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Tài liệu này là một nghiên cứu về chuyên đề mô hình aquponic. Aquaponic hiện là mô hình sản xuất đang được áp dụng nhiều tại các nước phát triển để tạo nguồn thực phẩm rau sạch, thủy sản sạch. Ngoài ra Mô hình auqaponic được áp dụng ở những khu vực đô thị, hay khan hiếm về nước. Tài liệu hoàn toàn bằng tiếng anh, nên đòi hỏi người đọc cần có khả năng tốt về ngoại ngữ này.
International Biodeterioration & Biodegradation 85 (2013) 693e700 Contents lists available at SciVerse ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod Effects of feeding frequency and photoperiod on water quality and crop production in a tilapiaewater spinach raft aquaponics system Jung-Yuan Liang a, Yew-Hu Chien a, b, * a b Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan Center of Marine Biotechnology and Bioenvironment, National Taiwan Ocean University, Keelung, Taiwan a r t i c l e i n f o a b s t r a c t Article history: Received 25 December 2012 Received in revised form 30 March 2013 Accepted 31 March 2013 Available online May 2013 A factorial arrangement of treatments, photoperiods for water spinach Ipomoea aquatica (12-h or 24-h light per day) X feeding frequencies for red tilapia Oreochromis sp (an equal daily ration evenly fed 6, or times at 4-h, 6-h or 12-h interval, respectively) were assigned to 12 tanks Each tank was an aquaponics system containing fish and raft-supporting plant Water loss in weeks was 3.3%, due to leaf transpiration mainly and evaporation Water quality remained safe and stable No fish died Overall average weight gain was 43.9% for fish and 169.0% for plant 24-h light resulted in 2.4% higher fish growth, 12% higher plant growth and lower accumulation of all nitrogen and phosphate species in water than 12-h light Increased feeding frequency favored stable and good water quality and fastened fish growth and plant growth by as much as 4.9% and 11%, respectively Ó 2013 Elsevier Ltd All rights reserved Keywords: Fish waste water Aquaponics Photoperiod Feeding frequency Tilapia Water spinach Introduction Aquaculture is the culture of aquatic organisms, commonly referred as animals, in a designated water body The water needs to be treated whenever toxicants in it have built up beyond animal’s safe level Toxicants such as ammonia and nitrite are derived from decomposition of unconsumed feed and metabolites or waste of the animals Hydroponics is the culture of aquatic plants in soilless water where nutrients for plant’s growth come entirely from a formulated fertilizer Aquaponics (a portmanteau of the terms aquaculture and hydroponics) integrates aquaculture and hydroponics into a common closed-loop eco-culture where a symbiotic relationship is created in which water and nutrients are recirculated and reused, concomitantly fully utilized and conserved In aquaponics system, waste organic matters from aquaculture system, which can become toxic to animals, are converted by microbes into soluble nutrients for the plants and simultaneously, hydroponics system has already treated the water and recirculates back to aquaculture system with cleansed and safe water for the animals Besides its ecological merits, aquaponics system can obtain extra * Corresponding author Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan Tel.: þ886 24622192x5204; fax: þ886 24625393 E-mail address: yhchien@mail.ntou.edu.tw (Y.