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

Authors: G. Singh; Madhulika Bhati; T. R. Rathod; U. K. Tomar
Journal: Physiology Journal
 (2014)

---

## Abstract

Leaf water potential (Ψ
l), net photosynthesis rate (P
N), transpiration rate (E), stomatal conductance (g
S), 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 Ψ
l, P
N, E
,and g
S. Relatively greater increase in E than P
N 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. P
N was more sensitive to environment than E. 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.


---

## Body

## 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 inA. thaliana corroborated the prevalence of crosstalk across P and Fe [7]. In addition, Zn deficiency induced accumulation of P in barley, whereas Pi deficiency inA. thaliana resulted in the suppression of high-affinity Zn transporterZIP9 [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 inEucalyptus camaldulensis Dehnh.,Acacia nilotica (L.) Willd. ex Delile andDalbergia 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 2
m
×
2
m
×
2
m) 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 ofAcacia nilotica,Dalbergia sissoo, andEucalyptus camaldulensis from a single provenance were planted in July 1998 in the lysimeters of 2
×
2
×
2 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 forEucalyptus/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 T
1: irrigation of seedlings with municipal effluent at 1/2 PET; T
2: irrigation of seedlings with municipal effluent at 1 PET; T
3: irrigation of seedlings with municipal effluent at 2 PET, and T
4: 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 37.3
±
0.5 (mean ± SE) cm and 0.5
±
0.0 cm inE. camaldulensis, 37.8
±
2.1 cm and 0.5
±
0.1 cm inA. nilotica and 49.8
±
0.3 cm, and 0.5
±
0.0 cm inD. 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 (
P
N
), transpiration rate (
E
), and stomatal resistance (
R
) were recorded with open system of portable CO2 Gas Analyzer, Model CI-301 (CT-301 PS0), CID Inc., Vancouver, USA. Stomatal conductance (
g
S
) 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 P
N
/
E. 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.


## 2.1. Site Description

Experiment was conducted in nonweighing in-filled type of lysimeters of capacity 8 m3 (i.e., size of 2
m
×
2
m
×
2
m) 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 ofAcacia nilotica,Dalbergia sissoo, andEucalyptus camaldulensis from a single provenance were planted in July 1998 in the lysimeters of 2
×
2
×
2 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 forEucalyptus/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 T
1: irrigation of seedlings with municipal effluent at 1/2 PET; T
2: irrigation of seedlings with municipal effluent at 1 PET; T
3: irrigation of seedlings with municipal effluent at 2 PET, and T
4: 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 37.3
±
0.5 (mean ± SE) cm and 0.5
±
0.0 cm inE. camaldulensis, 37.8
±
2.1 cm and 0.5
±
0.1 cm inA. nilotica and 49.8
±
0.3 cm, and 0.5
±
0.0 cm inD. 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 (
P
N
), transpiration rate (
E
), and stomatal resistance (
R
) were recorded with open system of portable CO2 Gas Analyzer, Model CI-301 (CT-301 PS0), CID Inc., Vancouver, USA. Stomatal conductance (
g
S
) 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 P
N
/
E. 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 (Figure1). 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 (P
<
0.05) concentration of N, P, K, Ca, Mg, Cu, Fe, Mn, and Zn than in the canal water (T
4) irrigated seedlings across the species (Table 1). Uptake and accumulation of the above-mentioned nutrients increased (P
<
0.01) with irrigation quantity from T
1 to T
3. When T
1 and T
4 treatments were compared, concentration of Na in all the species, Ca and Mg inE. camaldulensis and K inD. sissoo seedlings were the lowest in T
1, whereas other nutrients were the lowest in the seedlings of T
4 treatment. Concentrations of K, Ca, Mg, Cu, and Mn did not differ (P
>
0.05) 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 T
1) 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 (P
<
0.05) inA. nilotica, N and P were the highest inD. sissoo and Mn was the highest inE. camaldulensis seedlings. We observed nonsignificant differences in Ca and Cu concentrations betweenE. camaldulensis andD. sissoo and in Mn concentration betweenA. nilotica andD. sissoo (DMRT). The concentrations of these nutrients were adequate to high (N, Ca, Mg, Cu, and Fe inE. camaldulensis, Mg, K, Fe, Zn, and Cu inA. nilotica, and P, Ca, Cu, Fe, and Zn inD. sissoo) except in treatment T
4 (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 T
3 was 1.7 and 3.5-fold inE. camaldulensis, 1.8- and 2.2-fold inA. nilotica and 1.9- and 2.0-fold inD. sissoo. Concentrations of K, Ca, Mg, and Na were 1.1- to 1.3-fold inE. camaldulensis, 1.5- to 1.9-fold inA. nilotica and 1.5- to 3.0-fold inD. 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 ofE. 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−1DM
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*
F
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 P
<
0.01 except for Cu, which was significant at P
<
0.05 for species but not significant (NS) at P
>
0.05 for treatment and species (S) × treatment (T) interaction.
The same letter in the same column means no significant difference (P
>
0.05) between treatments (all three species).Differences in relative uptake of nutrients affected the treatments order (T
1 to T
4) with increasing nutrients concentration ratios (Figure 2). The ratios of K : N, Mg : Na, Zn : Mn, and Mg : Mn differed (P
<
0.01) due to both tree species and treatments. But significant variation (P
<
0.05) in Fe : Mn ratio was observed only between the species. Species × treatment interactions were also significant (P
<
0.01). Among the treatments, the highest K : N, K: Mg and Ca, and Fe : Mn ratios were in T
4, 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 T
3, Mg : Na in T
4, K : Ca, and Mg in T
2, and Zn : Mn in T
3 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.Ratios of mineral elements concentrations and their relationship withP
N, E, and g
S  (
×
10
-
2
) influenced by tree species and level of municipal effluent application. Error bars are ±SE of three replicates. T1, T2, and T
3 are municipal effluent irrigation levels of 1
/
2 PET, 1 PET, and 2 PET, respectively.
(a)
(b)
(c)
(d)
(e)


