Journal of Oceanology and Limnology   2021, Vol. 39 issue(6): 2267-2280     PDF
Institute of Oceanology, Chinese Academy of Sciences

Article Information

SONG Xiaoyue, ZHOU Yi, ZENG Jiangning, SHOU Lu, ZHANG Xiaomei, YUE Shidong, GAO Wei, FENG Weihua, WANG Zhifu, DU Ping
Distinct root system acclimation patterns of seagrass Zostera japonica in sediments of different trophic status: a research by X-ray computed tomography
Journal of Oceanology and Limnology, 39(6): 2267-2280

Article History

Received Oct. 30, 2020
accepted in principle Nov. 28, 2020
accepted for publication Dec. 4, 2020
Distinct root system acclimation patterns of seagrass Zostera japonica in sediments of different trophic status: a research by X-ray computed tomography
Xiaoyue SONG1, Yi ZHOU2, Jiangning ZENG1, Lu SHOU1, Xiaomei ZHANG2, Shidong YUE2, Wei GAO3, Weihua FENG4, Zhifu WANG4, Ping DU1     
1 Key Laboratory of Marine Ecosystem Dynamics, Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou 310012, China;
2 CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
3 College of Tropical Crops, Hainan University, Haikou 570228, China;
4 Key Laboratory of Engineering Oceanography, Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou 310012, China
Abstract: Conspecific seagrass living in differing environments may develop different root system acclimation patterns. We applied X-ray computed tomography (CT) for imaging and quantifying roots systems of Zostera japonica collected from typical oligotrophic and eutrophic sediments in two coastal sites of northern China, and determined sediment physicochemical properties that might influence root system morphology, density, and distribution. The trophic status of sediments had little influence on the Z. japonica root length, and diameters of root and rhizome. However, Z. japonica in oligotrophic sediment developed the root system with longer rhizome node, deeper rhizome distribution, and larger allocation to below-ground tissues in order to acquire more nutrients and relieve the N deficiency. And the lower root and rhizome densities of Z. japonica in eutrophic sediment were mainly caused by fewer shoots and shorter longevity, which was resulted from the more serious sulfide inhibition. Our results systematically revealed the effect of sediment trophic status on the phenotypic plasticity, quantity, and distribution of Z. japonica root system, and demonstrated the feasibly of X-ray CT in seagrass root system research.
Keywords: Zostera japonica    root system    acclimation pattern    sediment    trophic status    X-ray computed tomography    

Seagrass are angiosperms unique in the marine environment, and seagrass meadows provide important habitats for other marine organisms. Around 70 seagrass species have adapted to neritic environments around the world (Short et al., 2007), including coastal rocky, sandy, and muddy substrates, and estuaries, bays, lagoons, mangrove forests, and coral reefs. Seagrass root and rhizome systems contribute to this adaptability, since they anchor against water flow and enable the exploitation of sediment-associated nutrients (Stapel et al., 1996; Rubio et al., 2007). Their relatively simple branching may also minimize the distance for oxygen transport from shoots to roots (Kiswara et al., 2009). Meanwhile, sulfide, and especially dissolved H2S, is highly toxic for seagrass (Pedersen and Kristensen, 2015; Martin et al., 2019). Seagrass can aerate their below-ground tissues and lose oxygen from roots to prevent sulfide intrusion from the surrounding sediment (Connell et al., 1999; Koren et al., 2015; Martin et al., 2019). Oxygen dynamics of seagrass roots alter sediment redox conditions and influence benthic biogeochemical processes (Martin et al., 2019), causing differences between bacterial communities associated with the roots and those associated with bulk sediments of seagrass meadows (Isaksen and Finster, 1996; Jensen et al., 2007; Ettinger et al., 2017). In addition, iron plaque formation on roots and rhizomes has been observed in seagrass and other aquatic macrophytes with implication for sulfide intrusion (Povidisa et al., 2009).

There are distinct acclimation strategies among root systems of different seagrass species. For instance, in coastal zones of northern China, Zostera marina inhabits sediment and Phyllospadix iwatensis inhabits rocky areas. P. iwatensis develops a more complex root system with denser root hairs and inflated root ends, facilitating its attachment to a rocky substrate (Li et al., 2019). Root architecture differs markedly between seagrass species, where relatively long-lived and slow-growing species are characterized by short internodes, fewer unbranched roots per node, and dense root hair; meanwhile, faster-growing species have more numerous roots per node (Kiswara et al., 2009). Belowground biomass of different seagrass species in the same meadow show considerable vertical stratification within sediments, where larger species tend to extend deeper into the sediments than smaller ones (Duarte et al., 1998). This likely reduces potential interspecific competition for sediment resources, especially in the uppermost layers (Duarte et al., 1998). Some species show unique root system adaptations, such as Posidonia oceanica adults commonly lacking root hairs and regularly forming flourishing mycorrhizae through a specific association with a single pleosporalean fungus (Borovec and Vohník, 2018; Vohník et al., 2019).

Seagrass have the capacity to acclimate their morphological, physiological, and mechanical traits to their local conditions, which is regarded as its phenotypic plasticity (Jensen and Bell, 2001; Bercovich et al., 2019). Seagrass leaves can acclimate to low light conditions though a variety of physiological and morphological mechanisms, like enlarging chloroplast density and specific leaf area, to enhance photosynthetic capacity (McDonald et al., 2016; Beca-Carretero et al., 2019). Conspecific seagrass in distinct environments will also develop different root system acclimations patterns. Seagrass could achieve advantages in acquiring nutrients, by root architecture and root plasticity, in contrasting sediment types that differ in nutrient availability, such as carbonaceous nutrient-poor sediments as well as muddy and nutrient-rich sediments (Erftemeijer and Middelburg, 1993; Kamp-Nielsen et al., 2002). The root systems of two temperate seagrass, Posidonia australis and Posidonia sinuosa, display architectural and morphological plasticity with season, and to a lesser extent, nutrient addition (Hovey et al., 2012). Similarly, root lengths and nitrogen-obtaining patterns of seagrass in an offshore atoll differed from the same species in continental habitats in South China Sea (Jiang et al., 2019).