-H Chien) 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.ibiod.2013.03.029 economic advantages: saving cost (input) on water treatment for aquaculture system, saving another cost on formulated fertilizer for hydroponics system and benefit from double outputs, harvest of animal and plant, by a single input, fish feed Tilapia is the most commonly used fish in aquaponics systems (Rakocy et al., 2006) for their high availability, fast growing, stress and diseases resistant and easy adaptation to indoor environment (Hussain, 2004) Water spinach or swamp cabbage Ipomoea aquatica is a semiaquatic tropical macrophyte and commonly grown as a leaf vegetable in East and Southeast Asia It has hollow stems, rooting at the nodes and flourishes naturally in waterways or moist soil It requires little care to grow and hence low cost and popular in Taiwan It has been found effective in treating aquaculture waste water (Li and Li, 2009) and eutrophic water with undesirable levels of nitrogen and/or phosphorus (Hu et al., 2008) Nutrients dynamics are quite complex in aquaponics system (Seawright et al., 1998) In such system, feed is the primary source of nutrients which are eventually tied up as the biomass of animal, plant and microbes or stayed free in water When no discharge, no nutrients are output until the animal and plant are harvested as economic crops Through microbial decomposition, the insoluble fish metabolite and unconsumed feed are converted into soluble nutrients which then can be absorbed by plant Therefore, plant growth and production are indirectly related to feeding strategies, fish metabolic condition and microbial activity While plant removes the soluble nutrients, water 694 J.-Y Liang, Y.-H Chien / International Biodeterioration & Biodegradation 85 (2013) 693e700 is filtered Consequently, water quality or safe guard of fish growth and production depends highly on the disposal capacity of the plant Besides the above factors which affect the nutrient availability for plant and fish, system designs, plant and fish species and other physical factors such as temperature, light sources and photoperiod all add up the managing complexity for a steady state of nutrient flow, which can be essential for the stable and predictable production of fish and plant in aquaponics system Since photoperiod affects photosynthesis and plant growth, the increase of photoperiod may also increase the removal capacity of nutrients in aquatic macrophyte Some studies have showed that the growth and productivity of floating aquatic macrophytes are directly related to the intensity and amount of light, so are the absorption rates for nutrients (Gopal, 1987; Urbanc-Bercic and Gaberscik, 1989) Petrucio and Esteves (2000) found that h longer photoperiod a day enabled two aquatic macrophytes to reduce more nutrients from the water Feeding frequency can affect feed intake of fish, quantity of uneaten feed, feed utilization efficiency and consequently, metabolite and excreta of fish and water quality In an intensive culture of fingerling walleye Stizostedion vitreum, Phillips et al (1998) found that higher frequency feeding resulted in higher daily dissolved oxygen (DO) and lower total ammonia nitrogen Postlarval Ayu Plecoglossus altivelis with higher feeding frequency at lower feeding rate had higher survival and growth (Cho et al., 2003) When fed at 10% body weight daily, newly weaned Australian snapper Pagrus auratus fed times a day had higher growth and lower size heterogeneity than fed and times a day (Tucker et al., 2006) Therefore, in the present study we investigate under a constant nutrient input, namely, the feeding rate, if increasing photoperiod can increase plant production, concomitantly, plant’s filtering capacity and nutrient concentration in water and also if increasing feeding frequency can even out through time, fish metabolite and excrete, concomitantly, stabilize water quality and increase fish production Materials and methods The experiment had a factorial arrangement of treatments, namely, feeding frequencies for red tilapia Oreochromis sp X photoperiods for water spinach I aquatica Forsk Illumination was 12 h or 24 h daily An equal daily ration was evenly fed to the fish 2, or times at 12-h, 6-h and 4-h interval, respectively Each treatment had replicates or experimental units The experiment was completed in weeks Each experiment unit had an orange plastic tank (115 cm L  102 cm W  99 cm H), filled with 1000 L freshwater and stocked