Growth and physiological responses of submerged plant Vallisneria natans to water column ammonia nitrogen and sediment copper

Zhengjie Zhu; Siyuan Song; Pengshan Li; Nasreen Jeelani; Penghe Wang; Hezhong Yuan; Jinghan Zhang; Shuqing An; Xin Leng

doi:10.7717/peerj.1953

Growth and physiological responses of submerged plant Vallisneria natans to water column ammonia nitrogen and sediment copper

Zhengjie Zhu^1,2, Siyuan Song^1,2, Pengshan Li^1,2, Nasreen Jeelani^1,2, Penghe Wang^1,2, Hezhong Yuan^2,3, Jinghan Zhang², Shuqing An^1,2, Xin Leng ^1,2

1School of Life Science and Institute of Wetland Ecology, Nanjing University, Nanjing Jiangsu, PR China

2Nanjing University Ecology Research Institute of Changshu, Changshu Jiangsu, PR China

3School of Environmental Science and Engineering, Nanjing University of Information Science and Technology, Nanjing Jiangsu, PR China

DOI: 10.7717/peerj.1953

Published: 2016-04-21
Accepted: 2016-03-29
Received: 2016-02-16

Academic Editor: Vijai Kumar Gupta

Subject Areas: Biosphere Interactions, Ecology, Ecotoxicology, Environmental Sciences, Plant Science
Keywords: Ammonia nitrogen, Sediment copper, Submerged plant, Vallisneria natans

Copyright: © 2016 Zhu et al.
Licence: This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

Cite this article: Zhu Z, Song S, Li P, Jeelani N, Wang P, Yuan H, Zhang J, An S, Leng X. 2016. Growth and physiological responses of submerged plant Vallisneria natans to water column ammonia nitrogen and sediment copper. PeerJ 4:e1953 https://doi.org/10.7717/peerj.1953

The authors have chosen to make the review history of this article public.

Abstract

Background. The decline of submerged plant populations due to high heavy metal (e.g., Cu) levels in sediments and ammonia nitrogen (ammonia-N) accumulation in the freshwater column has become a significant global problem. Previous studies have evaluated the effect of ammonia-N on submerged macrophytes, but few have focused on the influence of sediment Cu on submerged macrophytes and their combined effects.

Methods. In this paper, we selected three levels of ammonia-N (0, 3, and 6 mg L⁻¹) and sediment Cu (25.75 ± 6.02 as the control, 125.75 ± 6.02, and 225.75 ± 6.02 mg kg⁻¹), to investigate the influence of sediment Cu and ammonia-N on submerged Vallisneria natans. We measured the relative growth rate (RGR), above- and below- ground biomass, chlorophyll, non-protein thiol (NP-SH), and free proline.

Results and Discussion. The below-ground biomass of V. natans decreased with increasing Cu sediment levels, suggesting that excessive sediment Cu can result in significant damage to the root of V. natans. Similarly, the above-ground biomass significantly decreased with increasing ammonia-N concentrations, indicating that excessive water ammonia-N can cause significant toxicity to the leaf of V. natans. In addition, high ammonia-N levels place a greater stress on submerged plants than sediment Cu, which is indicated by the decline of RGR and chlorophyll, and the increase of (NP-SH) and free proline. Furthermore, high sediment Cu causes ammonia-N to impose greater injury on submerged plants, and higher sediment Cu levels (Cu ≥ 125.75 mg kg⁻¹) led to the tolerant values of ammonia-N for V. natans decreasing from 6 to 3 mg L⁻¹. This study suggests that high sediment Cu restricts the growth of plants and intensifies ammonia-N damage to V. natans.

Introduction

Rapid worldwide economic and industrial development has resulted in eutrophication and heavy metal pollution of freshwater bodies, which has subsequently led to the deterioration of these aquatic environments (Cheung et al., 2003; Zhang et al., 2012). Vegetation restoration has emerged as an effective way of improving water quality by reducing eutrophication and removing heavy metals from soil and wastewater, because macrophytes have tremendous capacity of absorbing nutrients and toxic metals from polluted soil and water (Gupta & Chandra, 1998; Meagher, 2000; Ali, Bernal & Ater, 2004; Nixon, 2009). However, large numbers of pioneer plants have been blindly planted for ecological restoration. Successful ecological restoration depends on planting submerged macrophytes below the tolerant levels of water ammonia-N and sediment-Cu.

At low levels, copper (Cu) is an essential trace element for a variety of cells and tissues in submerged plants, but at high concentrations, it can cause phytotoxicity (Ali, Bernal & Ater, 2002; Ali, Bernal & Ater, 2004; Ali et al., 2015). Excessive Cu accumulation results in detrimental effects on several physiological and biochemical processes in plants and can also inhibit growth (Påhlsson, 1989; Fernandes & Henriques, 1991; Wang et al., 2013). Moreover, heavy metals such as Cu are generally bound to particulate matter and eventually become incorporated into sediments rather than water columns (Wang et al., 2010b; Ng, 2015). Taking into account that previous studies generally focused on water column copper, the effects of excessive sediment Cu on macrophytes such as Vallisneria natans (V. natans) need to be investigated.

