Ecotoxicology 3, 235-247 (1994)

Chronic toxicity of cadmium in Pteris vittata, a roadside fern M A N I S H A G U P T A and S A N T H A D E V I Electron Microscopy Section, National Botanical Research Institute, Lucknow 226 001, India

Received 17 June 1993; revised and accepted 19 November 1993

The effect of Cd at concentrations of 0.1, 1.0, 2.5, 5.0 and 10.0 ppm on Pteris vittata, a commonly occurring roadside fern was studied during the entire life cycle from spore germination to spore production. Light, fluorescence, scanning and transmission electron microscopy were used to study the structural changes due to Cd. With increasing Cd concentrations the following changes were observed: inhibition of first and second generation spores, abnormal development of protonema, disintegration of thylakoid organization, delayed formation of reproductive elements and abnormal sporangia. As the Cd content of the plants increased with increasing concentration in the medium, biomass-specific concentrations of chlorophyll and carotenoids decreased. The gametophytic phase was more sensitive than the sporophytic stage to Cd. The differences noticed during the three stages of development could be useful as markers for Cd contamination in the environment. Keywords: cadmium; fern; uptake; morphology; ultrastructure. Introduction

Many pollutants can adversely affect the vital processes of embryogenesis, differentiation and morphogenesis, thereby leading to genotoxic and teratogenic effects (Nriagu 1988). Most information about environmental effects of pollutants stems from studies of laboratory animals; much less is known about the long-term impacts of pollutants on plants. In extrapolating data from laboratory tests for developmental toxicology to natural systems, it is essential to have information on dose- and time-dependent toxic effects for the entire life cycle. This may be even more important for organisms that have alternation of generations. Data on the response of the general parameters of plant functions along with the toxicant level in situ and the possible history of exposure could also prove useful for biomonitoring. In an early study with cadmium, differential responses by gametophytic and sporophytic stages of the aquatic ferm Ceratopteris were observed (Gupta et al. 1992). We present here a more detailed investigation of this aspect, using a widely occurring roadside fern, Pteris vittata. This fern was selected because it is considered a reliable bioindicator for metal pollution (Ho and Tai 1985). Pteris vittata also allows for a convenient spore-germination bioassay (Francis and Petersen 1983) for quantification of toxicity. Profuse formation of highly allergenic spores also makes this fern important from an environmental 0963-9292 (~ 1994 Chapman & Hall

236

Gupta and Devi

perspective (Singh and Devi 1989a). We studied the effect of different concentrations of cadmium on spore germination and gametophyte and sporophyte development through analysis of morphological and ultrastructural parameters. Materials and methods

For the experimental studies, spores of P. vittata L. were obtained from the National Botanical Research Institute, Lucknow; the spores were derived from plants that had no prior exposure to pollutants. For measurement of the percentage of spore germination, solid Knop's medium (Miller and Greany 1974) was amended with AnalaR CdCIE.2H20 (Loba-Chemie Indoaustranal Co., India) to obtain Cd concentrations of 0.1, 1.0, 2.5, 5.0 and 10.0 ppm. Control experiments were performed simultaneously. Experiments were conducted in a growth chamber under standard physiological conditions of 25 _+ 0.5°C, 16 : 8 light (1600 ftc fluorescent light, Philips) : dark photoperiod at ambient humidity. After completion of germination, only 500 spores were scored from each plate. For calculating the onset of the development of the sex organs, the date at which the earliest manifestation was observed was noted. The period between the release of antherozoids and the cessation of their movement was used as an index of antherozoid motility and viability. Plants were maintained in solid Knop's medium from gametophytic to adult sporophytic phases. Each plant was grown separately in a 250 ml beaker. Ten replicates were used for each concentration. Of these, three plants were harvested at random for measurement of metal, chlorophyll, protein and starch. When the leaves were mature, three plants were left for sporulation studies and four were used for studies of morphology and anatomy. ESTIMATIONS Dried samples were digested with a mixture of 10 N H2SO4and HNO3(5 : 1 v/v). Cd was measured using a Perkin Elmer 2380 Atomic Absorption Spectrophotometer. The detection limit was 0.002 ppm; the results were expressed as/~g Cd per g dry weight. Chlorophyll was extracted in 80% chilled acetone. Chlorophyll a and b contents were measured by spectrophotometry (Arnon 1949) and carotenoids by the method of Duxbury and Yentsch (1956). Protein was precipitated with trichloroacetic acid (TCA) and analysed according to Lowry et al. (1951). Starch was determined by the phenolH2SO4 method (Montgomery 1957). SPORE MORPHOLOGY Spore morphology was studied by mounting the mature fresh spores directly in distilled water. Measurements of size were taken by an occular micrometer after standardizing the error of the microscope with a stage micrometer. Only the longest equatorial diameter was measured. The methodology given by Nayar and Devi (1964, 1966) was followed for describing the spore morphology. LEAF AREA The leaf area of the juvenile leaves was measured on Area Measurement System Delta-T Devices (UK).

