Bioindication of Heavy Metals in a Waterfall Outow Using a Bryophyte Community

Huay Pah Lahd stream in Doi Suthep-Pui National Park, Thailand, is potentially vulnerable to nearby anthropogenic activities. In this study, we determined heavy metal accumulation in bryophyte tissue and their growth substrates. Enrichment factors (EFs) of heavy metals were employed to monitor concentrations in bryophyte tissue. Of eight bryophyte taxa investigated, Scopelophila cataractae showed the highest capacity to accumulate metals in tissue, particularly Fe, Zn, Cd and Cu in protonemata (8,026.7, 1,187.2, 16.9 and 530.1 mg kg -1 , respectively). Furthermore, the endangered and rare bryophyte taxa S. cataractae and Porella acutifolia were found intermingled with other urban and common aquatic bryophytes. These taxa might be considered sensitive warning organisms for heavy metal stress in stream ecosystems induced by environmental pollution. Because EFs of all heavy metals were < 2, this suggests that natural processes are the key source of heavy metals; furthermore, the environment of this National Park was identied as being heathy, and an important ecosystem buffer and biodiversity haven. the same period of time (June, during the rainy season) and at the same study site as in the previous study 19 . Narrow, light-green patches of the protonemata occur along the stone base (c. 50 cm height) in streams, together with colonies of other bryophytes such as H. involuta. The colony consisted of numerous lamentous protonema which produced shoots of S. cataractae with numerous gemma-like cells on the axils of younger leaves.


Introduction
A number of heavy metals in soil and water (e.g., zinc (Zn), copper (Cu), iron (Fe), etc.) are essential for growth and reproduction of biota. However, other metals and metalloids (e.g., cadmium (Cd), arsenic (As), mercury (Hg) etc.), even when present in low quantities in the growth medium, can adversely affect organism development, homeostasis, metabolism and reproduction.
Numerous studies have found that hazardous metallic elements have the ability to affect biota in terrestrial ecosystems via the food chain and bioaccumulation 1 .
In freshwater habitats, exposure to heavy metals and other contaminants results in a range of negative impacts, including loss of aquatic biota and ecosystem alterations 2 . On the other hand, many output streams from waterfalls have been reported to have low heavy metal contamination as they are located primarily in highland national parks or other parcels of land designated as protected by governments. The physical condition of a waterfall habitat, which has a high velocity and volume of water, can dilute heavy metal concentrations. In many locations, however, rapid development in the tourism industry and various anthropogenic activities along waterfall streams have resulted in elevated concentrations of heavy metals in waterfall ecosystems in recent years 3 .
Bryophytes are signi cant species in aquatic and related terrestrial ecosystems, especially those where heavy metal concentrations have been elevated from anthropogenic sources. Heavy metals can be absorbed and accumulated by bryophytes through their surfaces from growth media such as soil and rock. Scopelophila cataractae (Mitt.) Broth. (Pottiaceae), for example, experiences high tolerance and accumulation of Cd and Cu 4 . Mosses and liverworts have been identi ed as ecological bioindicators of a variety of pollution sources and environmental changes 5 . This implies that the presence of certain bryophyte taxa may be used to monitor the ecological status of harsh environments.
In this study, we explore bryophyte communities and heavy metal accumulation capabilities of bryophytes found in the out ow from the Pah Lahd waterfall, Doi Suthep-Pui National Park, Thailand. This National Park is adjacent to the city of Chiang Mai, and is likely subject to anthropogenic activities such as stream pollution. To evaluate heavy metals potentially available to plants from surface soil and to determine the contamination level of each metal for use in monitoring anthropogenic pollution in the study site, enrichment factors (EFs) were used. ¢ N, 98 o 55¢ 52.0¢¢ E), the waterfall is surrounded by mixed evergreen/deciduous forest, small villages and tourist activities. The water depth in the stream ranges from 0.2 to 0.6 m. In 2020 total annual rainfall, mean relative humidity and mean temperature in Chiang Mai Province were approximately 1,085.1 mm., 63.4% and 27.6 °C, respectively. Anthropogenic activities are thought to be a major cause of water pollution in the waterfall streams.

