Physiology Journal

Volume 2014 (2014), Article ID 545967, 15 pages

http://dx.doi.org/10.1155/2014/545967

Research Article

Physiological Responses to Nutrient Accumulation in Trees Seedlings Irrigated with Municipal Effluent in Indian Desert

G. Singh,¹ Madhulika Bhati,¹ T. R. Rathod,¹ and U. K. Tomar²

¹Division of Forest Ecology, Arid Forest Research Institute, New Pali Road, Jodhpur 342005, India

²Division of Forest Genetics and Tree Breeding, Arid Forest Research Institute, New Pali Road, Jodhpur 342005, India

Received 5 February 2014; Revised 25 June 2014; Accepted 2 July 2014; Published 24 July 2014

Academic Editor: Deyue Yu

Copyright © 2014 G. Singh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Leaf water potential (), net photosynthesis rate (), transpiration rate (), stomatal conductance (), and water use efficiency (WUE) are greatly influenced by the nutrient composition of water which is used for irrigating trees. The above-mentioned physiological variables and foliage mineral concentrations were observed for Eucalyptus camaldulensis, Acacia nilotica, and Dalbergia sissoo plants irrigated with municipal effluent (ME) at 1/2 PET (potential evapotranspiration; T₁), 1 PET (T₂), and 2 PET (T₃) rates and the control plants irrigated with canal water at 1PET (T₄). Increased mineral concentrations in order T₁ < T₂ < T₃ enhanced , , , and . Relatively greater increase in than reduced WUE. Available nutrient in ME enhanced physiological function in T₂, whereas reduced quantity of water lowered it in T₁ than in T₄ plants. Differential minerals uptake increased concentrations of N and P in D. sissoo, Mn in E. camaldulensis, and the rest in A. nilotica. was more sensitive to environment than . Enhanced mineral concentration through ME was beneficial but its differential uptake and accumulation influenced physiological functions and WUE. E. camaldulensis is better for high and continuous loading of effluent and A. nilotica is best for high nutrient uptake. D. sissoo is efficient water user.

1. Introduction

Land degradation and contamination of environment from a variety of anthropogenic sources such as smelters, power station industry, the application of metal-containing pesticides, fertilizers and sewage sludge are wide spread [1]. Metals/minerals released into environment do not only become irreversibly immobilized in soil components but are also toxic to animals, plants, and microorganisms [2]. Zinc, nickel, and copper are important constituents of pigments and enzymes. Cadmium, lead, mercury, and copper are toxic at high concentrations because of they disrupt enzyme functions, replace essential metals in pigments, or produce reactive oxygen species [3]. Although some plants have tremendous potential to hyperaccumulate minerals [4, 5], their excess accumulation could have adverse effect on the physiological functions thereby affecting growth and biomass production of various tree species when exposed to wastewater disposal. The problem is further aggravated due to the prevalence of crosstalk across different elements. Incidents of interaction between phosphorus and other macro-and microelements have been reported in crop species [6], whereas nutrient interactions in A. thaliana corroborated the prevalence of crosstalk across P and Fe [7]. In addition, Zn deficiency induced accumulation of P in barley, whereas Pi deficiency in A. thaliana resulted in the suppression of high-affinity Zn transporter ZIP9 [6, 8]. However, the studies pertaining to nutrient interaction have been confined largely to crop species or model plant system.

Unrelenting disposal of effluent of varying chemical constituents is responsible for contamination of land and water bodies, though increased water and nutrients availability by effluent disposal improve photosynthetic capacity of plants [9]. Municipal effluent is a precious resource available in dry regions and is rich in nutrients required for the plants. Rate of photosynthesis, carbon assimilation, and biomass production can be increased in tree by making available this water and nutrients to the nutrient poor soil of the desert region [10]. Though increased photosynthetic efficiency is the most important way of increasing productivity, simultaneous increase in mineral concentrations may affect the efficiency of the species towards efficient utilization of this resource [11]. Protective mechanism of plants by absorption and uptake of minerals from the soil reduces soil toxicity and safeguards environment [12]. But long term disposal may lead to excess accumulation of mineral in biological system and affect physiology and productivity [13]. The extent of influences both on the plant and soil needs to be assessed to avoid mineral toxicity during long term effluent application. The influences may be assessed by measuring foliage mineral concentration in and the physiological functions of tree seedlings used in plantation for efficient utilization of the effluent along with environmental and aesthetic benefits.

Present investigation was undertaken to monitor the effect of varying levels of municipal effluent on minerals accumulation in Eucalyptus camaldulensis Dehnh., Acacia nilotica (L.) Willd. ex Delile and Dalbergia sissoo Roxb. ex DC. seedlings, and the physiological responses in these seedlings in relation to the accumulated minerals. Objectives of this study were to monitor changes in physiological functions of tree seedlings influenced by the mineral accumulation due to municipal effluent irrigation/disposal.

2. Materials and Method

2.1. Site Description

Experiment was conducted in nonweighing in-filled type of lysimeters of capacity 8 m³ (i.e., size of ) at the experimental field of Arid Forest Research Institute, Jodhpur (26°45′N latitude and 72°03′E longitude), in Rajasthan, India. The climate of the site is characterized by hot and dry summer, hot rainy season, warm autumn, and cool winter. The mean annual rainfall of 1998, 1999, and 2000 was 420 mm and the mean annual pan evaporation was 2025 mm. Averages of minimum and maximum air temperatures of a month were 14.5°C and 25.0°C in January, which increased gradually to 34.4°C and 40.7°C, respectively, in May. The soil was loamy sand (coarse loamy, mixed, hyperthermic family of Typic Camborthides, according to US soil taxonomy) with 82% sand, 12% silt, and 6.0% clay. Soil organic matter was 0.13% and available PO₄-P, NO₃-N, and NH₄-N were 5.00, 6.00, and 4.50 mg kg⁻¹, respectively. Soil pH and electrical conductivity (EC) were 7.61 and 0.71 dSm⁻¹, respectively [14].