### 3.3. Leaf Water Relations

Leaf water potential (Ψ
l) was the highest (P
<
0.01) in the seedlings of T
3 across species (Table 2). The highest Ψ
l was inE. camaldulensis, but it did not differ withD. sissoo for Ψ
l (P
>
0.05, DMRT) across the treatments. The lowest Ψ
l was inA. nilotica.Dalbergia sissoo indicated the highest Ψ
l in T
2, whereasE. camaldulensis showed greater Ψ
l in T3 and T
4 treatments (DMRT). The Ψ
l was the highest (P
<
0.01) in January (December inE. camaldulensis) that decreased gradually to the lowest value in May and then rose in July-August (Table 3). The lowest Ψ
l was recorded forA. nilotica seedlings in most of the months. The Ψ
l was the highest forD. sissoo from April to August and that ofA. 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
P
N
E
gs
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
F 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);P
N: rate of photosynthesis P
N: (µmol CO2 m−2 s−1); E: rate of transpiration (mmol m−2 s−1); gs: stomatal conductance (×
10
-
3 mol m−2 s−1), and WUE: instantaneous water use efficiency (P
N/E). T1, T2, T3, and T4 are irrigation of seedlings with municipal effluent at 1
/
2 PET, 1 PET, 2 PET, and canal water at 1 PET, respectively. *
*Significant at P
<
0.01. The same letter in the same column means no significant difference (P
>
0.05) between treatments/species.Table 3
Leaf water potential (-MPa) of trees seedlings ofE. 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
F value
P value
df
MSE
F value
P 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 P
<
0.01 except in February and March when it was significant at P
<
0.05 for species × treatment interaction. M, month; S, species and T, treatments.


### 3.4. Stomatal Conductance

Stomatal conductance(
g
S
) across the species increased (P
<
0.01) in order T
1 < T
4 < T
2 < T3 (Table 2). Considering species, g
S was highest (P
<
0.01) inE. camaldulensis and lowest inA. nilotica. However, DMRT showed non-significant difference in g
S betweenE. camaldulensis andD. sissoo.D. sissoo indicated highest (P
<
0.05) g
S during April to August (66.59
±
2.44
×
10
-
3 mol m−2 s−1) 2000 (August 2000 in all treatments) and inE. amaldulensis in rest of the observations. From the lowest value in December/January, g
S increased by 1.8-fold in T
1, 1.7-fold in T
2 and T
3 and 1.6-fold in the seedlings of T
4 with wide temporal variation (Figure 3, left panels).Monthly changes in stomatal conductance (g
S
×
10
-
3, (a)) and transpiration rate (E, (b)) of tree seedlings irrigated with canal water and varying levels of municipal effluent during 1998-99 and 1999-00. Error bars are ±SE. T
1, T
2, T3, and T
4 are irrigation of seedlings with municipal effluent at 1
/
2 PET, 1 PET, 2 PET, and canal water at 1 PET, respectively.
(a)
(b)


