Toxicity of cadmium to soil microbial biomass and its activity: Effect of incubation time on Cd ecological dose in a paddy soil

Toxicity of cadmium to soil microbial biomass and its activity: Effect of incubation time on Cd ecological dose in a paddy soil 1 first-author †E-mail:liaomin@zju.edu.cn †E-mail:liaomin@zju.edu.cn liaomin@zju.edu.cn 2 2 1 School of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China School of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China School of Land Resources and Environment, Jiangxi Agricultural University, Nanchang 330045, China School of Land Resources and Environment, Jiangxi Agricultural University, Nanchang 330045, China Cadmium (Cd) is ubiquitous in the human environment and has toxic effect on soil microbial biomass or its activity, including microbial biomass carbon (C mic ), dehydrogenase activity (DHA) and basal respiration (BR), etc., C mic , DHA, BR were used as bioindicators of the toxic effect of Cd in soil. This study was conducted to determine the effects of Cd on soil microbial biomass and its activity in a paddy soil. The inhibition of microbial biomass and its activity by different Cd concentrations was described by the kinetic model (M1) and the sigmoid dose-response model (M2) in order to calculate three ecological doses of Cd: ED 50 , ED 10 and ED 5 . Results showed that M2 was better fit than M1 for describing the ecological toxicity dose effect of cadmium on soil microbial biomass and its activity in a paddy soil. M2 for ED values (mg/kg soil) of C mic , DHA, BR best fitted the measured paddy soil bioindicators. M2 showed that all ED values (mg/kg) increased in turn with increased incubation time. ED 50 , ED 10 and ED 5 of C mic with M2 were increased in turn from 403.2, 141.1, 100.4 to 1000.7, 230.9, 144.8, respectively, after 10 d to 60 d of incubation. ED 50 , ED 10 and ED 5 of DHA with M2 increased in turn from 67.6, 6.2, 1.5 to 101.1, 50.9, 41.0, respectively, after 10 d to 60 d of incubation. ED 50 , ED 10 and ED 5 of BR with M2 increased in turn from 149.7, 6.5, 1.8 to 156.5, 50.8, 35.5, respectively, after 10 d to 60 d of incubation. So the ecological dose increased in turn with increased incubation time for M2 showed that toxicity of cadmium to soil microbial biomass and its activity was decreased with increased incubation time. Cadmium Soil microbial biomass Basal respiration Dehydrogenase activity Ecological dose Paddy soil INTRODUCTION 1 Cadmium (Cd) is ubiquitous in the human environment and has been recognized as one of the most deleterious heavy metal pollutants (Robards and Worsfold, 1991 1991 ; Christine, 1997 1997 ). It may easily move from soil to food plants through root absorption and accumulate in their tissues (Oliver, 1997 1997 ). In this way, Cd may enter the food chain and affect human health (Adriano, 1986 1986 ). Among many heavy metals polluting soil, Cd is of concern because of its potentially harmful effects on not only humans and animals, but also the most adverse effects on microbial biomass and its activity which play an important role in the biological cycles of almost all the major plant nutrients cycling, soil nutrient cycling and in maintaining soil fertility (Smith, 1996 1996 ; Jose et al., 2002 2002 ; Yao et al., 2003 2003 ). Cd can also cause changes in the size, composition and activity of soil microbial community (Giller et al., 1998 1998 ). There were many studies on this topic (Brookes, 1995 1995 ; Nannipieri et al., 1997 1997 ; Giller et al., 1998 1998 ). However, little is known about the toxicity effect of Cd on the microbial biomass and its activity of paddy soils. Paddy soil widely distributed in China and Asia is important resource for food production. In China, more than 30 million hectares located mainly in the area south of the Changjiang River are important rice production regions (Zhang and Gong, 2003 2003 ) contaminated by Cd in widespread area. In response to the apparent need to easily quantify the influence of pollutants on microbe-mediated ecological processes in various ecosystems, the concept of an ecological dose (ED 50 ) was developed, which is the toxicant concentration that inhibits a microbe-mediated ecological process by 50% (Babich et al., 1983 1983 ). However, a 50% reduction in a basic ecological process may be too extreme for the continued functioning of a paddy soil and so, lower percentage values of inhibition (5%, 10% or 25%) equivalent to ED 5 , ED 10 or ED 25 must be established. These values may be more suitable criteria for protecting soil quality and for assessing the sensitivity of a paddy soil ecosystem subjected to heavy metals pollution stress (Doelman and Haanstra, 1989 1989 ; Kostov and Van, 2001 2001 ). Different researchers proposed several mathematical models for calculating the ecological dose value. Haanstra et al.