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Sewage sludge fertilization in larch seedlings: Effects on trace metal accumulation and growth performance
Ecological Engineering 77 (2015) 216–224
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Sewage sludge fertilization in larch seedlings: Effects on trace metal accumulation and growth performance Mohamed Bourioug a , Laurence Alaoui-Sehmer b , Xavier Laffray b , Mohammed Benbrahim c , Lotfi Aleya b, * , Badr Alaoui-Sossé b a b c
Jean-François Champollion University Center for Teaching and Research, Place Verdun, F-81000 Albi, France Université de Franche-Comté, Laboratoire de Chrono-Environnement, UMR CNRS 6249, Besançon, France RITTMO Agroenvironnement, ZA Biopôle, 37 rue de Herrlisheim, CS 80023, F-68025 Colmar Cedex, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 September 2014 Received in revised form 14 January 2015 Accepted 20 January 2015 Available online xxx
The spreading of sewage sludge (SS) among forest plantations may provide interesting results for firewood production. While sludges are good fertilizers, they may nevertheless contain trace metals, which can reduce productivity and lead to environmental risks. We investigated the effects of SS application on nutrient uptake and growth parameters in larch seedlings (Larix decidua) and determined trace metal and mineral distribution. Without incorporation into the soil, sludge was applied to the soil surface at three rates (0, 30 and 60 t dry weight DW ha1). The plants were harvested after 12 months. The results showed significantly increased nitrogen and phosphorus concentrations in the top soil layer in pots amended with sludge, whereas no changes appeared in the lower layers. Similar results were obtained for the Cu, Zn and Cd concentrations. However, no differences were observed for the other measured soil mineral elements. Nitrogen concentrations in needles increased with rising sewage sludge application rates, yet the sludge had no effect on the P, Mg, Zn, Pb and Cd concentrations. In addition, Cu accumulated only in the lateral roots of seedlings that received the highest sludge loading rate. Sludge application improved the net photosynthesis, which resulted in higher chlorophyll contents in the needles. Following application, the dry matter accumulation rate increased due to the excessive availability of N, whereas available mineral elements in the plant tissues were diluted. Furthermore, amending the soil with sewage sludge can promote a higher biomass yield which may result in an increased trace metal bioaccumulation capacity in plants. Though this investigation has established the benefits of municipal SS application, further studies are needed to assess the potential transfer of TM to groundwater and through the food chain. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Sewage sludge Larix decidua Trace metals Forest plantations
1. Introduction Wastewater plant operators will continue to face the challenge of disposing of millions of tons of sewage sludge (SS) generated each year (Japan: 70 million, China: 30 million, USA: 6 million and France: 1.2 million) (Matsubara and Itoh, 2006; McClellan and Halden, 2010; Kelessidis and Stasinakis, 2012; Legroux and Truchot, 2009). As SS has a high nutrient content, its increased use as a fertilizer has been recommended as the best practical environmental option for the management of this organic residue. In France about 73% of sludge produced is applied to agricultural land (Legroux and Truchot, 2009). However, sludge contains
* Corresponding author. Tel.: +33 3 81 66 57 64; fax: +33 3 81 66 57 97. E-mail address: lotfi[email protected] (L. Aleya). http://dx.doi.org/10.1016/j.ecoleng.2015.01.031 0925-8574/ ã 2015 Elsevier B.V. All rights reserved.
several potentially harmful constituents such as trace metals (TMs) and organic pollutants which may accumulate in agricultural soil over time (Lopez-Mosquera et al., 2000; McBride, 2003; Chopra et al., 2009). Once accumulated, TMs are highly persistent in the topsoil, can cause potential problems or be transferred through the food chain (Alloway et al., 1991), thus posing a threat to human health (Wang et al., 2003; Chopra et al., 2009). In the case of forest plantations receiving SS application, the risks are mitigated since farmed trees are not a direct part of the human food chain. In addition, this alternative application is promoted as it is thought to enhance both tree growth and wood production (Henry and Cole, 1997; Mosquera-Losada et al., 2001; Bramryd, 2001; Tsakou et al., 2003; Vaitkuté et al., 2010) and to improve several soil characteristics (Barzegar et al., 2002; Veeresh et al., 2003; Hussein, 2009). In France, application of SS to forest land is currently in the experimental phase and requires intensive monitoring of
M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
ecosystem components such as plant, soil, water and fauna, to identify any potential positive and/or negative effects on plant productivity and environmental integrity (Bourioug et al., 2015). The objectives of this investigation were to study the balance between the beneficial effects and eventual toxicity of SS applications on young larch seedlings (Larix decidua) by measuring the effects of two different sludge doses (30 and 60 t dry matter ha1). Several growth parameters, biomass productivity and nitrogen, as well as phosphorus were examined. TM uptake and transfer within plant tissues were also investigated.
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2.3. Photosynthesis measurement and chlorophyll content
The soil used in this experiment was a pseudo luvisol with dysmull, collected from a forested area located at Mélisey, Haute-Saône, France (47 7530 latitude, 6 5800 longitude). Soil samples were collected from within the top 20 cm of the soil layer and sieved through a 1 cm mesh. Aerobically digested SS from a domestic wastewater treatment plant in the village of Mélisey was also used. The physico-chemical characteristics of the soil and SS are provided in Table 1.
The CO2 assimilation rate (Pn), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) were measured using a Li-COR6400 portable photosynthesis system (LI-COR-6400, LI-COR Biosciences, Inc., Lincoln, Nebraska, USA) connected to a conifer chamber. Monthly measurements were obtained from June to September under the ambient conditions of the culture chamber (i.e., 25 C, 60% RH and 200 mmol m2 s1). Only the CO2 concentration was maintained at 400 mmol s1. In addition, needles were collected from both sides of the enclosed branches that were used for gas exchange measurements; the projected areas of the needles were digitally determined in the laboratory. After at least 5 min of steady readings in the chamber, the photosynthesis rate was recorded. All measurements were obtained from a single branch at the same distance from the stem apex. Data were recalculated according to the measured needle area. The collected needles were used to quantify the chlorophyll content, which was extracted using 100% dimethyl sulfamide (Robakowski et al., 2004) and was calorimetrically determined according to Barnes et al. (1992). The results were expressed as milligram of chlorophyll per gram of fresh matter (FM) (mg g1).
2.2. Plant material, growth conditions and experimental setup
2.4. Sampling
One-year-old L. decidua (European larch) seedlings were purchased from a local nursery. On December 22, 2008 the soil samples were placed in 7.5 l plastic pots, each one filled with 6.7 kg of the sampled soil (Fig. 1). During the first 30 days the pots were watered with distilled water three times per week to maintain a constant weight (70% of field capacity). SS was then applied to the soil surface without incorporation into the soil. The rate of sludge was equivalent to (30 t dry weight (DW) ha1: 30S) or twice (60 t DW ha1: 60S) the maximum accumulated amount permitted under French law (i.e., 30 t DW ha1 over a 10-year period). Pots without sludge (0S) were used as the control. Each configuration was replicated five times. The plants were grown for 12 months in a greenhouse with a 14 h photoperiod (200 mmol m2 s1), a temperature regime of 25/14 C (day/night) and 60% relative humidity.
At harvest, an aerial part of each plant was removed, which, by means of the Scholander chamber method, permitted immediate determination of water potential. The aerial part was then cut into separate organs, i.e., needles, the main stem and ramifications. Afterward, the root portion was carefully removed from the pot, separated from the adhering soil and washed with distilled water. The lateral roots were removed from the taproot after their lengths had been measured. Each sample was lyophilized. Also, the dry mass of each sample was determined before being reduced to a fine powder using a ball mill (Retsch MM 200, Germany). Soil samples were simultaneously collected from three levels in each pot as follows: (i) the sludge layer (Sl), corresponding to the upper layer of the pot with a depth of approximately 2 cm. This layer is primarily composed of organic matter that was previously contained in the sludge and formed via mineralization;
2. Materials and methods 2.1. Origin and characterization of soil and sludge
Fig. 1. Schematic representation of the experimental design.
