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Response of weeping willows to linear alkylbenzene sulfonate
Chemosphere 64 (2006) 43–48 www.elsevier.com/locate/chemosphere
Response of weeping willows to linear alkylbenzene sulfonate Xiaozhang Yu b
a,*
, Stefan Trapp
b,*
, Puhua Zhou c, Xiaoying Peng c, Xi Cao
c
a Department of Environmental Science, Hunan Agricultural University, Changsha 410128 Hunan, PR China Institute of Environment and Resources DTU, Technical University of Denmark, Building 115, DK-2800 Kongens Lyngby, Denmark c Department of Biotechnology, Hunan Agricultural University, Changsha 410128 Hunan, PR China
Received 9 June 2005; received in revised form 15 November 2005; accepted 18 November 2005 Available online 5 January 2006
Abstract Linear alkylbenzene sulfonate (LAS) is the most commonly used anionic surfactant in laundry detergents and cleaning agents. LAS compounds are found in surface waters and soils. The short-term acute toxicity of LAS to weeping willows (Salix babylonica L.) was investigated. Willow cuttings were grown in hydroponic solution spiked with LAS at 24.0 ± 1 °C for 192 h. The normalized relative transpiration of plants was used to determine toxicity. Severe reduction of the transpiration was only found for high doses of LAS (P240 mg l1). Chlorophyll contents in leaves of treated plants varied with the dose of LAS, but there was no significant linear correlation. The activities of the enzymes superoxide dismutases (SOD), catalase (CAT), and peroxidase (POD) were quantified at the end of experiments. At higher concentrations of LAS (P240 mg l1), the activities of SOD and CAT were decreased. The correlation between the dose of LAS and the POD activity in leaf cells was the highest of all enzyme assays (R2 = 0.5). EC50 values for a 50% inhibition of the transpiration of the trees were estimated to 374 mg l1 (72 h) and 166 mg l1 (192 h). Results from this experiment indicated that phytotoxic effects of LAS on willow trees are not expected for normal environmental conditions. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: LAS; Linear alkylbenzene sulfonate; Toxicity; Transpiration; Willows; Enzyme activity
1. Introduction Linear alkylbenzene sulfonate (LAS), the most important group of synthetic anionic surfactant today, was introduced in 1964 as the readily biodegradable replacement for highly branched alkylbenzene sulphonates (Holmstrup and Krogh, 2001). The European chemical industry estimated a total annual consumption volume of 338 ktons. Most of LAS European consumption is in household detergents (>80%). The remainder of the LAS (<20%) is used in industrial and institutional cleaners, textile processing as wetting, dispersing and cleaning agents, industrial pro-
* Corresponding authors. Tel.: +45 4525 1622; fax: +45 4593 2850 (S. Trapp). E-mail addresses: [email protected] (X. Yu), [email protected] (S. Trapp).
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.11.025
cesses as emulsifiers, polymerization and in the formulation of crop protection agents (Carlsen et al., 2002). In mainland China, the annual production of synthetic anionic surfactants is about 1.0 million tons and more than 60% of the total is LAS (Liu et al., 2003). The average use of LAS in Germany in the year 2000 was 386 g capita1 = 1.1 g capita1 d1 (Wind et al., 2004), while the average use of household water between 1990 and 2001 was 127 l capita1 d1 (UBA, 2005). This yields an average concentration of LAS in household wastewater (before sewage treatment) of 8.6 mg l1, which is in agreement with the data (1–15 mg l1 LAS in raw sewage) given by HERA (2004). During sewage treatment, most of the LAS is removed from the wastewater. Concentrations of LAS in rivers are in the microgram range, e.g. for the small German river Itter, values between 7 and 11 lg l1 were measured (Wind et al., 2004). However, raw wastewater may be used as irrigation water, thus
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X. Yu et al. / Chemosphere 64 (2006) 43–48
plants can be exposed to LAS in the milligram per liter range. Furthermore, wastewater may be treated in constructed wetlands or by willow stands. One objective of this study was therefore to determine the effect of LAS on exposed willows. High concentrations of LAS were found in sewage sludge after anaerobic treatment. In Denmark, the highest observed LAS concentration in sludge was 16.1 g kg1. LAS could therefore be found in soils that were treated with sewage sludge as a fertilizer. With an application rate of 2 tons per hectar, the concentrations in the top 15 cm of soil would typically be from 0.1 to 1 mg kg1, with higher concentrations immediately after application of sewage sludge (Bro-Rasmussen and Solbe´, 1999). In seven soil samples that were collected immediately after dosing of the fields with sludge, the concentration of LAS ranged from 2.5 to 40.3 mg kg1 (median 25 mg kg1) (Painter, 1992). In sludge-amended soils, LAS had a half-life of about 1 week and monitored concentrations were around 1 mg kg1 (max 1.4 mg kg1) at harvesting time (Painter, 1992). Sewage sludge application is therefore another likely pathway for the exposure of terrestrial plants to LAS. Toxic effects of chemicals on plants can be measured in different ways. In plants, environmental adversity often leads to the increased generation of reduced oxygen species and, consequently, superoxide dismutases (SOD), catalase (CAT), and peroxidase (POD) have been proposed to be important in plant stress tolerance (Tsang et al., 1991). Adequate defense against oxygen toxicity requires efficient scavenging of both O2 and of H2O2. Superoxide radicals (O2) are toxic by-products of oxidative metabolism (Fridovich, 1978). Their toxicity has been attributed to their interaction with hydrogen peroxide to form highly reactive hydroxyl radicals (OH), which are thought to be largely responsible for mediating oxygen toxicity in vivo. SOD are metalloproteins that catalyze the dismutation of superoxide radicals (O2). CAT and POD catalytically scavenge H2O2 and provide the necessary defenses (Fridovich, 1989). Furthermore, a decrease in chlorophyll content in green plants in response to environmental stress has been reported (Fodor, 2002). Other effects may be reduced growth and transpiration, or changes in water use efficiency. Trapp et al. (2000) specially designed a rapid acute phytotoxicity test for chemicals in water or in soil using the transpiration of basket willow (Salix viminalis) cuttings. Instead of the European basket willow, the Chinese weeping willow (Salix babylonica L.) was used in this study, which can be handled similar to basket willows. This study investigates the response of the native Chinese species Salix babylonica L. exposed to LAS, with the objectives to compare the toxic effects and to provide quantitative information whether the toxicity of LAS would allow wastewater treatment, sewage sludge application in willow stands, or phytoremediation of LAS with willows.