( 1985 1985 ) used a sigmoid curve to describe the inhibitions of urease and phosphatase activity as a function of the natural logarithm of the concentration of heavy metals, while Speir et al.( 1995 1995 ) used two models based on enzyme inhibition kinetics. In the present study the changes of different microbial indicators in a paddy soil contaminated by a wide range of Cd levels were measured to evaluate the effects of Cd toxicity on the sensitivity of soil microbial indicators. The microbial indicators used were microbial biomass carbon (C mic ), dehydrogenase activity (DHA) and basal respiration (BR). Statistical test of non-linear correlation was used to evaluate which of the previously mentioned models best fit the experimental data. We calculated the ED values for Cd using two models and compared changes in ED values in a paddy soil under different incubation periods MATERIALS AND METHODS 1 Soil 2 The laboratory incubation experiment at constant temperature of 25 °C was carried out with a paddy soil sampled from near Jinhua City, Zhejiang Province, China. The soil samples used were collected from the surface layer (0–15 cm) of an agricultural field used for paddy rice. After removal of surface water, the fresh soil was brought to laboratory immediately after the collection, hand picked to remove discrete plant residues and large soil animals (earth warms, etc.). Soil samples were homogenized and air-dried at room temperature to facilitate the sieving, passed through a 2-mm sieve and stored at 4 °C prior to analyze. A sub-sample of the soil was taken, air-dried, ground, and analyzed for various physicochemical characteristics listed in Table 1 1 . Below detection limit Parameters Value pH (H 2 O) 4.74 WHC (g/kg) 510 O. C. (g/kg) 15.25 Av. N (mg/kg) 106.40 Av. P (mg/kg) 13.34 Total content of Cd (mg/kg) BDL # # CEC (cmol/kg) 7.328 Sand (g/kg) 278 Silt (g/kg) 562 Clay (g/kg) 160 Soil texture Silt loam Soil treatments and incubation conditions 2 The pretreated soils passed though 2 mm sieve in portions of 500 g were oven-dried, placed into 1000 ml plastic beakers. Eighteen plastic beakers were prepared, containing six Cd levels of cadmium nitrate. All treatments were carried out in triplicates (6 treatments). The soil samples were first adjusted to their required level of moisture contents (40% of its water-holding capacity, WHC, which was calculated according to Table 1 1 ) by adding distilled water and pre-incubated for 14 d at 25 °C to stabilize microbial activity. After 14 d pre-incubation, placed into a set of beakers with designated amounts of Cd nitrate applied in solution form to achieve several final Cd concentrations in the soil: 0 (background), 10, 20, 40, 80, 150 mg/kg soil. The moisture contents in the treated soils were adjusted to 60% WHC and then incubated at 25 °C for 0 d, 10 d, 20 d, 40 d, 60 d, respectively. The soil moisture was kept at the same level (60% WHC) by adding distilled water at regular intervals throughout the incubation period. At the end of the incubation for 0 d, 10 d, 20 d, 40 d, 60 d, respectively, the soil samples were taken out and analyzed for C mic , DHA, BR. Soil microbial parameters assay 2 All of the following parameters results reported were averages of triplication assays and analysis was used to calculate ED values. Statistical analyses were performed using SPSS 11.0 software. Soil samples for the determination of C mic were taken by fumigation-extraction (FE) method based on the difference between C extracted with 0.5 mol/L K 2 SO 4 from chloroform (CHCl 3 )-fumigated and unfumigated soil samples using a K c factor of 0.45 (Vance et al., 1987 1987 ; Ghamry et al., 2000 2000 ; Milne and Haynes, 2004 2004 ). The contents of K 2 SO 4 -extracted C in the CHCl 3 -treated and untreated soils were determined using a Shimadzu TOC-500 carbon analyzer (Zhang and Zhang, 2003 2003 ). DHA was assayed by the method described by Zhu ( 1996a 1996a ; 1996b 1996b ). This method involved colorimetric determination (at 492 nm wavelength on a 722 type spectrophotometer) of 2,3,5-triphenyl formazan (TF) produced by the reduction of 2,3,5-Triphenyltetrazolium Chloride (TTC) by soil microorganisms. The absorbance value obtained photometrically was converted to TF using its standard curve. DHA was expressed by TF generated contents. BR was assayed by the method of seal incubation in erlenmeyer flask, described by Jin et al.