Effects of simulated acid rain on Shorea macroptera growth and selected soil chemical properties

There is dearth of information on the effect of acid deposition on Shorea macroptera in Malaysia. Thus, this study was conducted to investigate the potential effect of simulated acid rain (SAR) on S. macroptera growth and selected soil chemical properties. Six treatments were evaluated in this study. Growth variables of S. macroptera were observed for 90 days. After 90 days, seedlings and soil were sampled and analyzed using standard methods. The seedlings height decreased with decrease in SAR pH. Chlorosis and necrosis were observed for low SAR pH (pH 3.5 and 4) treatments and this observation explains the reduction of dry matter production of the plants subjected to these treatments. Regardless of treatment, K, Ca, Mg and Na contents in the plants and soil were statistically similar. A similar observation was found for soil exchangeable Fe, Cu, Zn, acidity, Al and H. Thus, it can be concluded that SAR pH of 3.5, 4 and 4.5 affects S. macroptera height, biomass and selected nutrient contents in soil. S. macroptera is susceptible to acid deposition and it could be considered as one of the bio-indicators in Malaysia. A field study is recommended to validate the findings of this study.


INTRODUCTION
Rapid industrialization and unsustainable agricultural practices are some of the possible causes of acid deposition in Malaysia (Ayers et al., 2002).According to Wang et al. (2004), application of urea and animal manure causes ammonia (NH 3 ) accumulation in the atmosphere and long term accumulation may cause acid deposition.Acid deposition has adverse effect on soils and plants.It can reduce plant growth and yield due to foliar injury, low nutrient availability in soils, or exposure of plants to toxic substances that are released from the soil (Liu et al., 2011).
For deciduous trees, slow height increment and low biomass were shown at simulated acid rain (SAR) pH of 2.0 (John et al., 2012).Foliar damages were recorded for red spruce and Brassica napus at SAR pH of 2.5 and 3.0.Mountain birch showed reduced seeds germination when exposed to SAR at pH 4.0 (Ernst, 2012).The SO 4 2-, H + , NO 3 -and NH 4 + input from acid deposition may reduce soil pH.Then, it leads to soil acidification.Higher acidity in soil also increased the solubility of heavy metal (Heij et al., 1991).Due to the serious effect of acid deposition on plants, there is a need for a scientific study of this global problem in Malaysia.
Shorea macroptera is one of the commercial Dipterocarps species in Malaysia.This timber species is classified as hardwood by Malayan Grading Rules *Corresponding author.E-mail: mohamadhilmiibrahim@gmail.com.(Symington, 2004) and commonly has high growth rate (ranging from 5.6 to 8.1 mm year -1 ).This tree is in high demand in local or international markets for timber (Ang and Maruyama, 1995).The growth rate of S. macroptera is mostly affected by the surrounding environment such as sunlight, moisture and nutrients (Manokaran and Kochummen, 1993).Thus, this research was conducted to: (i) determine the effect of simulated acid rain on S. macroptera growth and (ii) determine the effect of simulated acid rain on selected soil chemical properties.

Preparation of simulated acid rain
Original rainwater collected from our study area was used as a control (pH 6 ± 0.02) in this experiment.Preparation of simulated acid rain (SAR) was done using combination of nitric and sulphuric acid at a ratio of 3: 2 (v/v), with original rainwater to obtain pH of 3.5, 4.0, 4.5, 5.0, and 5.5.The ratio was taken from SO 2 and NO 3 composition in Malaysia (Ayers et al., 2002).The preparation of SAR was done using Liu et al. (2008) method.Application of SAR with different pH was carried out using a dripper at a velocity of 2.71 ml s -1 .About 618 ml of SAR was applied on the 90 th day of the study.Treatments were applied every 6 days to mimic the dry condition weather of Malaysia (MMD, 2008).All treatments were applied in the morning (between 7 to 9 am) because high temperature and high irradiances reduce plants' ability to neutralize acid deposition.However, 150 mL rainwater was applied once every 2 days to avoid dryness of seedlings.

Pot study
A pot experiment was conducted in a greenhouse at Universiti Putra Malaysia Bintulu Sarawak Campus (UPMKB) (03°12.301'N,113°04.032'E)Bintulu, Malaysia.S. macroptera plants were grown in a greenhouse to minimize infestation of pests and to also ensure uniformity of sunlight and water supply.S. macroptera seedlings were collected from a rehabilitated forest at UPMKB.The mean height of the seedlings ranged from 20 to 30 cm.The seedlings were grown for 7 months in a poly bag with 0.8 kg soils.Afterwards, the plants were selected based on height and number of leaves for subsequent experiment.
Nyalau series (Typicpaleudults) was sampled at 10 to 15 cm depth from an undisturbed area of UPMKB.The soil was air-dried and sieved to pass a 2.0 mm sieve.The soil was analysed for texture, water holding capacity, pH, cation exchange capacity (CEC), organic matter content, total organic C, total N, available P and exchangeable cation using standard procedures.A 6 kg airdried soil was weighed into a 20'' × 18'' polybag, watered at 70% field capacity with tap water and afterwards, transplanting was carried out.The poly bags were arranged in a Completely Randomized Design (CRD) with 6 treatments and 6 replications.

Plant growth measurement and soil analysis
Plant height, number of leaves, chlorophyll content, chlorosis and necrosis were determined every 30 days.At day 90, plant and soil samples were taken and analyzed using standard procedures.The soil samples were analyzed for pH (Tan, 2005), exchangeable cation and available P (Mehlich, 1953) and available NO 3 -and NH 4 + using distillation method (Keeney and Nelson, 1982).Exchangeable Hilmi et al. 1281 acidity, Al and H were determined using titration method (Rowell, 1994).Meanwhile for SO 4 2-, soil was extracted using 0.5 M NaHCO 3 and was further analyzed using Ion Chromatograph-Mass Spectrometry (IC-MS) (PerkinElmer Inc., Model AI300).
Sampled S. macroptera plants were partitioned into leaves, stems and roots before being oven-dried at 60°C until constant weight was attained.Then, the dried samples were weighed for dry matter production using a digital balance.The plant samples were ground, ashed, after which K, Ca, Mg and Na contents were determined using Atomic Absorption Spectrophotometer (AAS) (PerkinElmer Inc., Model AAnalyst 800) (Cottenie, 1980).P content in the plant parts was determined using the Blue method (Murphy and Riley, 1962).Kjeldahl method was used to determine total N (Bremner, 1965) whilst nutrients uptake were calculated using a formula (Pomares-Gracia and Pratt, 1987).

Statistical analysis
Analysis of variance (ANOVA) at p ≤ 0.05 was used to detect effect of treatments while means of the treatments were compared using Tukey's test.All statistical analyses were conducted using Statistical Analysis System Version 9.2 (SAS, 2001).

Basic characteristics of soil and SAR
The soil used in this study was acidic (pH w 4.84 and pH KCl 3.58).The CEC of the soil was low (Table 1).The organic matter and ash content of the soil were 6 and 94%, respectively.The soil was a sandy clay loam with low available P and exchangeable cation.The chemical characteristics of the SAR used in this study are shown in Table 2.

Effect of SAR on S. macroptera growth
The height of S. macroptera was affected by SAR (Table 3).Seedling height treated with pH 3.5 (T1), 4 (T2) and 4.5 (T3) was lower compared to that of T5 (pH 5.5) which showed the highest increase compared to the other treatments, including the control (pH 6 ± 0.02).The number of leaves treated with T1 (pH 3.5) and T2 (pH 4.0) was lower than those of T3, T4 and the control (pH 6 ± 0.2).At day 30, significant number of leaves treated with low SAR pH was lower than those of T5 and the control.However, on day 90, there was no difference between these treatments (Table 3).
There was an increase in chlorophyll content due to application of T1, T2, T3 and T4 particularly in T2 (pH 4) which produced a sharp increase in chlorophyll content within 90 days and this might be due to reflex action of the SAR (Figure 1).
Total biomass was significantly affected by pH of SAR (Table 4).T4 (pH 5) produced higher total biomass compared to the other treatments.The foliar biomass of T1 (pH 3.5) and T2 (pH 4.0) was lower than those of T3, T4 and T5.The stem and roots biomass was the lowest .at T3 (pH 4.5) (Table 4).However, T0 (pH 3.5) and T5 (pH 5.5) produced the highest biomass in the roots (4.34 g) and stems (7.00 g), respectively.
Leaf chlorosis and necrosis were observed at SAR pH (pH 3.5, 4.0 and 4.5) (Table 5).Three out of the 36 seedlings were affected.There was no significant  difference at p ≤ 0.05 in foliar N, K, and Na concentrations (Table 6).Perhaps this could be the effects of nutrient leaching in foliar.
Higher acidity decreased P and Ca in the leaves, and increased Zn concentrations (Table 6).T3 (pH 4.5) showed the highest increase in Ca and P of the leaves while the lowest was in T1 (pH 3.5).As SAR increased from pH 3.5 to pH 4.5, P concentration in leaves increased from 0.085 to 0.231%.Similar results were obtained for Ca (Table 6).Nitrogen, P, K, Ca and Na accumulation were relatively lower at lower SAR pH, compared to that of the control.Increase in SAR pH from 3.5 to 6.0 ± 0.2 decreased accumulation of N in the leaves.The highest K concentration in the stems was in T4 (pH 5) and the lowest in T0 (Table 7).Other treatments showed no significant difference.The accumulation of N (Table 7) was highest in T1 (pH 3.5) and lowest in T2 (pH 4), T3 (pH 4.5), and T4 (pH 5).
For Zn and P accumulation, no significant difference was observed among treatments.T4 showed the highest uptake for K and Ca while T3 caused the lowest Na accumulation compared to other treatments.No significant difference was observed in N, K, Ca, Na and Zn concentrations in the roots (Table 8).T0 (pH 6 ± 0.02) caused the highest increase in P concentrations compared to other treatments (Table 8).Regardless of SAR pH, N, Ca and Na concentrations in the roots were not significantly different (Table 8).T0 (6.0 ± 0.2) pH showed the highest P while T5 (pH 5.5) caused the lowest.Similarly to P accumulation, T5 caused the highest accumulation of P and T1 caused the lowest.Highest concentration of Zn was recorded in T0 (6.0 ± 0.2) and lowest in T3 (pH 4.5).
Lack of phosphorus in leaves, roots and stems could   also be one of the reasons for the low biomass of the test plants (Table 4).

Effect of SAR on soil cultivated with S. macroptera
There was no significant difference in soil pH and available P cultivated with S. macroptera (Table 9).However, for NH 4, it was significant where T2 (pH 4.0) recorded the lowest (11.68 mg kg -1 ) concentration whilst the highest (37.36 mg kg -1 ) was in T5 and T0 (pH 5.5 and pH 6.0 ± 0.02).Treatment 2 (pH 4.0) produced the most significant available NO 3 with highest value (22.42 mg kg -1 ) and T0 (pH 6.0 ± 0.02) produced the lowest value    (12.61 mg kg -1 ).The soils treated with T0 (pH 6.0 ± 0.02) were lower in terms of exchangeable acidity, Al, and H. Treatments 2 and T3 (pH 4.0 and pH 4.5) produced higher in exchangeable Al about 1.11 and 1.40 cmol kg -1 , respectively.
The highest H concentration in soils was in T1 (pH 3.5) with 1.29 cmol kg -1 whilst other treatments did not show any statistical difference.This treatment also produced the similar results in available SO 4 2 .With the exception of Mg, no significant differences were recorded for K, Ca and Na (Figures 2 and 3).The lowest concentration of exchangeable Mg was produced in T3 (pH 4.5) (0.26 cmolkg -1 ) and the highest in T5 (pH 5.0) (1.18 cmol kg -1 ).

DISCUSSION
In this study, increment of plant height of S. macroptera after 90 days exposed to SAR at T1 (pH 3.5) was 3.83 cm from initial day at 47.00 cm.It was 2.41 cm lower than T0 (pH 6.0 ± 0.2) at 6.24 cm or 38.61%.The value obviously showed role of different acidity level in reduction of plant height.A similar pattern on plant height reduction in forest and crop plant has been reported by Balasubramanian et al. (2007).This could probably be due to the cell division function which was retarded by lack of macronutrient in soil.Exposure of S. macroptera to SAR had a definite impact on decreasing number of leaves.The decrease was mostly in T2 (pH 4.0) (2.44) as compared to T0 (pH 6.0 ± 0.2) (4.67).Perhaps, this effect comes from stress mechanism of leaves when exposed to different acidity.According to Sonia and Khan (1996), leaf growth is affected by simulated acid rain by reducing transpiring area with essential nutrient uptake.
Biomass was substantially reduced by the acidity of SAR.The result of the present studies was same with the findings of Malek (1995), in Larix decidua Mill.S. macroptera seedlings recorded reduced in leaf, stem and root weight with SAR of T1 (pH 3.5) compared with pH T0 (pH 6.0 ± 0.2).Another study by Sonia and Khan (1996) showed that acid rain stress caused significant reduction in stem weight by slowing cell division and expansion.They also report increased rainwater acidity and decreased redistribution of photosynthesis, which affects root elongation.
The effects of SAR pH acidity on chlorosis and necrosis are related to leaves of S. macroptera.The symptoms include the plant having deficient nitrogen, phosphorus, potassium and magnesium.This is true, from the result on plant accumulation in Table 6.
Increasing SAR acidity does not affect soil pH.It probably buffers soil capacity and series of soil type that was used.Nyalau soil series is considered as acidic soil (Paramananthan, 2000); whenever acidic solution fills the soil, it has a tendency to control H + ions.Fertility on soil mainly depends on the macronutrient available in soils.Exchanging NH 4 + for the lower SAR treatments presents lower value compared to control.As stated by Walna et al. (2000), enzymatic activities in highest pH level are faster in influencing the growth of soil microorganism and mineralization activities.
Exchangeable H + recorded highest in lower pH than control.It showed more free hydrogen was deposited in soil surface.The H + ions come from the mixture of acid for preparing the SAR.This is also indicator for higher presence of Al and heavy metal on soil.The role of hydrogen ions is to hydrolyze water molecules to release macronutrient and the same time triggers the heavy metal to combine with Al, which is called oxylation formation process.This result will give toxicity to the soil directly (Cronan and Grigal, 1995).

Conclusion
S. macroptera is sensitive to simulated acid rain (SAR) and has the potential to be sensitive plant for acid deposition especially in Malaysia.However, soil chemical characteristics were slightly affected by SAR due to little Cmolkg -1 Treatment changes in its characteristics.In order to validate this data, long term experiment is suggested.
within same column with different letters was significantly different at p ≤ 0.05 (Tukey's test).
within same column with different letters was significantly different at p ≤ 0.05 (Tukey's test).
within same column with different letters was significantly different at p ≤ 0.05 (Tukey's test).
within same rows with different letters was significantly different at p ≤ 0.05 (Tukey's test).

Figure 2 .Figure 3 .
Figure 2. Effect of SAR on soil exchangeable Ca and Mg concentration.Note: Mean with different letters was significantly different at p ≤ 0.05 (Tukey's test).

Figure 4 .
Figure 4. Effect of SAR on soil exchangeable Cu concentration.Note: Mean with different letters was significantly different at p ≤ 0.05 (Tukey's test).

Figure 5 .
Figure 5.Effect of SAR on soil exchangeable Fe concentration.Note: Mean with different letters was significantly different at p ≤ 0.05 (Tukey's test).

Figure 6 .
Figure 6.Effect of SAR on soil exchangeable Zn concentration.Note: Mean with different letters was significantly different at p ≤ 0.05 (Tukey's test).

Table 2 .
Chemical characteristics of simulated acid rain used in pot study.

Table 3 .
Effect of SAR on height and number of leaves increment in S. macroptera.
Note: Mean with different letters was significantly different at p ≤ 0.05 (Tukey's test), ' = Mean comparison by day, '' = Mean comparison by SAR level, ''' = Interaction between day and SAR level.

Table 4 .
Effect of SAR on biomass of S. macroptera.
c Note: Mean within same column with different letters was significantly different at p ≤ 0.05 (Tukey's test).

Table 5 .
Effect of SAR on the occurrence of chlorosis and necrosis in S. macroptera.
Note: 36 = Total number of seedling, # = No increasing number of chlorosis and necrosis, * = Plant mortality.

Table 6 .
Effect of SAR on foliar nutrient concentration and accumulation in S. macroptera leaves.

Table 7 .
Effect of SAR on stems nutrient concentrations and accumulation in S. macroptera.

Table 8 .
Effect of SAR on roots nutrient concentrations and accumulation in S. macroptera

Table 9 .
Effect of SAR on selected soil chemical properties in S. macroptera.