Methods
Collection of plant, sediment and water samples.The eld excursion was done on June 15, 2020, after receiving authorization from Department of National Parks, Wildlife, and Plant Conservation of Thailand. All bryophyte specimens were collected in Huay Pah Lahd streams with a plastic spatula on wet rocks and the thin soil layer above the rock, which are the substrates for all bryophyte taxa. Individual plant samples were stored in clean plastic bags, labeled, and transported to the laboratory in ice-lled box as quickly as possible. Plant tissue was rinsed with deionized (DI) water for 30 s to remove excess soil, visible debris, ne stones and pebbles, loosely attached mineral particles, and tiny organic materials, then air-dried at room temperature and stored at 4°C until required 44 29 and Zhang and He 47 .
Soil samples were collected under bryophyte patches with a plastic spatula. A water sample was collected from a stream near the plant specimens in a 1 L polyethylene bottle and stored in an ice-lled container (4 o C). In the eld, pieces of rocks under bryophyte patches (only S. cataractae and P. acutifolia var. birmanica) were crushed to small sizes using a hammer. The rock materials were stored in self-locking polythene bags and sealed in double bags before being transported to the laboratory. All the plant experiments were carried out in accordance with relevant institutional, national, and international guidelines and legislation.
Water temperature and dissolved oxygen (DO) level were determined with a DO meter (HI 9147, Hanna Instruments, USA), pH with a LAB 850 set pH meter (Accumetâ AP115, USA), and water depth with a wooden ruler (2 m). Air temperatures at the sampling site were determined at the same time of water sampling with a digital thermometer.
Heavy metals analysis. Plant and soil samples were dried at 70°C for 3 days. Each sample was nely powdered to pass through a 250-mm mesh using an IKA mill. Rocks were dried at 110°C for 24 h and then ground to a powder using an abrasion testing machine. The crushed rocks were then sieved using a 75-mm mesh sieve. Plant material was placed in a vessel tube and digested with aqua regia (conc. 70% HNO 3 : 37% HCl = 1:3); the soil sample was digested with conc. 70% HNO 3  Data analyses. Enrichment factors (EFs) are used to assess the levels of an element potentially available to bryophytes from soil, and also to evaluate the contribution to metal content in bryophyte tissues from anthropogenic sources. It is calculated as follows, using the example of Fe: Where C n is the concentration of the metal 'n' in bryophyte or soil samples, and C Fe is the concentration of Fe determined before exposure. As proposed by Macedo-Miranda et al. 49 , EF is classi ed into four categories, EF £ 1, no contamination; 3 < EF < 5, slight contamination; 6 < EF £ 9, moderate contamination; and EF ³ 10, highly contamination.
On a Windows-based PC, statistical analysis was performed using SPSS® (SPSS, Chicago, IL). To identify signi cant differences in mean values, a one-way ANOVA and least signi cant difference (LSD) post hoc comparison were employed.