( 1998 1998 ). Soil samples for BR were placed in a sealed flask with a small bottle of 0.1 mol/L NaOH in it, incubated for 2 d at 25 °C. The CO 2 produced was measured after 48 h by using 0.1 mol/L HCl dripping into the 0.1 mol/L NaOH. BR was expressed as mg CO 2 -C/kg soil per hour by CO 2 content released. The CO 2 content was calculated by the formula described by Lu ( 2000 2000 ). Mathematical models 2 The kinetic model proposed by Speir et al.( 1995 1995 ) and the sigmoid dose-response model proposed by Haanstra et al.( 1985 1985 ) were used to calculate the ED values and evaluate the suitability of these models for describing Cd inhibition of soil microbiological and biochemical properties. The algebraic expression of kinetic model was: v = c /(1+ bi ) (Model 1) The constants b and c were always positive. The constant c represents the uninhibited value of the tested parameter, and the constant b depends on the curve slope. Model 1 describes the full inhibition of v (tested parameter) by i , the concentration of inhibitor (Cd concentration). For data fitting Model 1, it was possible to calculate the ecological dose values from the relationships: ED 50 =1/ b ; ED 10 =1/(9 b ); ED 5 =5/(95 b ) Model 1 describes the concave rectangular hyperbolic relationships between v and i ; the mathematical equation for the sigmoid dose-response model was: y = a /{1+exp[ b ( x − c )]} (Model 2) where y is the tested parameter, x the natural logarithm of Cd concentration, a the uninhibited value of y , b a slope parameter indicating the inhibition rate, and c the natural logarithm of ED 50 . The ED values were calculated using the following expressions: ED 50 =exp( c ); ED 10 =exp[ c −(2.2/ b )]; ED 5 =exp[ c −(2.9/ b )] Model 2 describes a logistic curve which is the relationship between the measured activity and the natural logarithm of the inhibitor concentration. The value of the coefficient of determination ( r 2 ) of the non-linear regression was only determined at P < 0.05. RESULTS 1 The ED 50 , ED 10 and ED 5 values calculated with the above two models for C mic , DHA and BR are shown in Tables 2 2 – 4 4 . These ED values were calculated for cadmium pollution soil after the different incubation periods mentioned (10 d, 20 d, 40 d and 60 d). Two mathematical models described the inhibition situation of C mic (Table 2 2 ). The values of the coefficient of determination ( r 2 ) were high (0.8562–0.9957) after the addition of Cd during the whole incubation period. The ED 50 values calculated with Model 1 (M1) were much higher than that of Model 2 (M2) in different incubation times, but in 60 d of incubation, they were near. While ED 10 and ED 5 values with M2 were higher than those with M1 for Cd stress paddy soil. The ED 50 , ED 10 and ED 5 values with M2 were all increased with increase of incubation time. In all levels of incubation time, the ED 50 , ED 10 and ED 5 values with M1 were near to each other, respectively, but all of them were unstable with incubation time going on. The ED 50 , ED 10 and ED 5 values in 20 d of incubation all were highest using M1 during the whole incubation period, respectively, while these highest values of M2 were observed in 60 d of incubation. After 10 d and 40 d of Cd exposure, respectively, the lowest values of ED 10 and ED 5 calculated with M1 were observed. ED 10 and ED 5 values of 10 d were equal to those of 40 d, respectively. So the change trend of M2 with incubation time was obvious and regular. Treatments Model r 2 ED 50 ED 10 ED 5 10 d of incubation M1 0.8562 1000.0 111.1 52.6 M2 0.9957 403.2 141.1 100.4 20 d of incubation M1 0.9545 1250.0 138.9 65.8 M2 0.9639 612.6 164.7 108.4 40 d of incubation M1 0.9077 1000.0 111.1 52.6 M2 0.9774 649.9 168.7 109.8 60 d of incubation M1 0.8859 1111.1 123.5 58.5 M2 0.8682 1000.7 230.9 144.8 For DHA, the coefficients of determination for the two models were from 0.7477 to 0.9812 during the whole incubation period. M1 and M2 described well the inhibition of DHA, except for 60 d after the addition of Cd, whose coefficients of determination were 0.7954, 0.7747 for M1 and M2, respectively (Table 3 3 ). In general, the ED 50 values predicted from the two models were very near correspondingly, except for 10 d of incubation, when there was greater discrepancy in the predicted ED 10 and ED 5 values. From 20 d to 60 d of incubation, The ED 50 , ED 10 and ED 5 for M1 were increased in turn with incubation time. While ED 50 , ED 10 and ED 5 values