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150
Stem height (cm)
120 b b
90
b b
b b
a
a
a
60 30 0 0
3 6 9 Months after sludge application
12
Fig. 2. Effects of sewage sludge on the growth (variation in height) of larch (Larix decidua) grown in control ( 0S) and amended ( 30S: 30 t DW ha1; 60S: 60 t DW ha1) soils. The data represent the mean of 5 repeated applications; the vertical bars indicate standard deviations (). The different letters indicate significant differences according to the Tukey test at p < 0.05.
(ii) the top soil level (Ts), corresponding to the soil layer at the top of the pot, i.e., immediately under the sludge layer, and with a depth of 5 cm; and (iii) the bottom soil level (Bs), corresponding to the soil layer at the bottom of the pot with a depth of 5 cm. The samples were oven-dried at 60 C for 48 h before being crushed and sieved to 2 mm. 2.5. Chemical analyses
2.6. Statistical analysis The data are presented with standard deviations (). Statistical analysis was performed via one-way analysis of variance (ANOVA) with a multiple-comparison test using Prism 5 (GraphPad Software). Data with probabilities of less than 5% (p < 0.05) were deemed statistically significant. 3. Results
Soil pH was measured in a 2:5 (w/v) ratio of soil to distilled water (Fuentes et al., 2007). The soil redox potential was determined using a Pt electrode inserted 10 cm below the soil surface. Also, the total carbon and total nitrogen in the soil and plants were determined using a dry combustion CNS analyzer (LECO CNS-2000). Approximately 1 g of powdered soil was digested in a 10 ml solution (1:3) (v/v) (HNO3 (65%)/HCl (37%)) at 150 C for 2 h on a hot plate. The sample volume was increased to 40 ml with distilled water. Approximately 0.1 g of the plant sample was added to 2 ml of HNO3 (65%) before digestion on a hot plate for 3 h at 125 C. The TM (i.e., Cu, Zn, Cd and Pb) and mineral element (i.e., P, K, Mg and Mn) concentrations were determined using inductively coupled plasma mass spectroscopy (ICP-MS). The obtained values are in accordance (within 5%) with the certified concentrations in the standard references (calcareous loam soil n 141R from BCR in Belgium, and Virginia tobacco leaves “CTA-VTL-2” in Poland), confirming the reliability of our analytical procedure.
3.1. Effects of SS application on soil 3.1.1. pH and redox potential Only the Ts pH was significantly affected by the SS application, becoming more acidic relative to the control (Table 2). However, no significant differences were found between the 30S and 60S treatments. The soil redox potential ranged from 367 to 422 mV and did not change due to SS additions. 3.1.2. Carbon, nitrogen and phosphorus changes The nitrogen (N) and phosphorus (P) concentrations for the different soil horizons are presented in Table 2. High N and P concentrations were found within the Sl. In addition, a significant enrichment in N and P was also found within the Ts levels from the 30S and 60S treatments compared to the 0S. The C:N ratio in this horizon was indicative of a higher N concentration and was significantly lower than the ratio in the control. In contrast, no changes in these elements were observed in the Bs levels.
60
c
Dry matter (g)
40 20
b a
0 -20
a
-40
b
b
-60 0S
30S
60S
Fig. 3. Changes in total dry matter partitioned to the aerial (white column) and root (black column) parts of the larch plants (Larix decidua) grown in control (0S) and amended (30S: 30 t DW ha1; 60S: 60 t DW ha1) soils after 12 months of growth (n = 5). The different letters indicate significant differences according to the Tukey test at p < 0.05. The vertical bars indicate standard deviations () for the 5 repetitions.
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3.1.3. Other mineral element and trace metal changes Concentrations of the other selected mineral elements and trace metals within the soils are summarized in Table 2. The sludge layers (Sl) with the 30S and 60S treatments exhibited higher TM concentrations compared to the underlying horizons. Cu, Zn and Cd concentrations in the Ts layer increased significantly due to the SS application. For the same horizon, the mineral element (K, Mg and Mn) and Pb concentrations exhibited no significant differences among the 0S, 30S and 60S treatments.
10 1000 800 3000
SS limitsa
M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
2 100 100 300
3.2.1. Seedling survival and growth parameters One year after the SS application, L. decidua exhibited a higher survival rate (98.3%). The stem height growth of the L. decidua seedlings receiving SS increased significantly compared to the control seedlings (Fig. 2). This increase appeared for both treatment rates after 7 months of culturing; however, between seedlings having received the 30S treatment as opposed to the 60S, no differences were found. Significant increases in biomass accumulation were observed in both the aerial and root parts compared to the control (Fig. 3).
a
French regulatory limits (08/01/98) for concentration of TMs in SS for agricultural use.
1.8 0.6 843.8 61.2 15.9 4.5 592.8 32.4 3.7 0.2 0.2 0.1 5.7 0.3 7.2 0.9 41.6 3.4 15.5 2.8 3.9 5.4 1.1 83.5 7.4 5.8 0.4 22.9 1.6 – – – 0.2 0.1 7.1 1.7 13.0 0.8 61.0 3.5 2.7 0.2 0.2 0.1 4.7 0.2 0.1 0.0 2.1 0.2 0.2 0.1 1.1 1.6 0.3 6.4 0.7 20.4 1.9 – 18.6 0.6 38.0 0.7 36.9 0.4 Cd (mg g DW) Cu (mg g1 DW) Pb (mg g1 DW) Zn (mg g1 DW) Mg (mg g1 DW) Mn (mg g1 DW) pH water (2:5) N total % C total % P total (mg g1 DW) P olsen % K (mg g1 DW) OM total % Ratio C:N DW (g l1) Clay % Loam % Sand %
Sewage sludge (SS) Soil
1
Parameters (unit)
Table 1 Characteristics of soil and sewage sludge used in this experiment. Data are the means SD for the 5 repetitions.