Zostera japonica is widespread along coastal areas of the North Pacific Ocean and its meadows support a variety of marine ecosystem functions (Miki, 1933; Abe et al., 2009; Zhang et al., 2019), for example, the Z. japonica meadow in the Huanghe (Yellow) River estuary is one of the largest seagrass meadows in China and a key habitat for many marine organisms in the National Nature Reserve of Huanghe River estuary (Zhang et al., 2019). Despite its broad distribution and significant ecological role, there has been no specific research into Z. japonica root systems. Multiple factors affect seagrass root systems, including hydrodynamics, illumination, nutrition, and substrate physicochemical characteristics. Previous investigations of typical Z. japonica meadows in China indicated clear differences in the ratio of above-ground biomass to below-ground biomass in different areas of distribution (Zhang et al., 2015, 2019). A better understanding of its root system acclimation to different substrates will be useful for the conservation and restoration of Z. japonica meadows and relevant seagrass ecosystem.

Previous investigations of seagrass root systems were carried out by rinsing attached sediment and using water to spread the roots for visual observation and analysis (Kiswara et al., 2009; Hovey et al., 2011; Jiang et al., 2019). Most of the time, root systems are treated simply as below-ground biomass, which results in the loss of in situ root system information. Several researchers have applied minirhizotron tubes to visualize and quantify the root standing crop, production, mortality, and lifespan of Z. marina roots (Johnson et al., 2016) and optical nanoparticle-based sensors to visualize oxygen dynamics around rhizomes and roots (Koren et al., 2015; Martin et al., 2019). In recent decades, X-ray computed tomography (CT) has been used for imaging and quantifying 3D root systems of various plants (Tracy et al., 2010; Koebernick et al., 2014; Blaser et al., 2018), but this has not yet been applied to seagrass root systems.

In this study, we hypothesis that Z. japonica root system in sediments of different trophic status developed different acclimation patterns, and the nutrient condition and sulfide content difference therein were the primary reasons causing that. We collected Z. japonica root and sediment samples from typical oligotrophic and eutrophic sediments and used X-ray CT imaging to observe the differences in root systems between these environments, then compared these with sediment physicochemical properties, with the aim of describing the mechanisms by which Z. japonica root system acclimated to common sediment types of distinct trophic states.

2 MATERIAL AND METHOD 2.1 Site description and sampling

Our samples were collected from Z. japonica meadows in a lagoon named Swan Lake in Weihai (37°21′24″N; 122°35′21″E) and Huiquan Bay in Qingdao (36°3′34″N; 120°21′5″E), Shandong Province, China (Fig. 1). Persistent meadows of Z. japonica exist in both locations. Swan Lake has eutrophic sediment due to sewage input and relative closed lagoon topography (Zhang et al., 2014, 2015), the sampling site was located near the south bank where the influence of sewage was heavier than other area. Whereas sediment in Huiquan Bay is comparatively oligotrophic (Zhang et al., 2020).

Fig.1 The locations of two Zostera japonica meadows and sampling sites of different sediment trophic status, Swan Lake (SL) (37°21′24″N; 122°35′21″E) and Huiquan Bay (HQ) (36°3′34″N; 120°21′5″E) in Shandong Peninsula of China The patches in small pictures represents the Z. japonica meadows, and black stars show the sampling sites in two areas.

Sampling of both locations was carried out during the summer of 2018 at 25–32 ℃ air temperature. We collected three samples 10-m apart in each location at low tide, with each sample consisting of a sediment core 11.5 cm in diameter and 15-cm deep. Cores were fixed in polyvinyl chloride tubes and transported to a laboratory at 4 ℃. Shoot density, total biomass, percentage of below-ground biomass, root length, and rhizome node length were obtained from a simultaneous investigation that sampled 10 quadrats (20 cm×20 cm) in each site, as described in Zhang et al.(2015, 2019). All shoots in each quadrat were placed in plastic bags and transported to the laboratory in darkness at low temperature.

2.2 X-ray computed tomography

The above-ground tissues of seagrass were removed and collected. Sediment cores were scanned using an industrial X-ray CT scanner (phoenix v|tome|x m, General Electric Company, U.S.A.) at 180 kV and 210 μA (40 W). A total of 1 800 equiangular projections were acquired through 360° with an exposure time of 2 000 ms, resulting in a scan time of 60 min for each core. The surfaces of cores were automatically recognized basing on the enormous difference between sediment and other objects, like air and polyvinyl chloride tubes. The acquired images were reconstructed using a filtered back-projection algorithm in VGSTUDIO MAX software (Volume Graphics Company, Germany). The achieved resolution of 3D tomogram was 80 μm.

CT images were processed using the "Rootine" method (Gao et al., 2019a, b). In brief, reconstructed 16-bit images were converted to 8-bit to reduce the image size in Fiji/ImageJ (National Institutes of Health, U.S.A). The converted images were filtered according to non-local means. "Tubeness" filters were then applied at multiple scales to group tubular rhizomes and roots according to diameter. "3D Hysteresis thresholding" was used to binarize the greyscale "Tubeness" images. "Size opening" was then used to remove isolated segmentation noise. The length of rhizomes and roots were quantified voxel by voxel (90 μm×90 μm×90 μm) using "Skeletonize (2D/3D)" and "Analyze Skeleton" plugins in Fiji/ImageJ. Then the relative densities were calculated, and the root-per-rhizome length was acquired through the division of total root length by total rhizome length. The volume of rhizomes and roots was calculated by counting the voxel numbers of objects, and the relative densities were calculated. The quotients of the total volumes and lengths of rhizomes and roots were equal to their average cross-sectional areas; if they were assumed to be round, the approximate diameters of rhizomes and roots could be calculated. It is worth noting that the obvious upright thicker lines in superficial layer, like those in HQ-1 and HQ-3, were sheathes of shoots, and were not calculated as rhizomes.

2.3 Analysis of nutrients of leaves and physicochemical properties of sediments

Zostera japonica leaves samples from 5 quadrats per site were lyophilized at -50 ℃ for 60 h, and carbon (C) and nitrogen (N) contents were measured using a PE2400 Series Ⅱ elemental analyzer. Phosphorus (P) content was determined by using the phosphomolybdenum blue method modified for particulate total P determination (Zhou et al., 2003). And then the relative C/N and N/P were calculated.

Since X-ray CT results indicated that the root systems of all six samples were in the top 10 cm, sediment physicochemical analysis was also carried out in this range. The top 10 cm of sediment in each core sample was sliced and evenly divided into five subsamples (0–2-cm, 2–4-cm, 4–6-cm, 6–8-cm, and 8–10-cm depth). Grain size distribution was analyzed using a Laser Particle Counter (LS13320, Beckman, USA).