2. Materials and methods 2.1. Trees and exposure regimes Weeping willow (Salix babylonica L.) was sampled from nature at the campus of the Hunan Agricultural University, China. Forty centimeter long tree cuttings were removed from mature specimens and all from a single tree. They were placed in buckets of tap water at room temperature of 15–20 °C under natural sunlight until new roots and leaves appeared. After a 2-month growth, pre-rooted cuttings were transferred to 250 ml Erlenmeyer flasks filled with approximately 200 ml modified ISO 8692 nutrient solution (Table 1). The flasks were all sealed with cork stoppers and play dough to prevent escape of water or chemicals, and wrapped with aluminum foil to inhibit algae growth. For each treatment concentration, six replicates were measured. The flasks were put in a climate chamber with a constant temperature of 24.0 ± 1 °C under continuous artificial light. The plants remained there 48 h to allow them to adapt to their new living environment. Then, the weight of the plant system was measured. Twenty-four hours later, the flasks with the trees were weighed again. By this, the transpiration was determined. Trees with similar transpiration were selected for the tests. The nutrient solution of these trees was exchanged to spiked solution, except for controls. Dodecyl-benzene sulfonic acid sodium salt (CH3(CH2)11C6H4SO3Na) was used in this study. The LAS used was p.a. grade (per analysis, in China: P95% purity). It had a chain length of C12 and the benzene ring was attached to C6. Six different treatment concentrations of LAS were applied (0, 30, 60, 120, 240, 360 and 480 mg l1). The weight of the flasks was measured daily. 2.2. Normalized relative transpiration The weight loss, compared to initial loss, was the toxicity criteria. To compare the toxic effect on cuttings with different initial transpiration (before the toxicant is added), the weight loss is expressed as relative transpiration. The transpiration was normalized with respect to the initial transpiration (to eliminate the necessity of finding cuttings with similar initial transpiration) and with respect to the transpiration of uncontaminated control cuttings (to include the effect of normal growth of the cuttings during the test). The mean normalized relative transpiration (NRT) was calculated by Table 1 Composition of the modified ISO 8692 nutrient solution Macronutrients (lmol l1)
Micronutrients (nmol l1)
NaNO3 MgCl2 Æ 6H2O CaCl2 Æ 2H2O MgSO4 Æ 7H2O KH2PO4 MaHCO3
HBO3 MnCl2 Æ 4H2O ZnCl2 CoCl2 Æ 6H2O CuCl2 Æ 2H2O NaMoO4 Æ 2H2O
2823.9 59.0 122.4 60.9 246.0 1785.5
2992.1 2097.0 22.0 6.3 0.1 28.9
X. Yu et al. / Chemosphere 64 (2006) 43–48
NRTðC; tÞ ¼
1 n 1 m
Pn T i ðC; tÞ=T i ðC; 0Þ Pi¼1 ; m j¼1 T j ð0; tÞ=T j ð0; 0Þ
where C is concentration (mg l1), t is time period (h), T is absolute transpiration (g h1), i is replicate 1, 2,. . ., n and j is control 1, 2,. . ., m. Controls always have the NRT of 100%; NRT < 100% shows inhibition of the trees’ transpiration, which is usually connected with other effects (reduced growth, necrosis of leaves, and, in severe cases, death). An inhibition of NRT of about 50% is typically accompanied by a complete inhibition of growth. Dead trees in this system still loose water, so NRT < 10% is in most cases a sign for fatal effects (Trapp and Karlson, 2001). 2.3. Chlorophyll measurement The chlorophyll content was determined spectrophotometrically in leaves from the top shoot. At the end of the experiments (192 h), plant leaves were cut into pieces, precisely weighed (0.5 g fresh weight) and placed in 25 ml flasks. Then, 80% acetone was added to the mark of 25 ml. Three separate flasks were conducted for each treatment group. Flasks were all placed in the dark for 24 h. During this period, flasks were shaken twice. The absorption of light at 645 and 663 nm was measured in a cell of optical path length of 10 mm against 80% acetone as a blank. The amount of chlorophyll a and chlorophyll b in plant leaves was calculated by the following formula of Maclachalam and Zalik (1963): ð12:3D663 0:86D645 Þ V ; d 1000 W ð19:3D645 3:60D663 Þ V Cb ¼ ; d 1000 W
Ca ¼
where Ca is the concentration of chlorophyll a (mg g1 FW), Cb is the concentration of chlorophyll b (mg g1 FW), D is the optical density (OD) at wave length indicated, V is the final volume (ml), W is fresh weight of leaf materials (g), and d is the length of the light path in cm. 2.4. Enzyme activity measurement The activities of the three protective enzymes SOD, CAT and POD were measured in fresh tissues of leaves at the end of the experiment. Fresh leaves were taken from the top shoot. 0.3 g of tissue materials (fresh weight) was precisely weighted and placed in a triturator. 1.4 ml of phosphate buffer solution (pH 7.8, containing NaH2PO4, Na2HPO4, PVPP, EDTA and mercapto-ethanol) was added before trituration. Triturating was performed at 4 °C and centrifuged at 8000 rpm for 15 min, the supernatant was stored at 4 °C and employed for the assay of enzymes. Three separate tubes were conducted for each enzyme analysis. SOD, POD and CAT activities in leaf cells of plants were determined spectrophotometrically according to Jin and Ding (1981).