### 3.5. Transpiration Rate

Rate of transpiration(
E
) varied (P
<
0.01) within months, species and treatments. Across the species, E was the lowest in the seedlings of T
1. Average E was 4% lesser in the seedlings of T
1, but it increased by 46% and 85% in the seedlings of T
2 and T
3 treatment, respectively, as compared to E value in T
4 treatment (Table 2). Average across treatments, E was the highest (P
<
0.01) in E
. camaldulensis and the lowest inA. nilotica seedlings.E. camaldulensis indicated the highest values (maximum of 7.80
±
0.26 mmol m−2 s−1 in August 2000) of E during January to April and July to September.A. nilotica indicated the highest (2.97
±
0.04 mmol m−2 s−1 in December 1999)E during December to February, whereasD. sissoo indicated the highest (4.97
±
0.10 mmol m−2 s−1 in June 2000) E 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. E ranged from 0.87 to 3.96 mmol m−2 s−1 in T
1, 1.40 to 5.60 mmol m−2 s−1 in T
2, 1.96 to 7.8 mmol m−2 s−1 in T
3, and 1.0 to 3.99 mmol m−2 s−1 in T
4 treatment, where the highest values were inE. camaldulensis seedlings. Species × treatment interaction was also significant (P
<
0.05).


### 3.6. Net Photosynthesis Rate

Repeated measure ANOVA indicated variations (P
<
0.01) in net photosynthesis rate (
P
N
) due to species, treatments, and months. Average P
N increased with quantity of applied effluent and seedlings of T
3 treatments indicated the highest P
N in all species. The lowest P
N was in the seedlings of T
1 in most of the months and in T
4 in April, May, August, and September. When compared with the seedlings of T
4, P
N increased by 34% and 66% in the seedlings of T
2 and T
3, respectively, whereas it was 5% less in T
1 treatment. Across the treatments, the highest and lowest values of P
N were inE. camaludensisis andA. nilotica, respectively (Table 2). However, tempral variation indicated the highest P
N inE. camaldulensis in most of the observations (maximum of 13.56
±
0.34 μmol CO2m−2 s−1 in August 2000, mean ± 1SE), inD. sissoo in April, May, June, and July (6.35
±
0.36 μmol CO2m−2 s−1) and inA. nilotica in October 2000 (5.2
±
0.05 μmol CO2  m−2 s−1 in T
3). There was a significant (P
<
0.01) seasonal pattern in P
N with two maxima, that is, August and again in March/April (Figure 4(a)). P
N value in August was 6.2- to 7.1-fold in T
1, 4.0- to 5.2-fold in T
2, 3.9- to 4.5-fold in T
3, and 4.0- to 5.7-fold in the seedlings of T
4 as compared to the respective P
N value in December/January. Seedlings ofD. sissoo showed the highest seasonal variations in P
N among the species.Monthly changes in net photosynthetic rate (P
N, (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. T
1, T2, T
3, and T
4 are irrigation of seedlings with municipal effluent at 1
/
2 PET, 1 PET, 2 PET, and canal water at 1 PET, respectively.
(a)
(b)


### 3.7. Instantaneous Water Use Efficiency

Repeated measure ANOVA showed significant (P
<
0.01) variation in water use efficiency (WUE), that is, P
N/E (μmol CO2mmol−1 H2O) between the species, treatments, and months. WUE was the highest (P
<
0.01) in T
4 and the lowest in T
3 seedlings across the species. However, WUE did not differ significantly between the seedlings of T
1 and T
2 treatments (DMRT). Among the tree species, the highest (P
<
0.05) WUE was in the seedlings ofD. sissoo and the lowest inA. 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 T
2, 1.7- to 2.6-fold in T3, and 1.9- to 2.2-fold in T
4 treatments. Relative increase in WUE was highest inD. sissoo (Figure 4(a)).


### 3.8. Correlations and Regressions

Nutrient concentrations in foliage of the seedlings were positively correlated (r
=
0.453 to 0.841, P
<
0.05, n
=
27) to total applied municipal effluent (except for P and Mn). Fe, P, Na, Mn and N showed positive (r
=
0.426 to 0.716, P
<
0.05) and K : N ratio showed negative (r
=
-
0.496, P
<
0.05) relationship with Ψ
l. Total quantity of applied effluent showed positive relationship with average Ψ
l, P
N, E
, and g
S (r
=
0.429 to 0.939, P
<
0.05). Concentrations of foliage N, P, Fe, Mn, and Zn (r
=
0.385 to 0.728, P
<
0.05) were positively correlated with average P
N. Average E showed positive correlations (r
=
0.451 to 0.715, P
<
0.05) with N, Ca, Mg, Fe, Mn, and Zn concentrations, but we did not find relations (P
>
0.05) of these nutrients with stomatal conductance. Ratios of mineral elements did not show significant relationship with physiological variables except for negative correlation of P
N (r
=
-
0.412, P
<
0.05) with K : N; and WUE with K : N (r
=
-
0.465, P
<
0.05), Mg : Na (r
=
-
0.554, P
<
0.01), and Mg : Mn (r
=
-
0.488, P
<
0.01). WUE showed negative correlations (r
=
-
0.435 to −0.684, P
<
0.05) with foliage nutrients concentration in tree seedlings.Regressions equations (irrespective of species and treatments) between physiological functions and foliage nutrients concentration showed nonlinear relationships (P
<
0.05). Nitrogen and P concentrations showed linear relations to P
N and WUE, respectively (Table 4). Net photosynthesis and transpiration rates showed linear relations with slope value of 1.266, 1.067, and 1.605 forE. camaldulensis,A. nilotica, andD. sissoo (Supplementary Figure 1) Both P
N and E increased (P
<
0.05) with increase in nutrient concentrations except Cu, which did not indicate any relation with E. WUE was influenced by foliage biochemistry resulting in variations in P
N/E 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 (P
<
0.05) P
N positively but their respective concentration of greater than 22.26 g kg−1, 2.76 g kg−1, and 1146 mg kg−1 reduced P
N (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 E.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
a
0
b
1
b
2
b
3
R
2
SE
F value
P value
Rate of photosynthesis (P
N)
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 (gs)
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 (P
>
0.05) relations have not been shown. a
0, b
1, b
2, and b
3 are regression constants. R
2: coefficient of determination and SE: standard error.Figure 5
Relationship of mineral concentrations with average rate of net photosynthesis (P
N, μmol CO2m−2 s−1), transpiration (E, 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.


