LANDFILL LEACHATE ECOTOXICITY EXPERIMENTS USING LEMNA MINOR S. M. MACKENZIE ∗, S. WAITE, D. J. METCALFE and C. B. JOYCE Biogeography and Ecology and Research Group, University of Brighton, Cockcroft Building, Lewes Road, Brighton, BN2 4GJ, U.K. ( ∗ author for correspondence, e-mail:
[email protected])
(Received 13 December 2001; accepted 7 May 2002)
Abstract. The effects of varying concentrations of landfill leachate on the growth, frond area, chlorophyll content and fluorescence of four strains of Lemna minor were assessed. Growth fluorescence and frond chlorophyll content decreased after seven days exposure to leachate, although responses differed between the strains and end parameters. A L. minor bioassay was used to assess leachate toxicity and the effectiveness of a constructed wetland treatment system and pre-treatment aeration and settlement in reducing toxicity. Pre-treatments were found to significantly reduce toxicity, so their incorporation in any treatment system may increase pollutant stripping. Keywords: bioassay, bioremediation, chlorophyll fluorescence, constructed wetlands, ecotoxicity, landfill leachate, Lemna minor
1. Introduction Constructed wetlands are increasingly used for the remediation of landfill leachate (Mulamooti et al., 1999). They can provide low cost, long-term treatment of leachate and may function as a ‘protective buffer’, reducing the impact of seepage into the surrounding ground and surface waters. If constructed wetlands are to provide a viable treatment option, their design must be related to the characteristics and phytotoxicity of the leachate, which must therefore be reliably assessed. In some circumstances pre-treatment, e.g. aeration, settlement or dilution, may be necessary to ensure that the remedial and treatment capacity of the receiving wetland is not damaged. Leachates are a complex mix of inorganic and organic chemicals. Typically they have high biological and chemical oxygen demands and high concentrations of ammonia, chloride and heavy metals (Cameron and Koch, 1980; Chu et al., 1994; Robinson and Barr, 1999). Their toxicity has most often been studied using laboratory assays with fish, daphnids, macroinvertebrates and algae (Cameron and Koch, 1980; Baun et al., 1999); relatively little research has involved wetland plants (Clement and Merlin, 1995). In this present study we used a small floating aquatic plant Lemna minor L.; Lemna species have been extensively used as a representative aquatic higher plant in ecotoxicological studies (Wang, 1990; Hardman et Water, Air, and Soil Pollution: Focus 3: 171–179, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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al., 1993, Devare and Bahidir, 1994; Sallenave and Fomin, 1997). In addition to being a widely distributed species, native to the U.K., L. minor is easily cultivated and standard test procedures have been established (OECD, 1998; Environment Canada, 1999; LemnaTec GmbH, 2000). In this present study, L. minor bioassays were used to assess the toxicity of landfill leachate and the reduction of toxicity with pre-treatment stages including aeration and passage through an artificial wetland system. Four strains of L. minor were exposed to a range of treated and untreated landfill leachate samples. Toxicity was determined by the changes in leaf chlorophyll content, chlorophyll fluorescence, frond number and total leaf area. Growth inhibition is the most common measure of toxicity in L. minor assays (Wang, 1990), providing an overall measure of effects operating at physiological and cellular levels. Analysis of chlorophyll content and fluorescence allows the toxic effects operating at lower physiological levels to be assessed. Chlorophyll fluorescence provides a non-destructive measurement of photosystem II efficiency (Marwood et al., 2001; Hendry and Grime, 1993) while leaf chlorophyll content reflects the balance of its synthesis and breakdown and provides an indirect measurement of the total photosynthetic capacity.