45
2.4.1. Assay of SOD activity The reaction mixture (3 ml) was composed of 13 mM methionine, 0.075 mM NBT, 0.1 mM EDTA, 0.002 mM riboflavin, and 0.1 ml of enzyme extract in 50 mM phosphate buffer (pH 7.8). The mixture in the tube was placed on a rotating tube holder at 25 °C for 10 min. The absorbance was measured at 550 nm in a cell of optical path length of 10 mm against the reaction mixture without enzyme extract. The unit of SOD activity (U g1) was defined as the amount of enzyme, which caused 50% inhibition of the initial rate of reaction in the absence of the enzyme. 2.4.2. Assay of CAT activity The enzyme extract (0.1 ml) was added to 2 ml assay mixture (50 mM Tris–HCl buffer pH 6.8, containing 5 mM H2O2). The reaction was stopped by adding 0.1 ml 20% titanic tetrachloride after 1 min at 37 °C. The absorbance of the reaction solutions was measured at 405 nm in a cell of optical length of 5 mm against the reaction mixture without enzyme extract. One unit of CAT activity (U g1) was defined as the amount of CAT, which decomposed 1 lmol hydrogen peroxide in 1 min at 37 °C. 2.4.3. Assay of POD activity The reaction mixture (3 ml) was composed of 100 mM potassium phosphate buffer (pH 7.0), 20 mM guaiacol, 65 mM H2O2 and 0.1 ml enzyme extract. Changes in absorbance were recorded at 470 nm in a cell of optical length of 10 mm against the reaction mixture without enzyme extract at 25 °C for 3 min. One activity unit of POD (U g1) was defined as the amount of enzyme that caused an increase of 0.001 absorbance per minute. 3. Results and discussion 3.1. Phytotoxicity of LAS to weeping willows in hydroponic solution The measured absolute transpiration of weeping willows grown in hydroponic solution spiked with various levels of LAS is given in Table 2. The results were converted to normalized relative transpiration NRT (Fig. 1). The correlation between the LAS dosage and the NRT was high (R2 = 0.96). Negligible reduction of the NRT was found after 192 h for weeping willows exposed to 30 mg l1 LAS. Twenty-five percent reduction of the NRT was found for the treatment groups exposed to 60 and 120 mg l1 LAS. A reduction of 60% was found for 240 mg l1. Chlorosis of leaves was not observed during the duration of the test, but the color of plant roots changed from white to brown. When exposed to high doses of LAS (P360 mg l1), weeping willows showed severe toxic symptoms after a short time (72 h), e.g. strong reduction in normalized relative transpiration and chlorosis of leaves. All treated willows in the treatment groups with 360 and 480 mg l1 were obviously dead at the end of the
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X. Yu et al. / Chemosphere 64 (2006) 43–48
Table 2 Absolute transpiration (g h1) of willow trees growing in hydroponic solution spiked with LAS. Values are the mean of six replicates, in parenthesis: standard deviation Conc. (mg l1)
Initiala mean
0 30 60 120 240 360 480
0.79 0.55 0.46 0.39 0.35 0.37 0.29
24 h (0.051) (0.043) (0.019) (0.011) (0.013) (0.156) (0.024)
0.80 0.57 0.46 0.39 0.32 0.30 0.22
48 h (0.101) (0.044) (0.049) (0.063) (0.031) (0.110) (0.045)
0.80 0.59 0.48 0.40 0.32 0.27 0.17
72 h (0.138) (0.045) (0.089) (0.099) (0.063) (0.060) (0.036)
0.80 0.59 0.43 0.35 0.25 0.17 0.13
96 h (0.168) (0.05) (0.100) (0.104) (0.094) (0.045) (0.064)
0.75 0.55 0.40 0.32 0.22 0.14 0.07
(0.163) (0.071) (0.102) (0.104) (0.084) (0.042) (0.051)
120 h
144 h
160 h
192 h
0.75 0.50 0.36 0.30 0.16 0.11 0.05
0.76 0.48 0.32 0.27 0.13 0.06 0.03
0.80 0.49 0.34 0.29 0.14 0.05 0.02
0.73 0.49 0.32 0.28 0.13 0.04 0.02
(0.114) (0.049) (0.119) (0.136) (0.074) (0.046) (0.052)
(0.090) (0.058) (0.132) (0.146) (0.065) (0.020) (0.009)
(0.099) (0.078) (0.151) (0.184) (0.072) (0.013) (0.005)
(0.084) (0.083) (0.126) (0.178) (0.054) (0.018) (0.002)
Initial values are the absolute transpiration of weeping willows before the toxicant is added.
NRT(%)
a
Mean
120 100 80 60 40 20 0
0
24
48
72
96 120 144 168 192 Time (h)
Control
30 mg/l
60 mg/l
240 mg/l
360 mg/l
480 mg/l
120 mg/l
Fig. 1. Normalized relative transpiration NRT (%) of weeping willows (Salix babylonica L.) growing in hydroponic solution spiked with LAS. Values are the mean of six replicates.
experiment, their transpiration had dropped to below 10% of that of control trees. 3.2. Effects of LAS on the content of chlorophylls in plant leaves The measured chlorophyll a and chlorophyll b contents of leaves are shown in Fig. 2. Chlorophyll contents of leaves varied with the treated plants exposed to different doses of LAS. The highest contents of chlorophyll a and chlorophyll b in leaves were found for the treated plants exposed to 30 mg l1, with values of 0.77 and 0.25 mg g1 fresh weight (FW), respectively, whereas for untreated plants, chlorophyll a and chlorophyll b were 0.45 and 0.16 mg g1 FW. In the treatments exposed to the 60 and
Ca
3.3. Effects of LAS on the activities of protective enzymes in plant leaves The measured activities of SOD, CAT and POD in the leaf cells of weeping willows are shown in Figs. 3 and 4. When willows were exposed to 6120 mg l1 LAS, SOD and CAT activities in the leaf cells of treated plants remained at the same level as in the untreated plants, implying the detoxification system of leaf cells functioned well. At higher concentrations of LAS (P240 mg l1), the activities of SOD and CAT had decreased compared with the untreated plants. No leaves were available in the treatments exposed to LAS (360 and 480 mg l1) at the end of experiments (192 h), therefore activities of SOD and CAT could not be measured. The activity of POD in leaf cells varied with the different doses of LAS. At the treatment of 30 mg l1, POD activity in leaf cells was higher than that in leaf cells of untreated plants, implying LAS may increase the presence of POD in leaf cells. POD is also a catalyst that can speed up protoplasm carbonization, therefore
Cb
400
0.8 U/g FW
Conc. mg/g FW
1
120 mg l1 LAS, the content of chlorophyll in leaves showed a near equal level. In the treatment group with 240 mg l1, 22.2% and 18.8% reduction of the chlorophyll a and chlorophyll b in leaves were found in comparison with the untreated plants, respectively, but visible toxic symptoms, e.g. chlorosis of leaves, were not observed. When willows were exposed to high doses of LAS (P360 mg l1), chlorophyll contents in leaves were not measured due to the death of plants.
0.6 0.4
CAT 200 100
0.2 0
SOD
300
0
30
60 120 240 360 Conc. of LAS (mg/l)
480
Fig. 2. Effects of LAS on the content of chlorophylls in the leaves (mg g1 FW) of weeping willows (Salix babylonica L.). Values are the mean of three replicates, error bars represent standard deviation. Ca = chlorophyll a, Cb = chlorophyll b.
0
0
30
60 120 240 360 Conc. of LAS (mg/l)
480
Fig. 3. Effects of LAS on the activities of superoxide dismutases (SOD), catalase (CAT) in leaf cells (U g1 FW) of weeping willows (Salix babylonica L.). Values are the mean of three replicates, error bars represent standard deviation.