Soil limitsa
3.2. Effects of sludge applications on larch seedlings
3.2.2. Water status and concentrations of nutrients and TMs Significant increases in the N content (%) were observed in both the needles and lateral roots of the treated seedlings, whereas no pronounced changes in P concentrations were observed (Table 3). In the needles, the N content was approximately twice as high as in the lateral roots for all experimental setups. However, no differences in water potential were found for either of the two SS applications after 12 months of culturing. The water potentials ranged between 1.27 and 1.05 MPa. Also, K exhibited the same pattern as Mn. The concentrations of these elements increased significantly in both the needles and lateral roots for the highest SS application rate. There was no treatment effect on the Mg, Zn, Pb and Cd concentrations. The Cu concentrations were significantly higher in the lateral roots of the 60S seedlings. The Cu ratio between the lateral roots and needles was 5.2, 4.7 and 10.4 for the 0S, 30S and 60S treatments, respectively. The mineral element and TM contents of all the plants are presented in Fig. 4. The total amount of these elements increased significantly in treated seedlings. In addition, with the exception of Cu, there were no significant differences between the two SS application rates. 3.2.3. CO2 assimilation (Pn) and chlorophyll content The effects of SS on photosynthetic parameters are shown in Fig. 5. Pn increased significantly with increasing SS application rates; the average increases relative to the control were 30% and 47% in 30S and 60S plants, respectively. In contrast, the stomatal conductance (Gs) and intercellular CO2 (Ci) levels decreased significantly compared to the control plants except for Gs measured in September, which increased compared to the control. Furthermore, Pn decreased during the June–September campaign by 39%, 36% and 35% in larch grown with the 0S, 30S and 60S treatments, respectively. The Chlorophyll a contents increased significantly in the needles of the larch grown in SS-amended soil compared to those grown in unaltered soil. The Chlorophyll a contents were 53% and 54% higher in 30S and 60S plants, respectively, compared to 0S plants (Fig. 6). The Chlorophyll a remained constant; no differences between the treatments were detected. 4. Discussion This study indicates that the SS applications used showed positive effects on seedling survival and growth, associated with
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Table 2 Properties of different soil horizons for pots 12 months after sewage sludge application (Sl: sludge level; Ts: top soil level; Bs: bottom soil layer) for the control (0S) and amended (30S: 30 Mg dry weight (DW) ha1; 60S: 60 Mg DW ha1) soils. Data are the means SD for the 5 repetitions. Values followed by different letters differ significantly (p < 0.05). 30S
0S
60S
pH
Sl Ts Bs
– 4.8 0.1b 4.5 0.3a
5.8 0.1 4.3 0.1a 4.4 0.2a
5.6 0.1 4.3 0.1a 4.5 0.2a
N mg g1 DW
Sl Ts Bs
– 1.3 0.1a 1.2 0.1a
22.8 5.5 2.3 0.6b 1.2 0.1a
26.2 4.5 2.7 1.0b 1.5 0.6a
C:N
Sl Ts Bs
– 17.1 1.2b 15.7 0.4a
7.6 0.2 12.5 1.3a 15.7 0.9a
7.3 0.2 11.4 2.4a 14.2 1.9a
P mg g1 DW
Sl Ts Bs
– 0.1 0.0a 0.1 0.0a
10.2 0.7 0.6 0.1b 0.1 0.0a
9.2 0.8 1.1 0.2c 0.2 0.1a
K mg g1 DW
Sl Ts Bs
– 1.6 0.1a 1.5 0.2a
1.8 0.1 1.4 0.1a 1.3 0.1a
1.9 0.2 1.5 0.2a 1.5 0.1a
Mg mg g1 DW
Sl Ts Bs
– 2.4 0.1a 2.3 0.1a
3.9 0.2 2.4 0.1a 2.2 0.1a
3.6 0.3 2.3 0.2a 2.4 0.1a
Mn mg g1 DW
Sl Ts Bs
– 0.1 0.0a 0.1 0.0a
0.6 0.1 0.1 0.0a 0.1 0.0a
0.7 0.2 0.1 0.0a 0.1 0.0a
Cu mg g1 DW
Sl Ts Bs
– 7.9 0.3a 7.9 0.4a
799.4 34.0 21.3 1.3b 7.6 0.4a
689.9 57.2 33.6 4.2c 8.4 0.4a
Zn mg g1 DW
Sl Ts Bs
– 35.2 1.4a 35.2 1.5a
543.4 40.1 42.3 2.7b 34.7 1.1a
486.6 35.9 49.6 3.5c 35.7 0.7a
Pb mg g1 DW
Sl Ts Bs
– 18.8 1.3a 19.5 2.4a
46.9 1.2 20.5 0.4a 18.2 1.1a
32.5 3.9 19.5 1.1a 18.8 0.5a
Cd mg g1 DW
Sl Ts Bs
– 0.1 0.0a 0.1 0.0a
2.1 0.3 0.2 0.0b 0.1 0.1a
1.7 0.2 0.2 0.0b 0.1 0.0a
organic matter, total nitrogen and mineral enrichment in the soil. Twelve months after sludge application, the C:N ratio was significantly lower in the Ts level of the 30S and 60S plants, whereas no changes appeared in the Bs level. This decrease was primarily due to the high nitrogen supply in the sludge. In fact, the
upper soil layers were enriched in total nitrogen by up to 1.8 times for 30S and 2.1 times for 60S. Since the Bs layer remained unchanged we can conclude that after one year of culturing no downward leaching occurred. Similar results have been presented by Egiarte et al. (2005) and Mañas et al. (2010).
Table 3 Mineral element and trace metal concentrations in needles and lateral roots of larch plants (Larix decidua) after 12 months of growth in control (0S) and amended (30S: 30 Mg DW ha1; 60S: 60 Mg DW ha1) soils. Data are the means SD for the 5 repetitions. Values followed by different letters differ significantly (p < 0.05). Needles
N (%) P (mg g1) K (mg g1) Mg (mg g1) Mn (mg g1) Cu (mg g1) Zn (mg g1) Pb (mg g1) Cd (mg g1) TMs (mg) %a a
% of Cu from total TMs.
Lateral roots
0S
30S
60S
0S
30S
60S
2.1 0.2a 1.2 0.2a 7.4 1.3a 1.4 0.4a 2.1 0.3a 3.8 1.3a 49.6 9.4a 1.0 0.5a 0.7 0.4a 55.2 9.1a 6.9
2.8 0.5b 1.4 0.4a 8.1 3.0a 1.1 0.2a 3.0 0.6a 4.3 0.5a 66.1 13.1a 1.1 0.5a 0.8 0.1a 72.4 12.4a 5.9
3.7 0.4c 1.1 0.2a 14.3 4.0b 1.2 0.4a 4.8 1.6b 3.9 1.1a 57.9 19.3a 1.2 0.3a 1.0 0.2a 63.9 19.7a 6.1
1.2 0.1a 0.9 0.1a 4.3 0.3a 0.8 0.1a 0.4 0.0a 19.9 3.0a 89.6 13.0a 2.2 0.2a 0.9 0.4a 112.6 10.9a 17.7
1.7 0.1b 1.1 0.2a 5.0 0.5a 0.8 0.2a 0.5 0.2a 20.4 6.1a 96.1 26.2a 2.3 0.2a 0.6 0.3a 119.5 31.5a 17.1
1.9 0.2b 1.1 0.2a 5.7 0.6b 0.9 0.1a 0.9 0.2b 40.6 18.5b 100.6 14.7a 2.3 0.4a 0.7 0.3a 144.4 27.3a 28.1
M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
c
6
Pn (µmol CO2 m-2 s -1)
221
b
c
a
c
b a
4
c
b a
a
b
2
0
Gs (mol m -2s-1)
0.15
c b
0.10
c b
a
b
a
b
b
0.05
b
a a
0 0.00
400 Ci (µmol mol -1)
c
c b
a
c b
300
a
b b
a
b a
200 100 0 0S 30S 60S 0S 30S 60S 0S 30S 60S 0S 30S 60S June July September August
Fig. 4. Changes in the mineral element and trace metal contents of Larix decidua (entire plant) grown in control (0S) and amended (30S: 30 t DW ha1; 60S: 60 t DW ha1) soils after 12 months of growth (n = 5). The data are presented in standard box-plots. The box is delimited below and above by the lower and upper quartiles, respectively; the thick middle-line represents the median. The lower and upper whiskers represent the sample minimum and maximum, respectively. The different letters above the boxes indicate significant differences according to the Tukey test at p < 0.05.
The average concentrations of TMs in the SS were 843.8, 592.8, 15.9 and 1.8 mg g1 DW for Cu, Zn, Pb and Cd, respectively. Thus, this sewage sludge qualified for application onto the land. In contrast, the mean detected levels of Cu, Zn, Pb and Cd in the soil of Mélisey were 7.1, 61, 13, and 0.2 mg g1 DW, respectively. These concentrations were well below the authorized limits for SS application, which were 100, 300, 100 and 2 mg g1 DW for Cu, Zn, Pb and Cd, respectively. Cu, Zn and Cd contents were even higher in the sludge than in the soil, and the level in Ts of 30S and 60S treatments increased significantly for both elements. Many authors (Baize et al., 2006; Jalali and Khanlari, 2006) have reported that the surface horizon of amended soil showed a significant increase in Cu, Zn, Pb and Cd levels, suggesting that these elements were immobilized in stable chemical forms with organic matter. In the case of Pb, the Mélisey soil was naturally quite high which explains why no increase in this element was detected in the upper layers of the pots, whether the treatment was 30S or 60S. Higher accumulation in the dry matter was possibly due to the increased vegetative growth that resulted from greater photosynthetic activity, enhanced under the effect of SS additions. In fact, the increased N content in the soil altered that found in the needles; this change would become more apparent after analysis of
photosynthesis rates. In addition, a significant increase was observed in the total N content in the needles and lateral roots of the treated plants, the consequence of increased assimilation of the N provided by the sludge which, in turn, enriched the Ts layers. Furthermore, the N rate in needles was 2.1% in the control (0S) (reported in the dry matter) and 2.8% and 3.7% for the 30S and 60S treatments, respectively. According to Bonneau (1988), 2.3% N is the upper limit of the optimum range for larch. Therefore, the larch receiving SS had mean needle N concentrations that exceeded the optimal range. Numerous studies have demonstrated the positive effect of sludge application on photosynthesis rates (Antolín et al., 2010; Song and Lee, 2010; Song et al., 2013). Specifically, organic matter has been shown to increase CO2 assimilation by increasing the chlorophyll content and interfering with the enzyme activities that are involved in carbohydrate metabolism later used in the photosynthesis cycle (Nardi et al., 2002). Increasing photosynthetic activity causes a decrease in the pool of substrate-sequestering carbon, which reduces stomatal conductance and limits plant transpiration (Jarvis and Davies, 1998). Regardless of the photosynthesis rate, the plant water status remained constant; no differences between the treatments were detected. The photosynthesis rates of both the control (0S) and the treated
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160
800
b 80
a 40
K (mg plant -1)
b
600
a 200 0
200
100
b Mg (mg plant -1)
b
160 120 80
a
40
60
a
20
3
6
c
a
b
Zn (mg plant -1)
b
2
a 2
0
300
150
b
200
a
b
4
0
b
b
40
0
100
b
80
0
1
b
400
0
b Cd (µg plant -1)
Pb (µg plant -1)
Cu (mg plant -1)
Mn (mg plant -1)
P (mg plant -1)
b 120
b
100
a 50
0
0
0S
30S
60S
0S
30S
60S
Fig. 5. Changes in the CO2 assimilation rate (Pn), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) of Larix decidua grown in control (0S) and amended (30S: 30 t DW ha1; 60S: 60 t DW ha1) soils. The data are presented in standard box-plots. The box is delimited below and above by the lower and upper quartiles, respectively; the thick middle-line represents the median. The lower and upper whiskers represent the sample minimum and maximum, respectively. The different letters above the boxes indicate significant differences according to the Tukey test at p < 0.05.
plants (30S and 60S) remained relatively constant between June and July before declining significantly to low levels during the last two sampling periods. The widespread decrease in Pn for all experimental setups was an indicator of needle progressive senescence, which was expected since larch is a deciduous conifer. This phenomenon is attributed to the process of nitrogen remobilization, which is strongly associated with the subsequent degradation of chlorophyll and protein reserves (He and Jin, 1999), including RuBisCO (Jiang et al., 1993). Moreover, SS applications significantly increased the total P concentrations in the pots’ upper layers whereas no change was observed below, once again indicating absence of downward leaching. Warman and Termeer (2005) reported that organic
amendments must be mineralized in order to release enough available P, an often slow process in newly amended soils. The water-soluble P contents of the soils were relatively low even with large amounts of SS added (Krogstad et al., 2005). Therefore, only a small fraction of the total P remained available to the plants, thus explaining the less than optimal phosphorus contents in the larch needles (0.22–0.3%) (Bonneau, 1988). Compared to the control seedlings, a substantial increase was observed in the K and Mn concentrations in needles and lateral roots of plants having received the 60S treatment. Nevertheless, no significant enrichment was detected in the topsoil, whereas the lower layers exhibited a depletion in these elements in the treated
M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
223
10 b
b
Chlorophyll content (mg g-1 FM)
8
6
a
4 a a
2
a
0 Chl a
Chl b
Fig. 6. Changes in the chlorophyll contents of needles from larch plants (Larix decidua) grown in control (0S: white column) and amended (30S: 30 t DW ha1 – gray column; 60S: 60 t DW ha1 – black column) soils from the first photosynthesis rate measurement (June). The different letters above the columns indicate significant differences according to the Tukey test at p < 0.05. The vertical bars indicate standard deviations () for the 5 repetitions.
soils at harvest time. Also, the SS applications had no effect on Mg concentrations in tissues. This may be attributed to the fact that SS contains low concentrations of K, Mn and Mg (Lopez-Mosquera et al., 2000; Munir and Bayan, 2004). It is therefore not surprising that the mineral state did not differ according to treatment. Plant growth was accelerated by the excessive use of N. Furthermore, the available mineral elements were diluted in the plant tissues because the dry matter yield was lower for the control than for the SS treatments. Since the treated plants yielded a greater biomass, their tissue concentrations of these elements became diluted (Bhattacharya et al., 2009). Finally, the increase in TM contents did not affect plant growth; throughout the experiment growth was not restricted. The results indicate that higher biomass accumulation is an important factor in enhancing the efficiency of firewood production. Thus, SS-amended soils can promote higher biomass yields which increase the heavy metal bioaccumulation capacity of the plants. 5. Conclusion By increasing the nitrogen supply, sewage sludge may have indirect effects on plant growth. Any bioaccumulated TM would be diluted in the presence of greater biomass tissue, at which point no adverse effects would occur. In this study, SS application increased the nutrient and TM amounts in the soils which were less susceptible to leaching toward deeper soil layers. However, a distinction should be made between the sludge rates with respect to nutrient availability in order to avoid excessive application of sludge. Since the differences between the 30S and 60S treatments were not significant, the lower rate should be sufficient to fertilize young larch. Though this study establishes the benefits of municipal SS and demonstrates that the risks involved are minimal, further studies are needed to determine long-term TM behavior in the soil–plant continuum, with emphasis on assessing the potential transfer into groundwater and from there into the food chain. Acknowledgments The authors are grateful to the French Agency for Environment and Energy Management (ADEME – France), the Regional Council (Conseil Régional) of Franche-Comté and the Rhone-Mediterranean and Corsica Water Agency (Agence de l'Eau Rhône-Méditerranée & Corse) for their financial support.
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Legroux, J.P., Truchot, C., 2009. Bilan de dix années d'application de la réglementation relative à l'épandage des boues issues du traitement des eaux usées. Rapport n 771CGAAER du ministère de l'écologie, du développement et de l'aménagement durables (direction de l'eau) et du ministère de l'agriculture et de la pèche (direction générale de la forêt et des affaires rurales). French. Lopez-Mosquera, M.E., Moiron, C., Carral, E., 2000. Use of dairy industry sludge as fertilizer for grassland in northwest Spain: heavy metal levels in the soil and plants. Resour. Conserv. Recycl. 30, 95–109. Mañas, P., Castro, E., Vila, P., de las Heras, J., 2010. Use of waste materials as nursery growing media for Pinus halepensis production. Eur. J. Forest Res. 129, 521–530. Matsubara, M., Itoh, T., 2006. The present situation and future issues on benefical use of sewage sludge. Saisei to riyo (Association for Utilization of Sewage Sludge) 29, 19–27. McBride, M.B., 2003. Toxic metals in sewage sludge-amended soils: has promotion of beneficial use discounted the risks. Adv. Environ. Res. 8, 5–19. McClellan, K., Halden, R.U., 2010. Pharmaceuticals and personal care products in archived U.S. biosolids from the 2001 EPA national sewage sludge survey. Water Res. 44, 658–668. Mosquera-Losada, M.R., Lopez-Diaz, L., Rigueiro-Rodriguez, A., 2001. Sewage sludge fertilisation of a silvopastoral system with pines in northwestern Spain. Agrofor. Syst. 53, 1–10. Munir, J.M., Bayan, M.A., 2004. Changes in soil fertility and plant uptake of nutrients and heavy metals in response to sewage sludge application to calcareous soils. Agron. J. 3, 229–236. Nardi, S., Pizzeghello, D., Muscolo, A., Vianello, A., 2002. Physiological effects of humic substances on higher plants. Soil Biol. Biochem. 34, 1527–1536.