## 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 (Figure1). 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 (P
<
0.05) concentration of N, P, K, Ca, Mg, Cu, Fe, Mn, and Zn than in the canal water (T
4) irrigated seedlings across the species (Table 1). Uptake and accumulation of the above-mentioned nutrients increased (P
<
0.01) with irrigation quantity from T
1 to T
3. When T
1 and T
4 treatments were compared, concentration of Na in all the species, Ca and Mg inE. camaldulensis and K inD. sissoo seedlings were the lowest in T
1, whereas other nutrients were the lowest in the seedlings of T
4 treatment. Concentrations of K, Ca, Mg, Cu, and Mn did not differ (P
>
0.05) 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 T
1) 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 (P
<
0.05) inA. nilotica, N and P were the highest inD. sissoo and Mn was the highest inE. camaldulensis seedlings. We observed nonsignificant differences in Ca and Cu concentrations betweenE. camaldulensis andD. sissoo and in Mn concentration betweenA. nilotica andD. sissoo (DMRT). The concentrations of these nutrients were adequate to high (N, Ca, Mg, Cu, and Fe inE. camaldulensis, Mg, K, Fe, Zn, and Cu inA. nilotica, and P, Ca, Cu, Fe, and Zn inD. sissoo) except in treatment T
4 (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 T
3 was 1.7 and 3.5-fold inE. camaldulensis, 1.8- and 2.2-fold inA. nilotica and 1.9- and 2.0-fold inD. sissoo. Concentrations of K, Ca, Mg, and Na were 1.1- to 1.3-fold inE. camaldulensis, 1.5- to 1.9-fold inA. nilotica and 1.5- to 3.0-fold inD. 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 ofE. 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−1DM
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*
F
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 P
<
0.01 except for Cu, which was significant at P
<
0.05 for species but not significant (NS) at P
>
0.05 for treatment and species (S) × treatment (T) interaction.
The same letter in the same column means no significant difference (P
>
0.05) between treatments (all three species).Differences in relative uptake of nutrients affected the treatments order (T
1 to T
4) with increasing nutrients concentration ratios (Figure 2). The ratios of K : N, Mg : Na, Zn : Mn, and Mg : Mn differed (P
<
0.01) due to both tree species and treatments. But significant variation (P
<
0.05) in Fe : Mn ratio was observed only between the species. Species × treatment interactions were also significant (P
<
0.01). Among the treatments, the highest K : N, K: Mg and Ca, and Fe : Mn ratios were in T
4, 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 T
3, Mg : Na in T
4, K : Ca, and Mg in T
2, and Zn : Mn in T
3 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.Ratios of mineral elements concentrations and their relationship withP
N, E, and g
S  (
×
10
-
2
) influenced by tree species and level of municipal effluent application. Error bars are ±SE of three replicates. T1, T2, and T
3 are municipal effluent irrigation levels of 1
/
2 PET, 1 PET, and 2 PET, respectively.
(a)
(b)
(c)
(d)
(e)