X. Yu et al. / Chemosphere 64 (2006) 43–48
3.5. Determination of effect concentrations (EC50) and comparison to other results
U/g FW
50 40
POD
30 20 10 0
0
30
60
120
240
360
480
Conc. of LAS (mg/l)
Fig. 4. Effects of LAS on the activity of peroxidase (POD) in leaf cells (U g1 FW) of weeping willows (Salix babylonica L.). Values are the mean of three replicates, error bars represent standard deviation.
may stimulate plant growth. When willows were exposed to LAS (60 and 120 mg l1), POD activity was the same as in the leaf cells of untreated plants. With increasing LAS dose (P240 mg l1), POD activity in leaf cells was significantly reduced. The results from this test indicate that the activity of POD was more susceptible to the changes in LAS doses than that of SOD and CAT. Liu et al. (2004) also found that POD was the major enzyme to protect aquatic plants (Pistia stratiotes L., Lemna paucicostata L., Azolla imbricate, and Spirogyra sp.) from LAS injury. 3.4. Comparison of toxic effects Fig. 5 shows the results of all measured toxic effects plotted together in one graph. For the enzyme assays and the chlorophyll A, all replicates are shown (n is between 11 and 14), while for the NRT, the mean of six replicates is displayed for each dose. Additionally, the linear trend line is given, and the R2 value. All linear trends were significant, except the trend for chlorophyll A (a = 5%), judged by the critical R for given n (Sachs, 1992). The best correlation was obtained for the normalized transpiration (note that the correlation is slightly higher if the logarithm of the effect data is used). The enzyme assays and the chlorophyll content were measured in the leaves. LAS was probably not or to a minor extend translocated to leaves. This could be an explanation for the smaller effect of LAS on these parameters.
1250 1000 750
R 2 = 0.96
500
47
The effect concentrations EC10 and EC50 are defined by the concentration of a chemical, which produces 10% or 50% of the maximum possible response for that chemical. In our case, the EC10 and the EC50 correspond to the dose, at which the NRT is inhibited 10% or 50%. For the fit of effect concentrations, the NRT for each replicate was derived by dividing the change of initial transpiration by the average change of controls. This was done to get a higher degree of freedom (n = 42). A sigmoidal curve was fitted to the NRT data, assuming log-normal toxicity data distribution (Andersen, 1994), in order to estimate EC10 and EC50. Table 3 gives the estimated values at different exposure periods. The inhibition of the transpiration increased with time. The results from this study can be compared to earlier results with terrestrial plants. From laboratory tests with terrestrial plants, Jensen et al. (2001) reported EC50 values of 16–316 mg/kg (soil) for terrestrial plants using the growth of plants as a sensitive endpoint. For Sinapis alba, Avena sativa and Brassica rapa, EC10 values were at 200, 50 and 90 mg kg1, respectively. They also found that at an average soil concentration of 5–15 mg kg1, LAS did not seem to be detrimental to the soil ecosystem in the long term. A large number of data for LAS toxicity to plants, soil fauna, soil microorganisms and microbial soil processes has been collected for the terrestrial environmental risk assessment in Denmark (Kloepper-Sams et al., 1996; Jensen, 1999; Elsgaard et al., 2001; Holmstrup and Krogh, 2001; Jensen et al., 2001). A predicted non-effect concentration of LAS (PNEC) in soils of 5.2 mg kg1 was estimated from these Danish studies (Bro-Rasmussen and Solbe´, 1999), while a PNEC for soil organisms of 4.6 mg kg1 was reported in HERA (2004). Field observations indicated that the application of LAS-containing sludge generally stimulated the microbial activity and, hence, the abundance of soil fauna and growth of plants (Jensen, 1999; Jensen et al., 2001; Brandt et al., 2003). In this investigation, when exposed to low dose of LAS (30 mg l1), treated willows had a higher transpiration in comparison with untreated willows, and no toxic effects were observed at the low doses. It should be mentioned, that terrestrial plants seem less sensitive than some aquatic species: a wide range of toxicity of LAS towards algae was
2
R = 0.29 2 R = 0.40 2 R 2 = 0.45 R = 0.50
250 0 0
100
200
300
400
500
LAS mg/l
Chlo A
POD
CAT
SOD
NRT o/oo
Fig. 5. Comparison of all toxic effects; linear regression with R2 values added. Chlo A: chlorophyll A in mg/kg; POD, CAT, SOD: enzyme assays in U/g (see methods section); NRT: normalized relative transpiration after 72 h, here in per mill o/oo.
Table 3 Calculated EC10 and EC50 values (mg l1 LAS) for weeping willows exposed 72 and 192 h to LAS in hydroponic solution; confidence interval CI for a = 5% Period (h)
Parameter
mg l1
Lower CI
Upper CI
72 72 192 192
EC10 EC50 EC10 EC50
87 374 54 166
32 272 16 102
233 513 182 270
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reported in literature. Most data fall between 0.1 and 100 mg l1 (growth, population density, reproduction, mobility, emergence and mortality were the endpoints). Typical ranges of EC50 values are 1–100 mg l1 for fresh water species and <1 to 10 mg l1 for marine species (Painter, 1992). LC50 values have been found in the range of 1– 10 mg l1 when Daphnia magna were exposed to LAS homologues between C10 and C13 (Painter, 1992; Kusk and Petersen, 1997). A 96 h LC50 value of 1.8 mg l1 was found for C13 LAS in a study with fathead minnow (Pimephales promelas) (Macek and Slight, 1977). 4. Conclusions The toxicity tests in hydroponic solution using the normalized relative transpiration as a variable showed that the short-term acute toxicity of LAS to weeping willows was strongly related to the dosage. Severe signs of toxicity were only found at the treatment groups exposed to high doses of LAS. Most sensitive toxicity parameters were transpiration and POD. The results indicate that phytotoxic effects of LAS on willow trees are unlikely to occur under normal environmental conditions. Acknowledgments This work was supported by a research foundation from the Hunan Agricultural University for scientists (Grant no. 04PT02). Thanks to Yong Lei, Ling Li, Shengzhuo Huang, Jin Feng, Liang Chen, Yawen Tang, and Junjie Lei for their technical assistance. Thanks to Ole Kusk for help with the EC estimations. References Andersen, H., 1994. Statistical methods for evaluation of toxicity of wastewater. M.Sc. Thesis 7/94. Institute for Mathematical Statistic and Operation Analysis. Technical University of Denmark (in Danish). Brandt, K.K., Krogh, P.H., Sorensen, J., 2003. Activity and population dynamics of heterotrophic and ammonia-oxidising microorganisms in soil surrounding sludge spiked with LAS: a field study. Environ. Toxicol. Chem. 22, 821–829. Bro-Rasmussen, F., Solbe´, J., 1999. Executive summary of the SPT workshop in cooperation with the Danish EPA, Copenhagen 19/20 April 1999. Carlsen, L., Metzon, M.B., Kjelsmark, J., 2002. LAS in the terrestrial environment. Sci. Tot. Environ. 290, 225–230. Elsgaard, L., Petersen, S.O., Debosz, K., 2001. Effects and risk assessment of LAS in agricultural soil. 2. Effects on soil microbiology as influenced by sewage sludge and incubation time. Environ. Toxicol. Chem. 20, 1664–1672.