Ammonia-N is an important nutrient source to submerged macrophytes at low concentrations, but it can be toxic at higher levels (Best, 1980; Smolders, Van Riel & Roelofs, 2000; Cao et al., 2007; Ellis, Craft & Stanford, 2015). Damaging concentrations of ammonia-N can inhibit photosynthesis, trigger oxidative stress, and cause water loss in plants (Smolders, Van Riel & Roelofs, 2000; Cao, Ni & Xie, 2004; Li, Cao & Ni, 2007; Neuberg et al., 2010). Furthermore, the toxicity of ammonia-N in water bodies was significantly influenced by high contents of Cu because Cu caused the accumulation of excess ${NH}_{4}^{+}$ in the cytosol (Llorens et al., 2000; Mazen, 2004). However, although previous studies have commonly evaluated the effect of ammonia-N on submerged macrophytes, few have focused on the combined effects of ammonia-N and sediment Cu.

As seen in other polluted freshwater bodies, the ecosystem of the Huai River in China has been severely degraded by excessive pollutant discharge that produced high heavy metal contents, including Cu, and excessive ammonia-N levels in the water column (Zhang et al., 2010; Xia et al., 2011; Yuan et al., 2015a). The Huai River reportedly has ammonia-N concentrations up to 29.70 mg L⁻¹, and sediment Cu concentrations up to 208.8 mg kg⁻¹ (Ren et al., 2015; Yuan et al., 2015b). In this study, V. natans, a ubiquitous submerged plant in the Huai River, was selected as the treatment subject. Changes in plant growth and distribution (Britto & Kronzucker, 2002; Xie, An & Wu, 2005) and fluctuations of many metabolites such as chlorophyll (Prasad et al., 2001), non-protein thiol (NP-SH) (Maserti et al., 2005), and proline (Ashraf & Foolad, 2007) are important indexes to measure the response of a plant to environmental stress. Thus, the relative growth rate (RGR), above- and below-ground biomasses, chlorophyll, NP-SH, and free proline were selected to test (1) growth response of V. natans to sediment Cu and water column ammonia-N and (2) physiological response of V. natans to sediment Cu and water column ammonia-N.

Materials and Methods

Plant materials and culture

Vallisneria natans were collected from the downstream region (34°53′22″N, 113°41′13″E) of the Suoxu River, the tributary of the Huai River in Henan Province in central China. Uniform-sized plants (31.54 ± 6.32 cm tall, 5.03 ± 0.67 g fresh weight) were chosen for the experiment. The sediments used for the treatments were riverside soils containing 1.08% organic matter, 600 mg kg⁻¹ total N, and 25.75 mg kg⁻¹ Cu. The well water from the riverside contained 1.4 mg L⁻¹ total N and 0.36 mg L⁻¹ ammonia-N. In addition, nine large buckets (top diameter 84 cm, bottom diameter 67 cm, height 85 cm), 54 small basins (top diameter 12 cm, bottom diameter 8.7 cm, height 9.9 cm), and standard solutions of Cu (CuSO₄⋅5H₂O) and ${NH}_{4}^{+}$ solutions (NH₄Cl) were used.

Treatments

Given that ammonia-N enrichment in the Huai River varied from 0.02 to 15.43 mg L⁻¹ (Ren et al., 2015) and V. natans cannot survive levels higher than 8 mg L⁻¹ (Zhu et al., 2015), we selected three levels of ammonia-N in this study (0, 3, and 6 mg L⁻¹; L^N, M^N, and H^N, respectively). In addition, three levels of Cu in sediment (control and Cu added at levels of 100 and 200 mg kg⁻¹; L^Cu, M^Cu, and H^Cu, respectively) were selected based on the finding that the Cu sediment concentration in the polluted Huai River can reach 208.8 mg kg⁻¹ (Yuan et al., 2015b). The three levels of ammonia-N content in the water column and three levels of Cu in the sediment produced 9 experimental treatments (L^NL^Cu, L^NM^Cu, L^NH^Cu, M^NL^Cu, M^NM^Cu, M^NH^Cu, H^NL^Cu, H^NM^Cu, and H^NH^Cu). Each treatment was replicated three times.