Fresh sediment subsamples were lyophilized at -50 ℃ for 60 h and the water contents were determined by calculating their weight loss percentage. The lyophilized samples were sieved through 150-μm mesh prior to organic matter analysis. For total organic carbon (TOC) analysis, a sample was acidified with excessive 1-mol/L HCl to remove carbonates, and organic carbon content was determined using an elemental analyzer (PE2400 Series Ⅱ, PerkinElmer, Norwalk, CT, USA). Total nitrogen (TN) contents were determined using the same elemental analyzer. Total phosphorus (TP) and acid volatile sulfide (AVS) contents were determined using phosphomolybdenum blue (Zhou et al., 2003) and methylene blue spectrophotometric methods (Bradley and Stolt, 2006), respectively.

2.4 Statistical analysis

Shoot density, total biomass, percentage of below-ground biomass, root length, rhizome node length, root-per-rhizome length, densities and diameters of total roots and rhizomes, and leaf nutrient elements composition of Z. japonica in Huiquan Bay and Swan Lake were compared using a t-test in SPSS v. 20 (IBM, U.S.A). Three-way mixed-effect ANOVA including site, depth, and random effect core was adopted to analyze the density differences of root length, root volume, rhizome length, and rhizome volume of Z. japonica, and TOC, TN, TP, and AVS of sediments, respectively. Based on the results of above analysis, two-way mixed-effect ANOVA including depth and random effect core was adopted to analyze rhizome length, and rhizome volume densities in each site. Differences of those statistical analysis were considered significant at P < 0.05.

3 RESULT 3.1 Shoot, rhizome, and root characteristics of Z. japonica

The shoot, rhizome, and root characteristics of Z. japonica in Huiquan Bay and Swan Lake were compared and presented in Table 1 & Supplementary Table S2. Based on collections of Z. japonica from quadrats, shoot density, and total biomass were about 2.5-fold greater in Huiquan Bay than Swan Lake (shoot density: t(18)=2.965, P < 0.05; total biomass: t(18)=4.064, P < 0.05). Meanwhile, the percentage of below-ground biomass of Z. japonica in Huiquan Bay was significantly higher than that in Swan Lake by the 1.3-fold (t(18)=3.044, P < 0.05). There was no statistical difference between the root lengths of Z. japonica in two sites (t(18)=-0.323, P > 0.05). However, the rhizome node length of Z. japonica in Huiquan Bay was significantly longer than that in Swan Lake (t(18)=2.712, P < 0.05). Based on CT scans of cores, root and rhizome densities were about 4.3- and 3.7-fold higher in Huiquan Bay than Swan Lake (root length density: t(4)=4.198, P < 0.05; root volume density: t(4)=4.004, P < 0.05; rhizome length density: t(4)=2.926, P < 0.05; rhizome volume density: t(4)=2.847, P < 0.05). The root-per-rhizome length of Z. japonica in Huiquan Bay was not statistically different from that in Swan Lake (t(4)=-0.146 4, P > 0.05). Moreover, there were no significant differences between the diameters of both root and rhizome of Z. japonica in two research sites (root diameter: t(4)=-0.696 4, P > 0.05; rhizome diameter: t(4)=-0.455 6, P > 0.05).

Table 1 Shoot, rhizome, and root characteristics of Zostera japonica
3.2 Nutrient elements composition of Z. japonica leaves

The C, N, and P contents and the C/N, N/P of Z. japonica leaves in Huiquan Bay and Swan Lake were presented in Table 2. There were no significant differences between the C and P contents of Z. japonica leaves in two sites (C: t(8)=-0.483 7, P > 0.05; P: t(8)=0.062, P > 0.05). However, the N content of Z. japonica leaves in Huiquan Bay was lower than that in Swan Lake (t(8)=4.639, P < 0.01). Furthermore, the C/N of Z. japonica leaves in Huiquan Bay was significantly higher than that from Swan Lake (t(8)=7.313 7, P < 0.01); however, the N/P of Z. japonica leaves in Huiquan Bay was significantly lower (t(8)=-2.794, P < 0.05).

Table 2 Nutrient elements compostion of Zostera japonica leaves
3.3 Root system images and distribution

The 3D X-ray CT data were projected to show the vertical distribution of roots and rhizomes in two dimensions (Fig. 2). The roots were showed in thinner lines of yellow color, and rhizomes were showed in thicker lines of purple color. Generally, below-ground materials were denser at Huiquan Bay than Swan Lake and also distributed deeper in the sediment (Fig. 3; Table 3).

Fig.2 Zostera japonica root systems visualized as 2D vertical projection of the 3D data extracted from X-ray CT images, which were collected from Huiquan Bay (HQ) and Swan Lake (SL), respectively Bold purple lines represent rhizome and fine yellow lines represent roots.
Fig.3 Root (a, b) and rhizome (c, d) densities of Zostera japonica from Huiquan Bay (HQ) and Swan Lake (SL) in different layers Different letters (a, b) indicates significantly difference among depths in the same site at P < 0.05, that revealed by two-way mixed-effect ANOVA and multiple comparison.
Table 3 Results of three-way mixed-effect ANOVA of Zostera japonica root and rhizome densities in Huiquan Bay (HQ) and Swan Lake (SL)

In Swan Lake, maximum root length density occurred in the top layer at 3.42±1.28 mm/cm3 (Fig. 3a & Supplementary Table S2). Conversely, in Huiquan Bay, maximum root density occurred in the 4–6-cm layer at 11.45±1.61 mm/cm3. In Swan Lake, the main proportion of root length (85.92%) was located in the upper layers of 0–6 cm, while in Huiquan Bay, the main proportion (77.27%) was observed in the 6–10-cm layers (Fig. 3a). According to corresponding result of the three-way ANOVA for mixed effects including core (Table 3), significant difference existed between the root length densities of Z. japonica in Huiquan Bay and Swan Lake, however, they were not affected by depth (site: F(1, 19)=16.461, P < 0.001; depth: F(4, 19)=0.920, P > 0.05). Root volume density followed a similar statistical pattern to root length density (Fig. 3b) (site: F(1, 19)=16.690, P < 0.001; depth: F(4, 19)=0.934, P > 0.05), given that root diameters were similar between sites (Table 1).