## 3.3. Leaf Water Relations

Leaf water potential (Ψ
l) was the highest (P
<
0.01) in the seedlings of T
3 across species (Table 2). The highest Ψ
l was inE. camaldulensis, but it did not differ withD. sissoo for Ψ
l (P
>
0.05, DMRT) across the treatments. The lowest Ψ
l was inA. nilotica.Dalbergia sissoo indicated the highest Ψ
l in T
2, whereasE. camaldulensis showed greater Ψ
l in T3 and T
4 treatments (DMRT). The Ψ
l was the highest (P
<
0.01) in January (December inE. camaldulensis) that decreased gradually to the lowest value in May and then rose in July-August (Table 3). The lowest Ψ
l was recorded forA. nilotica seedlings in most of the months. The Ψ
l was the highest forD. sissoo from April to August and that ofA. 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
P
N
E
gs
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
F 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);P
N: rate of photosynthesis P
N: (µmol CO2 m−2 s−1); E: rate of transpiration (mmol m−2 s−1); gs: stomatal conductance (×
10
-
3 mol m−2 s−1), and WUE: instantaneous water use efficiency (P
N/E). T1, T2, T3, and T4 are irrigation of seedlings with municipal effluent at 1
/
2 PET, 1 PET, 2 PET, and canal water at 1 PET, respectively. *
*Significant at P
<
0.01. The same letter in the same column means no significant difference (P
>
0.05) between treatments/species.Table 3
Leaf water potential (-MPa) of trees seedlings ofE. 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
F value
P value
df
MSE
F value
P 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 P
<
0.01 except in February and March when it was significant at P
<
0.05 for species × treatment interaction. M, month; S, species and T, treatments.


## 3.4. Stomatal Conductance

Stomatal conductance(
g
S
) across the species increased (P
<
0.01) in order T
1 < T
4 < T
2 < T3 (Table 2). Considering species, g
S was highest (P
<
0.01) inE. camaldulensis and lowest inA. nilotica. However, DMRT showed non-significant difference in g
S betweenE. camaldulensis andD. sissoo.D. sissoo indicated highest (P
<
0.05) g
S during April to August (66.59
±
2.44
×
10
-
3 mol m−2 s−1) 2000 (August 2000 in all treatments) and inE. amaldulensis in rest of the observations. From the lowest value in December/January, g
S increased by 1.8-fold in T
1, 1.7-fold in T
2 and T
3 and 1.6-fold in the seedlings of T
4 with wide temporal variation (Figure 3, left panels).Monthly changes in stomatal conductance (g
S
×
10
-
3, (a)) and transpiration rate (E, (b)) of tree seedlings irrigated with canal water and varying levels of municipal effluent during 1998-99 and 1999-00. Error bars are ±SE. T
1, T
2, T3, and T
4 are irrigation of seedlings with municipal effluent at 1
/
2 PET, 1 PET, 2 PET, and canal water at 1 PET, respectively.
(a)
(b)


## 3.5. Transpiration Rate

Rate of transpiration(
E
) varied (P
<
0.01) within months, species and treatments. Across the species, E was the lowest in the seedlings of T
1. Average E was 4% lesser in the seedlings of T
1, but it increased by 46% and 85% in the seedlings of T
2 and T
3 treatment, respectively, as compared to E value in T
4 treatment (Table 2). Average across treatments, E was the highest (P
<
0.01) in E
. camaldulensis and the lowest inA. nilotica seedlings.E. camaldulensis indicated the highest values (maximum of 7.80
±
0.26 mmol m−2 s−1 in August 2000) of E during January to April and July to September.A. nilotica indicated the highest (2.97
±
0.04 mmol m−2 s−1 in December 1999)E during December to February, whereasD. sissoo indicated the highest (4.97
±
0.10 mmol m−2 s−1 in June 2000) E 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. E ranged from 0.87 to 3.96 mmol m−2 s−1 in T
1, 1.40 to 5.60 mmol m−2 s−1 in T
2, 1.96 to 7.8 mmol m−2 s−1 in T
3, and 1.0 to 3.99 mmol m−2 s−1 in T
4 treatment, where the highest values were inE. camaldulensis seedlings. Species × treatment interaction was also significant (P
<
0.05).


## 3.6. Net Photosynthesis Rate

Repeated measure ANOVA indicated variations (P
<
0.01) in net photosynthesis rate (
P
N
) due to species, treatments, and months. Average P
N increased with quantity of applied effluent and seedlings of T
3 treatments indicated the highest P
N in all species. The lowest P
N was in the seedlings of T
1 in most of the months and in T
4 in April, May, August, and September. When compared with the seedlings of T
4, P
N increased by 34% and 66% in the seedlings of T
2 and T
3, respectively, whereas it was 5% less in T
1 treatment. Across the treatments, the highest and lowest values of P
N were inE. camaludensisis andA. nilotica, respectively (Table 2). However, tempral variation indicated the highest P
N inE. camaldulensis in most of the observations (maximum of 13.56
±
0.34 μmol CO2m−2 s−1 in August 2000, mean ± 1SE), inD. sissoo in April, May, June, and July (6.35
±
0.36 μmol CO2m−2 s−1) and inA. nilotica in October 2000 (5.2
±
0.05 μmol CO2  m−2 s−1 in T
3). There was a significant (P
<
0.01) seasonal pattern in P
N with two maxima, that is, August and again in March/April (Figure 4(a)). P
N value in August was 6.2- to 7.1-fold in T
1, 4.0- to 5.2-fold in T
2, 3.9- to 4.5-fold in T
3, and 4.0- to 5.7-fold in the seedlings of T
4 as compared to the respective P
N value in December/January. Seedlings ofD. sissoo showed the highest seasonal variations in P
N among the species.Monthly changes in net photosynthetic rate (P
N, (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. T
1, T2, T
3, and T
4 are irrigation of seedlings with municipal effluent at 1
/
2 PET, 1 PET, 2 PET, and canal water at 1 PET, respectively.
(a)
(b)


## 3.7. Instantaneous Water Use Efficiency

Repeated measure ANOVA showed significant (P
<
0.01) variation in water use efficiency (WUE), that is, P
N/E (μmol CO2mmol−1 H2O) between the species, treatments, and months. WUE was the highest (P
<
0.01) in T
4 and the lowest in T
3 seedlings across the species. However, WUE did not differ significantly between the seedlings of T
1 and T
2 treatments (DMRT). Among the tree species, the highest (P
<
0.05) WUE was in the seedlings ofD. sissoo and the lowest inA. 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 T
2, 1.7- to 2.6-fold in T3, and 1.9- to 2.2-fold in T
4 treatments. Relative increase in WUE was highest inD. sissoo (Figure 4(a)).


## 3.8. Correlations and Regressions

Nutrient concentrations in foliage of the seedlings were positively correlated (r
=
0.453 to 0.841, P
<
0.05, n
=
27) to total applied municipal effluent (except for P and Mn). Fe, P, Na, Mn and N showed positive (r
=
0.426 to 0.716, P
<
0.05) and K : N ratio showed negative (r
=
-
0.496, P
<
0.05) relationship with Ψ
l. Total quantity of applied effluent showed positive relationship with average Ψ
l, P
N, E
, and g
S (r
=
0.429 to 0.939, P
<
0.05). Concentrations of foliage N, P, Fe, Mn, and Zn (r
=
0.385 to 0.728, P
<
0.05) were positively correlated with average P
N. Average E showed positive correlations (r
=
0.451 to 0.715, P
<
0.05) with N, Ca, Mg, Fe, Mn, and Zn concentrations, but we did not find relations (P
>
0.05) of these nutrients with stomatal conductance. Ratios of mineral elements did not show significant relationship with physiological variables except for negative correlation of P
N (r
=
-
0.412, P
<
0.05) with K : N; and WUE with K : N (r
=
-
0.465, P
<
0.05), Mg : Na (r
=
-
0.554, P
<
0.01), and Mg : Mn (r
=
-
0.488, P
<
0.01). WUE showed negative correlations (r
=
-
0.435 to −0.684, P
<
0.05) with foliage nutrients concentration in tree seedlings.Regressions equations (irrespective of species and treatments) between physiological functions and foliage nutrients concentration showed nonlinear relationships (P
<
0.05). Nitrogen and P concentrations showed linear relations to P
N and WUE, respectively (Table 4). Net photosynthesis and transpiration rates showed linear relations with slope value of 1.266, 1.067, and 1.605 forE. camaldulensis,A. nilotica, andD. sissoo (Supplementary Figure 1) Both P
N and E increased (P
<
0.05) with increase in nutrient concentrations except Cu, which did not indicate any relation with E. WUE was influenced by foliage biochemistry resulting in variations in P
N/E 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 (P
<
0.05) P
N positively but their respective concentration of greater than 22.26 g kg−1, 2.76 g kg−1, and 1146 mg kg−1 reduced P
N (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 E.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
a
0
b
1
b
2
b
3
R
2
SE
F value
P value
Rate of photosynthesis (P
N)
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 (gs)
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 (P
>
0.05) relations have not been shown. a
0, b
1, b
2, and b
3 are regression constants. R
2: coefficient of determination and SE: standard error.Figure 5
Relationship of mineral concentrations with average rate of net photosynthesis (P
N, μmol CO2m−2 s−1), transpiration (E, 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 ofE. camaldulensis,A. nilotica, andD. sissoo seedlings. Because of essential in nature, these nutrients were transported to and accumulated (P
<
0.01) in foliar parts with increased effluent quantity from T
1 to T3. Greater concentrations of the most of the nutrients in the seedlings in T
2 and N, P, Fe, and Zn in T
1 as compared to the seedlings in T4 treatment (despite same quantity of water in T2 and half quantity of water in T
1 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 Ψ
l and ratios of the nutrient concentrations in plant system. The highest concentration of Ca, Mg, K, Na, Cu, Fe, and Zn inA. nilotica seedlings particularly basic cations (Table 1) was related to reduced Ψ
l and physiological functions by increasing solute concentration. Lesser concentrations of these nutrients inE. camaldulnsis andD. sissoo were due to dilution effects because these species had much broader leaf blades (and increased growth and biomass) thanA. nilotica [32]. Relatively greater accumulation of Fe as compared to Mn (high Fe: Mn ratio) inA. 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 inE. 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 forPopulus angustifolia [35, 36].IncreasedΨ
l in the seedlings from T1 to T
3 was positively influenced by increased level of effluent application-soil water availability [37]. However, higher (P
<
0.05) Ψ
l in the seedlings of T4 as compared to the seedlings of T
1 treatment was due to two-fold higher water applied to T
4. A difference of 1.14 to 1.35 MPa between the lowest and highest Ψ
l in the seedlings ofE. camaldulensis as compared to those of 0.83 to 1.24 MPa inA. nilotica and 0.35 to 0.59 MPa inD. sissoo was due to higher E in former than in the latter two species (Table 3). High Ψ
l during December and January indicated low water loss or reduced E 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 Ψ
l from January to May in all the three species indicated negative relations between Ψ
l and these environmental factors.


### 4.2. Foliage Nutrients and Gas Exchange

Application of municipal effluent had no toxic effect onP
N, E
, and g
S 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 T
2 and T
3 treatments than in the control (T
4) further suggested that leaf function was unimpaired by the municipal effluent irrigation. Reduced P
N, E
, and g
S together with Ψ
l 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 P
N has been reported in a two-year-oldPicea ruben seedlings at water potential averaging −2.45 MPa [40]. Greater values (P
<
0.05) of P
N, E
, and g
S in T4 than in T1 seedlings (except in February, 1999, March, May, August, and October, 2000, for P
N, February and May 1999, and February to June, 2000, forE, and May 2000 for g
S) were due to two-fold water applied [41]. Despite of similar level of irrigation (1PET) increased values of the physiological variables in T
2 as compared to the seedlings of T
4 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 inCedrela 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 P
N and E in the seedlings of T
3. This increase in P
N, E and g
S was positively related to Ψ
l and foliage N and other nutrients as observed inPseudotsuga menziesii (Mirb.) Franco. [43]. However, the higher value of g
S was not paralleled by increased P
N or E, which may reflect a partial limitation in foliage biochemistry or leaf structure and varying effects on these physiological variables [44].Linear/nonlinear increase inP
N with nutrient concentrations suggests a close link of photosynthetic capacity with nutrient supply, but simultaneous increases in E and g
s (Figure 5) are indicative of rapid growth and biomass production [45]. A decline/saturation, after an initial increase in P
N and E 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 inCitrus limonia Osbeck andOlea 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 T
3 seemed to facilitate P
N to a greater extent than E evidenced by increased WUE as observed inD. sissoo discussed later (Supplementary Figure 1). A 4.0- to 7.1-fold variation in P
N compared to 2.2- to 4.0-fold variation in E among the months further indicated greater sensitivity of P
N to foliage chemistry as well as environmental factors. Increase in P
N, E
, and g
S 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 P
N, E
, and g
S inE. camaldulesis were the effects of lower Ψ
l 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 inPseudotsuga menziesii [49]. Drops in P
N and E 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 influencedP
N and E 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 E than on P
N. Increase in P
N was associated with increase in E and g
S from T
1 to T
3 treatments, but greater increase in E as compared to P
N 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 (T
4 treatment) was due increased water availability which enhanced E to a greater extent (increased by 46% in T2 and 85% in T
3) than P
N (increased by 34% in T2 and 66% in T3). Higher (P
<
0.01) WUE inD. sissoo than the other species in most of the months (Figure 4(b)) was due to enhanced foliage N and P concentrations with greater positive influence on P
N than on E. ThusD. sissoo was able to maintain high rates of P
N with relatively low g
S and E 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 E and P
N. Lowest WUE inA. 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 P
N
/
E ratio. This type of species-specific response in WUE had also been observed inVismia japurensis,Bellucia grossularioides, andLaetia 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 P
N : E ratio (WUE).


## 4.1. Foliage Nutrients and Water Relations

The results of this study showed beneficial effects of municipal effluent on the physiological functions ofE. camaldulensis,A. nilotica, andD. sissoo seedlings. Because of essential in nature, these nutrients were transported to and accumulated (P
<
0.01) in foliar parts with increased effluent quantity from T
1 to T3. Greater concentrations of the most of the nutrients in the seedlings in T
2 and N, P, Fe, and Zn in T
1 as compared to the seedlings in T4 treatment (despite same quantity of water in T2 and half quantity of water in T
1 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 Ψ
l and ratios of the nutrient concentrations in plant system. The highest concentration of Ca, Mg, K, Na, Cu, Fe, and Zn inA. nilotica seedlings particularly basic cations (Table 1) was related to reduced Ψ
l and physiological functions by increasing solute concentration. Lesser concentrations of these nutrients inE. camaldulnsis andD. sissoo were due to dilution effects because these species had much broader leaf blades (and increased growth and biomass) thanA. nilotica [32]. Relatively greater accumulation of Fe as compared to Mn (high Fe: Mn ratio) inA. 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 inE. 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 forPopulus angustifolia [35, 36].IncreasedΨ
l in the seedlings from T1 to T
3 was positively influenced by increased level of effluent application-soil water availability [37]. However, higher (P
<
0.05) Ψ
l in the seedlings of T4 as compared to the seedlings of T
1 treatment was due to two-fold higher water applied to T
4. A difference of 1.14 to 1.35 MPa between the lowest and highest Ψ
l in the seedlings ofE. camaldulensis as compared to those of 0.83 to 1.24 MPa inA. nilotica and 0.35 to 0.59 MPa inD. sissoo was due to higher E in former than in the latter two species (Table 3). High Ψ
l during December and January indicated low water loss or reduced E 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 Ψ
l from January to May in all the three species indicated negative relations between Ψ
l and these environmental factors.


## 4.2. Foliage Nutrients and Gas Exchange

Application of municipal effluent had no toxic effect onP
N, E
, and g
S 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 T
2 and T
3 treatments than in the control (T
4) further suggested that leaf function was unimpaired by the municipal effluent irrigation. Reduced P
N, E
, and g
S together with Ψ
l 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 P
N has been reported in a two-year-oldPicea ruben seedlings at water potential averaging −2.45 MPa [40]. Greater values (P
<
0.05) of P
N, E
, and g
S in T4 than in T1 seedlings (except in February, 1999, March, May, August, and October, 2000, for P
N, February and May 1999, and February to June, 2000, forE, and May 2000 for g
S) were due to two-fold water applied [41]. Despite of similar level of irrigation (1PET) increased values of the physiological variables in T
2 as compared to the seedlings of T
4 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 inCedrela 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 P
N and E in the seedlings of T
3. This increase in P
N, E and g
S was positively related to Ψ
l and foliage N and other nutrients as observed inPseudotsuga menziesii (Mirb.) Franco. [43]. However, the higher value of g
S was not paralleled by increased P
N or E, which may reflect a partial limitation in foliage biochemistry or leaf structure and varying effects on these physiological variables [44].Linear/nonlinear increase inP
N with nutrient concentrations suggests a close link of photosynthetic capacity with nutrient supply, but simultaneous increases in E and g
s (Figure 5) are indicative of rapid growth and biomass production [45]. A decline/saturation, after an initial increase in P
N and E 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 inCitrus limonia Osbeck andOlea 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 T
3 seemed to facilitate P
N to a greater extent than E evidenced by increased WUE as observed inD. sissoo discussed later (Supplementary Figure 1). A 4.0- to 7.1-fold variation in P
N compared to 2.2- to 4.0-fold variation in E among the months further indicated greater sensitivity of P
N to foliage chemistry as well as environmental factors. Increase in P
N, E
, and g
S 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 P
N, E
, and g
S inE. camaldulesis were the effects of lower Ψ
l 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 inPseudotsuga menziesii [49]. Drops in P
N and E 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 influencedP
N and E 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 E than on P
N. Increase in P
N was associated with increase in E and g
S from T
1 to T
3 treatments, but greater increase in E as compared to P
N 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 (T
4 treatment) was due increased water availability which enhanced E to a greater extent (increased by 46% in T2 and 85% in T
3) than P
N (increased by 34% in T2 and 66% in T3). Higher (P
<
0.01) WUE inD. sissoo than the other species in most of the months (Figure 4(b)) was due to enhanced foliage N and P concentrations with greater positive influence on P
N than on E. ThusD. sissoo was able to maintain high rates of P
N with relatively low g
S and E 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 E and P
N. Lowest WUE inA. 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 P
N
/
E ratio. This type of species-specific response in WUE had also been observed inVismia japurensis,Bellucia grossularioides, andLaetia 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 P
N : E ratio (WUE).


## 5. Conclusions and Recommendation

Irrigating tree seedlings with municipal effluent showed positive influence on nutrient accumulation and physiological functions, that is,Ψ
l, P
N, E
, and g
S. Enhanced P
N together with E and g
S with increased water and nutrient from T
1 to T3 indicated a fast growth in the tree seedlings. Increase in physiological functions in T
2 as compared to T
4 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 inA. nilotica and positively inE. 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 P
N and E 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 inA. 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 P
N and lesser E (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.



---

*Source: 545967-2014-07-24.xml*