The Cu treatments were created by adding a CuSO₄ solution to the original soil samples. The treatments were made by taking a standard solution of Cu [CuSO₄⋅5H₂O] that had 9.7656 g CuSO₄⋅5H₂O and adding purified water to amount to 1 L in a volumetric flask (Cu²⁺ 2500 mg L⁻¹). The low Cu treatment (L^Cu) consisted of 500 g soil, the medium Cu treatment (M^Cu) had 500 g soil with 20 ml CuSO₄ solution added, and the high Cu treatment (H^Cu) was made by combining 40 ml CuSO₄ solution with 500 g of soil. The three Cu concentration levels were calculated (L^Cu 25.75 ± 6.02 mg kg⁻¹, M^Cu 125 ± 6.02 mg kg⁻¹, H^Cu 225 ± 6.02 mg kg⁻¹) according to the Cu concentration measured in the original soil (25.75 ± 6.02 mg kg⁻¹). Each Cu treatment level had three ammonia-N concentrations (0, 3, and 6 mg L⁻¹) that were created by adding a certain amount of NH₄Cl solution.

The experiment began on 20 June 2014 and lasted for two weeks. Each small basin was filled with sediments and wrapped by plastic wrap, and then single plant was placed in each prepared basin through a little hole of the plastic wrap. The plastic wrap was used to avoid the removing of the Cu from sediments to water column. Three buckets were used for each sediment Cu level and nine buckets were utilized for the three levels of sediment Cu. Well water was added to each large bucket to create a water depth of 60 cm. Six small basins filled with the same level of sediment Cu were placed in one large bucket. Nine treatments were made by adding the three ammonia-N concentrations (0, 3, and 6 mg L⁻¹) to the buckets with the three levels of sediment Cu. The buckets were then randomly positioned outside where there were no shade differences. The ammonia-N concentration of each large bucket was monitored daily and kept constant by adding an appropriate amount of NH₄Cl solution. During the experiment, the concentration of ammonia-N in the water column ranged from 1.6 to 3.1 mg L⁻¹ in the M^N treatments, with an average of 2.4 mg L⁻¹, and 4.2 to 6.1 mg L⁻¹ in the H^N treatments, with an average of 5.2 mg L⁻¹. Water temperature was kept at 25.1–31.5 °C and underwater light intensity at noon ranged from 20,700 to 39,400 lux during the experimental period. The periphyton and phytoplankton in large bucket were removed through 100-mesh sieves every day. After the experiment, the Cu²⁺ concentration in the water from each large bucket was sampled and all levels were <0.01 mg L⁻¹.

Harvest and chemical analysis

The plants were harvested after 1 and 2 weeks of treatments. Three small basins from each treatment were randomly selected for measurement at each harvest time. After harvest, the periphyton attached to the plant leaves was removed with a soft brush. The plants were washed with purified water, and dry with blotting paper carefully. A whole plant was weighed, above- and below-ground portions were separated, and the fresh weights were recorded. The leaves were placed in an ice bath to obtain the content of chlorophyll, NP-SH, and free proline. The biomass, chlorophyll, NP-SH, and free proline content were measured in the first harvest and only the biomass was measured in the second harvest. To measure the biomass, above- and below-ground portions were separated, and the fresh weights were recorded.

The Cu concentration of the original soil was measured using inductively coupled plasma atomic emission spectrometry (ICP-MS 7700x, Agilent Technologies, USA). The Cu²⁺ concentration of the water body in the large bucket was measured by plasma atomic emission spectrometry (ME-ICP02, ALS Minerals/ALS Chemex Co. Ltd, Guangzhou, China). The concentration of ammonia-N was measured with a HACH DR 2800 Spectrophotometer (HACH Company, Loveland, CO, USA). Relative growth rate (RGR) was calculated as $RGR = ({InW}_{2} - {InW}_{1}) ∕ t$ , where W₁ and W₂ indicate the mean fresh weight at the first and second week, respectively, and t isthe growth time in days. In order to obtain the concentration of chlorophyll, 200 mg of sample was extracted using 25 ml 95% ethanol in the dark for 24 h at room temperature. The leaf chlorophyll concentration was measured by UV–vis spectroscopy, and the absorbance of the extracts was determined at 645 and 663 nm wavelengths (Lichtenthaler, 1987). The chlorophyll concentration was calculated using the equation described by (Arnon (1949); Su et al., 2012). Non protein thiol was determined following the method of Sedlak & Lindsay (1968). The molar extinction coefficient of 13,100 at 412 nm was used to estimate the thiol content and the values were expressed in nmol mg⁻¹ of protein (Patra & Swarup, 2000). The free proline concentration was determined by the rapid colorimetric method described by (Bates, Waldren & Teare, 1973). The concentration of chlorophyll, NP-SH, and free proline of the leaves was calculated on the basis of fresh weight.

Data analysis

Growth indications (RGR and above- and below-ground biomass) and physiological indexes (chlorophyll, NP-SH, and free proline) were analyzed with one-way ANOVA. In addition, a two-way ANCOVA, with sediment Cu and ammonia-N as main factors and plant characteristics (RGR, above- and below-ground biomass, chlorophyll, NP-SH, and free proline) as covariates, was used to test the effects of sediment Cu and ammonia-N on plants. All statistical analyses were performed in SPSS19.0 software (SPSS, Chicago, IL, USA).