There was no statistical difference between the diameters of rhizomes in two sampling sites (Table 1). The maximum rhizome length density of Z. japonica in Huiquan Bay was 3.67±0.24 mm/cm3 at 2–4 cm, compared to 1.61±0.58 mm/cm3 at 0–2 cm in Swan Lake. In Huiquan Bay, the rhizome at the 0–6 cm accounted for 87.47% of the total rhizome length, however, the rhizome at 0–4 cm accounted for 86.38% of the total rhizome length in Swan Lake (Fig. 3c). According to corresponding result of the three-way ANOVA for mixed effects including core (Table 3), there was a significant difference between the rhizome length densities of Z. japonica in Huiquan Bay and Swan Lake, and which was also significantly affected by depth effect (site: F(1, 19)=8.988, P < 0.01; depth: F(4, 19)=3.479, P < 0.05). On the basis of the two-way ANOVA and multiple comparison for mixed effects including core, in Huiquan Bay, although there was no statistical difference among that rhizome length densities of different depths (F(4, 8)=2.238, P > 0.05), the rhizome volume densities in layers of 2–4 cm was significantly higher than in 8–10-cm layers (Fig. 3d) (F(4, 8)=4.278, P < 0.05); meanwhile, in Swan Lake, the rhizome length and volume densities in 0–4-cm layers were both higher than those in 6–10-cm layers (Fig. 3c & d) (rhizome length density: F(4, 8)=6.845, P < 0.05; rhizome volume density: F(4, 8)=3.900, P < 0.05).

3.4 Physicochemical properties of sediments 3.4.1 Grain size distribution

There was no significant difference between Huiquan Bay and Swan Lake in sediment type. The sediments of top 4 cm in two sites both mainly consisted of the sand and gravel component (Huiquan Bay: 90.28%±9.73%; Swan Lake: 69.39%±21.79%), and the main components of sediments in 4–10 cm were silt (Huiquan Bay: 43.90%±18.26%; Swan Lake: 44.87%±11.47%) and sand and gravel (Huiquan Bay: 51.71%±24.86%; Swan Lake: 34.57%±12.65%).

3.4.2 Total organic carbon and total nitrogen

The quantification and analysis of the chemical properties were presented in Fig. 4, Table 4, & Supplementary Table S1. Swan Lake sediment had accumulated more organic matters. TOC and TN contents at Swan Lake were significantly higher than at Huiquan Bay (TOC: F(1, 19)=464.296, P < 0.001; TN: F(1, 19)=389.057, P < 0.001). For the same layer, TOC content of sediment at Swan Lake was about 6.2-fold greater than at Huiquan Bay, and TN content was 12.1-fold greater. There were no significant effects of depth on the TOC and TN contents of all samples (TOC: F(4, 19)=2.522, P > 0.05; TN: F(4, 19)=1.323, P > 0.05). As the great disparity in organic matter and nitrogen contents, the sediments in Swan Lake and Huiquan Bay were regarded as eutrophic and oligotrophic, respectively.

Fig.4 Total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), and acid volatile sulfide (AVS) contents in sediments of Zostera japonica core samples from Huiquan Bay (HQ) and Swan Lake (SL)
Table 4 Three-way mixed-effect ANOVA of chemical properties of sediments in Zostera japonica meadows in Huiquan Bay (HQ) and Swan Lake (SL)
SupplementaryTableS 1 Chemical index values in sediments of Zostera japonica meadows from Huiquan Bay (HQ) and Swan Lake (SL)
3.4.3 Total phosphorus and acid volatile sulfate

TP content at Swan Lake was significantly lower than at Huiquan Bay (F(1, 19)=241.975, P < 0.001), and 2.6-fold greater at Huiquan Bay in corresponding layers. Depth has significant effect on TP in three-way ANOVA (F(4, 19)=4.315, P < 0.05).

In Huiquan Bay, the minimum and maximum average AVS contents respectively appeared at 0–2 cm and 8–10 cm (Fig. 4). The maximum AVS content in Swan Lake appeared at 2–4 cm, and the minimum was in 8–10 cm (Fig. 4). The AVS in Swan Lake was significantly higher than that in Huiquan Bay (F(1, 19)=15.964, P < 0.01), and no significant effect of depth on AVS was observed according to the two-way ANOVA (F(4, 19)=0.92, P < 0.05).


By the use of X-ray CT technology for the rhizomes and roots of Z. japonica in oligotrophic and eutrophic sediments, we found the significant differences in root system morphology, density, and distribution. Through the analysis combing chemical properties of sediments and nutrients of seagrass leaves, the reasons contributing to the difference of root and rhizome acclimation patterns in sediments of distinct trophic status were addressed. In addition, the advantages and disadvantages of the application of X-ray CT technology in seagrass research are discussed.

4.1 Similarities and differences of Z. japonica root system acclimation characteristics

Here, we compared the morphology, density, and distribution characteristics of roots and rhizomes of Z. japonica in oligotrophic and eutrophic sediments. The trophic status of sediments had little influence on the root length, root and rhizome diameters of Z. japonica, which was demonstrated by Table 1. This result was consistent with the study of Kiswara et al. (2009) on root systems of six tropical seagrass species in Indonesian waters, which suggested that differences in sediment nutrient availability had little effect on root architecture parameters, such as length and diameter of per root order. However, the similarity in root lengths from sediments with different trophic status was inconsistent with several researches suggesting that seagrass in oligotrophic environment tended to own longer root length (Cabaço et al., 2009; Jiang et al., 2019). The longer root of seagrass is with a wider diameter allow the storage of carbohydrates and nitrogenous compounds (Vermaat, 2009). In our research, Z. japonica had not adopted these traits to acclimate the oligotrophic sediment. The comparison of our and previous researches suggested that under difference of sediment tropic status, the seagrass phenotype plasticity in root system differs among seagrass species.

However, Z. japonica in oligotrophic sediment owned longer rhizome node (Table 1). This result was consistent with the previous study on Z. noltii (Cabaço et al., 2009). There is obvious positive correlation between the node length and growing speed of seagrass (Kiswara et al., 2009; Vermaat, 2009). Thus, the longer Z. japonica node length showed its relative rapid expanding rate of rhizomes in oligotrophic sediment. In addition, Z. japonica rhizome preferred to constrict its range to upper sediment layer, and its rhizome vertical distribution in oligotrophic sediment was wider. Although the mixed-effect ANOVA indicated the influence of depth on rhizome volume densities in both oligotrophic and eutrophic sediments, depth only had significant influence on the rhizome length density in eutrophic sediment of Swan Lake (Table 3). In addition, the length density was the preferable indicator for the root system distribution characteristics (Li et al., 2014; Gao et al., 2019a). Thus, the Z. japonica rhizome in oligotrophic sediment distributed relatively deeper than that in eutrophic sediment.