2.2. Sampling, Preservation and Analysis of the Effluent

Samples of municipal effluent were collected and analyzed as described earlier [14, 15]. Samples were analyzed for pH, electrical conductivity, chemical oxygen demand, biochemical oxygen demand, macro- and micronutrients, total dissolved salts, total solids, and total suspended solids [16]. Nitrogen (N) and phosphorus (P) were analyzed following standard procedure [17]. Calcium (Ca), magnesium (Mg), potassium (K), sodium (Na), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) were estimated by the aqua-regia method of Jackson [17] followed by a measurement of concentrations using an atomic absorption spectrophotometer (model-3110, Perkin-Elmer, Boesch, Huenenberg, Switzerland). Municipal effluent was alkaline (pH 7.60 to 8.02); whereas electrical conductivity ranged from 0.91 to 2.14 dSm⁻¹as described earlier [14]. Biochemical and chemical oxygen demand ranged between 36 and 56 mg L⁻¹ and 190 and 270 mg L⁻¹, respectively. Availability of NH₄-N, NO₃-N, PO₄-P, K, Fe, Cu, Mn, and Zn was always higher in municipal effluent than in the canal water. Calcium and iron were highest in concentrations among the basic cations and micronutrients, respectively. The ratios of K : N, K : Ca and Mg, Mg : Na, Mg : Mn, Fe : Mn, and Zn : Mn in municipal effluent were 0.04, 0.21, 0.31, 0.91, 9.09, and 1.22, respectively (see Supplementary Table 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/545967). These effluent parameters increased during summer (due to high temperature and concentration) and decreased during rainy season (because of addition of runoff water), but highest concentration of NO₃-N during monsoon was due to its addition from the suburban area and the fertilized field through runoff water [14].

2.3. Plantation and Experimental Design

Nursery raised one-year-old seedlings of Acacia nilotica, Dalbergia sissoo, and Eucalyptus camaldulensis from a single provenance were planted in July 1998 in the lysimeters of  m³ capacity, which were filled with soil up to 185 cm leaving 15 cm space for irrigation. There was one seedling in each lysimeter. The plantation was done in completely randomized design with three replications. Irrigation with municipal effluent was initiated in the first week of September 1998 after seedling establishment. Irrigation was based on the potential evapotranspiration (PET) calculated by multiplication of pan evaporation (Class A evaporation pan fixed at the site) rate and pan coefficient (i.e., 0.70) considering the crop coefficient value of 1.2 to 1.5 for Eucalyptus/Alfaalfa [18–21]. Water uses by tree plantation was considered not less than 1.5 times that of agriculture crop or about 1.25 times of Class A pan [22]. Four treatments comprised : irrigation of seedlings with municipal effluent at 1/2 PET; : irrigation of seedlings with municipal effluent at 1 PET; : irrigation of seedlings with municipal effluent at 2 PET, and : irrigation of seedlings with canal water (potable water with low mineral concentration) at 1 PET as control. At the time of treatment application, average seedling heights and collar diameters (12 plants) were (mean ± SE) cm and  cm in E. camaldulensis,  cm and  cm in A. nilotica and  cm, and  cm in D. sissoo, respectively.

2.4. Observation Recording

Leaf water potential (LWP) was measured monthly on leaf discs in a leaf chamber (L-52; Wescor, Logan, Utah, USA) connected to a dew point microvoltmeter (Wescor HR-33T) between 0500 and 0700 hr from December 1998 to November 1999 before the reirrigation of the seedlings in each treatment. Leaf disc of 0.5 cm diameter was punched out from the attached leaves (without leaf abrasion) and was transferred into a leaf chamber and after 15 minutes of equilibration the water potential was determined [23]. The discs were collected at the time of observation recording for each measurement. Net photosynthetic rate , transpiration rate , and stomatal resistance were recorded with open system of portable CO₂ Gas Analyzer, Model CI-301 (CT-301 PS0), CID Inc., Vancouver, USA. Stomatal conductance was calculated as 1/stomatal resistance. These physiological variables were recorded between 10 : 00 and 11 : 00 hrs and at one-month interval from December 1998 to November 2000 (24 months). All these observations were recorded on leaves of middle canopy of the seedlings in three replicates. Self-shading within the cuvette was minimised by ensuring that the leaves did not overlap. Instantaneous water use efficiency (WUE) was calculated as . Atmospheric CO₂ concentration during the experiment period was 380 ppm.

2.5. Mineral Nutrient Analysis

Irrigation quality criteria of municipal effluent and canal water were assessed as described earlier [14, 15]. Leaf samples from the 24-month-old planted seedling were collected in June 2000, washed with tap water, and then rinsed with distilled water. The leaf samples were then oven-dried at 80°C, ground in a palvnizer, and digested with triacid mixture (HNO₃ : H₂SO₄ : HClO₄ in 10 : 4 : 1 ratio). Concentration of K, Ca, Mg, Na, Cu, Fe, Mn, and Zn was determined using atomic absorption spectrophotometer [17]. Measurement of N and P content was performed after wet digestion with 12 mL H₂SO₄ and two Kjeltab (Cu/3.5) catalyst tablets at 350°C for half an hour and estimated using UV-VIS spectrophotometer model 117 at 490 and 420 nm wavelengths, respectively [17].

2.6. Statistical Analysis

Data were statistically analyzed using SPSS statistical package. There were three species and four treatments; hence, the foliage nutrient data were analysed using a two way ANOVA. Tree species and treatments were the factors. Since the physiological data were recorded repeatedly at one-month interval, these data were analysed using repeated measure ANOVA. Physiological parameters per month were the response variables. Month was the within subject factor and tree species and treatments were the between subject factors. Before analysis, average data of these variables were log or reciprocal of square root transformed for normality [24] and homocedasticity [25] in order to make valid statistical inferences about population relationships. Duncan Multiple Range Tests (DMRT) were also performed on each set of data for homogeneous subsetting for treatments and species. Pearson’s correlation was performed to monitor the relations of foliage nutrients concentrations with the physiological variables and total effluent applied. Regressions were performed to observed relations between 24 months average physiological parameters and foliage mineral concentrations.