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Response of weeping willows to linear alkylbenzene sulfonate Xiaozhang Yu b
a,*
, Stefan Trapp
b,*
, Puhua Zhou c, Xiaoying Peng c, Xi Cao
c
a Department of Environmental Science, Hunan Agricultural University, Changsha 410128 Hunan, PR China Institute of Environment and Resources DTU, Technical University of Denmark, Building 115, DK-2800 Kongens Lyngby, Denmark c Department of Biotechnology, Hunan Agricultural University, Changsha 410128 Hunan, PR China
Received 9 June 2005; received in revised form 15 November 2005; accepted 18 November 2005 Available online 5 January 2006
Abstract Linear alkylbenzene sulfonate (LAS) is the most commonly used anionic surfactant in laundry detergents and cleaning agents. LAS compounds are found in surface waters and soils. The short-term acute toxicity of LAS to weeping willows (Salix babylonica L.) was investigated. Willow cuttings were grown in hydroponic solution spiked with LAS at 24.0 ± 1 °C for 192 h. The normalized relative transpiration of plants was used to determine toxicity. Severe reduction of the transpiration was only found for high doses of LAS (P240 mg l1). Chlorophyll contents in leaves of treated plants varied with the dose of LAS, but there was no significant linear correlation. The activities of the enzymes superoxide dismutases (SOD), catalase (CAT), and peroxidase (POD) were quantified at the end of experiments. At higher concentrations of LAS (P240 mg l1), the activities of SOD and CAT were decreased. The correlation between the dose of LAS and the POD activity in leaf cells was the highest of all enzyme assays (R2 = 0.5). EC50 values for a 50% inhibition of the transpiration of the trees were estimated to 374 mg l1 (72 h) and 166 mg l1 (192 h). Results from this experiment indicated that phytotoxic effects of LAS on willow trees are not expected for normal environmental conditions. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: LAS; Linear alkylbenzene sulfonate; Toxicity; Transpiration; Willows; Enzyme activity
1. Introduction Linear alkylbenzene sulfonate (LAS), the most important group of synthetic anionic surfactant today, was introduced in 1964 as the readily biodegradable replacement for highly branched alkylbenzene sulphonates (Holmstrup and Krogh, 2001). The European chemical industry estimated a total annual consumption volume of 338 ktons. Most of LAS European consumption is in household detergents (>80%). The remainder of the LAS (<20%) is used in industrial and institutional cleaners, textile processing as wetting, dispersing and cleaning agents, industrial pro-
* Corresponding authors. Tel.: +45 4525 1622; fax: +45 4593 2850 (S. Trapp). E-mail addresses: [email protected] (X. Yu), [email protected] (S. Trapp).
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.11.025
cesses as emulsifiers, polymerization and in the formulation of crop protection agents (Carlsen et al., 2002). In mainland China, the annual production of synthetic anionic surfactants is about 1.0 million tons and more than 60% of the total is LAS (Liu et al., 2003). The average use of LAS in Germany in the year 2000 was 386 g capita1 = 1.1 g capita1 d1 (Wind et al., 2004), while the average use of household water between 1990 and 2001 was 127 l capita1 d1 (UBA, 2005). This yields an average concentration of LAS in household wastewater (before sewage treatment) of 8.6 mg l1, which is in agreement with the data (1–15 mg l1 LAS in raw sewage) given by HERA (2004). During sewage treatment, most of the LAS is removed from the wastewater. Concentrations of LAS in rivers are in the microgram range, e.g. for the small German river Itter, values between 7 and 11 lg l1 were measured (Wind et al., 2004). However, raw wastewater may be used as irrigation water, thus
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X. Yu et al. / Chemosphere 64 (2006) 43–48
plants can be exposed to LAS in the milligram per liter range. Furthermore, wastewater may be treated in constructed wetlands or by willow stands. One objective of this study was therefore to determine the effect of LAS on exposed willows. High concentrations of LAS were found in sewage sludge after anaerobic treatment. In Denmark, the highest observed LAS concentration in sludge was 16.1 g kg1. LAS could therefore be found in soils that were treated with sewage sludge as a fertilizer. With an application rate of 2 tons per hectar, the concentrations in the top 15 cm of soil would typically be from 0.1 to 1 mg kg1, with higher concentrations immediately after application of sewage sludge (Bro-Rasmussen and Solbe´, 1999). In seven soil samples that were collected immediately after dosing of the fields with sludge, the concentration of LAS ranged from 2.5 to 40.3 mg kg1 (median 25 mg kg1) (Painter, 1992). In sludge-amended soils, LAS had a half-life of about 1 week and monitored concentrations were around 1 mg kg1 (max 1.4 mg kg1) at harvesting time (Painter, 1992). Sewage sludge application is therefore another likely pathway for the exposure of terrestrial plants to LAS. Toxic effects of chemicals on plants can be measured in different ways. In plants, environmental adversity often leads to the increased generation of reduced oxygen species and, consequently, superoxide dismutases (SOD), catalase (CAT), and peroxidase (POD) have been proposed to be important in plant stress tolerance (Tsang et al., 1991). Adequate defense against oxygen toxicity requires efficient scavenging of both O2 and of H2O2. Superoxide radicals (O2) are toxic by-products of oxidative metabolism (Fridovich, 1978). Their toxicity has been attributed to their interaction with hydrogen peroxide to form highly reactive hydroxyl radicals (OH), which are thought to be largely responsible for mediating oxygen toxicity in vivo. SOD are metalloproteins that catalyze the dismutation of superoxide radicals (O2). CAT and POD catalytically scavenge H2O2 and provide the necessary defenses (Fridovich, 1989). Furthermore, a decrease in chlorophyll content in green plants in response to environmental stress has been reported (Fodor, 2002). Other effects may be reduced growth and transpiration, or changes in water use efficiency. Trapp et al. (2000) specially designed a rapid acute phytotoxicity test for chemicals in water or in soil using the transpiration of basket willow (Salix viminalis) cuttings. Instead of the European basket willow, the Chinese weeping willow (Salix babylonica L.) was used in this study, which can be handled similar to basket willows. This study investigates the response of the native Chinese species Salix babylonica L. exposed to LAS, with the objectives to compare the toxic effects and to provide quantitative information whether the toxicity of LAS would allow wastewater treatment, sewage sludge application in willow stands, or phytoremediation of LAS with willows.