The trophic status of sediments influenced the rhizome and root densities of Z. japonica, those in oligotrophic sediment were obviously higher (Table 1). The three-way ANOVA indicated that site, which was featured with distinct sediment trophic status, was the dominant effect on rhizome and root densities of Z. japonica (Table 3). This is consistent with previous studies showing that seagrass species in low-nutrient habitats exhibited more extensive root systems to enhance their potential for acquiring nutrients from the sediments (Perez et al., 1994; Touchette and Burkholder, 2000; Hovey et al., 2011). However, the Z. japonica root system of higher density in oligotrophic sediment mainly reflected its corresponding higher shoot density. The significant difference of below-ground biomass percentages (Table 1) contributed to explain the distinct root system densities in two sites, but that of seagrass in oligotrophic sediment was merely 1.32-fold than eutrophic sediment. The root and rhizome length densities of Huiquan Bay were about 4.3- and 3.2- fold than those of Swan Lake in respective, thus shoot density should be the primary effect leading to the distinct root system densities. Meanwhile, the root-per-rhizome lengths without significant difference in two sites (Table 1) meant the below-ground parts of seagrass in either site had not allocated more to their root development, and that helped to support the significance of shoot density for the difference of root system density. Besides the shoot density, the root system longevity difference between two Z. japonica meadows was the other significant factor leading to their distinct densities. According to our previous research on these two seagrass meadows, about 6% shoots in the Swan Lake were recruited by overwintering rhizomes, but that percentage in Huiquan Bay reached 66% (Zhang et al., 2020).

4.2 Reasons causing different Z. japonica root system acclimation characteristics

In this study, Z. japonica in oligotrophic sediment endured N deficiency, and the sulfide, as a phytotoxin, threated the living of Z. japonica in eutrophic sediment. Seagrass derive N and P from sediment pore water (especially ammonium) and the water column (most nitrate), and the ratios of carbon, nitrogen and phosphorous (C꞉N꞉P) of seagrass leaves tend towards 550꞉30꞉1, which was called "seagrass Redfield ratios" (Atkinson and Smith, 1983). Duarte (1990) suggested that when N and P contents of seagrass leaves are lower than 1.8% and 0.2%, respectively, the plants are strongly nutrient limited, but not vice versa. In our study, the P contents of Z. japonica leaves in both sites were higher than 0.2% (Table 2), and the N/P of leaves from both sites were obviously lower than 30, thus it did not appear to be restricted by P. And the leaf N content and C/N in Swan Lake suggested that Z. japonica in the eutrophic sediment hadn't suffered N deficiency, but corresponding values in Huiquan Bay demonstrated that Z. japonica in the oligotrophic sediment was under N deficiency. The sulfide of higher concentration resulted in more serious intrusion to Z. japonica in eutrophic sediment. Sulfide intrusion in seagrass is widespread in all climate zones where seagrass are growing. The sulfide toxicity and physiological stress can lead to deleterious effects of seagrass like growth performance reduction and dieback (Cambridge et al., 2012; Apostolaki et al., 2018). The research reviewed the sensitivity thresholds for sulfide of different plant species showed that the sulfide toxicity threshold levels of Zostera was 200– 1 800 μmol/L (Lamers et al., 2013). Converted to the unit in our research, with the corresponding sediment moisture content, that range was about 1.6–14.4 μg/g. Because the sulfide contents in eutrophic sediment of Swan Lake surpassed that range in general (Fig. 4, Supplementary Table S2), the damage by this phytotoxin would be more severe.

SupplementaryTableS 2 Root and rhizome densities values of Zostera japonica in Huiquan Bay (HQ) and Swan Lake (SL)

Here, we addressed the reasons caused the differences of morphology, density, and distribution characteristics of Z. japonica root system in sediments of distinct trophic status. The development of seagrass rhizome and root and its phenotypic plasticity are closely affected by nutrients available (Kiswara et al., 2009; Hovey et al., 2012). The relative rapid expanding rate, which reflect as its longer node of Z. japonica rhizome in oligotrophic sediment should be interpreted by the lower nitrogenous nutrients availability (Table 4). The explanation of N deficiency impact was consistent with previous study on Z. noltii which owned longer internodes at low intertidal with less organic matter, lower N content (Cabaço et al., 2009). It is reasonable to infer that Z. japonica in oligotrophic sediment relatively speeded up its root system development through high rhizome expanding rate in order to acquire more nutrients. Moreover, the relative deeper distribution of Z. japonica rhizome in oligotrophic sediment (Table 3; Fig. 3) should be attributed to N deficiency impact. The deeper rhizome distributing range of seagrass facilitate it to absorb nutrients from wider space (Delgard et al., 2016), that also indicated its urgency to relieve N deficiency. Meanwhile, the relative shorter rhizome node and shallower rhizome distribution in eutrophic sediment could be interpreted by the oxygen transport convenience. Seagrass transport oxygen to belowground part to support aerobic root respiration and protect against sulfide intrusion (Connell et al., 1999; Martin et al., 2019). Even though the root length did not differ significantly, the shorter rhizome node and shallower rhizome distribution in eutrophic sediment shortened the distance from seagrass leaves to root tips, and facilitated Z. japonica to respirate and defend sulfide in sediment with more sulfide accumulation.

The shoot density difference of Z. japonica, which was the primary reason caused different root system densities, was resulted from the more serious sulfide inhibition in eutrophic sediment (Table 4; Fig. 4), rather than the effect of N deficiency in oligotrophic sediment. According to the investigation for Z. japonica meadow near the east bank of Swan Lake (Zhang et al., 2015), the leaf N content surpassed 1.8% and C/N value was less than 18.3, which indicated that Z. japonica in this site hadn't endured N deficiency. However, the Z. japonica density surpassed 3 356 shoots/m2, and was significantly larger than the 2 346 shoots/m2 in our research. Hence, the impact of N deficiency was not the reason of higher shoot density in oligotrophic sediment. Although there was no sediment sulfide content data of Zhang's investigation (Zhang et al., 2015), our past survey in Swan Lake had recorded the sediment AVS contents in Z. japonica meadow near the east bank (Song, 2018), which was just about one third of the AVS level of Z. japonica meadow sediment near the south bank in this study. Comparing the shoot densities and AVS contents in Huiquan Bay and the east and south banks of Swan lake, the sulfide inhibition was demonstrated to be the main reason resulting in the less shoots in eutrophic sediment. The finding on seagrass shoots reduction by sulfide was consistent with other researches that demonstrated seagrass meadow deterioration with increased loading of organic matter and sulfide accumulation (Borum et al., 2005; Calleja et al., 2007).