3. Results

3.1. Environmental Factors

Rainfall was 588.5 mm and total pan evaporation was 5420 mm during December 1998 to November 2000 showing high water deficit. Air temperature, photosynthetically active radiation (PAR), and vapour pressure deficit (VPD) varied between the months (Figure 1). Averages of minimum and maximum air temperatures of a month increased from the lowest at 08 : 00 hr to the highest at 13 : 00 hr and decreased in the evening (17 : 00 hr). Vapour pressure deficit (VPD) increased from 1 290 Pa in January, 1999, to 4660 Pa in April, 1999. PAR was highest at midday (13 : 00) and oscillated between 811 μmol m⁻² s⁻¹ in December, 1999, to 2140 μmol m⁻² s⁻¹ in May, 2000 (Figure 1).

Figure 1: Monthly changes in environmental variables. (a) Rainfall, (b) vapour pressure deficit (VPD), and (c) photosynthetically active radiations (PAR).

3.2. Foliage Nutrient Concentrations

Seedlings irrigated with municipal effluent at T₂ and T₃ levels had higher () concentration of N, P, K, Ca, Mg, Cu, Fe, Mn, and Zn than in the canal water () irrigated seedlings across the species (Table 1). Uptake and accumulation of the above-mentioned nutrients increased () with irrigation quantity from to . When and treatments were compared, concentration of Na in all the species, Ca and Mg in E. camaldulensis and K in D. sissoo seedlings were the lowest in , whereas other nutrients were the lowest in the seedlings of treatment. Concentrations of K, Ca, Mg, Cu, and Mn did not differ () between the seedlings of T₁ and T₄ treatments (DMRT) despite of twofold water applied in latter than in the former treatment (Supplementary Table 2). There was 2% (Mg in ) to 2.9-fold (Mn in T₃) increase in nutrient concentration in ME irrigated seedlings than the respective concentrations in the seedlings of T₄ treatment (Supplementary Table 2). Across the treatments, the concentrations of K, Ca, Mg, Na, Fe, Cu, and Zn were the highest () in A. nilotica, N and P were the highest in D. sissoo and Mn was the highest in E. camaldulensis seedlings. We observed nonsignificant differences in Ca and Cu concentrations between E. camaldulensis and D. sissoo and in Mn concentration between A. nilotica and D. sissoo (DMRT). The concentrations of these nutrients were adequate to high (N, Ca, Mg, Cu, and Fe in E. camaldulensis, Mg, K, Fe, Zn, and Cu in A. nilotica, and P, Ca, Cu, Fe, and Zn in D. sissoo) except in treatment (i.e., N and Cu in marginal concentrations) when compared with the reported literatures [27, 29, 32]. Compared to the nutrient concentrations in the seedlings of T₄, the increase in N and P concentrations in the seedlings of was 1.7 and 3.5-fold in E. camaldulensis, 1.8- and 2.2-fold in A. nilotica and 1.9- and 2.0-fold in D. sissoo. Concentrations of K, Ca, Mg, and Na were 1.1- to 1.3-fold in E. camaldulensis, 1.5- to 1.9-fold in A. nilotica and 1.5- to 3.0-fold in D. sissoo. The increase in Cu, Fe, Mn and Zn was 1.2- to 3.4-fold, 2.0- to 2.3-fold and 2.0- to 2.3-fold in the respective species.

Table 1: Foliage nutrient concentration in 24-month-old planted seedlings of E. camaldulensis (Ec), A. nilotica (An), and Dalbergia sissoo (Ds) irrigated with varying levels of municipal effluent. Mean ± SE of three replications in parentheses.

S
T
N
P
K
Ca
Mg
Na
Cu
Fe
Mn
Zn

g kg−1 DM
mg kg⁻¹ DM

Ec
T₁
20.67fg
0.96fg
11.64f
11.09g
3.66h
1.39g
26.80b
661.69f
169.00b
30.66e

(0.36)
(0.05)
(0.64)
(0.78)
(0.05)
(0.02)
(1.29)
(18.50)
(12.15)
(1.32)

T₂
24.71de
1.23ef
12.63ef
16.31def
4.03h
1.62def
28.40b
825.30e
404.00a
32.53e

(0.79)
(0.09)
(0.39)
(0.51)
(0.08)
(0.06)
(0.42)
(17.03)
(25.75)
(1.13)

T₃
31.72b
1.56cd
15.06de
19.95bc
5.34c
2.06b
28.60b
959.30cd
458.00a
43.50d

(0.63)
(0.06)
(0.65)
(0.64)
(0.43)
(0.05)
(2.60)
(27.22)
(19.20)
(2.06)

T₄
18.92g
0.45h
13.02ef
17.73cde
4.16gh
1.93bc
23.40ab
570.20g
134.70b
26.87e

(1.05)
(0.03)
(0.65)
(0.63)
(0.16)
(0.04)
(2.01)
(18.57)
(10.89)
(0.72)

An
T₁
22.67ef
0.49h
18.41c
17.56bc
4.87de
1.42fg
42.02b
699.00f
64.01cd
45.02d

(0.56)
(0.07)
(0.43)
(2.10)
(0.12)
(0.04)
(2.58)
(20.05)
(6.87)
(3.20)

T₂
25.20cd
0.78gh
21.22bc
19.87bc
5.97b
1.78cd
53.01ab
898.01de
78.02cd
60.33b

(1.23)
(0.02)
(0.55)
(0.35)
(0.17)
(0.08)
(2.28)
(26.86)
(3.80)
(1.60)

T₃
36.72a
1.01fg
23.41ab
22.00ab
8.87a
2.90a
76.80a
1477.01a
117.02bc
78.40a

(1.27)
(0.03)
(2.11)
(0.47)
(0.32)
(0.08)
(2.22)
(21.21)
(5.79)
(2.18)

T₄
20.78fg
0.45h
12.34ef
14.67ef
4.67ef
1.68de
38.40b
684.02f
52.01d
39.62d

(1.13)
(0.03)
(1.52)
(0.37)
(0.07)
(0.13)
(1.30)
(17.34)
(2.26)
(0.52)

Ds
T₁
27.87c
3.70a
12.71ef
13.78fg
2.29i
1.33g
34.80b
575.92g
66.00cd
39.45d

(0.45)
(0.04)
(1.04)
(0.59)
(0.04)
(0.05)
(1.18)
(15.81)
(3.66)
(2.38)

T₂
31.67b
4.10a
13.78ef
15.78def
4.70h
1.53efg
43.70b
705.34f
77.80cd
51.30c

(0.25)
(0.11)
(0.36)
(0.56)
(0.07)
(0.07)
(2.63)
(25.31)
(2.45)
(1.04)