Cadmium toxicity in Pteris vittata, a roadside fern

237

ELECTRON MICROSCOPY Materials were fixed in 2.5% glutaraldehyde, followed by 1% osmium tetroxide. For scanning electron microscopy (SEM), specimens were dried in a critical point drier, coated with gold in an ion sputter coater and examined by JEOL-JSM Stereo Scanning Electron Microscope at 30 ° tilt and 10 kV. For transmission electron microscopy (TEM), the material was embedded in epon araldite, sections cut on a LKB Nova Ulramicrotome using glass knives, stained with aqueous uranyl acetate and lead citrate and examined under a Philips CM-10 Transmission Electron Microscope. FLUORESCENCE MICROSCOPY Autofluorescence was studied by mounting the material directly in distilled water. Slides were viewed under a Leitz Labourlux Fluorescence Microscope fitted with an 12 excitation filter (BP 450-490), with a beam splitting mirror (RKP 510) and suppression filter (LP 515) or with an excitation filter (BP 340-380), with a beam splitting mirror (RKP 400) and suppression filter (LP 430). Photographic records were made with a Leitz Vario Orthomat camera on Agfa B/W film DIN(24), ASA(200) or DIN(24), ASA(400). STATISTICAL ANALYSIS The data was subjected to two-way analysis of variance. In cases where significant F-ratio values were obtained, Duncan's multiple range test was used to detect differences between the mean values of the treatments. A simple linear regression coefficient (r) was calculated wherever necessary (Gomez and Gomez 1984).

Results

SPORE GERMINATION AND GAMETOPHYTE Germination of P. vittata spores started in all the concentrations after approximately 7 days. The germination rate decreased with increasing Cd concentration (p < 0.01); a 50% decrease in germination (LCso) corresponded to a cadmium concentration of 2.5 ppm (Fig. 1). The variations in the gametophytic types in response to the Cd treatments are shown in Table 1. In the development of protonema, various abnormalities, such as stunting, branching and elongation were seen at different concentrations of Cd (Fig. 2A-C). A profuse proliferation of non-cordate thalli (Fig. 2C) was noted up to a Cd concentration of 2.5 ppm. In 2.5 and 5.0 ppm concentrations of Cd, some protonema gave rise to a cluster of cells after three- to four-celled stages (Fig. 2D). Typically one of the cells of these clusters gave rise to a cylindrical minute apogamous sporophyte carrying an apical meristem and a group of scale-like appendages (Fig. 2E and F). These sporophytes survived for only 20-25 days. They failed to regenerate even after being transferred to a Cd-free culture medium. The production of antheridia was not affected by Cd at a concentration lower than 10 ppm, but antheridia and archegonia formation were delayed at 10 ppm (Fig. 5A). The antherozoids were motile for 3 min in 5 and 10 ppm and for 5 min in the control. The rhizoids of Cd-treated thalli were irregular and swollen (Fig. 2C). The rupture