with M2 were all increased in turn with incubation time. The ED 10 and ED 5 values with M2 were higher than those of M1 at all spans of incubation time, respectively, except for 10 d of incubation. The ED 50 values of M1 were lower than those of M2 in 20 d of incubation, higher in other incubation days. From 20 d to 60 d of incubation, the predicted ED 10 and ED 5 values were higher (about two to seven times), respectively, with M2 than with M1. While in 10 d incubation, the predicted ED 10 and ED 5 values with M1 were about three or six times that with M2, respectively. Treatments Model r 2 ED 50 ED 10 ED 5 10 d of incubation M1 0.8567 178.6 19.8 9.4 M2 0.9812 67.6 6.2 1.5 20 d of incubation M1 0.8513 66.2 7.4 3.5 M2 0.9128 69.1 13.0 7.7 40 d of incubation M1 0.8038 103.1 11.5 5.4 M2 0.8147 95.5 48.3 38.9 60 d of incubation M1 0.7954 119.0 13.2 6.3 M2 0.7477 101.1 50.9 41.0 For soil microbial basal respiration (BR), the r 2 values for M1 and M2 also were very high, except for at the 10 d of incubation with M1 (Table 4 4 ). In this case the low Cd inhibition of soil microbial basal respiration and dispersion of data produced a poor fit to the mathematical models used. At 10 d of incubation, great discrepancy was observed between the ED values predicted by the two models. M2 gave better fitted the measured data. With this model, the ED values for the Cd stress of paddy soil were lower in the values of r 2 with M1 than with M2 in all stretches of incubation time. This finding may reflect reality because the r 2 values for this model are very high in all cases. r 2 of M2 were from 0.9373 to 0.9967. ED 50 , ED 10 and ED 5 values of M2 were all increased with incubation time, but these values of M1 were very near in all cases, respectively, except for 10 d of incubation. For M2 in 10 d of incubation, ED 50 , ED 10 and ED 5 values were the lowest, while they were the highest in 60 d of incubation, respectively. For M1 in 10 d of incubation, the ED 50 , ED 10 and ED 5 values did not exist, while these values were completely equal in 20 d and 40 d of incubation, respectively. Then, in 60 d of incubation, they were increased, respectively. So M1 could not describe well the inhibition of soil microbial basal respiration under Cd stress in the paddy soil, though their r 2 values were very high (from 0.8265 to 0.9256) except for 10 d of incubation. n.f.: the model did not fit the experimental data, r 2 values are calculable but the level of significance for the regression was not P < 0.05 Treatments Model r 2 ED 50 ED 10 ED 5 10 d of incubation M1 * * n.f. – – – M2 0.9967 149.7 6.5 1.8 20 d of incubation M1 0.9256 163.9 18.2 8.6 M2 0.9813 150.4 7.1 2.7 40 d of incubation M1 0.8265 163.9 18.2 8.6 M2 0.9752 154.8 43.8 29.3 60 d of incubation M1 0.9205 208.3 23.1 11.0 M2 0.9373 156.5 50.8 35.5 DISCUSSION 1 Tables 2 2 – 4 4 show that ED 50 , ED 10 and ED 5 values calculated with M1 were near with incubation time, respectively, except for DHA and BR in 10 d of incubation, while M2 showed that all ED values were increased in turn with prolongation of incubation time. This presumably was the reason that M1 was a full inhibition model. For M2, heavy metal cadmium could have an aging characteristic after entering soil, resulting in lower availability and lower toxicity for Cd with prolongation of incubation time (Martinez et al., 2003 2003 ). Results showed that compared to M1, M2 describes better the ecological toxicity dose effect of cadmium on soil microbial biomass and its activity in the paddy soil. M2 for ED values of C mic , DHA, BR best fitted the measured paddy soil bioindicators. Table 2 2 shows that M1 ED 50 values were very high (more than 1000 mg Cd/kg soil) and nearly the same with incubation time, which could be because higher Cd dose imposed full inhibition on the microbial metabolic processes and microbial growth. ED 50 values with M2 increased in turn with incubation time. This was possibly due to the slow increase in cadmium resistance in soil microorganisms (Bewley and Stotzky, 1983 1983 ) under relatively lower cadmium concentration. M1 ED 10 and ED 5 values were also very near, respectively, regardless of incubation time. But M2 ED 10 and ED 5 values were increased in turn from 20 d to 60 d of incubation time. In most cases, the soil microbiological indicators measured decreased with increasing Cd concentrations, which indicated that Cd had toxic effect on both the microbial metabolic processes and microbial growth. The microbial indicators with M2 decreased with the incubation time for ED 10 and ED 5 of DHA, while