T₃
37.68a
5.70a
20.14c
18.78bcd
5.32c
2.01b
51.60ab
1023.50b
138.59b
63.45b

(0.66)
(0.40)
(0.59)
(0.37)
(0.07)
(0.05)
(2.25)
(62.86)
(2.05)
(2.39)

T₄
19.23g
2.80b
13.78ef
10.71g
1.79i
1.38g
24.50b
515.40g
60.30cd
29.70e

(0.47)
(0.05)
(0.29)
(0.52)
(0.11)
(0.08)
(0.61)
(15.67)
(1.48)
(1.63)

Tow way ANOVA*

S
39.64
966.5
34.99
20.57
484.6
25.73
9.39
67.37
345.3
119.96

T
203.3
136.1
26.44
19.13
255.5
90.31
2.93 NS
221.8
112.1
123.68

S × T
7.10
9.09
7.54
7.40
32.72
8.80
1.82 NS
6.56
3.50
5.19

ANOVA results are significant at except for Cu, which was significant at for species but not significant (NS) at for treatment and species (S) × treatment (T) interaction.

The same letter in the same column means no significant difference () between treatments (all three species).

Differences in relative uptake of nutrients affected the treatments order ( to ) with increasing nutrients concentration ratios (Figure 2). The ratios of K : N, Mg : Na, Zn : Mn, and Mg : Mn differed () due to both tree species and treatments. But significant variation () in Fe : Mn ratio was observed only between the species. Species × treatment interactions were also significant (). Among the treatments, the highest K : N, K: Mg and Ca, and Fe : Mn ratios were in , and Mg : Na, Zn : Mn and Mg : Mn were in T₂ treatments (Figure 2). The lowest ratios of K : N, Fe: Mn, and Zn: Mn were in the seedlings of , Mg : Na in , K : Ca, and Mg in , and Zn : Mn in treatments. Among the species, ratios of K : N, K : Ca and Mg, Mg : Na, Fe : Mn, Mg : Mn, and Zn : Mn ranged from 0.4 to 0.9, 0.6 to 1.1, 1.3 to 4.5, 2.1 to 13.2, 10.1 to 102.7, and 0.1 to 0.8 (Figure 2), respectively.

(a)

(b)

(c)

(d)

(e)

Figure 2: Ratios of mineral elements concentrations and their relationship with , , and    influenced by tree species and level of municipal effluent application. Error bars are ±SE of three replicates. T₁, T₂, and are municipal effluent irrigation levels of PET, 1 PET, and 2 PET, respectively.

3.3. Leaf Water Relations

Leaf water potential () was the highest () in the seedlings of across species (Table 2). The highest was in E. camaldulensis, but it did not differ with D. sissoo for (, DMRT) across the treatments. The lowest was in A. nilotica. Dalbergia sissoo indicated the highest in , whereas E. camaldulensis showed greater in T₃ and treatments (DMRT). The was the highest () in January (December in E. camaldulensis) that decreased gradually to the lowest value in May and then rose in July-August (Table 3). The lowest was recorded for A. nilotica seedlings in most of the months. The was the highest for D. sissoo from April to August and that of A. nilotica from September to October.

Table 2: Average values of physiological variables. Values are mean of 12 data (across treatments) for species and 9 data (across species) for treatments.

Species/treatment
Physiological variables

LWP

s
WUE

Average values across municipal effluent treatments for species

E. camaldulensis
−2.01a
4.72a
3.06a
42.96a
1.56b

A. nilotica
−2.20b
3.82c
2.63c
39.04b
1.44c

D. sissoo
−1.99a
4.53b
2.67b
42.41a
1.66a

Average values across tree species for municipal effluent treatment

T₁
−2.28d
3.35d
2.05d
34.45d
1.57b

T₂
−2.01b
4.70b
3.01b
42.25b
1.55b

T₃
−1.81a
5.84a
3.95a
50.63a
1.48c

T₄
−2.15c
3.52c
2.13c
38.56c
1.62a

with significant level of two-way ANOVA

Species
147.99**
1683.38**
709.21**
61.38**
336.17**

Treatment
335.54**
7552.44**
7335.46**
483.82**
61.14**

Species × treatment
19.81**
86.79**
22.29**
9.16**
25.38**

LWP: leaf water potential (MPa, Mega Pascal); : rate of photosynthesis : (mol CO₂ m⁻² s⁻¹); E: rate of transpiration (mmol m⁻² s⁻¹); s: stomatal conductance ( mol m⁻² s⁻¹), and WUE: instantaneous water use efficiency (/). T₁, T₂, T₃, and T₄ are irrigation of seedlings with municipal effluent at  PET, 1 PET, 2 PET, and canal water at 1 PET, respectively. Significant at . The same letter in the same column means no significant difference () between treatments/species.

Table 3: Leaf water potential (-MPa) of trees seedlings of E. camaldulensis (Ec), A. nilotica (An), and Dalbergia sissoo (Ds) irrigated with varying levels of municipal effluent. Mean ± SE of three replications in parentheses.

S
T
December
January
February
March
April
May
June
July
August
September
October
November

Ec
T₁
1.65
1.71
1.92
2.09
2.65
3.00
2.76
2.76
2.38
2.48
2.65
2.10

(0.04)
(0.03)
(0.04)
(0.05)
(0.05)
(0.02)
(0.04)
(0.05)
(0.09)
(0.06)
(0.06)
(0.04)

T₂
1.38
1.43
1.60
1.93
2.15
2.52
2.11
1.97
2.29
2.35
2.35
1.48

(0.06)
(0.02)
(0.05)
(0.03)
(0.04)
(0.04)
(0.04)
(0.05)
(0.05)
(0.04)
(0.03)
(0.03)

T₃
1.00
1.14
1.43
1.72
1.81
2.20
2.05
1.65
1.50
1.84
2.07
1.33

(0.04)
(0.05)
(0.02)
(0.02)
(0.04)
(0.05)
(0.08)
(0.04)
(0.05)
(0.04)
(0.06)
(0.02)