Gupta and Devi

238 50

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o f rhizoids, discharging their contents back into the m e d i u m , was seen in 10 p p m in C d - t r e a t e d thalli. Ultrastructure analysis of the cordate g a m e t o p h y t e s in the C d - t r e a t e d ferns s h o w e d disruption of the tonoplast and slight shrinkage of starch grains ( 0 . 1 5 - 1 . 2 ~ m ) as c o m p a r e d to the control (0.44-3.4/~m) (Fig. 3 A and B). In some thalli, plastids w e r e also e l o n g a t e d and fused. T h e a p o g a m o u s s p o r o p h y t e s s h o w e d e x t r e m e agglutination of the cytoplasm leading to distortion o f the organelles (Fig. 3C). T h e thylakoids had d e g e n e r ated and the starch grains were rudimentary.

Table 1. Change in the occurrence of gametophytic types (percent of the control) due to Cd treatment Medium Cd (ppm) 0.0 0.1

1.0 2.5 5.0 10.0

Cordate

Reversible

Irreversible

-

-

3~ + 0.34 9d + 0.57 45 a + 0.86 21 b -+ 0.57 17b~ + 0.57

23 a + 1.32 24ab + 1.21 19c + 0.34

23b + 2.42 36 a + 0.69

100 a

97b + 0.57 91 c +- 0.57 32d + 0.57 31 e + 0.86 28f --- 0.57

Apogamous

(-) Absent. Each value is the arithmetic mean _+ SE of triplicates. Similar letters above the values indicate that the means do not differ significantly at the 5% level according to Duncan's multiple range test.

Cadmium toxicity in Pteris vittata, a roadside fern

239

Fig. 2. Control and Cd-treated prothalli (scale = 100/~m). (A) Control protonema, (B) control thallus showing the meristem formation and the normal elongate rhizoids, (C) 2.5 ppm Cd-treated thallus showing branching and abnormal swollen rhizoids, (D) thallus treated with 5 ppm concentration of Cd, showing cluster of bulbous cells, (E) apogamous sporophyte produced from 10 ppm Cd-treated cultures, (F) a portion of the tip of the sporophyte showing appendages and meristematic cells.

240

Gupta and Devi Table 2. Leaf area (cm2) of the control and Cd-treated juvenile leaves of P. vittata

