TI is the tolerance index (Sneller et al. 1999), which corresponds to: TI ¼ Response at elevated SDS concentration/ Response under control conditions. Response was measured in terms of growth rate and chlorophyll concentration. SDS determination SDS concentrations were determined both in plants and in media. Plants (3 g fresh weight) and growth media (10 ml) were sampled after 3 and 7 days of treatment, and frozen until analysis. Additional control experiments (without plants) were run to test the adsorption of SDS from the solution onto the walls of the bowls. SDS concentration was determined by the methylene blue reaction. The medium samples were shaken and diluted with H2O (1.5 ml medium: 40 ml H2O). Homogenised plants or 10 ml of media samples were added with 100 ml of distilled water and the solution poured into a separatory funnel, added with 10 ml of phosphate buffer (0.07 M, pH 10) and 5 ml of methylene blue solution (0.35 g/l), 15 ml of

chloroform and 2 ml of H2O2 (40 volumes). The solution was shaken for 1 min, and then the organic phase was recovered and added with 110 ml of distilled water and 5 ml of methylene blue (0.35 g/l) dissolved in acidic solution (6.5 ml 96% H2SO4 in one litre distilled water). The organic phase was once more shaken for 1 min until it separated from the non organic one. It was poured into a flask using a funnel containing a ball of cotton imbibed with chloroform. The filtration was repeated three times, and then the filtrate added with chloroform up to 50 ml total volume. SDS concentration was measured spectrophotometrically (UVIKON 860-Spectra) at 650 nm. Different concentrations of methylene blue were used as a standard. The concentration of detergent was expressed in ppm of SDS in the medium and in the plant extracts.

Results Effect of SDS on growth and chlorophyll content The presence of SDS in the media affected the growth of Azolla; in fact, the biomass of plants, treated for 7 days with SDS concentrations from 10 ppm to 100 ppm, was inhibited (Figure 1) to a maximum of 30% in plants exposed to 50 and 100 ppm SDS. No difference in biomass from the beginning to the end of the treatment was detected in plants grown in the presence of 2.5 ppm SDS (Figure 1). The EC50 was 11.8 ppm. Lemna was tolerant to the lower SDS concentrations, whereas 25, 50 and 100 ppm SDS decreased its growth by 5%, 12% and 25%, respectively (Figure 2). The EC50 was 51.6 ppm. EI on growth are reported in Table I and indicate that SDS affected the fern more than the duckweed. Chlorophyll content was reduced in both Azolla (6% at 2.5 ppm and 60% at 100 ppm) and Lemna (15% at 10 ppm and 89% at 100 ppm SDS) (Figures 3, 4). No difference in chlorophyll content compared with the control was detected in Lemna plants treated with 2.5 ppm SDS. Differences in chlorophyll a/b ratio were detected in all treated plants (Tables II and III).

Figure 1. Growth of Azolla in medium added with SDS.

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Aquatic macrophytes and SDS

SDS concentration in growth media and in the plants The removal of SDS from the medium by Azolla was almost complete in 7 days (Table IV). Bioaccumulation of the detergent was already detected after 3 days in plants grown in the presence of 50 and 100 ppm SDS. At the seventh day of growth, SDS was detected only in plants grown in 100 ppm SDS. Duckweed was not able to completely remove the SDS from the media (Table V). Bioaccumulation of the detergent was detected after 3 days in the plants grown with 50 and 100 ppm SDS, and after 7 days with 10, 25, 50 and 100 ppm SDS (Table V).

Figure 2. Growth of Lemna in medium added with SDS.

Discussion The presence of considerable amounts of detergents in the environment affects water quality and plant Table III. Chlorophyll a/b ratio and EI of SDS on Lemna. Figure 3. Chlorophyll content in Azolla after 7 days of treatment.

SDS treatment (ppm) 0 2.5 10 25 50 100

Chl a/b

EI

2.39 2.45 3.87 4.08 1.51 1.05

0 0.02 0.01 0.12 0.65 0.67

Table IV. SDS content in the media and in Azolla.

Figure 4. Chlorophyll content in Lemna after 7 days of treatment.

Table I. EI of SDS on the growth of Azolla and Lemna. SDS treatment (ppm) 0 2.5 10 25 50 100

EI Azolla

EI Lemna

0 0.16 0.32 0.37 0.46 0.49

0 0.03 0.11 0.16 0.23 0.41

0 2.5 10 25 50 100

0 2.5 10 25 50 100

Chl a/b

EI

2.48 2.43 2.51 4.01 5.70 4.24

0 0.72 0.94 0.93 0.96 0.97

SDS in plants after 3 days (mg/g FW)

SDS in medium after 7 days (ppm)


0 0 0.5 2.2 5.7 81.8

0 0 0 0 86.1 228.1

0 0.7 0.1 2.1 4.6 69.8

0 0 0 0 0 248.8

FW, fresh weight. Table V. SDS content in the media and in Lemna.

SDS treatment (ppm)

Table II. Chlorophyll a/b ratio and EI of SDS on Azolla. SDS treatment (ppm)

SDS treatment (ppm)


0 2.5 10 25 50 100





0 0.4 7.6 25 49 91.5

0 0 0 0 219.7 86.4

0 1.8 3.5 21.2 46 62.4

0 0 4.4 109.2 488.3 2607

FW, fresh weight.

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growth, as in the case of the macrophytes tested in this work. In fact, SDS inhibited the growth of both species mainly at higher concentrations. Based on comparison of EI, Lemna seems to be more tolerant to SDS than Azolla. SDS can also affect chlorophyll content and the chlorophyll a/b ratio, thus negatively acting on photosynthetic activity. Nevertheless, these plants, especially the fern, can remove the detergent from the media. Concerning the removal of SDS, a different behaviour was observed, because the duckweed accumulates the surfactant, whereas the fern probably degrades it, at least at lower concentrations. In fact, no SDS was detected in Azolla plants at the end of the treatment except for plants grown in 100 ppm SDS. This may be ascribed to the possible role played by the bactobionts in SDS degradation. It has been reported that Arthrobacter sp. is able to completely degrade SDS (Margesin & Schinner 1998), and this genus has been found in the leaf cavities of Azolla (Forni et al. 1989). Based on the removal rate and the ability to degrade SDS, Azolla may be considered for future applications in phytoremediation. Acknowledgements The authors thank Dr. Roberto Braglia for assistance in the laboratory.

References Dirilgen N, Ince N. 1995. Inhibition effect of the anionic surfactant SDS on duckweed, Lemna minor with considerations of growth and accumulation. Chemosphere 31:4185–4196.

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