Results

Growth indicators of V. natans

Both ammonia-N and sediment Cu had significant effects on RGR (p < 0.01), but the influence of ammonia-N was more dramatic (p < 0.001, Table 1). The RGR was significantly lower in the M^NL^Cu and H^NL^Cu treatments than in the ammonia control (L^NL^Cu) (Fig. 1). Compared to the Cu control (L^NL^Cu), RGR was significantly decreased in the L^NM^Cu and L^NH^Cu treatments (Fig. 1). Moreover, the highest ammonia-N and Cu combination (H^NH^Cu) resulted in the lowest value of RGR.

Table 1:

Two-way ANOVA results (F value) for the relative growth rate (RGR), above-ground biomass, below-ground biomass, chlorophyll, non-protein thiol (NP-SH), and free proline of Vallisneria natans using water column ammonia-N and sediment Cu as dependent variables.

Dependent variable	Ammonia-N	Sediment Cu	Ammonia-N × Sediment Cu
RGR	112.67^∗∗∗	7.70^∗	0.48^ns
Above-ground biomass	109.51^∗∗∗	0.68^ns	2.32^ns
Below-ground biomass	0.98^ns	4.38^∗	0.15^ns
Chlorophyll	9.86^∗∗∗	4.27^∗	0.20^ns
NP-SH	25.80^∗∗∗	5.24^∗	0.86^ns
Free proline	16.95^∗∗∗	1.34^ns	0.23^ns

DOI: 10.7717/peerj.1953/table-1

Notes:

Statistical significance indicated through asterisk(s): ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05, ns p > 0.05.

Relative growth rates (RGR) of nine treatments across two harvest times of Vallisneria natans. — Figure 1: Relative growth rates (RGR) of nine treatments across two harvest times of *Vallisneria natans*.
Mean and standard errors of three replicates were shown; different letters represent significant difference at p < 0.05 between treatments. Abbreviations are the same to those shown in Table 2.

Download full-size image

DOI: 10.7717/peerj.1953/fig-1

The above-ground biomass differed significantly among the ammonia-N treatments (p < 0.001), whereas the below-ground biomass showed significant variation among the sediment Cu groups (p < 0.05, Table 1). The below-ground biomass decreased significantly with increasing Cu sediment levels when water column ammonia-N levels were constant (Fig. 2A). Similarly, the above-ground biomass also decreased with an increasing water column ammonia-N concentration when the sediment Cu levels were stable (Fig. 2B).

Figure 2: Biomass of nine treatments at the first harvest.
(A) Below-ground biomass (g) and (B) Above-ground biomass (g) of *Vallisneria natans* of nine treatments from the first harvest. Mean and standard errors of three replicates were shown; different letters represent significant difference at p < 0.05 between treatments. Abbreviations are the same to those shown in Table 2.

Download full-size image

DOI: 10.7717/peerj.1953/fig-2

Physiological indexes of V. natans

Chlorophyll

Both ammonia-N and sediment Cu had significant effects on chlorophyll (p < 0.05), but the influence of ammonia-N was more dramatic (p < 0.001, Table 1). Increasing levels of both water column ammonia-N and sediment Cu levels led to lower chlorophyll concentrations. The lowest chlorophyll content corresponded to the treatment with the highest ammonia-N and sediment Cu levels (H^NH^Cu, Fig. 3).

NP-SH

Compared to the ammonia control (L^NL^Cu), NP-SH significantly increased in the medium and high ammonia-N treatments (M^NL^Cu and H^NL^Cu, Table 2). In addition, when compared to the Cu control (L^NL^Cu), NP-SH significantly increased in the medium and high Cu treatments (L^NM^Cu and L^NH^Cu, Table 2). The NP-SH concentration was more sensitive to ammonia-N (p < 0.001) than to sediment Cu (p < 0.05, Table 1), although it was significantly affected by both factors.

Table 2:

Non-protein thiol (NP-SH) and free proline contents of Vallisneria natans in nine treatments obtained from the first harvest.

Treatment	L^NL^Cu	L^NM^Cu	L^NH^Cu	M^NL^Cu	M^NM^Cu	M^NH^Cu	H^NL^Cu	H^NM^Cu	H^NH^Cu
NP-SH (µmol g⁻¹)	3.50 ± 0.61d	4.16 ± 0.31cd	4.44 ± 0.85bc	4.55 ± 0.18bc	5.26 ± 0.46ab	5.53 ± 0.19a	5.47 ± 0.48a	5.59 ± 0.42a	5.60 ± 0.21a
Free proline (µg g⁻¹)	23.28 ± 3.69c	25.65 ± 4.69c	41.02 ± 27.56bc	54.03 ± 9.88ab	62.2 ± 15.91ab	62.31 ± 8.38ab	64.68 ± 10.79ab	71.77 ± 15.97a	73.71 ± 21.77a

DOI: 10.7717/peerj.1953/table-2

Notes:

L: Low
M: Medium
H: High
^N: Nitrogen
^Cu: Copper, L^N (0 mg N L⁻¹), M^N (3 mg N L⁻¹), H^N (6 mg N L⁻¹), L^Cu (25.75 mg Cu kg⁻¹), M^Cu (125.75 mg Cu kg⁻¹), H^Cu (225.75 mg Cu kg⁻¹)

Mean and standard error of three replicates is shown; different letters represent significant difference at p < 0.05.

Free proline

Free proline levels were primarily determined by ammonia-N (p < 0.001), whereas the impact of sediment Cu was negligible (p > 0.05, Table 1). The free proline content in the medium and high ammonia-N treatments (M^NL^Cu and H^NL^Cu) was significantly higher than in the ammonia control (L^NL^Cu, Table 2). Sediment Cu level showed little effect on the free proline content, and the higher sediment Cu did not produce a greater free proline content when the ammonia-N content was constant (Table 2).

Discussion

A reduction in RGR and biomass of submerged plants has been reported under high ammonia-N (Wang et al., 2008; Zhang et al., 2013) and Cu concentrations (Srivastava et al., 2006; Xue et al., 2010). Our study shows that the below-ground biomass of V. natans decreased with increasing Cu sediment levels, indicating that excessive sediment Cu (heavy metal toxicity) could result in significant damage to the root of V. natans. This injury, which can include stunted roots and poor growth initiation, might result in low water and nutrient uptake and a disturbance in metabolism (Påhlsson, 1989). In contrast, above-ground biomass significantly decreased with increasing ammonia-N concentrations, indicating that excessive water ammonia-N can cause significant toxicity to the leaf of V. natans. Previous studies showed that ammonia-N was the preferred N-source for submerged plants to uptake N through the shoots (Cedergreen & Madsen, 2003; Racchetti et al., 2010). In summary, our experiment showed that above-ground biomass was affected by water ammonia-N and below-ground biomass by sediment Cu. Thus, the RGR of V. natans was more affected by water ammonia-N than sediment Cu, indicating that the more dramatic leaf as compared to root reduction may result in the decrease of RGR.

Several studies have demonstrated a reduction in chlorophyll content under Cu stress in a variety of aquatic plants, including Potamogeton pusillus (Monferrán et al., 2009), Elodea canadensis (Malec et al., 2009), and Lemna sp. (Geoffroy, Frankart & Eullaffroy, 2004). The present study showed a similar trend, with a clear reduction in the chlorophyll content of V. natans after exposure to sediment Cu, thus supporting the findings of earlier studies on submerged plants (e.g., Geoffroy, Frankart & Eullaffroy, 2004; Malec et al., 2009). Damage from sediment Cu likely results from high Cu contents distorting chlorophyll structure and thereby inhibiting the synthesis of photosynthetic pigment (Prasad et al., 2001). Previous studies showed that excessive ammonia decreases total chlorophyll in aquatic plants such as Myriophyllum (Saunkaew, Wangpakapattanawong & Jampeetong, 2011) and Egeria densa (Su et al., 2012). Our results support these early findings and demonstrate that total chlorophyll in V. natans was also reduced by high ammonia-N. Ammonia-N appears to affect total chlorophyll in aquatic plants by damaging the photosynthetic system and inhibiting photosynthesis (Wang et al., 2008).

NP-SH, a class of low-molecular-weight-SH compounds, has been considered as an important plant defense source in response to heavy metals, including Cu (Maserti et al., 2005). Our results suggest that NP-SH content increased with rising sediment Cu levels. These results are supported by previous studies in which NP-SH significantly increased with higher sediment Cu levels (Morelli & Scarano, 2004; Srivastava et al., 2006; Fernández et al., 2014). NP-SH may induce resistance to heavy metals by protecting labile macromolecules against attack by the formation of free radicals in metabolic reactions and its oxidative stress (Patra, Swarup & Dwivedi, 2001; Mishra et al., 2006). Like sediment Cu, excess ammonia-N also leads to NP-SH accumulation in V. natans. The present results are in agreement with previous studies in which ammonia-N induced an increase of NP-SH content in submerged plants (Wang et al., 2010a). Therefore, the accumulation of the NP-SH likely indicates that plants are being stressed by sediment Cu and water ammonia-N.

Proline is a common free amino acid in plant tissues that contributes to osmotic adjustment, detoxification of reactive oxygen species, and protection of membrane integrity (Sharma & Dietz, 2006; Ashraf & Foolad, 2007). The accumulation of proline under heavy metal stress conditions has been reported in aquatic macrophytes such as Salvinia natans (Mohan & Hosetti, 2006), Lemna gibba (Megateli, Semsari & Couderchet, 2009), and Najas indica (Singh et al., 2010). Our results also indicate that proline levels increased in V. natans in response to excess sediment Cu. Proline-Cu complexes likely enhance the tolerance of plants to heavy metals by reducing free metal ion activities through the formation of metal–proline complexes (Xiong, Liu & Geng, 2006; Szabados & Savouré, 2010). Proline accumulation can also increase dramatically in response to rising ammonia concentrations in aquatic environments (Xu et al., 2012; Lee et al., 2013). We also found that proline accumulated in V. natans under high water column ammonia-N conditions. Previous studies revealed that excess ammonia-N in plant tissues caused cellular and whole plant water imbalance by decreasing Ca²⁺ and K⁺ uptake (Britto & Kronzucker, 2002; Roosta & Schjoerring, 2007). The accumulation of proline may prevent water loss by sustaining cell turgor, maintaining membrane integrity, and inhibiting protein denaturation (Hong et al., 2000; Kim et al., 2004; Neuberg et al., 2010).

Most growth indicators and physiological indexes of V. natans are significantly correlated with the concentration of water column ammonia-N and sediment Cu (p < 0.05). We also found that ammonia-N concentration (p < 0.001) played a more crucial role in affecting plant growth indicators and physiological indexes than sediment Cu (p < 0.05). Moderate to high levels of sediment Cu enhanced the toxicity of water column ammonia-N, and even moderate ammonia-N content yielded negative RGR when exposed to moderate levels of sediment Cu.

This study provides new and important insights into potential methods of ecological restoration after an environment has been damaged by heavy metals. Experiments assessing ammonia-N stress on V. natans showed that high ammonia-N content (>8 mg L⁻¹) in the water column lead to severe plant damage (Zhu et al., 2015). Compared to previous studies, our experiments evaluated relatively lower ammonia-N content in the water column and further narrowed the tolerant water ammonia-N content to <6 mg L⁻¹. Moreover, this study suggests that cross effect of various factors are non-neglectful, even this cross effect seems less significant. Moderate to high sediment Cu levels intensify ammonia-N stress on submerged plants and yield much lower tolerant water ammonia-N content (<3 mg L⁻¹) for V. natans.

Supplemental Information

Raw data

DOI: 10.7717/peerj.1953/supp-1

Download

[1] Ali NA, Bernal MP, Ater M. 2002. Tolerance and bioaccumulation of copper in Phragmites australis and Zea mays. Plant and Soil 239(1):103-111

[2] Ali NA, Bernal MP, Ater M. 2004. Tolerance and bioaccumulation of cadmium by Phragmites australis grown in the presence of elevated concentrations of cadmium, copper, and zinc. Aquatic Botany 80(3):163-176

[3] Ali S, Shahbaz M, Shahzad AN, Fatima A, Khan HAA, Anees M, Haider MS. 2015. Impact of copper toxicity on stone-head cabbage (Brassica oleracea var. capitata) in hydroponics. PeerJ PrePrints 3:e1029

[4] Arnon DI. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24:1-15

[5] Ashraf M, Foolad M. 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59(2):206-216

[6] Bates L, Waldren R, Teare I. 1973. Rapid determination of free proline for water-stress studies. Plant and Soil 39(1):205-207

[7] Best EP. 1980. Effects of nitrogen on the growth and nitrogenous compounds of Ceratophyllum demersum. Aquatic Botany 8:197-206

[8] Britto DT, Kronzucker HJ. 2002. ${NH}_{4}^{+}$ toxicity in higher plants: a critical review. Journal of Plant Physiology 159(6):567-584

[9] Cao T, Ni L, Xie P. 2004. Acute biochemical responses of a submersed macrophyte, Potamogeton crispus L., to high ammonium in an aquarium experiment. Journal of Freshwater Ecology 19(2):279-284

[10] Cao T, Xie P, Ni LY, Wu AP, Zhang M, Wu SK, Smolders AJP. 2007. The role of ${NH}_{4}^{+}$ toxicity in the decline of the submersed macrophyte Vallisneria natans in lakes of the Yangtze River basin, China. Marine and Freshwater Research 58(6):581-587

[11] Cedergreen N, Madsen TV. 2003. Nitrate reductase activity in roots and shoots of aquatic macrophytes. Aquatic Botany 76(3):203-212

[12] Cheung K, Poon B, Lan C, Wong M. 2003. Assessment of metal and nutrient concentrations in river water and sediment collected from the cities in the Pearl River Delta, South China. Chemosphere 52(9):1431-1440

[13] Ellis BK, Craft JA, Stanford JA. 2015. Long-term atmospheric deposition of nitrogen, phosphorus and sulfate in a large oligotrophic lake. PeerJ 3:e841

[14] Fernandes JC, Henriques FS. 1991. Biochemical, physiological, and structural effects of excess copper in plants. The Botanical Review 57(3):246-273

[15] Fernández R, Fernández-Fuego D, Bertrand A, González A. 2014. Strategies for Cd accumulation in Dittrichia viscosa(L.) Greuter: role of the cell wall, non-protein thiols and organic acids. Plant Physiology and Biochemistry 78:63-70

[16] Geoffroy L, Frankart C, Eullaffroy P. 2004. Comparison of different physiological parameter responses in Lemna minor and Scenedesmus obliquus exposed to herbicide flumioxazin. Environmental Pollution 131(2):233-241

[17] Gupta M, Chandra P. 1998. Bioaccumulation and toxicity of mercury in rooted-submerged macrophyte Vallisneria spiralis. Environmental Pollution 103(2):327-332

[18] Hong Z, Lakkineni K, Zhang Z, Verma DPS. 2000. Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiology 122(4):1129-1136

[19] Kim TH, Lee BR, Jung WJ, Kim KY, Avice JC, Ourry A. 2004. De novo protein synthesis in relation to ammonia and proline accumulation in water stressed white clover. Functional Plant Biology 31(8):847-855

[20] Lee BR, Muneer S, Park SH, Zhang Q, Kim TH. 2013. Ammonium-induced proline and sucrose accumulation, and their significance in antioxidative activity and osmotic adjustment. Acta Physiologiae Plantarum 35(9):2655-2664

[21] Li H, Cao T, Ni L. 2007. Effects of ammonium on growth, nitrogen and carbohydrate metabolism of Potamogeton maackianus A. Benn. Fundamental and Applied Limnology/Archiv Für Hydrobiologie 170(2):141-148

[22] Lichtenthaler HK. 1987. Chlorophyll fluorescence signatures of leaves during the autumnal chlorophyll breakdown. Journal of Plant Physiology 131(1–2):101-110

[23] Llorens N, Arola L, Blade C, Mas A. 2000. Effects of copper exposure upon nitrogen metabolism in tissue cultured Vitis vinifera. Plant Science 160(1):159-163

[24] Malec P, Maleva M, Prasad MNV, Strzałka K. 2009. Copper toxicity in leaves of Elodea canadensis Michx. Bulletin of Environmental Contamination and Toxicology 82(5):627-632

[25] Maserti BE, Ferrillo V, Avdis O, Nesti U, Di Garbo A, Catsiki A, Maestrini PL. 2005. Relationship of non-protein thiol pools and accumulated Cd or Hg in the marine macrophyte Posidonia oceanica (L.) Delile. Aquatic Toxicology 75(3):288-292

[26] Mazen AMA. 2004. Accumulation of four metals in tissues of Corchorus olitorius and possible mechanisms of their tolerance. Biologia Plantarum 48(2):267-272

[27] Meagher RB. 2000. Phytoremediation of toxic elemental and organic pollutants. Current Opinion in Plant Biology 3(2):153-162

[28] Megateli S, Semsari S, Couderchet M. 2009. Toxicity and removal of heavy metals (cadmium, copper, and zinc) by Lemna gibba. Ecotoxicology and Environmental Safety 72(6):1774-1780

[29] Mishra S, Srivastava S, Tripathi RD, Govindarajan R, Kuriakose SV, Prasad MNV. 2006. Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa monnieri L. Plant Physiology and Biochemistry 44(1):25-37

[30] Mohan BS, Hosetti BB. 2006. Phytotoxicity of cadmium on the physiological dynamics of Salvinia natans L. grown in macrophyte ponds. Journal of Environmental Biology 27(4):701-704

[31] Monferrán MV, Agudo JAS, Pignata ML, Wunderlin DA. 2009. Copper-induced response of physiological parameters and antioxidant enzymes in the aquatic macrophyte Potamogeton pusillus. Environmental Pollution 157(8):2570-2576

[32] Morelli E, Scarano G. 2004. Copper-induced changes of non-protein thiols and antioxidant enzymes in the marine microalga Phaeodactylum tricornutum. Plant Science 167(2):289-296

[33] Neuberg M, Pavlíková D, Pavlík M, Balík J. 2010. The effect of different nitrogen nutrition on proline and asparagine content in plant. Plant, Soil and Environment 56(7):305-311

[34] Ng W. 2015. Ammonium interference reduced copper uptake by formaldehyde-crosslinked Sargassum sp. seaweed. PeerJ PrePrints 3:e1506

[35] Nixon SW. 2009. Eutrophication and the macroscope. Hydrobiologia 629(1):5-19

[36] Påhlsson AMB. 1989. Toxicity of heavy metals (Zn, Cu, Cd, Pb) to vascular plants. Water, Air, and Soil Pollution 47(3–4):287-319

[37] Patra R, Swarup D. 2000. Effect of lead on erythrocytic antioxidant defence, lipid peroxide level and thiol groups in calves. Research in Veterinary Science 68(1):71-74

[38] Patra RC, Swarup D, Dwivedi SK. 2001. Antioxidant effects of α tocopherol, ascorbic acid and L-methionine on lead induced oxidative stress to the liver, kidney and brain in rats. Toxicology 162(2):81-88

[39] Prasad M, Malec P, Waloszek A, Bojko M, Strzałka K. 2001. Physiological responses of Lemna trisulca L. (duckweed) to cadmium and copper bioaccumulation. Plant Science 161(5):881-889

[40] Racchetti E, Bartoli M, Ribaudo C, Longhi D, Brito LE, Naldi M, Viaroli P. 2010. Short term changes in pore water chemistry in river sediments during the early colonization by Vallisneria spiralis. Hydrobiologia 652(1):127-137

[41] Ren LJ, Wen T, Pan W, Chen YS, Xu LL, Yu LJ, An SQ. 2015. Nitrogen removal by ecological purification and restoration engineering in a polluted river. CLEAN—Soil, Air, Water 43(12):1565-1573

[42] Roosta HR, Schjoerring JK. 2007. Effects of ammonium toxicity on nitrogen metabolism and elemental profile of cucumber plants. Journal of Plant Nutrition 30(11):1933-1951

[43] Saunkaew P, Wangpakapattanawong P, Jampeetong A. 2011. Growth, morphology, ammonium uptake and nutrient allocation of Myriophyllum brasiliense Cambess. under high ${NH}_{4}^{+}$ concentrations. Ecotoxicology 20(8):2011-2018

[44] Sedlak J, Lindsay RH. 1968. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Analytical Biochemistry 25:192-205

[45] Sharma SS, Dietz KJ. 2006. The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress. Journal of Experimental Botany 57(4):711-726

[46] Singh R, Tripathi RD, Dwivedi S, Kumar A, Trivedi PK, Chakrabarty D. 2010. Lead bioaccumulation potential of an aquatic macrophyte Najas indica are related to antioxidant system. Bioresource Technology 101(9):3025-3032

[47] Smolders A, Van Riel M, Roelofs J. 2000. Accumulation of free amino acids as an early indication for physiological stress (nitrogen overload) due to elevated ammonium levels in vital Stratiotes aloides L. stands. Archiv Für Hydrobiologie 150(1):169-175

[48] Srivastava S, Mishra S, Tripathi RD, Dwivedi S, Gupta DK. 2006. Copper-induced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (Lf) Royle. Aquatic Toxicology 80:405-415

[49] Su S, Zhou Y, Qin JG, Wang W, Yao W, Song L. 2012. Physiological responses of Egeria densa to high ammonium concentration and nitrogen deficiency. Chemosphere 86:538-545

[50] Szabados L, Savouré A. 2010. Proline: a multifunctional amino acid. Trends in Plant Science 15(2):89-97

[51] Wang Q, Li Z, Cheng S, Wu Z. 2010b. Influence of humic acids on the accumulation of copper and cadmium in Vallisneria spiralis L. from sediment. Environmental Earth Sciences 61(6):1207-1213

[52] Wang P, Wang C, Ouyang P, Qian J, Shi R. 2013. Physiological responses of Vallisneria spiraslis L. induced by different hydraulic conditions when exposed to copper and nitrogen. African Journal of Biotechnology 10(38):7441-7452

[53] Wang C, Zhang SH, Wang PF, Hou J, Li W, Zhang WJ. 2008. Metabolic adaptations to ammonia-induced oxidative stress in leaves of the submerged macrophyte Vallisneria natan (Lour.) Hara. Aquatic Toxicology 87:88-98

[54] Wang C, Zhang SH, Wang PF, Li W, Lu J. 2010a. Effects of ammonium on the antioxidative response in Hydrilla verticillata (Lf) Royle plants. Ecotoxicology and environmental safety 73(2):189-195

[55] Xia J, Zhang YY, Zhan C, Ye AZ. 2011. Water quality management in China: the case of the Huai River Basin. Water Resources Development 27(01):167-180

[56] Xie Y, An S, Wu B. 2005. Resource allocation in the submerged plant Vallisneria natans related to sediment type, rather than water-column nutrients. Freshwater Biology 50(3):391-402

[57] Xiong ZT, Liu C, Geng B. 2006. Phytotoxic effects of copper on nitrogen metabolism and plant growth in Brassica pekinensis Rupr. Ecotoxicology and Environmental Safety 64(3):273-280

[58] Xu J, Zhang J, Zhao C, Li C, Xie H, Wang S. 2012. Effect of ammonia stress on physiological and biochemical character of Phragmites australis in constructed wetland. In: Digital Manufacturing and Automation (ICDMA), 2012 Third International Conference on. IEEE, Vol. 2012. Piscataway: IEEE. 343-346

[59] Xue PY, Li GX, Liu WJ, Yan CZ. 2010. Copper uptake and translocation in a submerged aquatic plant Hydrilla verticillata (Lf) Royle. Chemosphere 81(9):1098-1103

[60] Yuan HZ, Pan W, Ren LJ, Liu EF, Shen J, Geng QF, An SQ. 2015a. Species and biogeochemical cycles of organic phosphorus in sediments from a river with different aquatic plants located in Huaihe river watershed, China. International Journal of Phytoremediation 17(3):215-221