Medium Cd(ppm) 0.0 5.4 a + 1.7

0.1 2.8 b

+0.23

1.0

2.5

5.0

10.0

1.7¢ +0.11

1.6¢ +0.11

1.5e +0.0

1.4c +0.57

Each value is the arithmetic mean + sE of triplicates. Similarletters abovethe valueindicate that the means do not differ significantlyat the 5% level according to Duncan's multiple range test. JUVENILE SPOROPHYTE Cadmium strongly affected sporophyte production at 1.0 ppm or higher levels (p < 0.05, r = 0.768). In the controls and 0.1 ppm Cd-treated samples sporophytes emerged after approximately 90 days and after approximately 100 days at higher concentrations. Ninety-nine percent of the thalli produced sporophytes in the controls and 95% in 0.1 and 1.0 ppm Cd-treated thalli. These values were reduced to 80% in 2.5 and 5 ppm and 30% in 10 ppm Cd-treated (p < 0.01). With the exception of size, there was no change in the morphology of the treated sporophytes. The density of the root system decreased at higher Cd concentrations. The leaf area decreased to 74% in 10 ppm Cd-treated group as compared to the control group (p < 0.01) (Table 2). Adnation of the leaflets with the petioles was seen in all the treatments with Cd concentrations ~> 2.5 ppm. ADULT PLANTS In the control, new leaves were produced starting approximately 120 days after spore germination. After approximately 1 year, the fifth and sixth leavves produced fertile pinnae, with elongate and continuous sori along their margins. Mature plants of P. vittata treated with 5 and 10 ppm Cd showed stunted growth, while plants treated with lower concentrations showed normal growth as did the controls. The leaf surface of these plants showed open stomata in the control group (Fig. 3D), while 80% of the stomata were partially closed in the 2.5 ppm Cd-treated group and completely closed in the 5 ppm Cd-treated group (Fig. 3E). From 2.5 ppm Cd onwards there was a reduction in the amount of wax deposition accompanied by thickening of the guard cells (1700-1800/zm), the intensity of which increased with higher Cd concentration (2000-2200 #m). In the control group, root hairs were long and straight (16-22 mm), whereas in the 10 ppm Cd-treated group they were longer and curled (80-92 mm). Mature roots in the 10 ppm Cd-treated groups as a whole were growing into club-like structures towards the surface. Sori on sporophyte fronds from the 0.1 and 5 ppm Cd treatments were discontinuous. Curling of the fronds was noted at and above 2.5 ppm Cd treatment. The mid-rib of the terminal pinnae extended beyond the lamina at 10 ppm Cd. The frequency of abnormal sporangia also increased simultaneously with the increase of Cd concentration in the medium (p < 0.01). At 5 and 10 ppm Cd, the sporangia were often smaller in size, were produced in clusters and contained abnormal tetrads and diads. Most of the sporangia (99%) in the 10 ppm concentration of Cd were abnormal (Fig. 5B). The abnormalities included poor development of tapetum, sporogenous tissue

Cadmium toxicity in Pteris vittata, a roadside fern

241

Fig. 3. TEM micrographs of control and treated thalli (scale = 10/.tm): (A) control showing normal chloroplast and intact tonoplast (arrow), (B) cordate gametophyte showing disruption of tonoplast (arrow), shrunken starch grains which appeared osmiophilic in 10 ppm Cd, (C) agglutination of the cytoplasm in the cylindrical sporophyte treated with 10 ppm Cd. SEM micrographs (scale = 100/xm): (D) stomata of the control leaf, (E) closed stomata of a corresponding stage in 5 ppm Cd-treated leaf of the sporophyte.

Gupta and Devi

242

Fig. 4. (A) Control sporangium of P. vittata showing UV fluorescence of the annular wall and spores in the sporangium (autofluoresence) on UV excitation. (B) Sporangium of P. vittata (5 ppm Cd) showing absence of fluorescence of the annular wall and spores in the sporangium (autofluorescence) on U V excitation. (Scale = 100 p m . )

and annulus (Fig. 4A and B). Additionally, the number of spores per sorus was lower and the mean diameter of the spores was smaller as the Cd content in the medium increased (p < 0.01, r = -0.813) (Table 3). Shrunken spores with poor ornamentation were very common in 5 and 10 ppm Cd. The number of spores that were viable was reduced as the concentration of Cd increased with 88% spore germination in the 10 ppm Cd treatment as compared to the control (p < 0.01). Anatomical studies of petioles in the 10 ppm Cd treatment revealed the presence of fewer tannins; these petioles also had poor development of sclerenchyma in the cortex, compared to the control. However, we found no change in fluorescence intensity of vascular tissue in the treated material. Table 3. Effect of Cd concentration on spore diameter (~m) Medium Cd(ppm) 0.0

0.1

1.0

2.5

5.0

10.0

71.3 a +3.17

68.3 b +2.13

62.6 c +3.81

53.6 d +3.35

51.0 e +3.29

50.1 e +3.23

Each value is the arithmetic mean + SE of triplicates. Similar letters above the values indicate that the means do not differ significantly at the 5% level according to Duncan's multiple range test.

Cadmium toxicity in Pteris vittata, a roadside fern

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Chronic toxicity of cadmium in Pteris vittata, a roadside fern.

: The effect of Cd at concentrations of 0.1, 1.0, 2.5, 5.0 and 10.0 ppm on Pteris vittata, a commonly occurring roadside fern was studied during the e...
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