2. Materials and methods 2.1. Trees and exposure regimes Weeping willow (Salix babylonica L.) was sampled from nature at the campus of the Hunan Agricultural University, China. Forty centimeter long tree cuttings were removed from mature specimens and all from a single tree. They were placed in buckets of tap water at room temperature of 15–20 °C under natural sunlight until new roots and leaves appeared. After a 2-month growth, pre-rooted cuttings were transferred to 250 ml Erlenmeyer flasks filled with approximately 200 ml modified ISO 8692 nutrient solution (Table 1). The flasks were all sealed with cork stoppers and play dough to prevent escape of water or chemicals, and wrapped with aluminum foil to inhibit algae growth. For each treatment concentration, six replicates were measured. The flasks were put in a climate chamber with a constant temperature of 24.0 ± 1 °C under continuous artificial light. The plants remained there 48 h to allow them to adapt to their new living environment. Then, the weight of the plant system was measured. Twenty-four hours later, the flasks with the trees were weighed again. By this, the transpiration was determined. Trees with similar transpiration were selected for the tests. The nutrient solution of these trees was exchanged to spiked solution, except for controls. Dodecyl-benzene sulfonic acid sodium salt (CH3(CH2)11C6H4SO3Na) was used in this study. The LAS used was p.a. grade (per analysis, in China: P95% purity). It had a chain length of C12 and the benzene ring was attached to C6. Six different treatment concentrations of LAS were applied (0, 30, 60, 120, 240, 360 and 480 mg l1). The weight of the flasks was measured daily. 2.2. Normalized relative transpiration The weight loss, compared to initial loss, was the toxicity criteria. To compare the toxic effect on cuttings with different initial transpiration (before the toxicant is added), the weight loss is expressed as relative transpiration. The transpiration was normalized with respect to the initial transpiration (to eliminate the necessity of finding cuttings with similar initial transpiration) and with respect to the transpiration of uncontaminated control cuttings (to include the effect of normal growth of the cuttings during the test). The mean normalized relative transpiration (NRT) was calculated by Table 1 Composition of the modified ISO 8692 nutrient solution Macronutrients (lmol l1)
Micronutrients (nmol l1)
NaNO3 MgCl2 Æ 6H2O CaCl2 Æ 2H2O MgSO4 Æ 7H2O KH2PO4 MaHCO3
HBO3 MnCl2 Æ 4H2O ZnCl2 CoCl2 Æ 6H2O CuCl2 Æ 2H2O NaMoO4 Æ 2H2O
2823.9 59.0 122.4 60.9 246.0 1785.5
2992.1 2097.0 22.0 6.3 0.1 28.9
X. Yu et al. / Chemosphere 64 (2006) 43–48
NRTðC; tÞ ¼
1 n 1 m
Pn T i ðC; tÞ=T i ðC; 0Þ Pi¼1 ; m j¼1 T j ð0; tÞ=T j ð0; 0Þ
where C is concentration (mg l1), t is time period (h), T is absolute transpiration (g h1), i is replicate 1, 2,. . ., n and j is control 1, 2,. . ., m. Controls always have the NRT of 100%; NRT < 100% shows inhibition of the trees’ transpiration, which is usually connected with other effects (reduced growth, necrosis of leaves, and, in severe cases, death). An inhibition of NRT of about 50% is typically accompanied by a complete inhibition of growth. Dead trees in this system still loose water, so NRT < 10% is in most cases a sign for fatal effects (Trapp and Karlson, 2001). 2.3. Chlorophyll measurement The chlorophyll content was determined spectrophotometrically in leaves from the top shoot. At the end of the experiments (192 h), plant leaves were cut into pieces, precisely weighed (0.5 g fresh weight) and placed in 25 ml flasks. Then, 80% acetone was added to the mark of 25 ml. Three separate flasks were conducted for each treatment group. Flasks were all placed in the dark for 24 h. During this period, flasks were shaken twice. The absorption of light at 645 and 663 nm was measured in a cell of optical path length of 10 mm against 80% acetone as a blank. The amount of chlorophyll a and chlorophyll b in plant leaves was calculated by the following formula of Maclachalam and Zalik (1963): ð12:3D663 0:86D645 Þ V ; d 1000 W ð19:3D645 3:60D663 Þ V Cb ¼ ; d 1000 W
Ca ¼
where Ca is the concentration of chlorophyll a (mg g1 FW), Cb is the concentration of chlorophyll b (mg g1 FW), D is the optical density (OD) at wave length indicated, V is the final volume (ml), W is fresh weight of leaf materials (g), and d is the length of the light path in cm. 2.4. Enzyme activity measurement The activities of the three protective enzymes SOD, CAT and POD were measured in fresh tissues of leaves at the end of the experiment. Fresh leaves were taken from the top shoot. 0.3 g of tissue materials (fresh weight) was precisely weighted and placed in a triturator. 1.4 ml of phosphate buffer solution (pH 7.8, containing NaH2PO4, Na2HPO4, PVPP, EDTA and mercapto-ethanol) was added before trituration. Triturating was performed at 4 °C and centrifuged at 8000 rpm for 15 min, the supernatant was stored at 4 °C and employed for the assay of enzymes. Three separate tubes were conducted for each enzyme analysis. SOD, POD and CAT activities in leaf cells of plants were determined spectrophotometrically according to Jin and Ding (1981).
45
2.4.1. Assay of SOD activity The reaction mixture (3 ml) was composed of 13 mM methionine, 0.075 mM NBT, 0.1 mM EDTA, 0.002 mM riboflavin, and 0.1 ml of enzyme extract in 50 mM phosphate buffer (pH 7.8). The mixture in the tube was placed on a rotating tube holder at 25 °C for 10 min. The absorbance was measured at 550 nm in a cell of optical path length of 10 mm against the reaction mixture without enzyme extract. The unit of SOD activity (U g1) was defined as the amount of enzyme, which caused 50% inhibition of the initial rate of reaction in the absence of the enzyme. 2.4.2. Assay of CAT activity The enzyme extract (0.1 ml) was added to 2 ml assay mixture (50 mM Tris–HCl buffer pH 6.8, containing 5 mM H2O2). The reaction was stopped by adding 0.1 ml 20% titanic tetrachloride after 1 min at 37 °C. The absorbance of the reaction solutions was measured at 405 nm in a cell of optical length of 5 mm against the reaction mixture without enzyme extract. One unit of CAT activity (U g1) was defined as the amount of CAT, which decomposed 1 lmol hydrogen peroxide in 1 min at 37 °C. 2.4.3. Assay of POD activity The reaction mixture (3 ml) was composed of 100 mM potassium phosphate buffer (pH 7.0), 20 mM guaiacol, 65 mM H2O2 and 0.1 ml enzyme extract. Changes in absorbance were recorded at 470 nm in a cell of optical length of 10 mm against the reaction mixture without enzyme extract at 25 °C for 3 min. One activity unit of POD (U g1) was defined as the amount of enzyme that caused an increase of 0.001 absorbance per minute. 3. Results and discussion 3.1. Phytotoxicity of LAS to weeping willows in hydroponic solution The measured absolute transpiration of weeping willows grown in hydroponic solution spiked with various levels of LAS is given in Table 2. The results were converted to normalized relative transpiration NRT (Fig. 1). The correlation between the LAS dosage and the NRT was high (R2 = 0.96). Negligible reduction of the NRT was found after 192 h for weeping willows exposed to 30 mg l1 LAS. Twenty-five percent reduction of the NRT was found for the treatment groups exposed to 60 and 120 mg l1 LAS. A reduction of 60% was found for 240 mg l1. Chlorosis of leaves was not observed during the duration of the test, but the color of plant roots changed from white to brown. When exposed to high doses of LAS (P360 mg l1), weeping willows showed severe toxic symptoms after a short time (72 h), e.g. strong reduction in normalized relative transpiration and chlorosis of leaves. All treated willows in the treatment groups with 360 and 480 mg l1 were obviously dead at the end of the