with fish at 467 Æ 30 g each or around 3.7 kg mÀ3 Constant aeration was provided at tank bottom by a membrane disc diffuser (LTD-325/325 mm, Aquatek, Kaohsiung, Taiwan), which had a membrane diameter of 32.5 cm and provided an intensive air throughput of 0.02e0.12 CMM 1e3 mm diameter air bobbles A piece of 3-cm thick polyethylene raft covered almost entire water surface except a 15 cm  15 cm corner cut open, allowing an automatic feeder release pellet feed into the water A cut plant stem 25.1 Æ 3.7 cm or 7.8 Æ 0.5 g was wrapped around with layers of sponge, stuffed in a black plastic ring (4.5-cm D) then fit into one of the 63 evenly distributed round holes Total plant biomass on a raft was 490.2 Æ 5.5 g Part of a stem was submerged to expose its first bottom node, allowing for root initiation A piece of coarse screen (2.54 cm mesh) was secured 20 cm below the polyethylene raft to prevent the plant root from possible disturbance by the fish Each tank was encased in a 200-cm tall wooden framework, which a timer, a feeder and an illumination device could be fixed onto A near-sunlight 28-W 115-cm T5 tube was used for illumination, hanging 25 cm above plant top and its height was adjusted as the plant grew Top and sides of the framework were covered with black vinyl to obstruct the interference of illumination from outside Each day same ration of feed for all experimental units was hand loaded in the funnel of a feeder Coupled with a timer, the feeder released feed 2, or times a day at 12 h, h or h interval, respectively, as designated in the experimental design In this week period, daily ration was gradually decreased from 5% to 3% fish biomass as fish grew A commercial tilapia feed was used, which contained 25% crude protein, 3% crude fat, 12% crude ash, 6.5% crude fiber, 2% acid insoluble and 11% moisture No water was added or exchanged throughout the experiment Water was sampled weekly and monitored for pH (HM-20P, DKK-TOA, Tokyo), dissolved DO and temperature (Oxi 330i, WTW GmbH, Weilheim, Germany) and electrical current (EC) (750II conductivity/TDS monitor, Myron L Company, Carlsbad, CA) Total ammonia-N (TAN), nitrite-N, nitrate-N, total nitrogen, soluble phosphate-P, total phosphorus were analyzed by flow injection analyzer (FIA) (Flow SolutionÔ FS3100, O I Analytical, College Station, TX) The absorbance wavelengths used and methods based for the analysis of those substances were 640 nm and phenol hypochlorite method (Solorzano, 1969) for ammonia nitrogen, 543 nm and Pink azo dye method (APHA, 1992) for nitrite nitrogen, 543 nm and CdeCu reduction method (Bendschneider and Robinson, 1952; Strickland and Parsons, 1972; APHA, 1992) for nitrate nitrogen, 543 nm and CdeCu reduction method (Strickland and Parsons, 1972; APHA, 1992) by Grasshoff et al (1983) for total nitrogen, 885 nm and molybdenum blue method (Strickland and Parsons, 1972) for phosphate and 885 nm and method by Grasshoff et al (1983) and Strickland and Parsons (1972) for total phosphorus Analysis of five days’ Biochemical Oxygen Demand (BOD5) was based by Sawyer et al (2003) Biomass of fish and plant was measured at 0, and wk In wk 2, plant 25 cm above the raft was cut, weighed and harvested In wk 4, all fish and whole plant were harvested Fish weight gain (%) was calculated as the ratio of average individual fish weight in wk or wk to average individual fish weight in wk Plant weight gain (%) in wk was calculated as the ratio of the biomass of cut part/initial biomass and plant weight gain (%) in wk further added the ratio of the biomass of whole plant/initial biomass Three-way ANOVAs were performed to determine time effect, the effects of photoperiod and feeding frequency and their interaction on fish and plant growth and water parameters Duncan’s multiple range test (DMRT) was used to compare differences among levels of a factor The significant level applied to all analyses was set to 5% SAS version 9.0 software (SAS