T₄
1.57
1.63
1.83
2.01
2.04
2.81
2.19
2.19
2.32
2.42
2.47
1.62

(0.05)
(0.03)
(0.04)
(0.04)
(0.05)
(0.04)
(0.07)
(0.06)
(0.07)
(0.04)
(0.06)
(0.03)

An
T₁
1.95
1.92
2.24
2.75
2.80
3.19
2.87
2.47
2.10
2.14
2.01
2.01

(0.06)
(0.03)
(0.04)
(0.14)
(0.04)
(0.06)
(0.06)
(0.05)
(0.06)
(0.06)
(0.06)
(0.07)

T₂
1.85
1.81
1.98
2.23
2.37
2.82
2.51
2.01
1.93
1.98
1.90
1.87

(0.02)
(0.02)
(0.03)
(0.04)
(0.05)
(0.10)
(0.03)
(0.05)
(0.04)
(0.06)
(0.04)
(0.04)

T₃
1.80
1.73
1.89
2.03
2.15
2.63
2.30
1.93
1.82
1.92
1.89
1.82

(0.03)
(0.02)
(0.05)
(0.09)
(0.02)
(0.08)
(0.04)
(0.07)
(0.03)
(0.04)
(0.04)
(0.04)

T₄
1.93
1.91
2.13
2.55
2.61
2.93
2.83
2.32
2.02
2.10
1.99
1.96

(0.02)
(0.02)
(0.04)
(0.05)
(0.03)
(90.1)
(0.02)
(0.02)
(0.07)
(0.05)
(0.03)
(0.05)

Ds
T₁
1.96
1.87
1.95
2.13
2.23
2.39
2.31
2.09
2.01
2.29
2.28
2.09

(0.02)
(0.05)
(0.04)
(0.03)
(0.04)
(0.04)
(0.02)
(0.03)
(0.03)
(0.02)
(0.02)
(0.03)

T₂
1.79
1.57
1.73
1.89
2.07
2.27
2.18
1.92
1.90
2.20
2.07
1.87

(0.03)
(0.05)
(0.03)
(0.06)
(0.05)
(0.03)
(0.01)
(0.02)
(0.02)
(0.02)
(0.03)
(0.02)

T₃
1.63
1.40
1.62
1.77
1.89
2.22
2.01
1.85
1.73
1.89
1.82
1.71

(0.05)
(0.04)
(0.02)
(0.06)
(0.04)
(0.03)
(0.06)
(0.08)
(0.02)
(0.02)
(0.04)
(0.04)

T₄
1.91
1.82
1.84
2.07
2.21
2.26
2.22
1.97
1.98
2.19
2.17
2.01

(0.04)
(0.04)
(0.04)
(0.06)
(0.03)
(0.03)
(0.03)
(0.01)
(0.02)
(0.03)
(0.04)
(0.02)

Repeated Measure ANOVA*

Tests of within subjects effects

Tests of between subjects effects

df
MSE
value
value

df
MSE
value
value

M
11
2.507
435.84
<0.001

S
2
1.948
147.99
<0.001

M × S
22
0.372
64.69
<0.001

T
3
4.417
335.54
<0.001

M × T
33
0.031
5.32
<0.001

S × T
6
0.261
19.81
<0.001

M × S × T
66
0.041
7.06
<0.001

ANOVA results are significant at except in February and March when it was significant at for species × treatment interaction. M, month; S, species and T, treatments.

3.4. Stomatal Conductance

Stomatal conductance across the species increased () in order < < < T₃ (Table 2). Considering species, was highest () in E. camaldulensis and lowest in A. nilotica. However, DMRT showed non-significant difference in between E. camaldulensis and D. sissoo. D. sissoo indicated highest () during April to August ( mol m⁻² s⁻¹) 2000 (August 2000 in all treatments) and in E. amaldulensis in rest of the observations. From the lowest value in December/January, increased by 1.8-fold in , 1.7-fold in and and 1.6-fold in the seedlings of with wide temporal variation (Figure 3, left panels).

(a)

(b)

Figure 3: Monthly changes in stomatal conductance (, (a)) and transpiration rate (, (b)) of tree seedlings irrigated with canal water and varying levels of municipal effluent during 1998-99 and 1999-00. Error bars are ±SE. , , T₃, and are irrigation of seedlings with municipal effluent at PET, 1 PET, 2 PET, and canal water at 1 PET, respectively.

3.5. Transpiration Rate

Rate of transpiration varied () within months, species and treatments. Across the species, was the lowest in the seedlings of . Average was 4% lesser in the seedlings of , but it increased by 46% and 85% in the seedlings of and treatment, respectively, as compared to value in treatment (Table 2). Average across treatments, was the highest () in . camaldulensis and the lowest in A. nilotica seedlings. E. camaldulensis indicated the highest values (maximum of  mmol m⁻² s⁻¹ in August 2000) of during January to April and July to September. A. nilotica indicated the highest ( mmol m⁻² s⁻¹ in December 1999) E during December to February, whereas D. sissoo indicated the highest ( mmol m⁻² s⁻¹ in June 2000) in May and June. Rate of transpiration was the lowest in December/January (Figure 3(b)). It increased in March and April and decreased again in May before approaching the highest value in August. ranged from 0.87 to 3.96 mmol m⁻² s⁻¹ in , 1.40 to 5.60 mmol m⁻² s⁻¹ in , 1.96 to 7.8 mmol m⁻² s⁻¹ in , and 1.0 to 3.99 mmol m⁻² s⁻¹ in treatment, where the highest values were in E. camaldulensis seedlings. Species × treatment interaction was also significant ().

3.6. Net Photosynthesis Rate

Repeated measure ANOVA indicated variations () in net photosynthesis rate due to species, treatments, and months. Average increased with quantity of applied effluent and seedlings of treatments indicated the highest in all species. The lowest was in the seedlings of in most of the months and in in April, May, August, and September. When compared with the seedlings of , increased by 34% and 66% in the seedlings of and , respectively, whereas it was 5% less in treatment. Across the treatments, the highest and lowest values of were in E. camaludensisis and A. nilotica, respectively (Table 2). However, tempral variation indicated the highest in E. camaldulensis in most of the observations (maximum of  μmol CO2 m⁻² s⁻¹ in August 2000, mean ± 1SE), in D. sissoo in April, May, June, and July ( μmol CO2 m⁻² s⁻¹) and in A. nilotica in October 2000 ( μmol CO₂  m⁻² s⁻¹ in ). There was a significant () seasonal pattern in with two maxima, that is, August and again in March/April (Figure 4(a)). value in August was 6.2- to 7.1-fold in , 4.0- to 5.2-fold in , 3.9- to 4.5-fold in , and 4.0- to 5.7-fold in the seedlings of as compared to the respective value in December/January. Seedlings of D. sissoo showed the highest seasonal variations in among the species.