BR and C mic were same as DHA, except for 10 d of incubation. This was presumably due to the decrease with time of the substrates easily available to microorganisms (Moreno et al., 1999 1999 ). The ED values obtained for Cd were much greater than the maximum concentration of Cd permitted in soils by EU (European Union) legislation (3 mg/kg soil). However, for safety, a value one tenth or one thousandth that of ED 50 (depending on the statistical reliability of this value) was proposed as a maximum limit for heavy metal in soils (Doelman and Haanstra, 1989 1989 ). Other authors used the ED 10 and ED 5 values to propose regulatory criteria for heavy metals in soils (Scott and Pedersen, 1995 1995 ). These ED 10 and ED 5 values may be more suitable indicators of the sensitivity of an ecosystem to a given stress, because a 50% reduction in a basic ecological process may be too extreme for its continued functioning (Babich et al., 1983 1983 ). Discrepancy observed in ED 50 values calculated from the two models can be due to the great elongation of the initial part of the curve of M1 and thus small variation in the rate of decrease of the studied bioindicator can produce a large variation in ED 50 values calculated from this model. M1 is a full inhibition model and the minimum possible value of the studied bioindicator is always higher than zero, except for soil microbial basal respiration. In general, for this experiment, the addition of Cd to the soil increased the inhibitory effects on biological parameters. The addition of lower Cd could stimulate soil microbial biomass and its metabolic activity. The first time addition of Cd to soil can obviously inhibit soil microbial biomass and its metabolic activity, then favoring a selection of Cd resistant microorganisms with prolongation of addition time (Kandeler et al., 2000 2000 ). Furthermore, soil organic matter contributes to adsorption of Cd and thus restricts the movement of this heavy metal through the soil profile (Saviozzi et al., 1983 1983 ). In general, the ED values increased noticeably over time after soil Cd treatment. Doelman and Haanstra ( 1984 1984 ) and Speir et al.( 1995 1995 ) also reported an increase in ED 50 over time for different heavy metals, when the inhibition of soil microbial parameters by soil heavy metal pollution was studied. It is possible that this is due to an increase in Cd resistance in microorganisms as observed by other authors (Bewley and Stotzky, 1983 1983 ). C mic had been suggested as possible indicators of soil environmental quality in toxicity assays (Yao et al., 2000 2000 ). DHA is related to a group of intracellular enzymes present in active soil microorganisms. Enzymatic activity had been used as an index of overall microbiological activity in toxicity assays (Nannipieri et al., 1997 1997 ). DHA was largely affected by increasing Cd concentration in the paddy soil after 10 d, perhaps because metals cannot be taken up by soil and accumulated inside microorganisms at once. Differences in C availability in the media had been shown to have profound effect on the toxicity of Cd (Brynhildsen et al., 1988 1988 ). In 60 d, however, the soil had higher ED 50 . Perhaps this is due to either a complexation of Cd with the humic substances or a different evolution of the soil microbial community. In general, the soil microbial basal respiration measurements fit M2 better (Table 4 4 ). This model assumes that a portion of the measured activity is not affected by high soil Cd concentrations. This means that the full inhibition of the measured activity is never 100% of the control value (Dixon and Webb, 1979 1979 ). In conclusion this study suggests that the addition of Cd to the paddy soil increased the inhibitory toxic effect on microbial function, a finding which may be of great use for the paddy soils with severe Cd contamination. Examples of such soils would be non-agricultural soils near smelters or mines. However, Cd bioavailability in paddy soils can change with time. The values of the ecological dose that produce a specific level of inhibition of soil microbial parameters, are suitable for setting a maximum limit of Cd concentration in order to avoid irreversible effects on soil functionality. However, for other soils a different indicator may produce a better fit to these models. So to safeguard the “health” of the soil, it is necessary to evaluate multiple indicators when using ED x values as a criterion to set a maximum permissible concentration of a contaminant in soil. 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