Institute, Inc., Cary, NC) was used for statistical analysis Results and discussion 3.1 System setup (Fig 1) Raft aquaponics can be the most simple and least cost aquaponics system The essential elements of an aquaponic system as suggested by Rakocy et al (2006) are fish-rearing tanks, a settleable and suspended solids removal component, a biofilter, a hydroponic component and a sump In raft aquaponics if the plant and its supporting media such as gravel and coarse sand can provide sufficient biofiltration, a separate biofilter is not needed (Rakocy et al., 2006) Solids removal component is highly recommended by Rakocy et al (2006) since if otherwise the organic materials from fish fecal waste and unconsumed feed accumulate, deposit and decompose anaerobically in tank bottom, the reduced toxic products can deteriorate water and harm the fish In our system, the upwelling afloat from disk membrane diffuser kept the solids J.-Y Liang, Y.-H Chien / International Biodeterioration & Biodegradation 85 (2013) 693e700 695 Blidariu and Grozea (2011): an aquaculture system that incorporates the treatment and reuse of water with less than 10% of total water volume replaced per day Water replacement can vary with the system setup In a Nile tilapia and lettuce aquaponics system where fish culture tanks, aquaponic channels, netting tanks, clarifier and sump were open to evaporation, 1.4% of the total system water was added daily to compensate the evaporation and transpiration losses (Al-Hafedh et al., 2008) The water consumption for fish production in the present study was 0.020 m3 kgÀ1 or 20.0 L kgÀ1 when calculating from the following data on per tank base: initial fish biomass 3.7 kg, overall average fish weight gain 43.9%, initial water volume 1000 L, water loss 3.3% The other water consumption data from previous studies are as the following: extensive fish culture >5 m3 kgÀ1 and semi-intensive fish culture 2.5 m3 kgÀ1 (both cited by Al-Hafedh et al., 2008), aquaponics by AlHafedh et al (2008) 0.32 m3 kgÀ1 and aquaponics by Rakocy et al (1997) 0.25 m3 kgÀ1 Fig A tilapiaewater spinach raft aquaponics system setup suspended and the effective aeration from the diffuser could mineralize the organic particles so that little deposition was observed in tank bottom at the end of experiment The level of polyethylene raft descended average 3.3 cm so that the water loss in wk was estimated only about 3.3% or about 0.1% dÀ1 Since water surface was afloat with polyethylene raft, only the feeding corner area (15 cm  15 cm) was open to evaporation Other part of the water loss could be attributed to transpiration at the leaf surface, which could be quite limited, too Aquaponic system is a recirculating aquaculture system (RAS) as defined by 3.2 Treatment effects on fish survival and growth and plant growth (Fig 2) Both feeding frequency (FF) (no of meals dÀ1) and photoperiod (PP) (illumination h dÀ1) had no effect on fish mortality since no fish died throughout the experiment Increased FF favored fish growth since in wk fish weight gain (WG) for dÀ1 was already 1.2% and 2.2% higher than WG for dÀ1 and dÀ1, respectively, but there was no difference between WG for dÀ1 and for dÀ1 FF effect on growth became even more pronounce in wk that dÀ1 had 3.0% and 4.9% more WG than dÀ1 and dÀ1, respectively Generally, fish growth increases with feeding frequency In indoor, intensive fish culture systems, fish may be fed as many as times per day in order to maximize growth at optimum temperatures Feeding frequency d-1 d-1 d-1 Photoperiod 12h d-1 24h d-1 b 41.6 (1.9) 50 b 43.5 (1.9) a 46.5 (0.8) 40 30 b 20.0 (0.9) b 21.0 (0.9) a 22.2 (0.4) 20 60 Fish weight gain (%) Fish Weight gain(%) 60 a 45.1 (2.3) b 42.7 (3.5) 50 40 30 a 21.6 (1.1) b 20.5 (1.7) 20 10 10 180 c 164 (8) 160 140 c 133 (4) b 134 (4) a 138 (6) b 169 (9) a 175 (8) Plant weight gain(%) Plant weight gain (%) 200 b 163 (6) b 131 (2) a 175 (4) a 139 (3) 120 Week Week Fig Mean value and standard deviation (in parenthesis) of fish weight gain (upper) and plant weight gain (bottom) of the tilapiaewater spinach aquaponic system under the effects of feeding frequency (n ¼ 4) (left) and photoperiod (n ¼ 6) (right) during two sampling periods Mean values without sharing a common letter are significant different (p 0.05) 696 J.