(a)

(b)

Figure 4: Monthly changes in net photosynthetic rate (, (a)) and instantaneous water use efficiency (WUE, (b)) irrigated with canalwater and varying levels of municipal effluents during 1998-99 and 1999-00. Error bars are ±SE. , T₂, , and are irrigation of seedlings with municipal effluent at PET, 1 PET, 2 PET, and canal water at 1 PET, respectively.

3.7. Instantaneous Water Use Efficiency

Repeated measure ANOVA showed significant () variation in water use efficiency (WUE), that is, / (μmol CO2 mmol⁻¹ H₂O) between the species, treatments, and months. WUE was the highest () in and the lowest in seedlings across the species. However, WUE did not differ significantly between the seedlings of and treatments (DMRT). Among the tree species, the highest () WUE was in the seedlings of D. sissoo and the lowest in A. nilotica (Figure 4(b)). When compared with the lowest WUE (observed either in winter or in summer), a 2.1- to 2.6-fold greater WUE was observed in the seedlings of T₁, whereas the increase was 1.9- to 2.4-fold in , 1.7- to 2.6-fold in T₃, and 1.9- to 2.2-fold in treatments. Relative increase in WUE was highest in D. sissoo (Figure 4(a)).

3.8. Correlations and Regressions

Nutrient concentrations in foliage of the seedlings were positively correlated ( to 0.841, , ) to total applied municipal effluent (except for P and Mn). Fe, P, Na, Mn and N showed positive ( to 0.716, ) and K : N ratio showed negative (, ) relationship with . Total quantity of applied effluent showed positive relationship with average , , , and ( to 0.939, ). Concentrations of foliage N, P, Fe, Mn, and Zn ( to 0.728, ) were positively correlated with average . Average showed positive correlations ( to 0.715, ) with N, Ca, Mg, Fe, Mn, and Zn concentrations, but we did not find relations () of these nutrients with stomatal conductance. Ratios of mineral elements did not show significant relationship with physiological variables except for negative correlation of (, ) with K : N; and WUE with K : N (, ), Mg : Na (, ), and Mg : Mn (, ). WUE showed negative correlations ( to −0.684, ) with foliage nutrients concentration in tree seedlings.

Regressions equations (irrespective of species and treatments) between physiological functions and foliage nutrients concentration showed nonlinear relationships (). Nitrogen and P concentrations showed linear relations to and WUE, respectively (Table 4). Net photosynthesis and transpiration rates showed linear relations with slope value of 1.266, 1.067, and 1.605 for E. camaldulensis, A. nilotica, and D. sissoo (Supplementary Figure 1) Both and increased () with increase in nutrient concentrations except Cu, which did not indicate any relation with . WUE was influenced by foliage biochemistry resulting in variations in / ratio, which decreased with increase in nutrient concentration, but P concentration was positively related (Figure 5). WUE increased with increase in Mg : Na, Fe : Mn, Zn : Mn and Mg : Mn ratios but decreased when their ratio increased above 1.9, 6.67, 0.2, and 34.1, respectively. Increase in Ca, Na, and Fe concentrations influenced () positively but their respective concentration of greater than 22.26 g kg⁻¹, 2.76 g kg⁻¹, and 1146 mg kg⁻¹ reduced (Table 4, Figure 5). Likewise greater than 1.65 g kg⁻¹, 21.09 g kg⁻¹, and 2.76 g kg⁻¹ concentrations of P, Ca, and Na respectively, reduced .

Table 4: Regression equations∗ between mineral element concentrations and net photosynthesis rate, transpiration rate, stomatal conductance, and instantaneous water use efficiency as the dependent variables and mineral concentrations as independent variables.

Variable
Equation

SE
value
value

Rate of photosynthesis ()

N
Logarithm
−8.62930
3.97209
—
—
0.5319
0.7837
28.40
0.000

P
Sigmoid
1.69905
−0.23188
—
—
0.3166
0.2094
11.58
0.002

Ca
Cubic
12.29345
−2.00749
0.14362
−0.0031
0.3759
0.9434
4.62
0.011

Mg
Sigmoid
1.75381
−1.10272
—
—
0.1790
0.2295
5.45
0.027

Na
Quadratic
−8.92904
12.89990
−2.77332
—
0.7060
0.6338
28.82
0.000

Fe
Quadratic
−5.02941
0.01869
−0.00001
—
0.5924
0.7464
17.43
0.000

Mn
Inverse
5.97239
−1.44654
—
—
0.4325
0.8629
19.05
0.000

Zn
Power
1.25817
0.33138
—
—
0.1712
0.2306
5.16
0.031

K/N
Compound
6.71215
0.50522
—
—
0.1710
0.2306
5.16
0.032

Fe/Mn
Inverse
4.07304
2.71389
—
—
0.1638
1.0474
4.90
0.036

Rate of transpiration (E)

N
Linear
0.16705
0.09866
—
—
0.5111
0.5815
26.13
0.000

P
Cubic
0.85675
3.25457
−1.13713
0.1113
0.3623
0.6823
4.36
0.014

K
Sigmoid
1.45824
−6.13854
—
—
0.1242
0.2677
3.55
0.071

Ca
Cubic
12.73548
−2.24278
0.14900
0.002986
0.5185
0.6016
8.25
0.000

Mg
Sigmoid
1.45201
−1.17157
—
—
0.3397
0.2325
12.86
0.001

Na
Quadratic
−5.60495
7.83469
−1.57456
—
0.7883
0.3901
44.80
0.000

Fe
Sigmoid
1.96642
−729.363
—
—
0.6725
0.1637
51.33
0.000

Cu
Cubic
13.22883
−0.68615
0.01381
−0.000084
0.2865
0.7323
3.08
0.047

Mn
Inverse
4.01221
−108.593
—
—
0.4637
0.6090
21.61
0.000

Zn
Compound
1.86315
1.00904
—
—
0.2454
0.2485
8.13
0.008

Fe/Mn
Inverse
2.58987
2.01328
—
—
0.1711
0.7571
5.16
0.032

Zn/Mn
Inverse
2.68570
0.07460
—
—
0.1564
0.7638
4.63
0.041

Stomatal conductance (s)