4.3 Advantages and disadvantages of the application of X-ray CT technology

The study also represents the first use of X-ray CT scanning to produce 3D images and quantitative data of seagrass root systems, and demonstrates this approach as feasible for future research. The 3D imagine could provide visual impression of Z. japonica rhizomes and roots, and would be beneficial to grasp their morphological characteristics intuitively. The application of X-ray CT technology enhanced the efficiency of measuring seagrass roots and rhizomes, because the relevant scanning, recognition, measuring, and calculating work are carried out automatically. Meanwhile, the whole procedure was non-destructive for core sample, thus it revealed the distributing information of seagrass roots and rhizomes as real as natural state, and avoid possible manual harm to them in traditional measurement. Furthermore, the digitalization of seagrass below-ground tissues makes more accurate and delicate measuring to be feasible, for instance, the root and rhizome densities of Z. japonica in this research could be presented in hundreds of layers.

However, there are several disadvantages of X-ray CT technology emerged during our work, which remain to be considered or resolved in such researches of future. At first, the detection limitation of CT scanning was about 2–3 times the side length of voxel (Kaestner et al., 2006), that means the small roots whose diameter less than about 200 μm could not be recognized. However, the traditional approach by human eyes can recognize these small roots with ease. Meanwhile, though the imaging was powerful, the recognition function of software adopted could not automatically distinguish and acquire the detailed morphological characteristics like root length, rhizome node length, and roots amount of each node. Moreover, such information was supplemented by manual measurement work carried out synchronously in the same locations. Moreover, the size of core sample is restricted by the volume capacity of X-ray CT scanner. In spite of these disadvantages, the advantages of X-ray CT technology over traditional investigation methods be of benefit in exploring morphological, physiological, and ecological characteristics of seagrass below-ground tissues, and it still owns potential to be improved.


We used X-ray CT scanning to observe a difference in root and rhizome acclimation of Z. japonica living in oligotrophic and eutrophic sediments. Morphology measures, and distribution of rhizomes and roots demonstrated that Z. japonica in oligotrophic sediment, developed the root system with longer rhizome node, deeper rhizome distribution, larger allocation to below-ground tissues in order to acquire more nutrients and relieve the N deficiency, and the shorter rhizome node and shallower rhizome distribution of Z. japonica in eutrophic sediment facilitate its oxygen transport. The lower root and rhizome densities of Z. japonica in eutrophic sediment were mainly caused by fewer shoots and shorter longevity, which was resulted from the more serious sulfide inhibition. Moreover, to the best of our knowledge, this is the first study to use X-ray CT methods in this manner, and demonstrates their feasibility for future seagrass research.


All data generated and/or analyzed during this study are available upon request by contact with the first or corresponding author.


We are indebted to the help from Yumeng JIANG and Han YANG in Zhejiang Gongshang University, staff in the Environmental assessment laboratory of Key Laboratory of Engineering Oceanography, Second Institute of Oceanography, MNR, especially for their director, Dr. Hengtao XU. We also are grateful for our colleagues for their assistance during this work.

Abe M, Yokota K, Kurashima A, Maegawa M. 2009. Temperature characteristics in seed germination and growth of Zostera japonica Ascherson & Graebner from Ago Bay, Mie Prefecture, central Japan. Fisheries Science, 75(4): 921-927. DOI:10.1007/s12562-009-0123-z
Apostolaki E T, Holmer M, Santinelli V, Karakassis I. 2018. Species-specific response to sulfide intrusion in native and exotic Mediterranean seagrasses under stress. Marine Environmental Research, 134: 85-95. DOI:10.1016/j.marenvres.2017.12.006
Atkinson M S, Smith S V. 1983. C: N: P ratios of benthic marine plants. Limnology and Oceanography, 28(3): 568-574. DOI:10.4319/lo.1983.28.3.0568
Beca-Carretero P, Guihéneuf F, Winters G, Stengel D B. 2019. Depth-induced adjustment of fatty acid and pigment composition suggests high biochemical plasticity in the tropical seagrass Halophila stipulacea. Marine Ecology Progress Series, 608: 105-117. DOI:10.3354/meps12816
Bercovich M V, Schubert N, Saá A C A, Silva J, Horta P A. 2019. Multi-level phenotypic plasticity and the persistence of seagrasses along environmental gradients in a subtropical lagoon. Aquatic Botany, 157: 24-32. DOI:10.1016/j.aquabot.2019.06.003
Blaser S R G A, Schlüter S, Vetterlein D. 2018. How much is too much?—Influence of X-ray dose on root growth of faba bean (Vicia faba) and barley (Hordeum vulgare). PLoS One, 13(3): e0193669. DOI:10.1371/journal.pone.0193669
Borovec O, Vohník M. 2018. Ontogenetic transition from specialized root hairs to specific root-fungus symbiosis in the dominant Mediterranean seagrass Posidonia oceanica. Scientific Report, 8: 10773. DOI:10.1038/s41598-018-28989-4
Borum J, Pedersen O, Greve T M, Frankovich T A, Zieman J C, Fourqurean J W, Madden C J. 2005. The potential role of plant oxygen and sulphide dynamics in die-off events of the tropical seagrass, Thalassia testudinum. Journal of Ecology, 93(1): 148-158. DOI:10.1111/j.1365-2745.2004.00943.x
Bradley M P, Stolt M H. 2006. Landscape-level seagrass-sediment relations in a coastal lagoon. Aquatic Botany, 84(2): 121-128. DOI:10.1016/j.aquabot.2005.08.003
Cabaço S, Machás R, Santos R. 2009. Individual and population plasticity of the seagrass Zostera noltii along a vertical intertidal gradient. Estuarine, Coastal and Shelf Science, 82(2): 301-308. DOI:10.1016/j.ecss.2009.01.020
Calleja M L, Marbà N, Duarte C M. 2007. The relationship between seagrass (Posidonia oceanica) decline and sulfide porewater concentration in carbonate sediments. Estuarine, Coastal and Shelf Science, 73(3-4): 583-588. DOI:10.1016/j.ecss.2007.02.016
Cambridge M L, Fraser M W, Holmer M, Kuo J, Kendrick G A. 2012. Hydrogen sulfide intrusion in seagrasses from Shark Bay, Western Australia. Marine and Freshwater Research, 63(11): 1027-1038. DOI:10.1071/MF12022
Connell E L, Colmer T D, Walker D I. 1999. Radial oxygen loss from intact roots of Halophila ovalis as a function of distance behind the root tip and shoot illumination. Aquatic Botany, 63(3-4): 219-228. DOI:10.1016/S0304-3770(98)00126-0
Delgard M L, Deflandre B, Kochoni E, Avaro A, Cesbron F, Bichon S, Poirier D, Anschutz P. 2016. Biogeochemistry of dissolved inorganic carbon and nutrients in seagrass (Zostera noltei) sediments at high and low biomass. Estuarine, Coastal and Shelf Science, 179: 12-22. DOI:10.1016/j.ecss.2016.01.012
Duarte C M. 1990. Seagrass nutrient content. Marine Ecology Progress Series, 67(2): 201-207. DOI:10.3354/meps067201
Duarte C M, Merino M, Agawin N S R, Uri J, Fortes M D, Gallegos M E, Marbá N, Hemminga M A. 1998. Root production and belowground seagrass biomass. Marine Ecology Progress Series, 171: 97-108. DOI:10.3354/meps171097
Erftemeijer P L A, Middelburg J J. 1993. Sediment-nutrient interactions in tropical seagrass beds: a comparison between a terrigenous and a carbonate sedimentary environment in South Sulawesi (Indonesia). Marine Ecology Progress Series, 102: 187-198. DOI:10.3354/meps102187
Ettinger C L, Voerman S E, Lang J M, Stachowicz J J, Eisen J A. 2017. Microbial communities in sediment from Zostera marina patches, but not the Z. marina leaf or root microbiomes, vary in relation to distance from patch edge. PeerJ, 5: e3246. DOI:10.7717/peerj.3246
Gao W, Blaser S R G A, Schlüter S, Shen J B, Vetterlein D. 2019a. Effect of localised phosphorus application on root growth and soil nutrient dynamics in situ-comparison of maize (Zea mays) and faba bean (Vicia faba) at the seedling stage. Plant and Soil, 441(1): 469-483. DOI:10.1007/s11104-019-04138-2
Gao W, Schlüter S, Blaser S R G A, Shen J B, Vetterlein D. 2019b. A shape-based method for automatic and rapid segmentation of roots in soil from X-ray computed tomography images: Rootine. Plant and Soil, 441(1): 643-655. DOI:10.1007/s11104-019-04053-6
Hovey R K, Cambridge M L, Kendrick G A. 2011. Direct measurements of root growth and productivity in the seagrasses Posidonia australis and P. sinuosa. Limnology and Oceanography, 56(1): 394-402. DOI:10.4319/lo.2011.56.1.0394
Hovey R K, Cambridge M L, Kendrick G A. 2012. Season and sediment nutrient additions affect root architecture in the temperate seagrasses Posidonia australis and P. sinuosa. Marine Ecology Progress Series, 446: 23-30. DOI:10.3354/meps09483
Isaksen M F, Finster K. 1996. Sulphate reduction in the root zone of the seagrass Zostera noltii on the intertidal flats of a coastal lagoon (Arcachon, France). Marine Ecology Progress Series, 137: 187-194. DOI:10.3354/meps137187
Jensen S, Bell S. 2001. Seagrass growth and patch dynamics: cross-scale morphological plasticity. Plant Ecology, 155(2): 201-217. DOI:10.1023/A:1013286731345
Jiang Z J, Zhao C Y, Yu S, Liu S L, Cui L J, Wu Y C, Fang Y, Huang X P. 2019. Contrasting root length, nutrient content and carbon sequestration of seagrass growing in offshore carbonate and onshore terrigenous sediments in the South China Sea. Science of the Total Environment, 662: 151-159. DOI:10.1016/j.scitotenv.2019.01.175
Johnson M G, Andersen C P, Phillips D L, Kaldy J E. 2016. Zostera marina root demography in an intertidal estuarine environment measured using minirhizotron technology. Marine Ecology Progress Series, 557: 123-132. DOI:10.3354/meps11867
Kaestner A, Schneebeli M, Graf F. 2006. Visualizing three-dimensional root networks using computed tomography. Geoderma, 136(1-2): 459-469. DOI:10.1016/j.geoderma.2006.04.009
Kamp-Nielsen L, Vermaat J E, Wesseling I, Borum J, Geertz-Hansen O. 2002. Sediment properties along gradients of siltation in South-east Asia. Estuarine, Coastal and Shelf Science, 54(1): 127-137. DOI:10.1006/ecss.2001.0822
Kiswara W, Behnke N, van Avesaath P, Huiskes A H L, Erftemeijer P L A, Bouma T J. 2009. Root architecture of six tropical seagrass species, growing in three contrasting habitats in Indonesian waters. Aquatic Botany, 90(3): 235-245. DOI:10.1016/j.aquabot.2008.10.005
Koebernick N, Weller U, Huber K, Schlüter S, Vogel H J, Jahn R, Vereecken H, Vetterlein D. 2014. In situ visualization and quantification of three-dimensional root system architecture and growth using X-ray computed tomography. Vadose Zone Journal, 13(8): 1-10. DOI:10.2136/vzj2014.03.0024
Koren K, Brodersen K E, Jakobsen S L, Kühl M. 2015. Optical sensor nanoparticles in artificial sediments—a new tool to visualize O2 dynamics around the rhizome and roots of seagrasses. Environmental Science and Technology, 49(4): 2286-2292. DOI:10.1021/es505734b
Lamers L P M, Govers L L, Janssen I C J M, Geurts J J M, Van der Welle M E W, Van Katwijk M M, Van der Heide T, Roelofs J G M, Smolders A J P. 2013. Sulfide as a soil phytotoxin—a review. Frontiers in Plant Science, 4: 268. DOI:10.3389/fpls.2013.00268
Li H B, Ma Q H, Li H G, Zhang F S, Rengel Z, Shen J B. 2014. Root morphological responses to localized nutrient supply differ among crop species with contrasting root traits. Plant and Soil, 376(1-3): 151-163. DOI:10.1007/s11104-013-1965-9
Li H C, Zhang P D, Li W T, Yang X L, Hu C Y, Li C J. 2019. Quantitative distribution and ecological characteristics of seagrass beds in the coastal area of Moye Island, Yellow Sea. Marine Science, 43(4): 46-51. (in Chinese with English abstract)
Martin B C, Bougoure J, Ryan M H, Bennett W W, Colmer T D, Joyce N K, Olsen Y S, Kendrick G A. 2019. Oxygen loss from seagrass roots coincides with colonisation of sulphide-oxidising cable bacteria and reduces sulphide stress. ISME Journal, 13(3): 707-719. DOI:10.1038/s41396-018-0308-5
McDonald A M, Prado P, Heck K L, Fourqurean J W, Frankovich T A, Dunton K H, Cebrian J. 2016. Seagrass growth, reproductive, and morphological plasticity across environmental gradients over a large spatial scale. Aquatic Botany, 134: 87-96. DOI:10.1016/j.aquabot.2016.07.007
Miki S. 1933. On the sea-grasses in Japan (1): Zostera and Phyllospadix, with special reference to morphological and ecological characters. The Botanical Magazine, 47(564): 842-862. DOI:10.15281/jplantres1887.47.842
Pedersen M Ø, Kristensen E. 2015. Sensitivity of Ruppia maritima and Zostera marina to sulfide exposure around roots. Journal of Experimental Marine Biology and Ecology, 468: 138-145. DOI:10.1016/j.jembe.2015.04.004
Perez M, Duarte C M, Romero J, Sand-Jensen K, Alcoverro T. 1994. Growth plasticity in Cymodocea nodosa stands: the importance of nutrient supply. Aquatic Botany, 47(3-4): 249-264. DOI:10.1016/0304-3770(94)90056-6
Povidisa K, Delefosse M, Holmer M. 2009. The formation of iron plaques on roots and rhizomes of the seagrass Cymodocea serrulata (R. Brown) Ascherson with implications for sulphide intrusion. Aquatic Botany, 90(4): 303-308. DOI:10.1016/j.aquabot.2008.11.008
Rubio L, Linares-Rueda A, García-Sánchez M J, Fernández J A. 2007. Ammonium uptake kinetics in root and leaf cells of Zostera marina L. Journal of Experimental Marine Biology and Ecology, 352(2): 271-279. DOI:10.1016/j.jembe.2007.07.024
Short F, Carruthers T, Dennison W, Waycott M. 2007. Global seagrass distribution and diversity: a bioregional model. Journal of Experimental Marine Biology and Ecology, 350(1-2): 3-20.
Stapel J, Aarts T L, van Duynhoven B H M, de Groot J D, van den Hoogen P H W, Hemminga M A. 1996. Nutrient uptake by leaves and roots of the seagrass Thalassia hemprichii in the Spermonde Archipelago, Indonesia. Marine Ecology Progress Series, 134(1-3): 195-206. DOI:10.3354/meps134195
Touchette B W, Burkholder J M. 2000. Review of nitrogen and phosphorus metabolism in seagrasses. Journal of Experimental Marine Biology and Ecology, 250(1-2): 133-167. DOI:10.1016/S0022-0981(00)00195-7
Tracy S R, Roberts J A, Black C R, McNeill A, Davidson R, Mooney S J. 2010. The X-factor: visualizing undisturbed root architecture in soils using X-ray computed tomography. Journal of Experimental Botany, 61(2): 311-313. DOI:10.1093/jxb/erp386
Vermaat J E. 2009. Linking clonal growth patterns and ecophysiology allows the prediction of meadow-scale dynamics of seagrass beds. Perspectives in Plant Ecology, Evolution and Systematics, 11(2): 137-155. DOI:10.1016/j.ppees.2009.01.002
Vohník M, Borovce O, Kolaříková Z, Sudová R, Réblová M. 2019. Extensive sampling and high-throughput sequencing reveal Posidoniomyces atricolor gen. et sp. nov. (Aigialaceae, Pleosporales) as the dominant root mycobiont of the dominant Mediterranean seagrass Posidonia oceanica. MycoKeys, 55: 59-86. DOI:10.3897/mycokeys.55.35682
Zhang X M, Lin H Y, Song X Y, Xu S C, Yue S D, Gu R T, Xu S, Zhu S Y, Zhao Y J, Zhang S Y, Han G X, Wang A D, Sun T, Zhou Y. 2019. A unique meadow of the marine angiosperm Zostera japonica, covering a large area in the turbid intertidal Yellow River Delta, China. Science of the Total Environment, 686: 118-130. DOI:10.1016/j.scitotenv.2019.05.320
Zhang X M, Zhou Y, Liu P, Wang F, Liu B J, Liu X J, Xu Q, Yang H S. 2014. Temporal pattern in the bloom-forming macroalgae Chaetomorpha linum and Ulva pertusa in seagrass beds, Swan Lake lagoon, North China. Marine Pollution Bulletin, 89(1-2): 229-238. DOI:10.1016/j.marpolbul.2014.09.054
Zhang X M, Zhou Y, Liu P, Wang F, Liu X J, Yang H S. 2015. Temporal pattern in biometrics and nutrient stoichiometry of the intertidal seagrass Zostera japonica and its adaptation to air exposure in a temperate marine lagoon (China): Implications for restoration and management. Marine Pollution Bulletin, 94(1-2): 103-113. DOI:10.1016/j.marpolbul.2015.03.004
Zhang X M, Zhou Y, Xu S C, Wang P M, Zhao P, Yue S D, Gu R T, Song X Y, Xu S, Liu J X, Wang X D. 2020. Differences in reproductive effort and sexual recruitment of the seagrass Zostera japonica between two geographic populations in northern China. Marine Ecology Progress Series, 638: 65-81. DOI:10.3354/meps13248
Zhou Y, Zhang F S, Yang H S, Zhang S M, Ma X N. 2003. Comparison of effectiveness of different ashing auxiliaries for determination of phosphorus in natural waters, aquatic organisms and sediments by ignition method. Water Research, 37(16): 3 875-3 882. DOI:10.1016/S0043-1354(03)00267-7