-Y Liang, Y.-H Chien / International Biodeterioration & Biodegradation 85 (2013) 693e700 Feeding frequency d -1 d -1 d -1 Photoperiod 24h d -1 12h d -1 Total nitrogen (mgL-1) a a b 46.0 b 44.7 38.6 b (4.6) 40.9 c a b ab (5.8) (7.8) (10.0) 37.3 38.8 35.7 36.6 37.4 (1.3) (2.8) (5.1) (9.9) (13.6) a a 25.4 23.4 (2.5) (0.7) a a a 25.4 23.8 24.1 (1.0) (0.9) (0.4) b 35.1 (6.2) b 34.1 (5.3) a a 6.5 6.5 (0.1) (0.1) a a a 6.5 6.5 6.5 (0.1) (0.1) (0.1) 50 b b 33.5 32.0 (6.1) (7.9) a a a 21.5 21.3 20.6 (1.3) (0.8) (0.7) a a a 0.1 0.1 0.1 (0.1) (0.1 (0.1) a 20.5 (0.7) 30 a 39.9 (2.3) a 40.0 (0.4) a a 33.6 31.7 (2.5) (3.0) 40 Nitrate-N (mgL-1) a 38.5 (5.2) a 36.7 ab b a a a 33.5 33.0 32.4 32.6 (3.6) 32.0 (6.2) (3.2) (2.6) (3.6) (7.9) Nitrate-N (mgL-1) a 47.8 (1.7) a 46.3 (3.2) a 38.9 b (1.9) 35.1 (3.6) b 30.1 (6.7) a 20.9 (0.7) 20 10 a 0.1 (0) a 0.1 (0.1) 0 Week Week Fig Mean value and standard deviation (in parenthesis) of total nitrogen (upper) and nitrate-N (bottom) concentration in water of the tilapiaewater spinach aquaponic system under the effects of feeding frequency (n ¼ 4) (left) and photoperiod (n ¼ 6) (right) during two sampling periods Mean values without sharing a common letter are significant different (p 0.05) Photoperiod 24h d -1 12h d -1 8 7 a 3.2 (2.2) a a a 0.1 0.1 0.1 (0) (0) (0) a a a 0.8 0.7 0.8 (0.5) (0.2) (0.3) a 5.0 (1.8) a 4.7 (2.2) ab 3.1 (3.1) b 2.0 (2.4) a 1.1 (0.6) a 0.7 (0.4) Ammonia-N (mgL-1) Ammonia-N (mgL-1) Feeding frequency d -1 d -1 d -1 ab 3.0 (2.8) b 2.2 (1.4) a 5.2 (1.7) a 0.1 (0) a 0.1 (0) a 0.9 (0.2) a 0.6 (0.1) a 1.8 (1.6) a 1.5 (1.9) b 1.3 (1.2) b 1.6 (1.8) 0 1.6 a 1.3 (0.4) a a a 0.6 0.5 0.6 (0.2) (0.2) (0.2) a 0.8 (0.2) a 0.6 a (0.2) 0.5 (0.1) a a a 0.1 0.1 0.1 (0) (0 (0) 1.4 a 0.8 a (0.7) 0.7 (0.2) a 0.8 (0.6) a 0.5 (0.7) a 0.5 (0.2) Nitrite-N (mgL-1) Nitrite-N (mgL-1) a 6.2 (0.7) 1.2 1.0 a a 0.6 0.6 (0.1) (0.1) 0.8 a a 0.6 0.6 (0.2) (0.2) a a 1.0 (0.2) 0.9 (0.1) a 0.7 (0.2) 0.6 0.4 0.2 a 0.1 (0) a 0.1 (0) 0.0 Week Week Fig Mean value and standard deviation (in parenthesis) of ammonia-N (upper) and nitrite-N (bottom) concentration in water of the tilapiaewater spinach aquaponic system under the effects of feeding frequency (n ¼ 4) (left) and photoperiod (n ¼ 6) (right) during two sampling periods Mean values without sharing a common letter are significant different (p 0.05) J.-Y Liang, Y.-H Chien / International Biodeterioration & Biodegradation 85 (2013) 693e700 (Craig and Helfrich, 2002) Riche et al (2004) found that feeding tilapia at intervals shorter than the time required for the return of appetite can lead to gastric overload resulting in reduced absorption efficiency The return of appetite following a satiation meal, defined as the point that consumption is equivalent to the amount of the previous meal evacuated, is approximately h in Nile tilapia held at 28 C In the present study, the interval of the highest FF, dÀ1 was no shorter than h and the absorption efficiency should not be reduced In an intensive culture of fingerling walleye S vitreum, Phillips et al (1998) found that higher frequency feeding resulted in higher daily DO and lower TAN but had no effect on fish growth and size distribution In conclusion, higher FF with less feed given at a time can result in higher absorption efficiency and lower excretion into water, consequently, less nutrient accumulation in water Long PP increased fish growth since full day illumination (24 h dÀ1) resulted in 1.1% and 2.4% higher WG than half-day illumination (12 h dÀ1) in wk and wk 4, respectively The present results was not consistent with the results of