N
Compound
28.39680
1.01473
—
—
0.1622
0.2003
4.83
0.037

Na
Sigmoid
4.24489
−0.79956
—
—
0.2381
0.1910
7.81
0.009

Fe
Sigmoid
4.08435
−256.357
—
—
0.1420
0.2027
4.14
0.052

Mn
Sigmoid
3.9789
−22.8573
—
—
0.2967
0.1835
10.55
0.003

Instantaneous water use efficiency (WUE)

P
Linear
1.43590
0.04460
—
—
0.4673
0.0869
21.93
0.000

K
Compound
1.81403
0.98970
—
—
0.3293
0.0655
12.27
0.001

Ca
Compound
1.8993
0.98773
—
—
0.3669
0.0636
14.49
0.000

Mg
Compound
1.76437
0.97289
—
—
0.6349
0.0483
43.48
0.000

Na
Compound
1.86695
0.89376
—
—
0.4765
0.0579
22.75
0.000

Fe
Compound
1.83373
0.99979
—
—
0.5011
0.0565
25.10
0.000

Cu
Compound
1.71655
0.99728
—
—
0.3036
0.0667
10.90
0.002

Zn
Compound
1.71518
0.99764
—
—
0.2151
0.0708
6.85
0.014

K/N
Inverse
1.28251
0.13853
—
—
0.2370
0.1039
7.76
0.010

Mg/Na
Cubic
0.41192
1.46011
−0.54740
0.0606
0.4673
0.0905
6.72
0.002

Fe/Mn
Quadratic
1.34984
0.09269
−0.00715
—
0.7007
0.0665
28.10
0.000

Zn/Mn
Cubic
1.30110
2.78703
−6.07910
3.5243
0.2924
0.1044
3.17
0.043

Mg/Mn
Cubic
1.30661
0.02323
−0.00047
0.000003
0.6924
0.0688
17.26
0.000

Nonsignificant () relations have not been shown. , , , and are regression constants. R ²: coefficient of determination and SE: standard error.

Figure 5: Relationship of mineral concentrations with average rate of net photosynthesis (, μmol CO2 m⁻² s⁻¹), transpiration (, mmol m⁻² s⁻¹), and instantaneous water use efficiency in tree seedlings irrigated with varying levels of municipal effluent. The observed and estimated trend in values are shown by cross and solid line, respectively.

4. Discussion

4.1. Foliage Nutrients and Water Relations

The results of this study showed beneficial effects of municipal effluent on the physiological functions of E. camaldulensis, A. nilotica, and D. sissoo seedlings. Because of essential in nature, these nutrients were transported to and accumulated () in foliar parts with increased effluent quantity from to T₃. Greater concentrations of the most of the nutrients in the seedlings in and N, P, Fe, and Zn in as compared to the seedlings in T₄ treatment (despite same quantity of water in T₂ and half quantity of water in treatment) were due to the nutrients applied through municipal effluent. Relatively greater accumulation of Mn, Fe, Cu, and Zn (10% to 2.9-fold in effluent irrigated than in T₄) as compared to N, K, Ca, and Mg (2% to 93%) showed an increase in absorption and mobility of the former elements under increased level of effluent irrigation [26]. However, absence of any toxic effect on the tree seedlings showed that the nutrient concentrations were adequate [27–29] or lesser than the critical concentrations observed in other studies [30, 31]. The differential accumulation of nutrients varied with species characteristics influencing and ratios of the nutrient concentrations in plant system. The highest concentration of Ca, Mg, K, Na, Cu, Fe, and Zn in A. nilotica seedlings particularly basic cations (Table 1) was related to reduced and physiological functions by increasing solute concentration. Lesser concentrations of these nutrients in E. camaldulnsis and D. sissoo were due to dilution effects because these species had much broader leaf blades (and increased growth and biomass) than A. nilotica [32]. Relatively greater accumulation of Fe as compared to Mn (high Fe: Mn ratio) in A. nilotica indicated impairing effect of Ca and K reducing Mn concentration, an important constituent (together with Cu, Zn and Fe) of many enzymes influencing physiological function [33]. Higher plants have also evolved sophisticated antioxidant defense system and glyoxalase system to scavenge the oxidative effects of metals [34]. However, the lowest ratio of Fe : Mn in E. camaldulensis was an adaptation/defense mechanism through antioxidative systems (superoxide dismutase) for success of this species under high water availability or waterlogged conditions as observed for Populus angustifolia [35, 36].

Increased in the seedlings from T₁ to was positively influenced by increased level of effluent application-soil water availability [37]. However, higher () in the seedlings of T₄ as compared to the seedlings of treatment was due to two-fold higher water applied to . A difference of 1.14 to 1.35 MPa between the lowest and highest in the seedlings of E. camaldulensis as compared to those of 0.83 to 1.24 MPa in A. nilotica and 0.35 to 0.59 MPa in D. sissoo was due to higher in former than in the latter two species (Table 3). High during December and January indicated low water loss or reduced as a function of low PAR, VPD, low air temperature, and rainfall in January and February, 1999 (Figure 1). However, gradual increase in PAR, VPD, and air temperature with concomitant decrease in from January to May in all the three species indicated negative relations between and these environmental factors.