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X. Yu et al. / Chemosphere 64 (2006) 43–48
Table 2 Absolute transpiration (g h1) of willow trees growing in hydroponic solution spiked with LAS. Values are the mean of six replicates, in parenthesis: standard deviation Conc. (mg l1)
Initiala mean
0 30 60 120 240 360 480
0.79 0.55 0.46 0.39 0.35 0.37 0.29
24 h (0.051) (0.043) (0.019) (0.011) (0.013) (0.156) (0.024)
0.80 0.57 0.46 0.39 0.32 0.30 0.22
48 h (0.101) (0.044) (0.049) (0.063) (0.031) (0.110) (0.045)
0.80 0.59 0.48 0.40 0.32 0.27 0.17
72 h (0.138) (0.045) (0.089) (0.099) (0.063) (0.060) (0.036)
0.80 0.59 0.43 0.35 0.25 0.17 0.13
96 h (0.168) (0.05) (0.100) (0.104) (0.094) (0.045) (0.064)
0.75 0.55 0.40 0.32 0.22 0.14 0.07
(0.163) (0.071) (0.102) (0.104) (0.084) (0.042) (0.051)
120 h
144 h
160 h
192 h
0.75 0.50 0.36 0.30 0.16 0.11 0.05
0.76 0.48 0.32 0.27 0.13 0.06 0.03
0.80 0.49 0.34 0.29 0.14 0.05 0.02
0.73 0.49 0.32 0.28 0.13 0.04 0.02
(0.114) (0.049) (0.119) (0.136) (0.074) (0.046) (0.052)
(0.090) (0.058) (0.132) (0.146) (0.065) (0.020) (0.009)
(0.099) (0.078) (0.151) (0.184) (0.072) (0.013) (0.005)
(0.084) (0.083) (0.126) (0.178) (0.054) (0.018) (0.002)
Initial values are the absolute transpiration of weeping willows before the toxicant is added.
NRT(%)
a
Mean
120 100 80 60 40 20 0
0
24
48
72
96 120 144 168 192 Time (h)
Control
30 mg/l
60 mg/l
240 mg/l
360 mg/l
480 mg/l
120 mg/l
Fig. 1. Normalized relative transpiration NRT (%) of weeping willows (Salix babylonica L.) growing in hydroponic solution spiked with LAS. Values are the mean of six replicates.
experiment, their transpiration had dropped to below 10% of that of control trees. 3.2. Effects of LAS on the content of chlorophylls in plant leaves The measured chlorophyll a and chlorophyll b contents of leaves are shown in Fig. 2. Chlorophyll contents of leaves varied with the treated plants exposed to different doses of LAS. The highest contents of chlorophyll a and chlorophyll b in leaves were found for the treated plants exposed to 30 mg l1, with values of 0.77 and 0.25 mg g1 fresh weight (FW), respectively, whereas for untreated plants, chlorophyll a and chlorophyll b were 0.45 and 0.16 mg g1 FW. In the treatments exposed to the 60 and
Ca
3.3. Effects of LAS on the activities of protective enzymes in plant leaves The measured activities of SOD, CAT and POD in the leaf cells of weeping willows are shown in Figs. 3 and 4. When willows were exposed to 6120 mg l1 LAS, SOD and CAT activities in the leaf cells of treated plants remained at the same level as in the untreated plants, implying the detoxification system of leaf cells functioned well. At higher concentrations of LAS (P240 mg l1), the activities of SOD and CAT had decreased compared with the untreated plants. No leaves were available in the treatments exposed to LAS (360 and 480 mg l1) at the end of experiments (192 h), therefore activities of SOD and CAT could not be measured. The activity of POD in leaf cells varied with the different doses of LAS. At the treatment of 30 mg l1, POD activity in leaf cells was higher than that in leaf cells of untreated plants, implying LAS may increase the presence of POD in leaf cells. POD is also a catalyst that can speed up protoplasm carbonization, therefore
Cb
400
0.8 U/g FW
Conc. mg/g FW
1
120 mg l1 LAS, the content of chlorophyll in leaves showed a near equal level. In the treatment group with 240 mg l1, 22.2% and 18.8% reduction of the chlorophyll a and chlorophyll b in leaves were found in comparison with the untreated plants, respectively, but visible toxic symptoms, e.g. chlorosis of leaves, were not observed. When willows were exposed to high doses of LAS (P360 mg l1), chlorophyll contents in leaves were not measured due to the death of plants.
0.6 0.4
CAT 200 100
0.2 0
SOD
300
0
30
60 120 240 360 Conc. of LAS (mg/l)
480
Fig. 2. Effects of LAS on the content of chlorophylls in the leaves (mg g1 FW) of weeping willows (Salix babylonica L.). Values are the mean of three replicates, error bars represent standard deviation. Ca = chlorophyll a, Cb = chlorophyll b.
0
0
30
60 120 240 360 Conc. of LAS (mg/l)
480
Fig. 3. Effects of LAS on the activities of superoxide dismutases (SOD), catalase (CAT) in leaf cells (U g1 FW) of weeping willows (Salix babylonica L.). Values are the mean of three replicates, error bars represent standard deviation.
X. Yu et al. / Chemosphere 64 (2006) 43–48
3.5. Determination of effect concentrations (EC50) and comparison to other results
U/g FW
50 40
POD
30 20 10 0
0
30
60
120
240
360
480
Conc. of LAS (mg/l)
Fig. 4. Effects of LAS on the activity of peroxidase (POD) in leaf cells (U g1 FW) of weeping willows (Salix babylonica L.). Values are the mean of three replicates, error bars represent standard deviation.
may stimulate plant growth. When willows were exposed to LAS (60 and 120 mg l1), POD activity was the same as in the leaf cells of untreated plants. With increasing LAS dose (P240 mg l1), POD activity in leaf cells was significantly reduced. The results from this test indicate that the activity of POD was more susceptible to the changes in LAS doses than that of SOD and CAT. Liu et al. (2004) also found that POD was the major enzyme to protect aquatic plants (Pistia stratiotes L., Lemna paucicostata L., Azolla imbricate, and Spirogyra sp.) from LAS injury. 3.4. Comparison of toxic effects Fig. 5 shows the results of all measured toxic effects plotted together in one graph. For the enzyme assays and the chlorophyll A, all replicates are shown (n is between 11 and 14), while for the NRT, the mean of six replicates is displayed for each dose. Additionally, the linear trend line is given, and the R2 value. All linear trends were significant, except the trend for chlorophyll A (a = 5%), judged by the critical R for given n (Sachs, 1992). The best correlation was obtained for the normalized transpiration (note that the correlation is slightly higher if the logarithm of the effect data is used). The enzyme assays and the chlorophyll content were measured in the leaves. LAS was probably not or to a minor extend translocated to leaves. This could be an explanation for the smaller effect of LAS on these parameters.