El-Sayed and Kawanna (2004), who reared fingerling Nile tilapia Oreochromis niloticus under four photoperiod (light:dark, L:D) cycles (24L:0D, 18L:6D, 12L:12D and 6L:18D) at same feeding rate and feeding frequency for 90 days and found that fish performance was not significantly affected by photoperiods Since in all tanks the polyethylene raft blocked most illumination onto the water, the only difference in illumination resulted from the two PP was that the feeding corner of 24 h dÀ1 received twice illumination as 12 h dÀ1, which might somewhat help fish’s feeding and then the growth FF and PP had no interaction effect on fish growth Plant partial harvest in wk had already showed that fish FF helped for plant growth since plant WG increased with FF, namely, dÀ1 > dÀ1 > dÀ1 in both wk and wk In wk while the whole plant was harvest, dÀ1 had 11% and 6% more WG than dÀ1 and dÀ1, respectively Long illumination had positive effect on plant growth since PP at 24 h dÀ1 obtained 8% and 12% higher WG than 12 h dÀ1 in wk and wk 4, respectively It is comprehensible that longer illumination resulted in longer photosynthesis and faster plant growth but not comprehensible how higher FF can link to better plant growth 3.3 Treatment effects on nitrogen species (Figs and 4) In total nitrogen (TN), nitrate-N contributed around 88%, ammonia-N (TAN) 11% and nitrite-N < 1% Regardless of the treatments, the overall average TN, nitrate-N and TAN increased markedly until wk 2, slightly wk to and leveled off wk to 4, showing their accumulation had lessened The overall average nitrite-N reached its peak in wk 3, 0.9 mg LÀ1 and decreased to 0.6 mg LÀ1 in wk The ammonia (NH3) safe level for Nile tilapia is 0.42 mg LÀ1 (Stickney, 1979; Karasu Benli and Koksal, 2005) Since this safe ammonia level is expressed in terms of free NH3, it has to be transformed into TAN When the NH3 fraction from TAN at overall average pH 6.69 and temperature 29.6 C, 0.5% are accounted for, the TAN safe level for Nile tilapia is 84 mg LÀ1, which is far higher than the highest TAN in the present study Therefore, it can be concluded that our aquaponics system ammonia toxicity risk free In the present study, the highly oxidized environment made nitrite-N, the transitional nitrogen species in nitrification process unstable and in very low concentration, and probably insensitive to the treatment effect throughout the experiment TN, nitrate-N and TAN decreased with increased FF since wk or wk The adverse effect of FF on nitrogen species was most Photoperiod 12h d-1 24h d-1 a a 70.7 71.6 a a (7.3) (4.8) 63.6 61.8 a a (11.0) (10.6) 53.6 57.1 (3.6) (4.4) a a a 15.6 12.6 14.8 (4.9) (3.5) (1.7) a 36.4 b (8.7) b 25.4 22.0 (5.6) (1.2) Total phosphorus (mgL-1) Total phosphorus (mgL -1) Feeding frequency d-1 d-1 d-1 a a a 3.6 3.3 3.6 (0.1) (0.1) (0.1) Week Phosphorate (mgL-1) Phosphorate (mgL-1) a a a 3.4 3.3 3.4 (0.1) (0.1) (0.1) a a a 22.4 18.2 20.4 a a (4.2) a (3.2) (1.2) 13.3 12.6 11.3 (3.8) (1.1) (0.7) a ab 64.9 62.5 b (6.6) (3.4) 56.7 (9.4) a 73.2 (5.3) a 59.7 (7.7) a a 3.5 3.6 (0.1) (0.1) a a 45.7 42.3 a (7.6) (4.2) 40.7 (8.7) 697 a a 15.5 13.1 (3.1) (2.7) a 55.3 (6.5) a b 31.2 (9.8) 24.6 (5.8) a 47.7 (5.5) a a 3.3 3.4 (0.1) (0.1) a a 12.8 12.0 (3.4) (2.6) b 64.1 (8.4) a 65.7 b (4.8) 57.1 (6.8) b 38.1 (4.0) a a 21.3 19.4 (3.9) (2.5) Week Fig Mean value and standard deviation (in parenthesis) of total phosphorus (upper) and phosphate (bottom) concentration in water of the tilapiaewater spinach aquaponic system under the effects of feeding frequency (n ¼ 4) (left) and photoperiod (n ¼ 6) (right) during two sampling periods Mean values without sharing a common letter are significant different (p 0.05) 698 J.-Y Liang, Y.-H Chien / International Biodeterioration & Biodegradation 85 (2013) 693e700 pronounced on TN and TAN in wk when their concentrations were in the order of dÀ1 > dÀ1 > dÀ1 FF showed no effect on nitrite-N, possibly due to its low concentration,