Results And Discussion
Physicochemical properties of waterfall stream. Table 1 shows the results of the environmental parameters of the Huay Pah Lahd stream. Rainfall strongly affects physicochemical properties of the water because velocity, depth, and level increased, and nutrient run-off (and/or pollution run-off) changed during the sampling period in the rainy season. The water at the sampling site is shallow (0.4-1 m). Temperatures in water samples range from 20°C to 33°C, while water temperature along the bank of Pharadorn waterfall, located downstream of Romklao waterfall in Phu Hin Rong Kla National Park, is 25°C 6 . Low water temperature and low light intensity are important for bryophyte development and primary productivity; however, nutrient enrichment in plant media (e.g., soil, sediment) is the most important component for bryophyte growth 7 .
The quantities of nutrients such as ammonia-nitrogen (NH 3 -N), nitrate-nitrogen (NO 3 -N), total Kjeldahl nitrogen (TKN) and phosphate (PO 4 ) were below detectable levels, i.e., 1.01 mg L -1 , < 4.0 mg L -1 , and 0.02 mg L -1 , respectively. Increased concentrations of phosphorus and nitrogen compounds, which are limiting factors and essential nutrients for aquatic life, can cause eutrophication. However, dilution effects during the rainy season in lotic ecosystems is a key factor in causing low nutrient concentrations in water bodies 8 . Heavy metal concentrations (Cu, Cd, Zn, Fe, Cr, Pb and Mn) in the water sample were relatively low to undetectable except for Ni, which was detected at 0.056 mg L -1 . Furthermore, F¯ and Cl¯contents were < 0.15 and 6.2 mg L -1 , respectively, which are considered low according to the permissible limits set by the World Health Organization (WHO) at 0.6-1.5 mg L -1 and 250 mg L -1 , respectively 9 . Total organic carbon (TOC) content of the water sample is below the detection limit (< 0.05 mg L -1 ), which is due to the low content of contaminants in the water sources 10 .
Total solids is a direct measurement of the total mass of organic and inorganic particles suspended in water, as well as total dissolved ions in the water 11 . A high total solid content in waters is the most likely cause of increased total hardness. Other possible sources of increased hard water content include Ca, Mg and other heavy metals widely distributed in rocks and sediments 12 . Pah Lahd stream is naturally soft in this study (31.7 mg L -1 ) because it has very low amounts of total solids and minerals. Soft water is de ned as having a low amount of calcium carbonate in the water sample 13 .
In this study, the pH value of the stream was near neutral (6.75). Slightly acidic water (~pH 5-6) occurs naturally, enabling some heavy metals that are adsorbed to mineral surfaces to be dissolved in aquatic ecosystems, as well as enhancing mineral dissolution in sediment 14 . However, in this investigation, slightly acidic water (pH 6.5-6.9) was not shown to be detrimental to aquatic biota or the ecosystem. The DO level of the study site (5.03 mg L -1 ) was nearly equivalent to the DO water quality standard in a Thai waterfall stream (6 mg L -1 ); nevertheless, a reduction in DO levels might be linked to human activities, i.e., the presence of relatively large numbers of tourists and improper waste disposal 15 . High ow velocity and turbulence of a waterfall increases DO content 16 . Biological oxygen demand (BOD) is another important indicator of water quality, as it measures the quantity of oxygen required for microbial respiration and biological degradation of organic matter in water. Reduced BOD levels imply that the quantity of organic substances is promoting the growth of microbial populations, thus enhancing the available DO content for aquatic life. The current study revealed that the water sample had a low BOD level (<1 mg L -1 ), indicating that the waterfall ecosystem had good water quality 17 .
Bryophyte taxa in the study site. Based on their microhabitats and life modes, a total of eight bryophytes were collected from two major taxonomic groupings i.e., (1)  Heavy metal concentrations in bryophyte tissues and substrates. Bryophytes do not have true roots, stems, and leaves; rather, they possess multicellular rhizoids at the lowest part of the structure, which are responsible for water and nutrient absorption 30 . Bryophyte rhizoids facilitate uptake of available minerals and water to the stems through capillary action. Because bryophytes lack roots, they can readily absorb heavy metals throughout their entire surface of rhizoids 31 . Furthermore, phyllids (leaf-like structure) and thalli of bryophytes have highly absorbent surfaces and an absence of waxy cuticle over the laminal surfaces. As a consequence, cell walls easily absorb moisture and a wide range of minerals and metal ions from the water that ows over the plant 32 . Heavy metal accumulation in bryophyte tissues in this study appear in Table 2 cataractae protonema); and Mn from 143.3 mg kg -1 (S. cataractae gametophyte) to 504.6 mg kg -1 (C. prionophyllum gametophyte).
In this investigation, gametophytes of S. cataractae had considerably greater Cu accumulation (p < 0.05) or approximately 3.7-59 × than did other bryophytes, although protonema of S. cataractae had a slightly higher Cu concentration than gametophytes of S. cataractae (p > 0.05). Because it accumulated Cu primarily in gametophyte tissue, S. cataractae, often known as "rare Cu moss," is categorized as a hyperaccumulator 33 . Copper is an essential nutrient that is required for plant development and growth. This element plays a signi cant role in regulating physiological functions such as the photosynthetic and respiratory electron transport chains, nitrogen xation, protein metabolism, antioxidant production, the ROS defense system, cell wall metabolism, and hormone perception, and acts as an essential cofactor for numerous metalloproteins 34 (Table 3). Copper concentrations in substrates of S. cataractae and water were generally in the following order: sediment substrate > rock > water. According to recent research, growth substrate is a key source of heavy metals in stream environments. This may have led to increased absorption and accumulation of Al, Cu and Zn in S. cataractae gametophytes 19 . Cadmium and Zn concentrations in rock substrates of S. cataractae and P. acutifolia var. birmanica were low(0.5 and 0.3 mg kg -1 for Cd, and 34.9 and 31.2 mg kg -1 for Zn, respectively). Heavy metals (Cu, Fe and Mn) in rock substrates of P. acutifolia var. birmanica were found to be similar to in the rock substrates of S. cataractae. This comparable distribution of heavy metals in rock substrates may be attributed to the fact that they are located in a similar environment and so receive heavy metals from similar sources and mechanisms. This trend is consistent with the ndings of the previous study 19 .
Many bryophyte taxa have been tested for their tolerance and accumulation capabilities at both laboratory and eld scales. In this study, substantial Mn concentrations were detected in gametophytic tissues of C. prionophyllum, Bryum sp., H.

Conclusions
The presence of bryophytes in terrestrial and aquatic ecosystems has received increasing attention in recent decades, as they play an important role in healthy habitats including nutrient, water, heavy metal and carbon cycling; soil formation; and successional processes. The EFs (all heavy metals < 2) indicated that bryophytes from the study site were enriched with low concentrations of heavy metals due to natural processes; however, anthropogenic activities, e.g., nearby tourist and community activities, may have an impact on increased heavy metal content in the future. This is the rst report to show the heavy metal accumulating capacity of bryophyte communities. Furthermore, S. cataractae accumulated more Cu, Cd, Zn and Fe than other bryophyte taxa, suggesting that it might be the best bioindicator in this aquatic environment.