2. Materials and Methods 2.1. S OURCE AND CULTURE OF L EMNA MINOR STRAINS AND SOURCE OF LEACHATE
L. minor was collected from three sites in East Sussex, south-east England; two were from clean water sites (Lewes Brooks – LB and Plumpton – PL), the third was collected from the surface water within the vicinity of a landfill site (Beddingham – BD). A fourth cultivated strain (LT) was obtained from LemnaTec GmbH, a commercial supplier of L. minor for ecotoxicological tests. All stock strain cultures were maintained under constant illumination at 25 ◦ C and sub-cultured weekly into fresh growth medium (LemnaTec GmbH, 2000). Leachate 1 (Table I) was collected untreated from a landfill in East Sussex, U.K. Leachate 2 (Table I) samples were collected untreated from a landfill site in Somerset, U.K., and after both aeration and passage through a wetland system. The constructed wetland contained established plants of Phragmites australis (Cav.) Trin. Ex Steud. Growing in a bed of graded gravel. Leachate was actively aerated for c. 6 hours prior to top loading; discharge was into a holding pond before release into a natural watercourse. In experiment 1, all strains of L. minor were exposed to a range of Leachate 1 concentrations. In experiment 2 the LemnaTec GmbH strain (LT) was exposed to concentrations of untreated, aerated and wetland-treated Leachate 2 samples.
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TABLE I Chemical data for Leachate 1 collected from Beddingham Landfill Site and Leachate 2 collected from Dimmer Lane Landfill Site
NH3 –N mg l−1 Conductivity pH NO2 mg l−1 NO3 mg l−1 TOC mg l−1 Sus. solids mg l− COD mg l−1 Fe mg l−1 Mn mg l−1 Cr mg l−1 Zn mg l−1
Leachate 1
Leachate 2
290 6790 7.6 0.05 0.4 199 27 690 7 0.019 0.05 0.10
910 1330 7.7 0.025 >5 197 42 1250 2.0 0.28 0.09 0.12
2.2. T EST METHODOLOGY The toxicity tests were carried out according to LemnaTec GmbH (2000) protocols. Leachate was diluted with distilled water and nutrient solution to give final concentrations of 90, 45, 22, 11, 5.5 and 2.75%. A chromate positive standard was used (KCr2O7 at 20 mg l−1 ) as a reference toxicant (Wang, 1990) in addition to a distilled water control. Each test chamber contained 50 mls of solution. Four replicates of each dilution were inoculated with 10 uniformly sized fronds of Lemna minor. Average specific growth rate (µ) values were calculated as: lnxt 1 − lnxt 0 , t1 − t0 where xt 0 and xt 1 are respectively the initial number of fronds at the start and end of the experiment. Chlorophyll fluorescence was measured using a plant efficiency analyser (Hansatech, 1999). Chlorophyll fluorescence can be characterised by two parameters, Fm , maximum fluorescence and Fv , the increase in fluorescence; both are recorded following brief illumination of dark-adapted plants (Hansatech, 1995). The ratio Fv to Fm provides a measure of the efficiency of photosystem II (Marwood et al., 2001). A decrease in the ratio (Fv /Fm ) is likely to indicate that the plant is physiologically challenged (Miles, 1990; Judy et al., 1991; Marwood et al., 2001). Total frond chlorophyll concentration was determined spectrophotometrically, using the methods and equations of Lichentaler and Wellburn (1983). Leaf µ=
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Figure 1. Fluorescence ratio of four strains of Lemna minor after 7 days exposure to landfill leachate. Error bars are SD.
chlorophyll content has been shown to be sensitive to pollution and environmental contaminants (cf. Hendry and Grime, 1993). Results were analysed using one and two-way ANOVA. The concentration of leachate that causes a 50% reduction in growth (IC50 ) was calculated using regression analysis.
3. Results The results of the ANOVA for experiment 1 show that for all strains µ, mean frond area, Fv /Fm (Figure 1) and frond chlorophyll concentrations decreased significantly in response to increasing concentrations of Leachate 1 (Table II). With the exception of Fv /Fm , the pattern of response to leachate concentration differed between strains. This suggests leachate tolerance differed significantly between strains. Leachate 1 caused significant declines in growth rate, the BD (near landfill) strain was the least sensitive and had the highest IC50 value (28.6%). Growth rate IC50 values for LB, LT and PL strains were 19.4%, 18.4% and 13% respectively. Above 11% leachate concentration the BD strain maintained consistently greater frond areas and higher Fv /Fm ratios. A contrasting pattern was demonstrated for leaf chlorophyll content, which with the exception of the LT strain, declined rapidly as leachate concentration increased. Chlorophyll content decreased on exposure to 22% leachate by 84% in BD but only by 22% in LT strains (Table II). For all strains growth rate, frond area, fluorescence ratio and chlorophyll levels declined significantly in the presence of 20 mg l −1 of chromate (CrVI) (Table III); growth rates values were comparable to those plants exposed to 45% leachate.
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TABLE II Growth rate,a frond area,b Fv /Fm c and total chlorophyll d of the four strains of Lemna; Lemna BD, Lemna LT, Lemna PL, Lemna LB when exposed to Leachate 1. Data represent the mean ±S.E. Lemna strain
Leachate 1 concentration 0% 2.75%
5.5%
11%
22%
45%
90%
Average specific growth rate (µ) PL BD LT LB
0.15 ± 0.01 0.13 ± 0.014 0.16 ± 0.008 0.15 ± 0.09
0.09 ± 0.01 0.14 ± 0.018 0.13 ± 0.018 0.15 ± 0.015
0.09 ± 0.003 0.10 ± 0.005 0.10 ± 0.017 0.16 ± 0.005
0.06 ± 0.01 0.08 ± 0.018 0.07 ± 0.012 0.14 ± 0.012
0.01 ± 0.013 0.05 ± 0.032 0.05 ± 0.023 0.002 ± 0.00
0.01 ± 0.003 0.00 0.00 0.005 ± 0.00
0.00 0.00 0.00 0.002 ± 0.01
Mean frond area (cm2 ) PL BD LT LB
10.00 ± 1.7 23.20 ± 5.00 14.00 ± 0.80 10.70 ± 0.80
16.00 ± 0.5 20.50 ± 1.80 10.00 ± 1.50 11.70 ± 1.80
11.00 ± 2.6 16.00 ± 0.90 22.00 ± 12.0 12.00 ± 0.50
2.70 ± 0.6 16.00 ± 1.70 3.20 ± 0.60 9.00 ± 0.50
1.00 ± 0.0 10.03 ± 1.50 3.50 ± 1.50 1.70 ± 0.03
1.70 ± 0.3 13.70 ± 2.00 1.20 ± 0.25 2.00 ± 0.00
0.00 8.50 ± 0.50 1.50 ± 0.30 2.30 ± 0.03
Fluorescence ratio (Fv /Fm ) PL BD LT LB
0.85 ± 0.002 0.84 ± 0.001 0.84 ± 0.005 0.82 ± 0.004
0.85 ± 0.007 0.82 ± 0.06 0.84 ± 0.003 0.84 ± 0.008
0.84 ± 0.012 0.84 ± 0.004 0.78 ± 0.006 0.84 ± 0.08
0.78 ± 0.024 0.77 ± 0.012 0.79 ± 0.017 0.89 ± 0.004
0.17 ± 0.06 0.75 ± 0.03 0.52 ± 0.012 0.00
0.03 ± 0.03 0.24 ± 0.05 0.00 0.16 ± 0.024
0.00 0.14 ± 0.02 0.00 0.00
0.05 ± 0.02 1.80 ± 0.45 9.25 ± 0.35 0.00
0.00 0.25 ± 0.025 0.40 ± 0.35 0.00
0.00 0.00 0.25 ± 0.25 0.00
Total chlorophyll concentration (µg ml−1 cm−1 ) PL BD LT LB
13.15 ± 0.60 10.95 ± 2.25 11.80 ± 0.65 14.50 ± 1.10
8.90 ± 0.10 7.15 ± 0.85 10.50 ± 1.15 15.50 ± 0.60
7.20 ± 1.75 6.50 ± 0.40 11.40 ± 0.30 11.00 ± 0.45
6.20 ± 0.70 3.40 ± 1.25 10.55 ± 1.90 9.85 ± 0.75
a Two-way ANOVA df 7,127 , F = 294, p < 0.001. b Two-way ANOVA df 18,111 , F = 1.57, p < 0.05. c Two-way ANOVA df 18,127 , F = 2.2, p < 0.001. d Two-way ANOVA df 7,92 , F = 1.5, p < 0.001.
Values obtained for frond area were comparable to those of plants exposed to 90% leachate. Overall, BD appeared to be the most chromate tolerant strain. Exposure to untreated leachate 2 resulted in marked and significant declines in growth rate (Table IV). IC50 values for untreated, aerated and wetland-treated leachates were respectively