Robakowski, P., Wyka, T., Samardakiewicz, S., Kierzkowski, D., 2004. Growth, photosynthesis, and needle structure of silver fir (Abies alba Mill.) seedlings under different canopies. For. Ecol. Manag. 201, 211–227. Song, U., Lee, E.J., 2010. Ecophysiological responses of plants after sewage sludge compost applications. J. Plant Biol. 53, 259–267. Song, U., Waldman, B., Lee, E.J., 2013. Ameliorating topsoil conditions by biosolid application for a waste. Int. J. Environ. Res. 7, 1–10. Tsakou, A., Roulia, M., Christodoulakis, N.S., 2003. Growth parameters and heavy metal accumulation in Poplar tree cultures (Populus euramericana) utilising water and sludge from a sewage treatment plant. Bull. Environ. Contam. Toxcol. 71, 330–337. Vaitkuté, D., Baltrénaité, E., Booth, C.A., Fullen, M.A., 2010. Doses sewage sludge amendment to soil enhance the development of silver birch and Scots pine. Hung. Geogr. Bull. 59, 393–410. Veeresh, H., Tripathy, S., Chaudhuri, D., Ghosh, B.C., Hart, B.R., Powell, M.A., 2003. Changes in physical and chemical properties of three soil types in India as a result of amendment with fly ash and sewage sludge. Environ. Geol. 43, 513–520. Wang, Q.R., Cui, Y.S., Liu, X.M., Dong, Y.T., Christrie, P., 2003. Soil contamination and plant uptake of heavy metals at polluted sites in China. J. Environ. Sci. Health 38, 823–838. Warman, P.R., Termeer, W.C., 2005. Evaluation of sewage sludge, septic waste and sludge compost applications to corn and forage: yields and N, P and K content of crops and soils. Bioresour. Technol. 96, 955–961.
Contents lists available at ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Sewage sludge fertilization in larch seedlings: Effects on trace metal accumulation and growth performance Mohamed Bourioug a , Laurence Alaoui-Sehmer b , Xavier Laffray b , Mohammed Benbrahim c , Lotfi Aleya b, * , Badr Alaoui-Sossé b a b c
Jean-François Champollion University Center for Teaching and Research, Place Verdun, F-81000 Albi, France Université de Franche-Comté, Laboratoire de Chrono-Environnement, UMR CNRS 6249, Besançon, France RITTMO Agroenvironnement, ZA Biopôle, 37 rue de Herrlisheim, CS 80023, F-68025 Colmar Cedex, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 September 2014 Received in revised form 14 January 2015 Accepted 20 January 2015 Available online xxx
The spreading of sewage sludge (SS) among forest plantations may provide interesting results for firewood production. While sludges are good fertilizers, they may nevertheless contain trace metals, which can reduce productivity and lead to environmental risks. We investigated the effects of SS application on nutrient uptake and growth parameters in larch seedlings (Larix decidua) and determined trace metal and mineral distribution. Without incorporation into the soil, sludge was applied to the soil surface at three rates (0, 30 and 60 t dry weight DW ha1). The plants were harvested after 12 months. The results showed significantly increased nitrogen and phosphorus concentrations in the top soil layer in pots amended with sludge, whereas no changes appeared in the lower layers. Similar results were obtained for the Cu, Zn and Cd concentrations. However, no differences were observed for the other measured soil mineral elements. Nitrogen concentrations in needles increased with rising sewage sludge application rates, yet the sludge had no effect on the P, Mg, Zn, Pb and Cd concentrations. In addition, Cu accumulated only in the lateral roots of seedlings that received the highest sludge loading rate. Sludge application improved the net photosynthesis, which resulted in higher chlorophyll contents in the needles. Following application, the dry matter accumulation rate increased due to the excessive availability of N, whereas available mineral elements in the plant tissues were diluted. Furthermore, amending the soil with sewage sludge can promote a higher biomass yield which may result in an increased trace metal bioaccumulation capacity in plants. Though this investigation has established the benefits of municipal SS application, further studies are needed to assess the potential transfer of TM to groundwater and through the food chain. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Sewage sludge Larix decidua Trace metals Forest plantations
1. Introduction Wastewater plant operators will continue to face the challenge of disposing of millions of tons of sewage sludge (SS) generated each year (Japan: 70 million, China: 30 million, USA: 6 million and France: 1.2 million) (Matsubara and Itoh, 2006; McClellan and Halden, 2010; Kelessidis and Stasinakis, 2012; Legroux and Truchot, 2009). As SS has a high nutrient content, its increased use as a fertilizer has been recommended as the best practical environmental option for the management of this organic residue. In France about 73% of sludge produced is applied to agricultural land (Legroux and Truchot, 2009). However, sludge contains
* Corresponding author. Tel.: +33 3 81 66 57 64; fax: +33 3 81 66 57 97. E-mail address: lotfi[email protected] (L. Aleya). http://dx.doi.org/10.1016/j.ecoleng.2015.01.031 0925-8574/ ã 2015 Elsevier B.V. All rights reserved.
several potentially harmful constituents such as trace metals (TMs) and organic pollutants which may accumulate in agricultural soil over time (Lopez-Mosquera et al., 2000; McBride, 2003; Chopra et al., 2009). Once accumulated, TMs are highly persistent in the topsoil, can cause potential problems or be transferred through the food chain (Alloway et al., 1991), thus posing a threat to human health (Wang et al., 2003; Chopra et al., 2009). In the case of forest plantations receiving SS application, the risks are mitigated since farmed trees are not a direct part of the human food chain. In addition, this alternative application is promoted as it is thought to enhance both tree growth and wood production (Henry and Cole, 1997; Mosquera-Losada et al., 2001; Bramryd, 2001; Tsakou et al., 2003; Vaitkuté et al., 2010) and to improve several soil characteristics (Barzegar et al., 2002; Veeresh et al., 2003; Hussein, 2009). In France, application of SS to forest land is currently in the experimental phase and requires intensive monitoring of
M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
ecosystem components such as plant, soil, water and fauna, to identify any potential positive and/or negative effects on plant productivity and environmental integrity (Bourioug et al., 2015). The objectives of this investigation were to study the balance between the beneficial effects and eventual toxicity of SS applications on young larch seedlings (Larix decidua) by measuring the effects of two different sludge doses (30 and 60 t dry matter ha1). Several growth parameters, biomass productivity and nitrogen, as well as phosphorus were examined. TM uptake and transfer within plant tissues were also investigated.
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2.3. Photosynthesis measurement and chlorophyll content
The soil used in this experiment was a pseudo luvisol with dysmull, collected from a forested area located at Mélisey, Haute-Saône, France (47 7530 latitude, 6 5800 longitude). Soil samples were collected from within the top 20 cm of the soil layer and sieved through a 1 cm mesh. Aerobically digested SS from a domestic wastewater treatment plant in the village of Mélisey was also used. The physico-chemical characteristics of the soil and SS are provided in Table 1.
The CO2 assimilation rate (Pn), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) were measured using a Li-COR6400 portable photosynthesis system (LI-COR-6400, LI-COR Biosciences, Inc., Lincoln, Nebraska, USA) connected to a conifer chamber. Monthly measurements were obtained from June to September under the ambient conditions of the culture chamber (i.e., 25 C, 60% RH and 200 mmol m2 s1). Only the CO2 concentration was maintained at 400 mmol s1. In addition, needles were collected from both sides of the enclosed branches that were used for gas exchange measurements; the projected areas of the needles were digitally determined in the laboratory. After at least 5 min of steady readings in the chamber, the photosynthesis rate was recorded. All measurements were obtained from a single branch at the same distance from the stem apex. Data were recalculated according to the measured needle area. The collected needles were used to quantify the chlorophyll content, which was extracted using 100% dimethyl sulfamide (Robakowski et al., 2004) and was calorimetrically determined according to Barnes et al. (1992). The results were expressed as milligram of chlorophyll per gram of fresh matter (FM) (mg g1).
2.2. Plant material, growth conditions and experimental setup
2.4. Sampling
One-year-old L. decidua (European larch) seedlings were purchased from a local nursery. On December 22, 2008 the soil samples were placed in 7.5 l plastic pots, each one filled with 6.7 kg of the sampled soil (Fig. 1). During the first 30 days the pots were watered with distilled water three times per week to maintain a constant weight (70% of field capacity). SS was then applied to the soil surface without incorporation into the soil. The rate of sludge was equivalent to (30 t dry weight (DW) ha1: 30S) or twice (60 t DW ha1: 60S) the maximum accumulated amount permitted under French law (i.e., 30 t DW ha1 over a 10-year period). Pots without sludge (0S) were used as the control. Each configuration was replicated five times. The plants were grown for 12 months in a greenhouse with a 14 h photoperiod (200 mmol m2 s1), a temperature regime of 25/14 C (day/night) and 60% relative humidity.
At harvest, an aerial part of each plant was removed, which, by means of the Scholander chamber method, permitted immediate determination of water potential. The aerial part was then cut into separate organs, i.e., needles, the main stem and ramifications. Afterward, the root portion was carefully removed from the pot, separated from the adhering soil and washed with distilled water. The lateral roots were removed from the taproot after their lengths had been measured. Each sample was lyophilized. Also, the dry mass of each sample was determined before being reduced to a fine powder using a ball mill (Retsch MM 200, Germany). Soil samples were simultaneously collected from three levels in each pot as follows: (i) the sludge layer (Sl), corresponding to the upper layer of the pot with a depth of approximately 2 cm. This layer is primarily composed of organic matter that was previously contained in the sludge and formed via mineralization;
2. Materials and methods 2.1. Origin and characterization of soil and sludge
Fig. 1. Schematic representation of the experimental design.
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150
Stem height (cm)
120 b b
90
b b
b b
a
a
a
60 30 0 0
3 6 9 Months after sludge application
12
Fig. 2. Effects of sewage sludge on the growth (variation in height) of larch (Larix decidua) grown in control ( 0S) and amended ( 30S: 30 t DW ha1; 60S: 60 t DW ha1) soils. The data represent the mean of 5 repeated applications; the vertical bars indicate standard deviations (). The different letters indicate significant differences according to the Tukey test at p < 0.05.
(ii) the top soil level (Ts), corresponding to the soil layer at the top of the pot, i.e., immediately under the sludge layer, and with a depth of 5 cm; and (iii) the bottom soil level (Bs), corresponding to the soil layer at the bottom of the pot with a depth of 5 cm. The samples were oven-dried at 60 C for 48 h before being crushed and sieved to 2 mm. 2.5. Chemical analyses
2.6. Statistical analysis The data are presented with standard deviations (). Statistical analysis was performed via one-way analysis of variance (ANOVA) with a multiple-comparison test using Prism 5 (GraphPad Software). Data with probabilities of less than 5% (p < 0.05) were deemed statistically significant. 3. Results
Soil pH was measured in a 2:5 (w/v) ratio of soil to distilled water (Fuentes et al., 2007). The soil redox potential was determined using a Pt electrode inserted 10 cm below the soil surface. Also, the total carbon and total nitrogen in the soil and plants were determined using a dry combustion CNS analyzer (LECO CNS-2000). Approximately 1 g of powdered soil was digested in a 10 ml solution (1:3) (v/v) (HNO3 (65%)/HCl (37%)) at 150 C for 2 h on a hot plate. The sample volume was increased to 40 ml with distilled water. Approximately 0.1 g of the plant sample was added to 2 ml of HNO3 (65%) before digestion on a hot plate for 3 h at 125 C. The TM (i.e., Cu, Zn, Cd and Pb) and mineral element (i.e., P, K, Mg and Mn) concentrations were determined using inductively coupled plasma mass spectroscopy (ICP-MS). The obtained values are in accordance (within 5%) with the certified concentrations in the standard references (calcareous loam soil n 141R from BCR in Belgium, and Virginia tobacco leaves “CTA-VTL-2” in Poland), confirming the reliability of our analytical procedure.
3.1. Effects of SS application on soil 3.1.1. pH and redox potential Only the Ts pH was significantly affected by the SS application, becoming more acidic relative to the control (Table 2). However, no significant differences were found between the 30S and 60S treatments. The soil redox potential ranged from 367 to 422 mV and did not change due to SS additions. 3.1.2. Carbon, nitrogen and phosphorus changes The nitrogen (N) and phosphorus (P) concentrations for the different soil horizons are presented in Table 2. High N and P concentrations were found within the Sl. In addition, a significant enrichment in N and P was also found within the Ts levels from the 30S and 60S treatments compared to the 0S. The C:N ratio in this horizon was indicative of a higher N concentration and was significantly lower than the ratio in the control. In contrast, no changes in these elements were observed in the Bs levels.
60
c
Dry matter (g)
40 20
b a
0 -20
a
-40
b
b
-60 0S
30S
60S
Fig. 3. Changes in total dry matter partitioned to the aerial (white column) and root (black column) parts of the larch plants (Larix decidua) grown in control (0S) and amended (30S: 30 t DW ha1; 60S: 60 t DW ha1) soils after 12 months of growth (n = 5). The different letters indicate significant differences according to the Tukey test at p < 0.05. The vertical bars indicate standard deviations () for the 5 repetitions.
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3.1.3. Other mineral element and trace metal changes Concentrations of the other selected mineral elements and trace metals within the soils are summarized in Table 2. The sludge layers (Sl) with the 30S and 60S treatments exhibited higher TM concentrations compared to the underlying horizons. Cu, Zn and Cd concentrations in the Ts layer increased significantly due to the SS application. For the same horizon, the mineral element (K, Mg and Mn) and Pb concentrations exhibited no significant differences among the 0S, 30S and 60S treatments.
10 1000 800 3000
SS limitsa
M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
2 100 100 300
3.2.1. Seedling survival and growth parameters One year after the SS application, L. decidua exhibited a higher survival rate (98.3%). The stem height growth of the L. decidua seedlings receiving SS increased significantly compared to the control seedlings (Fig. 2). This increase appeared for both treatment rates after 7 months of culturing; however, between seedlings having received the 30S treatment as opposed to the 60S, no differences were found. Significant increases in biomass accumulation were observed in both the aerial and root parts compared to the control (Fig. 3).
a
French regulatory limits (08/01/98) for concentration of TMs in SS for agricultural use.
1.8 0.6 843.8 61.2 15.9 4.5 592.8 32.4 3.7 0.2 0.2 0.1 5.7 0.3 7.2 0.9 41.6 3.4 15.5 2.8 3.9 5.4 1.1 83.5 7.4 5.8 0.4 22.9 1.6 – – – 0.2 0.1 7.1 1.7 13.0 0.8 61.0 3.5 2.7 0.2 0.2 0.1 4.7 0.2 0.1 0.0 2.1 0.2 0.2 0.1 1.1 1.6 0.3 6.4 0.7 20.4 1.9 – 18.6 0.6 38.0 0.7 36.9 0.4 Cd (mg g DW) Cu (mg g1 DW) Pb (mg g1 DW) Zn (mg g1 DW) Mg (mg g1 DW) Mn (mg g1 DW) pH water (2:5) N total % C total % P total (mg g1 DW) P olsen % K (mg g1 DW) OM total % Ratio C:N DW (g l1) Clay % Loam % Sand %
Sewage sludge (SS) Soil
1
Parameters (unit)
Table 1 Characteristics of soil and sewage sludge used in this experiment. Data are the means SD for the 5 repetitions.
Soil limitsa
3.2. Effects of sludge applications on larch seedlings
3.2.2. Water status and concentrations of nutrients and TMs Significant increases in the N content (%) were observed in both the needles and lateral roots of the treated seedlings, whereas no pronounced changes in P concentrations were observed (Table 3). In the needles, the N content was approximately twice as high as in the lateral roots for all experimental setups. However, no differences in water potential were found for either of the two SS applications after 12 months of culturing. The water potentials ranged between 1.27 and 1.05 MPa. Also, K exhibited the same pattern as Mn. The concentrations of these elements increased significantly in both the needles and lateral roots for the highest SS application rate. There was no treatment effect on the Mg, Zn, Pb and Cd concentrations. The Cu concentrations were significantly higher in the lateral roots of the 60S seedlings. The Cu ratio between the lateral roots and needles was 5.2, 4.7 and 10.4 for the 0S, 30S and 60S treatments, respectively. The mineral element and TM contents of all the plants are presented in Fig. 4. The total amount of these elements increased significantly in treated seedlings. In addition, with the exception of Cu, there were no significant differences between the two SS application rates. 3.2.3. CO2 assimilation (Pn) and chlorophyll content The effects of SS on photosynthetic parameters are shown in Fig. 5. Pn increased significantly with increasing SS application rates; the average increases relative to the control were 30% and 47% in 30S and 60S plants, respectively. In contrast, the stomatal conductance (Gs) and intercellular CO2 (Ci) levels decreased significantly compared to the control plants except for Gs measured in September, which increased compared to the control. Furthermore, Pn decreased during the June–September campaign by 39%, 36% and 35% in larch grown with the 0S, 30S and 60S treatments, respectively. The Chlorophyll a contents increased significantly in the needles of the larch grown in SS-amended soil compared to those grown in unaltered soil. The Chlorophyll a contents were 53% and 54% higher in 30S and 60S plants, respectively, compared to 0S plants (Fig. 6). The Chlorophyll a remained constant; no differences between the treatments were detected. 4. Discussion This study indicates that the SS applications used showed positive effects on seedling survival and growth, associated with
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Table 2 Properties of different soil horizons for pots 12 months after sewage sludge application (Sl: sludge level; Ts: top soil level; Bs: bottom soil layer) for the control (0S) and amended (30S: 30 Mg dry weight (DW) ha1; 60S: 60 Mg DW ha1) soils. Data are the means SD for the 5 repetitions. Values followed by different letters differ significantly (p < 0.05). 30S
0S
60S
pH
Sl Ts Bs
– 4.8 0.1b 4.5 0.3a
5.8 0.1 4.3 0.1a 4.4 0.2a
5.6 0.1 4.3 0.1a 4.5 0.2a
N mg g1 DW
Sl Ts Bs
– 1.3 0.1a 1.2 0.1a
22.8 5.5 2.3 0.6b 1.2 0.1a
26.2 4.5 2.7 1.0b 1.5 0.6a
C:N
Sl Ts Bs
– 17.1 1.2b 15.7 0.4a
7.6 0.2 12.5 1.3a 15.7 0.9a
7.3 0.2 11.4 2.4a 14.2 1.9a
P mg g1 DW
Sl Ts Bs
– 0.1 0.0a 0.1 0.0a
10.2 0.7 0.6 0.1b 0.1 0.0a
9.2 0.8 1.1 0.2c 0.2 0.1a
K mg g1 DW
Sl Ts Bs
– 1.6 0.1a 1.5 0.2a
1.8 0.1 1.4 0.1a 1.3 0.1a
1.9 0.2 1.5 0.2a 1.5 0.1a
Mg mg g1 DW
Sl Ts Bs
– 2.4 0.1a 2.3 0.1a
3.9 0.2 2.4 0.1a 2.2 0.1a
3.6 0.3 2.3 0.2a 2.4 0.1a
Mn mg g1 DW
Sl Ts Bs
– 0.1 0.0a 0.1 0.0a
0.6 0.1 0.1 0.0a 0.1 0.0a
0.7 0.2 0.1 0.0a 0.1 0.0a
Cu mg g1 DW
Sl Ts Bs
– 7.9 0.3a 7.9 0.4a
799.4 34.0 21.3 1.3b 7.6 0.4a
689.9 57.2 33.6 4.2c 8.4 0.4a
Zn mg g1 DW
Sl Ts Bs
– 35.2 1.4a 35.2 1.5a
543.4 40.1 42.3 2.7b 34.7 1.1a
486.6 35.9 49.6 3.5c 35.7 0.7a
Pb mg g1 DW
Sl Ts Bs
– 18.8 1.3a 19.5 2.4a
46.9 1.2 20.5 0.4a 18.2 1.1a
32.5 3.9 19.5 1.1a 18.8 0.5a
Cd mg g1 DW
Sl Ts Bs
– 0.1 0.0a 0.1 0.0a
2.1 0.3 0.2 0.0b 0.1 0.1a
1.7 0.2 0.2 0.0b 0.1 0.0a
organic matter, total nitrogen and mineral enrichment in the soil. Twelve months after sludge application, the C:N ratio was significantly lower in the Ts level of the 30S and 60S plants, whereas no changes appeared in the Bs level. This decrease was primarily due to the high nitrogen supply in the sludge. In fact, the
upper soil layers were enriched in total nitrogen by up to 1.8 times for 30S and 2.1 times for 60S. Since the Bs layer remained unchanged we can conclude that after one year of culturing no downward leaching occurred. Similar results have been presented by Egiarte et al. (2005) and Mañas et al. (2010).
Table 3 Mineral element and trace metal concentrations in needles and lateral roots of larch plants (Larix decidua) after 12 months of growth in control (0S) and amended (30S: 30 Mg DW ha1; 60S: 60 Mg DW ha1) soils. Data are the means SD for the 5 repetitions. Values followed by different letters differ significantly (p < 0.05). Needles
N (%) P (mg g1) K (mg g1) Mg (mg g1) Mn (mg g1) Cu (mg g1) Zn (mg g1) Pb (mg g1) Cd (mg g1) TMs (mg) %a a
% of Cu from total TMs.
Lateral roots
0S
30S
60S
0S
30S
60S
2.1 0.2a 1.2 0.2a 7.4 1.3a 1.4 0.4a 2.1 0.3a 3.8 1.3a 49.6 9.4a 1.0 0.5a 0.7 0.4a 55.2 9.1a 6.9
2.8 0.5b 1.4 0.4a 8.1 3.0a 1.1 0.2a 3.0 0.6a 4.3 0.5a 66.1 13.1a 1.1 0.5a 0.8 0.1a 72.4 12.4a 5.9
3.7 0.4c 1.1 0.2a 14.3 4.0b 1.2 0.4a 4.8 1.6b 3.9 1.1a 57.9 19.3a 1.2 0.3a 1.0 0.2a 63.9 19.7a 6.1
1.2 0.1a 0.9 0.1a 4.3 0.3a 0.8 0.1a 0.4 0.0a 19.9 3.0a 89.6 13.0a 2.2 0.2a 0.9 0.4a 112.6 10.9a 17.7
1.7 0.1b 1.1 0.2a 5.0 0.5a 0.8 0.2a 0.5 0.2a 20.4 6.1a 96.1 26.2a 2.3 0.2a 0.6 0.3a 119.5 31.5a 17.1
1.9 0.2b 1.1 0.2a 5.7 0.6b 0.9 0.1a 0.9 0.2b 40.6 18.5b 100.6 14.7a 2.3 0.4a 0.7 0.3a 144.4 27.3a 28.1
M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
c
6
Pn (µmol CO2 m-2 s -1)
221
b
c
a
c
b a
4
c
b a
a
b
2
0
Gs (mol m -2s-1)
0.15
c b
0.10
c b
a
b
a
b
b
0.05
b
a a
0 0.00
400 Ci (µmol mol -1)
c
c b
a
c b
300
a
b b
a
b a
200 100 0 0S 30S 60S 0S 30S 60S 0S 30S 60S 0S 30S 60S June July September August
Fig. 4. Changes in the mineral element and trace metal contents of Larix decidua (entire plant) grown in control (0S) and amended (30S: 30 t DW ha1; 60S: 60 t DW ha1) soils after 12 months of growth (n = 5). The data are presented in standard box-plots. The box is delimited below and above by the lower and upper quartiles, respectively; the thick middle-line represents the median. The lower and upper whiskers represent the sample minimum and maximum, respectively. The different letters above the boxes indicate significant differences according to the Tukey test at p < 0.05.
The average concentrations of TMs in the SS were 843.8, 592.8, 15.9 and 1.8 mg g1 DW for Cu, Zn, Pb and Cd, respectively. Thus, this sewage sludge qualified for application onto the land. In contrast, the mean detected levels of Cu, Zn, Pb and Cd in the soil of Mélisey were 7.1, 61, 13, and 0.2 mg g1 DW, respectively. These concentrations were well below the authorized limits for SS application, which were 100, 300, 100 and 2 mg g1 DW for Cu, Zn, Pb and Cd, respectively. Cu, Zn and Cd contents were even higher in the sludge than in the soil, and the level in Ts of 30S and 60S treatments increased significantly for both elements. Many authors (Baize et al., 2006; Jalali and Khanlari, 2006) have reported that the surface horizon of amended soil showed a significant increase in Cu, Zn, Pb and Cd levels, suggesting that these elements were immobilized in stable chemical forms with organic matter. In the case of Pb, the Mélisey soil was naturally quite high which explains why no increase in this element was detected in the upper layers of the pots, whether the treatment was 30S or 60S. Higher accumulation in the dry matter was possibly due to the increased vegetative growth that resulted from greater photosynthetic activity, enhanced under the effect of SS additions. In fact, the increased N content in the soil altered that found in the needles; this change would become more apparent after analysis of
photosynthesis rates. In addition, a significant increase was observed in the total N content in the needles and lateral roots of the treated plants, the consequence of increased assimilation of the N provided by the sludge which, in turn, enriched the Ts layers. Furthermore, the N rate in needles was 2.1% in the control (0S) (reported in the dry matter) and 2.8% and 3.7% for the 30S and 60S treatments, respectively. According to Bonneau (1988), 2.3% N is the upper limit of the optimum range for larch. Therefore, the larch receiving SS had mean needle N concentrations that exceeded the optimal range. Numerous studies have demonstrated the positive effect of sludge application on photosynthesis rates (Antolín et al., 2010; Song and Lee, 2010; Song et al., 2013). Specifically, organic matter has been shown to increase CO2 assimilation by increasing the chlorophyll content and interfering with the enzyme activities that are involved in carbohydrate metabolism later used in the photosynthesis cycle (Nardi et al., 2002). Increasing photosynthetic activity causes a decrease in the pool of substrate-sequestering carbon, which reduces stomatal conductance and limits plant transpiration (Jarvis and Davies, 1998). Regardless of the photosynthesis rate, the plant water status remained constant; no differences between the treatments were detected. The photosynthesis rates of both the control (0S) and the treated
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M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
160
800
b 80
a 40
K (mg plant -1)
b
600
a 200 0
200
100
b Mg (mg plant -1)
b
160 120 80
a
40
60
a
20
3
6
c
a
b
Zn (mg plant -1)
b
2
a 2
0
300
150
b
200
a
b
4
0
b
b
40
0
100
b
80
0
1
b
400
0
b Cd (µg plant -1)
Pb (µg plant -1)
Cu (mg plant -1)
Mn (mg plant -1)
P (mg plant -1)
b 120
b
100
a 50
0
0
0S
30S
60S
0S
30S
60S
Fig. 5. Changes in the CO2 assimilation rate (Pn), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) of Larix decidua grown in control (0S) and amended (30S: 30 t DW ha1; 60S: 60 t DW ha1) soils. The data are presented in standard box-plots. The box is delimited below and above by the lower and upper quartiles, respectively; the thick middle-line represents the median. The lower and upper whiskers represent the sample minimum and maximum, respectively. The different letters above the boxes indicate significant differences according to the Tukey test at p < 0.05.
plants (30S and 60S) remained relatively constant between June and July before declining significantly to low levels during the last two sampling periods. The widespread decrease in Pn for all experimental setups was an indicator of needle progressive senescence, which was expected since larch is a deciduous conifer. This phenomenon is attributed to the process of nitrogen remobilization, which is strongly associated with the subsequent degradation of chlorophyll and protein reserves (He and Jin, 1999), including RuBisCO (Jiang et al., 1993). Moreover, SS applications significantly increased the total P concentrations in the pots’ upper layers whereas no change was observed below, once again indicating absence of downward leaching. Warman and Termeer (2005) reported that organic
amendments must be mineralized in order to release enough available P, an often slow process in newly amended soils. The water-soluble P contents of the soils were relatively low even with large amounts of SS added (Krogstad et al., 2005). Therefore, only a small fraction of the total P remained available to the plants, thus explaining the less than optimal phosphorus contents in the larch needles (0.22–0.3%) (Bonneau, 1988). Compared to the control seedlings, a substantial increase was observed in the K and Mn concentrations in needles and lateral roots of plants having received the 60S treatment. Nevertheless, no significant enrichment was detected in the topsoil, whereas the lower layers exhibited a depletion in these elements in the treated
M. Bourioug et al. / Ecological Engineering 77 (2015) 216–224
223
10 b
b
Chlorophyll content (mg g-1 FM)
8
6
a
4 a a
2
a
0 Chl a
Chl b
Fig. 6. Changes in the chlorophyll contents of needles from larch plants (Larix decidua) grown in control (0S: white column) and amended (30S: 30 t DW ha1 – gray column; 60S: 60 t DW ha1 – black column) soils from the first photosynthesis rate measurement (June). The different letters above the columns indicate significant differences according to the Tukey test at p < 0.05. The vertical bars indicate standard deviations () for the 5 repetitions.
soils at harvest time. Also, the SS applications had no effect on Mg concentrations in tissues. This may be attributed to the fact that SS contains low concentrations of K, Mn and Mg (Lopez-Mosquera et al., 2000; Munir and Bayan, 2004). It is therefore not surprising that the mineral state did not differ according to treatment. Plant growth was accelerated by the excessive use of N. Furthermore, the available mineral elements were diluted in the plant tissues because the dry matter yield was lower for the control than for the SS treatments. Since the treated plants yielded a greater biomass, their tissue concentrations of these elements became diluted (Bhattacharya et al., 2009). Finally, the increase in TM contents did not affect plant growth; throughout the experiment growth was not restricted. The results indicate that higher biomass accumulation is an important factor in enhancing the efficiency of firewood production. Thus, SS-amended soils can promote higher biomass yields which increase the heavy metal bioaccumulation capacity of the plants. 5. Conclusion By increasing the nitrogen supply, sewage sludge may have indirect effects on plant growth. Any bioaccumulated TM would be diluted in the presence of greater biomass tissue, at which point no adverse effects would occur. In this study, SS application increased the nutrient and TM amounts in the soils which were less susceptible to leaching toward deeper soil layers. However, a distinction should be made between the sludge rates with respect to nutrient availability in order to avoid excessive application of sludge. Since the differences between the 30S and 60S treatments were not significant, the lower rate should be sufficient to fertilize young larch. Though this study establishes the benefits of municipal SS and demonstrates that the risks involved are minimal, further studies are needed to determine long-term TM behavior in the soil–plant continuum, with emphasis on assessing the potential transfer into groundwater and from there into the food chain. Acknowledgments The authors are grateful to the French Agency for Environment and Energy Management (ADEME – France), the Regional Council (Conseil Régional) of Franche-Comté and the Rhone-Mediterranean and Corsica Water Agency (Agence de l'Eau Rhône-Méditerranée & Corse) for their financial support.
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