4.2. Foliage Nutrients and Gas Exchange

Application of municipal effluent had no toxic effect on , , and and seedlings grown with municipal effluent irrigation were capable of maintaining efficient photosynthetic activity throughout the growing season. Higher values of these physiological variables in the seedlings grown in and treatments than in the control () further suggested that leaf function was unimpaired by the municipal effluent irrigation. Reduced , , and together with in the seedlings of T₁ indicated negative impact of low water supply [38]. Relatively greater increase in these variables during August (monsoon period, Figures 3 and 4) further suggests that the seedlings of this treatment suffered of water stress [39]. A 15% decrease in has been reported in a two-year-old Picea ruben seedlings at water potential averaging −2.45 MPa [40]. Greater values () of , , and in T₄ than in T₁ seedlings (except in February, 1999, March, May, August, and October, 2000, for , February and May 1999, and February to June, 2000, for E, and May 2000 for ) were due to two-fold water applied [41]. Despite of similar level of irrigation (1PET) increased values of the physiological variables in as compared to the seedlings of were due to nutritional effects of municipal effluent (Figures 3 and 4). Carswell et al. [42] observed an enhanced rate of electron transport and velocity of carboxylation in Cedrela odorata seedlings at 5% rate of macro- and micronutrient supply compared to that at 1% rate. Highest level of irrigation and corresponding increase in water and nutrient supply induced absorption and transport of the nutrients to the seedling resulted in the highest and in the seedlings of . This increase in , and was positively related to and foliage N and other nutrients as observed in Pseudotsuga menziesii (Mirb.) Franco. [43]. However, the higher value of was not paralleled by increased or , which may reflect a partial limitation in foliage biochemistry or leaf structure and varying effects on these physiological variables [44].

Linear/nonlinear increase in with nutrient concentrations suggests a close link of photosynthetic capacity with nutrient supply, but simultaneous increases in and (Figure 5) are indicative of rapid growth and biomass production [45]. A decline/saturation, after an initial increase in and with increase in concentrations of Ca, Na, and Fe, was as a result of the effects of accumulated minerals and limitations due to other nutrients and their ratios [45, 46]. A reduction in net photosynthetic rate and stomatal conductance due to a toxic effect of Na⁺ has also been reported in Citrus limonia Osbeck and Olea europaea L. [47]. Though increases in N, K, Fe, and Mn concentrations were beneficial, but relatively greater increase in N and Mn than K and Fe, respectively, from T₁ to seemed to facilitate to a greater extent than evidenced by increased WUE as observed in D. sissoo discussed later (Supplementary Figure 1). A 4.0- to 7.1-fold variation in compared to 2.2- to 4.0-fold variation in among the months further indicated greater sensitivity of to foliage chemistry as well as environmental factors. Increase in , , and during monsoon and spring due to reduced VPD, PAR, and air temperature though rainfall suggests the effects of environmental factors in influencing physiological variables. Despite of lower nutrient concentrations except Mn (highest) higher , , and in E. camaldulesis were the effects of lower and tolerance to Mn because of scavenging system composed of antioxidants as reported for Mn-tolerant maize (Zea mays L.) [48]. Decrease in the values of these physiological variables during winter (due to plant senescence and reduced VPD and transpiration losses) and summer (due to increase in VPD, PAR, air temperature, desiccating wind velocity, and probably mineral concentrations) was similar to that in Pseudotsuga menziesii [49]. Drops in and as a function of high irradiance/temperature through stomatal control have also been reported by Van Assche and Clijsters [50] and Castillo et al. [51].

4.3. Foliage Nutrients and Water Use Efficiency

Nutrient concentration influenced and and thus instantaneous water use efficiency (WUE). A negative relation of nutrients concentration with WUE suggested impaired effects of K, Ca, Na, and Zn on than on . Increase in was associated with increase in and from to treatments, but greater increase in as compared to due to increased water and nutrient supply from T₁ to T₃ impaired WUE. Ewers et al. [52] observed an increase in transpiration rate in irrigated trees, relative to unirrigated trees by the effect of irrigation combined with fertilization. Low WUE in municipal effluent irrigated seedlings as compared to control ( treatment) was due increased water availability which enhanced to a greater extent (increased by 46% in T₂ and 85% in ) than (increased by 34% in T₂ and 66% in T₃). Higher () WUE in D. sissoo than the other species in most of the months (Figure 4(b)) was due to enhanced foliage and concentrations with greater positive influence on than on . Thus D. sissoo was able to maintain high rates of with relatively low and is considered to be tolerant to low moisture availability and has high WUE [50]. An inverse relation between K : N ratio and WUE (Table 3; Figure 5) also suggests foliar chemistry regulated variations in and . Lowest WUE in A. nilotica was due to relatively greater concentrations (than in other species) of basic cations together with Fe and Zn and lesser concentrations of P and Mn influencing ratio. This type of species-specific response in WUE had also been observed in Vismia japurensis, Bellucia grossularioides, and Laetia procera when treated with P, Ca, and gypsum [53]. After initial increase, a decrease in WUE with increase in ratios of Mg : Mn, Fe : Mn, and Zn : Mn suggested an adverse effect of Mg, Fe, and Zn on WUE at enhanced concentrations. It seemed that Mn played a part in stabilizing mineral ratio to maintain up right  :  ratio (WUE).

5. Conclusions and Recommendation

Irrigating tree seedlings with municipal effluent showed positive influence on nutrient accumulation and physiological functions, that is, , , , and . Enhanced together with and with increased water and nutrient from to T₃ indicated a fast growth in the tree seedlings. Increase in physiological functions in as compared to was the nutrient effects, whereas their increase in T₄ than in T₁ was the effect of water. Relatively higher and lower concentrations of basic cations and Fe influenced gas exchange negatively in A. nilotica and positively in E. camaldulensis, respectively, affecting WUE. A positive effect of N and P on net photosynthesis and that of K on transpiration rate influenced WUE in these seedlings. D. sissoo was efficient water user by maintaining up right ratio between and by accumulating higher N and P, and lower Mg, Na, and Fe concentrations than other mineral nutrients. Adequate concentration of Mg, Na, Fe and Zn enhanced physiological functions, but their higher concentrations adversely affected gas exchange and WUE. Conclusively, higher nutrient accumulation and low WUE in A. nilotica seedling were adaptations towards higher nutrient load and this species can safely be categorized as best soil ameliorator [32]. D. sissoo maintained relatively greater and lesser (a characteristic of efficient water user). E. camaldulensis maintained higher gas exchange by reducing concentration of basic cations and stabilizing Fe : Mn and Mg : Mn ratios and can be better species for long term disposal of municipal effluent.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

The authors are thankful to the Director of Arid Forest Research Institute, Jodhpur, for providing necessary facilities.

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