1250 1000 750
R 2 = 0.96
500
47
The effect concentrations EC10 and EC50 are defined by the concentration of a chemical, which produces 10% or 50% of the maximum possible response for that chemical. In our case, the EC10 and the EC50 correspond to the dose, at which the NRT is inhibited 10% or 50%. For the fit of effect concentrations, the NRT for each replicate was derived by dividing the change of initial transpiration by the average change of controls. This was done to get a higher degree of freedom (n = 42). A sigmoidal curve was fitted to the NRT data, assuming log-normal toxicity data distribution (Andersen, 1994), in order to estimate EC10 and EC50. Table 3 gives the estimated values at different exposure periods. The inhibition of the transpiration increased with time. The results from this study can be compared to earlier results with terrestrial plants. From laboratory tests with terrestrial plants, Jensen et al. (2001) reported EC50 values of 16–316 mg/kg (soil) for terrestrial plants using the growth of plants as a sensitive endpoint. For Sinapis alba, Avena sativa and Brassica rapa, EC10 values were at 200, 50 and 90 mg kg1, respectively. They also found that at an average soil concentration of 5–15 mg kg1, LAS did not seem to be detrimental to the soil ecosystem in the long term. A large number of data for LAS toxicity to plants, soil fauna, soil microorganisms and microbial soil processes has been collected for the terrestrial environmental risk assessment in Denmark (Kloepper-Sams et al., 1996; Jensen, 1999; Elsgaard et al., 2001; Holmstrup and Krogh, 2001; Jensen et al., 2001). A predicted non-effect concentration of LAS (PNEC) in soils of 5.2 mg kg1 was estimated from these Danish studies (Bro-Rasmussen and Solbe´, 1999), while a PNEC for soil organisms of 4.6 mg kg1 was reported in HERA (2004). Field observations indicated that the application of LAS-containing sludge generally stimulated the microbial activity and, hence, the abundance of soil fauna and growth of plants (Jensen, 1999; Jensen et al., 2001; Brandt et al., 2003). In this investigation, when exposed to low dose of LAS (30 mg l1), treated willows had a higher transpiration in comparison with untreated willows, and no toxic effects were observed at the low doses. It should be mentioned, that terrestrial plants seem less sensitive than some aquatic species: a wide range of toxicity of LAS towards algae was
2
R = 0.29 2 R = 0.40 2 R 2 = 0.45 R = 0.50
250 0 0
100
200
300
400
500
LAS mg/l
Chlo A
POD
CAT
SOD
NRT o/oo
Fig. 5. Comparison of all toxic effects; linear regression with R2 values added. Chlo A: chlorophyll A in mg/kg; POD, CAT, SOD: enzyme assays in U/g (see methods section); NRT: normalized relative transpiration after 72 h, here in per mill o/oo.
Table 3 Calculated EC10 and EC50 values (mg l1 LAS) for weeping willows exposed 72 and 192 h to LAS in hydroponic solution; confidence interval CI for a = 5% Period (h)
Parameter
mg l1
Lower CI
Upper CI
72 72 192 192
EC10 EC50 EC10 EC50
87 374 54 166
32 272 16 102
233 513 182 270
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reported in literature. Most data fall between 0.1 and 100 mg l1 (growth, population density, reproduction, mobility, emergence and mortality were the endpoints). Typical ranges of EC50 values are 1–100 mg l1 for fresh water species and <1 to 10 mg l1 for marine species (Painter, 1992). LC50 values have been found in the range of 1– 10 mg l1 when Daphnia magna were exposed to LAS homologues between C10 and C13 (Painter, 1992; Kusk and Petersen, 1997). A 96 h LC50 value of 1.8 mg l1 was found for C13 LAS in a study with fathead minnow (Pimephales promelas) (Macek and Slight, 1977). 4. Conclusions The toxicity tests in hydroponic solution using the normalized relative transpiration as a variable showed that the short-term acute toxicity of LAS to weeping willows was strongly related to the dosage. Severe signs of toxicity were only found at the treatment groups exposed to high doses of LAS. Most sensitive toxicity parameters were transpiration and POD. The results indicate that phytotoxic effects of LAS on willow trees are unlikely to occur under normal environmental conditions. Acknowledgments This work was supported by a research foundation from the Hunan Agricultural University for scientists (Grant no. 04PT02). Thanks to Yong Lei, Ling Li, Shengzhuo Huang, Jin Feng, Liang Chen, Yawen Tang, and Junjie Lei for their technical assistance. Thanks to Ole Kusk for help with the EC estimations. References Andersen, H., 1994. Statistical methods for evaluation of toxicity of wastewater. M.Sc. Thesis 7/94. Institute for Mathematical Statistic and Operation Analysis. Technical University of Denmark (in Danish). Brandt, K.K., Krogh, P.H., Sorensen, J., 2003. Activity and population dynamics of heterotrophic and ammonia-oxidising microorganisms in soil surrounding sludge spiked with LAS: a field study. Environ. Toxicol. Chem. 22, 821–829. Bro-Rasmussen, F., Solbe´, J., 1999. Executive summary of the SPT workshop in cooperation with the Danish EPA, Copenhagen 19/20 April 1999. Carlsen, L., Metzon, M.B., Kjelsmark, J., 2002. LAS in the terrestrial environment. Sci. Tot. Environ. 290, 225–230. Elsgaard, L., Petersen, S.O., Debosz, K., 2001. Effects and risk assessment of LAS in agricultural soil. 2. Effects on soil microbiology as influenced by sewage sludge and incubation time. Environ. Toxicol. Chem. 20, 1664–1672.
Fodor, F., 2002. Physiological responses of vascular plants to heavy metals. In: Prasad, M.N.V., Strzalka, K. (Eds.), Physiology and Biochemistry of Metal Toxicity and Tolerance in Plants. Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 149–177. Fridovich, I., 1978. The biology of oxygen radical. Science 39, 522–526. Fridovich, I., 1989. Superoxide dismutases. An adaptation to a paramagnetic gas. J. Biol. Chem. 264, 7761–7764. Holmstrup, M., Krogh, P.H., 2001. Effects and risk assessment of LAS in agricultural soil. 3. Sub lethal effects on soil invertebrates. Environ. Toxicol. Chem. 20, 1673–1679. HERA Human and Environmental Risk Assessment, 2004. Linear alkylbenzene sulfonates (LAS). (CAS No. 68411-30-3). Available from: