DSpace at VNU: Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River

DSpace at VNU: Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of...

STOTEN-20661; No of Pages 11 Science of the Total Environment xxx (2016) xxx–xxx Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River Thanh-Son Dao a,⁎, Vu-Nam Le b, Ba-Trung Bui c, Khuong V Dinh d,e, Claudia Wiegand f, Thanh-Son Nguyen c, Cong-Thanh Dao a, Van-Dong Nguyen b, Thi-Hien To b, Ly-Sy-Phu Nguyen b, Truong-Giang Vo b, Thi-My-Chi Vo a a Hochiminh City University of Technology, Vietnam National University – Hochiminh City, 268 Ly Thuong Kiet Street, District 10, Hochiminh City, Vietnam University of Science, Vietnam National University – Hochiminh City, 227 Nguyen Van Cu Street, District 5, Hochiminh City, Vietnam Institute for Environment and Resources, Vietnam National University – Hochiminh City, 142 To Hien Thanh Street, District 10, Hochiminh City, Vietnam d National Institute of Aquatic Resources, Technical University of Denmark, 2920 Charlottenlund, Denmark e Department of Freshwater Aquaculture, Nha Trang University, Nha Trang City, Vietnam f University Rennes1, UMR 6553 ECOBIO, Campus de Beaulieu, 35042 Rennes Cedex, France b c H I G H L I G H T S G R A P H I C A L A B S T R A C T • The sensitivity of a tropical daphnid Daphnia lumholtzi to Cu, Ni, Zn were assessed • Mekong River water was used to increase environmental realistic exposure scenarios • D lumholtzi showed higher sensitivity to metals than temperate Daphnia species • D lumholtzi is recommended for assessing toxicity of metals in tropical environments a r t i c l e i n f o Article history: Received 14 June 2016 Received in revised form August 2016 Accepted August 2016 Available online xxxx Keywords: Acute toxicity Life history traits Mekong River water Sensitivity Trace metals a b s t r a c t Metal contamination is one of the major issues to the environment worldwide, yet it is poorly known how exposure to metals affects tropical species We assessed the sensitivity of a tropical micro-crustacean Daphnia lumholtzi to three trace metals: copper (Cu), zinc (Zn) and nickel (Ni) Both, acute and chronic toxicity tests were conducted with metals dissolved in in situ water collected from two sites in the lower part of the Mekong River In the acute toxicity test, D lumholtzi neonates were exposed to Cu (3–30 μg L−1), Zn (50–540 μg L−1) or Ni (46–2356 μg L−1) for 48 h The values of median lethal concentrations (48 h-LC50) were 11.57–16.67 μg Cu L−1, 179.3–280.9 μg Zn L−1, and 1026–1516 μg Ni L−1 In the chronic toxicity test, animals were exposed to Cu (3 and μg L−1), Zn (50 and 56 μg L−1), and Ni (six concentrations from to 302 μg L−1) for 21 days The concentrations of μg Cu L−1 and μg Ni L−1 enhanced the body length of D lumholtzi but 46 μg Ni L−1 and 50 μg Zn L−1 resulted in a strong mortality, reduced the body length, postponed the maturation, and lowered the fecundity The results tentatively suggest that D lumholtzi showed a higher sensitivity to metals than related species in the temperate region The results underscore the importance of including the local species in ecological risk assessment in ⁎ Corresponding author E-mail address: dao.son@hcmut.edu.vn (T.-S Dao) http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 0048-9697/© 2016 Elsevier B.V All rights reserved Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx important tropical ecosystems such as the Mekong River to arrive at a better conservational and management plan and regulatory policy to protect freshwater biodiversity from metal contamination © 2016 Elsevier B.V All rights reserved Introduction Anthropogenic emissions from mining operations, industrial and agricultural activities have increased the metal concentrations in the environment so that they have become the common contaminants in aquatic ecosystems and a challenge to control (Tomasik and Warren, 1996; Schwarzenbach et al., 2010; Lanctot et al., 2016) Several metals are essential, while others not have a function in organisms, but all become toxic at a certain concentration (Wetzel, 2001) Metals are indestructible contaminants with high potential for bioaccumulation, in particular in their organic-metal form (e.g., Lau et al., 1998; Waykar and Shinde, 2011) and can be transferred to higher trophic levels of the food chain (Ikemoto et al., 2008) As exposure to metals impairs aquatic organisms such as aquatic crustaceans, insects and fishes, metal contamination has been identified as one of the major threat to freshwater biodiversity (Millennium Ecosystem Assessment, 2005; Dinh Van et al., 2013; Moldovan et al., 2013; Lanctot et al., 2016) Toxicity of dissolved metals to aquatic organisms such as microcrustacean and fish is regulated by several environmental parameters such as pH, alkalinity, dissolved organic carbon (DOC) and hardness (De Schamphelaere and Janssen, 2002; Hoang et al., 2004; Linbo et al., 2009; Ryan et al., 2009; Jo et al., 2010) The increase of pH and humic acid concentration in the test medium decreased bioavailability of Zn, thus reducing its toxicity to Daphnia magna (Paulauskis and Winner, 1988) Similarly, toxicity of metals decreased with increasing water hardness in daphnid species, e.g Ceriodaphnia dubia or Daphnia pulexpulicaria testing with Cu, Ni, Zn, and Cd (Naddy et al., 2015; Taylor et al., 2016) Considerable progress has been made on understanding the effects of trace metals on aquatic organisms, including daphnid species in temperate regions (reviewed by Grosell et al., 2002; Tsui and Wang, 2007) For examples, exposure to metals e.g., Cu, Ni, Zn, Cr, or Ag caused impairments of life history traits such as growth rate, maturity age, lifespan, reproduction, and survival in many temperate Daphnia species such as D magna, D pulex, D parvula, D ambigua and D obtusa (Winner and Farrell, 1976; Coniglio and Baudo, 1989; Munzinger, 1994; Bianchini and Wood, 2002; Pane and McGeer, 2004; Muyssen et al., 2006) Yet, a recent study has showed that there is a gap in knowledge of how tropical species deal with contaminants (Ghose et al., 2014) Few studies have investigated the responses of tropical zooplankton such as Daphnia species to metals (Vardia et al., 1988; Chishty et al., 2012; Dao et al., 2015; Bui et al., 2016) As mentioned above, among the trace metals, Cu, Zn and Ni, were commonly used to evaluate the chronically negative effects on zooplankton, e.g temperate daphnids However, the chronic effects of these metals, especially dissolved metals in field water, on tropical Daphnia lumholtzi have not been reported The Mekong River is one of the biggest rivers in the world with high level of anthropogenic activities such as hydropower plants, urbanization, transportation of goods, agriculture (Wilbers et al., 2014), aquaculture (Marcussen et al., 2014), and industrialization (Quyen et al., 1995) While the concentrations of most trace metals (e.g Ag, As, Cr, Co, Cu, Cd, Pb, Se, Sn, Zn) in water in the lower part of the Mekong River were relatively low (b 1.6 μg L−1; Ikemoto et al., 2008), a high level of anthropogenic activities in this region may pose a risk of metal contamination In fact, metal contaminations have been occurring locally in several places in the lower part of the Mekong River and its basin (e.g., Cenci and Martin, 2004) Despite this, the assessment of metal impacts on freshwater and tropical daphnids (e.g D lumholtzi) is neglected (but see Vardia et al., 1988; Chishty et al., 2012; Bui et al., 2016), especially upon chronic exposure (but see Dao et al., 2015) The direct application of ecological risk assessments based on toxicity tests of temperate model species such as D magna (Dave, 1984; De Schamphelaere et al., 2004, 2007) may not be relevant to extrapolate the risk in tropical regions such as the Mekong River For example, the Vietnamese regulations on surface water quality regarding trace metals for protection on aquatic life (QCVN-38, 2011) are not based on the toxicity tests with local species This may be problematic as tropical animals differ in key important life history traits such as faster life history comparing to temperate species thereby differing in the sensitivity to contaminants (Kwok et al., 2009; Dinh Van et al., 2014) Given that toxicity of metals depends on the presence of the dissolved organic matter, water hardness and alkalinity, these parameters should be taken into account in ecotoxicological studies (Ryan et al., 2009; Jo et al., 2010) To address these issues, we aim to test the sensitivity of a tropical crustacean species Daphnia lumholtzi to three essential metals: Cu, Ni and Zn at ecologically relevant concentrations (Jing et al., 2013; Onojake et al., 2015) in in situ water collected from two sites in Mekong River Daphnia lumholtzi was chosen as the study species as it is a key species in freshwater ecosystems in the lower basin of the Mekong River Cu, Zn and Ni were chosen to test their acute and chronic toxicity to Daphnia lumholtzi because of (i) these metals are among the most common metal contaminants in the Mekong River (Cenci and Martin, 2004; Bui et al., 2016; Dao et al., manuscript in preparation), and (ii) the availability of toxicity data of Cu, Zn and Ni on other daphnid species, especially D magna enabled comparisons and recommendations for ecological risk assessment programs in tropical countries like Vietnam The water samples collected from two sites were comprehensively analyzed for the environmental parameters and metal and pesticide contamination before using them for the acute and chronic toxicity tests We documented how exposures to metals affect key fitness-related traits in D lumholtzi such as survival, growth rate, maturation and fecundity Finally, recommendations for ecological risk assessment in tropical ecosystem are provided Materials and methods 2.1 Test solutions 2.1.1 Water samples collection Surface water was collected at sampling sites in Mekong River: site at Vinh Loc ferry-port, An Phu district and site at Tan Chau ferry-port, Tan Chau district, An Giang Province (Fig 1) The water samples were transferred to the Environmental Toxicology Laboratory, Institute for Environment and Resources in Hochiminh City and prepared for the experiments at the same day In the laboratory, the water samples were filtered through 0.45 μm syringe filter (Sartorius, Germany) and stored in pre-cleaned low density polyethylene plastic containers at °C prior to the tests 2.1.2 Water samples characteristics The filtered waters from each sampling site were analyzed for water quality parameters that may affect the bioavailability of dissolved metals and the survival and growth of Daphnia such as DOC, alkalinity and hardness, pH, trace metals and pesticides The DOC was analyzed with a total organic carbon (TOC) analyzer (TOC-5000, Shimadzu) according to APHA (2005) Total hardness was determined based on concentrations of Ca2 + and Mg2+ and the alkalinity was determined by titration method (APHA, 2005) The pH of water was measured with a pH meter (Metrohm 744) Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx Fig Mekong River in Vietnam and the positions of two sampling sites for the toxicity test, indicated as stars (1 is Vinh Loc: 10°50′54 N, 105°40′41 E, and is Tan Chau: 10°48′10 N, 105° 14′56 E) 2.1.2.1 Analysis of metals The pooled filtered waters (from 10 sub-samples per sampling site, Relyea and Diecks, 2012; Relyea, 2012; Dinh Van et al., 2013) for metal characterization were acidified with concentrated HNO3 (Merck) to pH b and used for dissolved metal characterization (APHA, 2005) with an inductively coupled plasma/mass spectrometry (ICP/MS - Agilent 7500, USA) ICP-MS operating conditions and parameters for metal analysis in samples are presented in the Supplementary A multi-element tuning solution was used to check accuracy of measurement (relative standard deviation, RSD b 5%, Agilent Technologies) The calibration curve was prepared using single stock solutions for each metal The concentrations of metals in mixed working standard solutions were prepared based on their estimated concentrations in water samples from preliminary semi-quantitative analysis The weighted calibration curves for each element with R2 N 0.999 were accepted for concentration calculation All samples and working standard solutions for calibration were spiked with 10 μg Scandium L−1 as internal standard to correct for instrument drift and physical interferences The percent recovery of the initial calibration verification standard should be 90– 110% for each element being determined 2.1.2.2 Analysis of pesticides Pooled water samples (based on 10 subsamplings from each storage tanks) for organochlorine pesticides (OCPs) and organophosphate pesticides (OPPs) characterization were taken and kept in dark glass bottles on ice in the field until analysis in the laboratory Water samples were filtered (Sartorius, Germany) to remove residual suspended particulates prior to liquid – liquid extraction (AOAC, 1996) OSPs in water samples were extracted with methylene chloride (DCM) and OPPs were extracted with mixture of DCM and hexane (15/85, v/v; Merck & Labscan Inc.) The mixture was shaken for 15 min, followed by phase separation The organic phase was transferred into a dry vial The extraction process was repeated times (AOAC, 1996; US EPA, 2008) The pooled extracts were concentrated by rotary evaporation then cleaned on a neutral silica solid phase extraction (SPE) column (Silica Gel 100/200 mesh) (US EPA, 1996 - Method 3630) The column was eluted with 40 mL of hexane and 30 mL of DCM with the flow rate of mL min−1 SPE extracts were concentrated by rotary evaporation and with a gentle stream of nitrogen and redissolved into mL hexane for injection to GC-ECD GC–ECD analysis was carried out on an Agilent 7890 (USA) with a DB – 5.625 capillary column (30 m length 0.25 mm i.d., 0.25 mm film thickness) The recoveries of OCPs and OPPs were 80–91% (SD b 5%) and 103–109% (SD b 5%), respectively The detection limits of OCPs and OPPs were 0.01 μg L−1 and 0.1 μg L−1, respectively OCPs standard mixture includes 13 compounds: 2,4,5,6 Tetrachloro-m-xylene, α-HCH, α-Chlordane, 4,4′-DDE, β-Endosulfan, Delta-HCH, Aldrin, Heptachlor epoxide, δ-Chlordane, Endrin aldehyde, Endosulfan sulfate, Endrin ketone, Decachlordiphenyl and OPPs standard mix includes compounds: Diazinon, Malathion, Parathion, Ethion, Trithion that were purchased from Sigma-Aldrich Co Laboratory blanks consisted of milipore water extracted and analyzed in the same way as samples and did not contain OCPs and OPPs 2.2 Toxicity test 2.2.1 Exposure solutions The Cu, Zn, Ni stocks were 1000 mg L− Cu, Zn, Ni in Nitric acid (HNO3 ~ 2–3%, Merck) From these stock solutions, exposure solutions with different concentrations of each metal were prepared using the filtered river water and exposure concentrations in one of the replicates of acute or chronic tests were determined when the tests terminated (see Table 2) During the toxicity tests, water temperature (WTW Oxi197i multi-detector), dissolved oxygen (DO, WTW 350i), and pH (Metrohm 744) were measured at the beginning and at the termination (for all tests) and also at the time of medium renewal (chronic tests) These physical and chemical characteristics were used to confirm if these parameters were favorable for D lumholtzi 2.2.2 Test organisms The tropical daphnid D lumholtzi was collected from Bac Ninh Province, Vietnam (Bui et al., 2016) and has been maintained in the Laboratory of Environmental Toxicology, Institute for Environment and Resources, Vietnam National University – Hochiminh City, for N2 years The Daphnia was raised in COMBO medium (Kilham et al., 1998), at 27 ± °C with a photoperiod of 12 h: 12 h light: dark cycle and the light intensity of around 1000 Lux The Daphnia was fed with a mixture of green alga (Chlorella sp.) cultured in COMBO medium and YCT (yeast, cerrophyl and trout chow digestion) prepared according to the U.S Environmental Protection Agency Method (US EPA, 2002) 2.2.3 Acute toxicity tests The 48-h static nonrenewal acute toxicity tests were conducted following the guidelines of the US EPA methods (US EPA, 2002) with two adjustments of: i) light regime (a photoperiod of 12 h:12 h light:dark at a light intensity of ca 1000 Lux) and ii) temperature (27 ± °C) for Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx tropical species Neonates of D lumholtzi (age ≤ 24 h) were used for testing Each treatment had four replicates and each replicate consists of 10 neonates in 40 mL of exposure solution in a 50-mL polypropylene cup Five to seven concentrations of metals were prepared for each metal exposure (Table 2) Controls were prepared by transferring the neonates into Mekong River water without metal addition The neonates were fed during the pre-exposure duration but starved during the tests (US EPA, 2002) We checked daily for dead organisms and removed them from the cups Dead of the animals was confirmed by observing the stop of the heart beat under a microscope Mortality data were used to determine median lethal concentrations (48 h-LC50) When the test terminated, we randomly took test solution in one of the four replicates (in each metal concentration) for the metal analysis by ICP/MS 2.2.4 Chronic tests Chronic tests were performed at the same condition as in acute toxicity test Based on the 48 h-LC50 values and previous investigation (Dao et al., 2015), the concentrations of metals (Cu, Zn, Ni) for chronic tests were chosen The metal concentrations in chronic tests were and μg Cu L− 1, 50 and 56 μg Zn L−1, and from to 302 μg Ni L− (Table 1) Also, the chosen concentrations of Cu, Zn and Ni have been found in natural water of the lower Mekong River (e.g μg Cu L−1; 57 μg Zn L−1, Dao et al., manuscript in preparation; 151 Ni μg L−1; Bui et al., 2016) Chronic tests were performed according to the APHA (2005) and Dao et al (2010) with minor modifications (see 2.2.3) Briefly, neonates (15 individuals per treatment) of D lumholtzi b24 h-age from 2nd to 3rd clutch were individually and independently incubated for each treatment in 50-mL polypropylene cups containing 20 mL control solution or exposure solutions Each treatment had 15 replicates (n = 15) Exposure solutions were renewed every second day The Daphnia were fed with a mixture of Chlorella (~ mg C L−1, approximately 140,000 cells mL−1) and YTC (~20 μL) just after the exposure solutions were renewed Life history traits of the Daphnia including mortality, maturation, and reproduction were scored daily Maturity age was defined as the day on which the first egg appeared in the brood chamber of the Daphnia Numbers of neonates per clutch of each mother daphnid were checked daily, removed from the cup with a glass pipet and counted for clutch size to evaluate the fecundity Reproduction was calculated as total accumulated offspring reproduced by all mother daphnids in each treatment Fecundity was defined as the average number of offspring in one clutch reproduced by one mother daphnid The chronic tests lasted for 21 days At test termination, living mother daphnids were immediately fixed with Lugol solution (Sournia, 1978) and body length was measured to the nearest μm, on a microscope (Olympus BX 51) coupled with a digital camera (DP 71) The body length was measured from the eye to the base of tail spine of the mothers 2.3 Data analyses Median lethal concentrations with 95% confidence intervals (95% CIs) were calculated by Toxcalc Program (Tidepool Scientific LLC Table Metal and pesticide concentrations in filtered field water from Mekong River BDL, below detection limits of the analytical methods, μg L−1 for BDLa, 0.1 μg L−1 for BDLb, and 0.01 μg L−1 for BDLc Dissolved metals (μg L−1) Site – Vinh Loc Site – Tan Chau Pesticides (μg L−1) Site – Vinh Loc Site – Tan Chau Al As Ba Fe Zn Cu Co Cr Mn Ni Se Mo Ag Cd Pb 25 BDLa BDLa BDLa BDLa BDLa BDLa BDLa BDLa BDLa 30 BDLa BDLa BDLa BDLa BDLa BDLa BDLa BDLa BDLa BDLa Tetrachloro-m-xylene Alpha-HCH Alpha-Chlordane 4,4′-DDE Beta-Endosulfan Delta-HCH Aldrin Heptachlor epoxide Gamma-Chlordane Endrin aldehyde Endosulfan sulfate Endrin ketone Decachlordiphenyl Diazinon Ethion Malathion Pazathion Trithion BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLc BDLc BDLc BDLc BDLc BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLb BDLc BDLc BDLc BDLc BDLc USA) Kruskal-Wallis test (Sigma Plot, version 12) was applied for calculation the significant difference of the maturation, fecundity and body length of D lumholtzi between control and metal exposure solutions To provide full overview of the sensitivity of the D lumholtzi to metals, we analyzed and documented the results separately for exposure solutions made from waters collected at each sampling site Results and discussion 3.1 Physical and chemical characteristics of field water from Mekong River All analyzed organic pesticides in filtered Mekong River water were below the detection levels of the equipment (Agilent 7890, USA; Table 2), including Tetrachloro-m-xylene, Alpha-HCH, 4,4′-DDE, BetaEndosulfan, Delta-HCH, Aldrin, Heptachlor epoxide, Gamma-Chlordane, Endrin aldehyde, Endosulfan sulfate, Endrin ketone, Decachlordiphenyl, Diazinon, Ethion, Malathion, Pazathion and Trithion Overall, concentrations of trace metals in filtered water from both sampling sites of the Mekong River were very low They ranged from to μg L−1 of Al, to μg L−1 of As, 25 to 30 μg L− of Ba, to μg L− of Fe and to μg L−1 of Zn Concentrations of other metals: Cu, Co, Cr, Mn, Ni, Se, Mo, Ag, Cd and Pb were below the detection levels of the ICP/MS, μg L−1 (Table 2) The concentrations of trace metals and pesticides in filtered Mekong River water in the current study were similar to those documented in a previous study at the same sampling locations (Bui et al., 2016) The As concentration (3 μg L−1) was ca 1000 times lower than the lowest concentration inducing acute negative effects on other daphnid species e.g D magna (3000 μg L− 1; Hoang et al., Table Concentrations of the Cu, Zn and Ni (μg L−1) confirmed by the ICP/MS in the acute and chronic tests with Daphnia lumholtzi Metals Concentrations of metals dissolved in river water from site 1, Vinh Loc Concentrations of metals dissolved in river water from site 2, Tan Chau Acute test Cu (μg L−1) Zn (μg L−1) Ni (μg L−1) 13, 15, 18, 19, 20 56, 156, 247, 343, 539 1087, 1403, 1659, 1985, 2090 3, 7, 8, 10,11,13,15 50, 87, 139, 192, 226, 476, 688 481, 766, 968, 1369, 1602, 1807 Chronic tests Cu (μg L−1) Zn (μg L−1) Ni (μg L−1) 56 6, 59, 302 50 5, 46, 225 Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx 2007) and D pulex (2500–3900 μg L−1; Shaw et al., 2007) Similarly, dissolved Zn (4 μg L−1) and aluminum (5 μg L−1) in the test water were considerably lower 48 h-LC50 values (61.8–130 μg Zn L− 1; 742– 1900 μg Al L−1) to the micro-crustacean, Ceriodaphnia dubia, reported elsewhere (Gostomski, 1990; Naddy et al., 2015) The pH of Mekong River water was 7.8 at both sampling sites (Table 3) However, the pH decreased to 6.8 after metals (Cu, Ni, Zn) were spiked into the test water During the toxicity tests the DO varied from 6.3 to 6.6 mg L−1 (ca 78–80% of saturated oxygen concentration; Wetzel, 2001) The DOC concentrations in the water from Vinh Loc (site 1) and Tan Chau (site 2) were 2.99 and 1.89 mg L−1, and hardness was 79 and 87 mg CaCO3 L−1, respectively (Table 3) Both pH and DO in the test water were within the favorable range for the growth and development of daphnids such as Daphnia magna, Daphnia pulex and Ceriodaphnia dubia (APHA, 2005; Ebert, 2005) However, lower pH could increase bioavailability and consequently toxicity of metals to daphnids, thus contribute as confounding factor The DOC concentrations of the river water (1.89–2.99 mg L−1, Table 3) were considerably lower than that in a previous study (DOC = 14.4–14.7 mg L− 1) in which samples were collected from a nearby location (Bui et al., 2016) The alkalinity (64–68 mg CaCO3 L−1) and hardness (79–87 mg CaCO3 L− 1) were only slightly different between the two sampling sites, and the water could be classified as moderately hard water (Villavicencio et al., 2005; Naddy et al., 2015) Probably, the DOC concentrations, alkalinity and hardness of water from Mekong River varied depending on the preceding meterological conditions but are in range with other tropical rivers (Villavicencio et al., 2005; Bui et al., 2016) 3.2 Acute effects of metals on Daphnia lumholtzi The 48 h-LC50 values for D lumholtzi incubated in Mekong River water ranged from 11.57 to 16.67 μg L−1 of Cu, 179.3 to 280.9 μg L−1 of Zn and 1026 to 1516 μg L−1 of Ni (Table 4, Supplementary 2) The 48 h-LC50 values were lower in the test with river water from site than that from site probably associated with the lower DOC concentration in water at site (1.89 mg L−1) compared to site (2.99 mg L−1) Overall, the toxicity order of the three metals to daphnids in our study decreased from Cu N Zn N Ni (Table 4) which is in line with previous investigations (e.g., Biesinger and Christensen, 1972; Wong, 1992; Vardia et al., 1988; Traudt et al., 2016) Bui et al (2016) reported 48 h-LC50 values for Cu of 6.15– 8.61 μg L−1, and 5.77–7.23 μg L−1 in two tropical micro-crustaceans, D lumholtzi and Ceriodaphnia cornuta, respectively, exposed to Cu spiked into Mekong River water These 48 h-LC50 values are two times lower than those from our study (Table 4) It seems that the higher alkalinity and hardness in the water used in the current study contributed to the lower toxicity of Cu compared to Bui et al (2016), despite their higher DOC In acute toxicity test with D lumholtzi exposed to Cu in dechlorinated tap water (pH 7–9, DOC 2–4 mg L−1, alkalinity and hardness 180 and 200 mg CaCO3 mg L−1), the 48 h-LC50 value of 54.6 μg Cu L−1 (Vardia et al., 1988) was higher than that in our study (Table 4) The higher alkalinity and hardness together with the possibly older age of D lumholtzi in the study of Vardia et al (1988), may have contributed to the lower sensitivity Chishty et al (2012) used several daphnid species Table Physical and chemical characteristics of the field water from Mekong River and the exposure solutions during the experiments Parameters Site – Vinh Loc Site – Tan Chau pH (in the field water) pH (in the test water after metal addition) Dissolved oxygen in the test water (mg L−1) Dissolved organic carbon (mg L−1) Hardness (mg CaCO3 L−1) Alkalinity of the field water (mg CaCO3 L−1) Alkalinity of the test water (mg CaCO3 L−1) 7.8 6.8–7.8 6.3–6.6 2.99 79 68 64–68 7.8 6.6–7.8 6.3–6.6 1.89 87 68 64–68 such as D lumholtzi, Moina, and Ceriodaphnia to test the acute toxicity of Zn, Pb and Cd dissolved in a natural water sample originating from a well (pH of 7.9, alkalinity and hardness of 512 and 582 mg CaCO3 L−1, respectively) In their studies, the 48 h-LC50 was 2300 μg Zn L−1 (to D lumholtzi), which is by a factor of 10 higher than that in our experiment (Table 4) Higher water hardness, pH and alkalinity as well as the use of adult daphnids may have contributed to this higher value However, lacking experimental details (age of the animals and rearing conditions) impede the comparison In acute toxicity tests of Cu in moderately hard water and similar range of DOC (1–3 mg L− 1), and pH (7–8) similar to our study, the values of 48 h-LC50 of D magna, D obtusa, and D pulex ranged from 60.3 to 156.1, 41.1 to 100.1 and 19.5 to 26 μg Cu L− 1, respectively, which are higher than in our study with D lumholtzi (Villavicencio et al., 2005; Traudt et al., 2016) In addition, Rodriguez and Arbildua (2012) found D magna with the 48 h-EC50 of 16.5 μg Cu L−1, under the test conditions of mL−1 of DOC, pH of 6.3 and hardness of 169 mg L−1 as CaCO3 Though the same authors reported similar 48 hLC50/EC50 value to our record, but the double hardness and lower pH in their study compared to ours revealed that D lumholtzi (from our study) appeared to be more sensitive to Cu than the other three temperate Daphnia species, D magna, D obtusa, and D pulex In COMBO medium (0.67 mg L−1 of DOC, hardness and alkalinity of 44 and 10 mg L−1 as CaCO3, respectively) the 48 h-LC50 of 1775 μg Ni L−1 for D lumholtzi (Dao et al., 2015) was a little higher than the 48 h-LC50 values of the current study (1026–1516 μg Ni L−1; Table 4) Pane et al (2003) reported a 48 h-LC50 of 1068 μg Ni L−1 for D magna in (soft) tap water, pH of 7.3–7.6 and total organic carbon (TOC) of 3.6 mg L−1 which was in range with the 48 h-LC50 from our study In moderately hard water and mg L−1 DOC, a 48 h-LC50 of 1633 μg Ni L−1 was attained for D magna (Traudt et al., 2016) Therefore, D lumholtzi and D magna seem to have a similar sensitivity regarding acute toxicity to Ni Vardia et al (1988) reported the 48 h-LC50 of D lumholtzi of 2290 μg Zn L−1, which is far higher than the 48 h-LC50 value in our study (Table 4) Again, this difference could be the consequence of higher hardness and the age tolerance to metal of the daphnids as mentioned above Comparing D lumholtzi 48 h-LC50 values for Zn of our study (179– 280 μg Zn L− 1, in moderately hard water, Table 4) to those of D magna (928 μg Zn L−1 in moderately hard water) and C dubia (102– 130 μg Zn L−1 in hard water) reveals an increase of sensitivity from D magna to D lumholtzi to C dubia despite the possible mitigating effect of water hardness (Naddy et al., 2015; Traudt et al., 2016) Notably, the Cu concentration of 200 μg L−1 is used as the safety level for protection of aquatic life (QCVN-38, 2011), but this Cu concentration is even 13 times higher than the 48-LC50 value of D lumholtzi exposed to Cu in this study Taking more safety factors into consideration (e.g 10 for intra species differences and 10 for the chronic exposure scenario) the QCVN-38 (2011) should be re-considered and adjusted for aquatic ecosystem protection To our knowledge this is the first report on the acute test of Ni and Zn spiked into field water to D lumholtzi, which together with previous results of Bui et al (2016), may be used for the developing of the metal Biotic Ligand Model (Di Toro et al., 2001; Villavicencio et al., 2005) with tropical micro-crustaceans 3.3 Chronic effects of metals on life history traits of Daphnia lumholtzi Several trace metals such as Zn and Cu are essential components of more than hundred enzymes and various biological functions (Walker et al., 1996) contributing to the function and regulation of many enzyme activities related to the fitness (health, growth and reproduction) in animals However, increasing metal concentrations at some point impair physiological functions, reduce fitness or even become lethal to organisms (Pane et al., 2003) Several nonexclusive mechanisms may underlie the metal-induced reduction of the fitness-related traits in exposed aquatic animals such as growth, age to maturation and fecundity: the Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx Table The values of 48 h median lethal concentrations (48 h-LC50) of Cu, Zn and Ni for daphnid species (without *); *, values of 48 h-EC50 (immobilization); **, values of 72 h-LC50 Species Metals 48 h-LC50 (95% CI) Test water Sources Daphnia lumholtzi Ceriodaphnia cornuta Daphnia lumholtzi Ceriodaphnia dubia Daphnia magna Daphnia magna Daphnia obtusa Daphnia pulex Ceriodaphnia reticulata Ceriodaphnia pulchella Daphnia magna Daphnia galeata Daphnia longispina Daphnia magna Daphnia ambigua Daphnia pulex Daphnia parvula Daphnia lumholtzi Daphnia lumholtzi Daphnia lumholtzi Daphnia lumholtzi Ceriodaphnia Moina Daphnia magna Daphnia magna Daphnia pulex Daphnia ambigua Ceriodaphnia dubia Ceriodaphnia dubia Daphnia lumholtzi Daphnia lumholtzi Daphnia lumholtzi Daphnia magna Daphnia magna Daphnia lumholtzi Daphnia lumholtzi Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Cu (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Zn (μg L−1) Ni (μg L−1) Ni (μg L−1) Ni (μg L−1) Ni (μg L−1) Ni (μg L−1) 6.15–8.61 5.77–7.23 54.6 16.6 60.3–156.1 100 41.1–100.1 19.5–26 13.3–17.7* 12.0–16.4* 26.8–53.2* 22.6* 9.89–11.9* 86.5** 67.7** 86** 72** 16.67 (15.92–17.38) 11.57 (10.97–12.07) 2290 2300 1400 1200 819 928 273 304 260 102–130 280.9 (257–306.6) 179.3 (162.4–198.2) 1775 1068 1633 1516 (1398–1616) 1026 (941.6–1114) Mekong river Mekong river Tap water Artificial medium Rivers and lakes Artificial medium Rivers and lakes Rivers and lakes Artificial medium Artificial medium Artificial medium Artificial medium Artificial medium Pond water Pond water Pond water Pond water Mekong river, site 1, Vinh Loc Mekong river, site 2, Tan Chau Tap water Water from a well Water from a well Water from a well Artificial medium Artificial medium Artificial medium Artificial medium Artificial medium Artificial medium Mekong river, site 1, Vinh Loc Mekong river, site 2, Tan Chau Artificial medium Tap water Artificial medium Mekong river, site 1, Vinh Loc Mekong river, site 2, Tan Chau Bui et al., 2016 Bui et al., 2016 Vardia et al., 1988 Naddy et al., 2015 Villavicencio et al., 2005 Traudt et al., 2016 Villavicencio et al., 2005 Villavicencio et al., 2005 Bossuyt and Janssen, 2005 Bossuyt and Janssen, 2005 Bossuyt and Janssen, 2005 Bossuyt and Janssen, 2005 Bossuyt and Janssen, 2005 Winner and Farrell, 1976 Winner and Farrell, 1976 Winner and Farrell, 1976 Winner and Farrell, 1976 This study This study Vardia et al., 1988 Chishty et al., 2012 Chishty et al., 2012 Chishty et al., 2012 Shaw et al., 2006 Traudt et al., 2016 Shaw et al., 2006 Shaw et al., 2006 Shaw et al., 2006 Naddy et al., 2015 This study This study Dao et al., 2015 Pane et al., 2003 Traudt et al., 2016 This study This study impairment of the respiratory function (Pane et al., 2003), the inhibition of the sodium uptake, impairing the osmotic imbalance (Grosell et al., 2002) inducing oxidative stress and an increase in the energy expense for detoxification (e.g., upregulation of costly metallothioneins or antioxidant mechanisms (Amiard et al., 2006; Dinh Van et al., 2013) Therefore, the maintenance cost is increased Furthermore, exposure to metals may also reduce the foraging activity, hence lowering energy intake (e.g., Janssens et al., 2014) Consequently, metal-exposed animals may suffer a lower growth and reproduction rate, or even mortality (e.g., Winner and Farrell, 1976; Pane and McGeer, 2004; Muyssen et al., 2006) 3.3.1 Effects on survival Mekong River water did not impair survival of D lumholtzi during three weeks of exposure (Fig 2a, b) Exposure to Cu caused mortality of 20% at μg L−1 and μg L−1 (Fig 2a, b) The concentration of 56 μg Zn L−1 in river water from site resulted in 16% mortality of daphnids whereas 50 μg Zn L−1 in river water from site caused 54% mortality (Fig 2c, d) Ni in water from sites and caused mortality of 14–27% at 5–59 μg Ni L−1 This metal induced 100% mortality on day 10 at 302 and 225 μg Ni L−1 for Ni dissolved in water from site and 2, respectively; Fig 2e, f) Comparing the vulnerability of four Daphnia species (D magna, D pulex, D parvula, D ambigua) to Cu in pond water (alkalinity of 110– 119 mg CaCO3 L−1; hardness of 130–160 mg CaCO3 L−1; and pH of 8.2–9.5) survival of the four Daphnia species slightly decreased at 20 μg Cu L−1 during weeks of incubation (Winner and Farrell, 1976), whereas D lumholtzi suffered already 20% mortality during 21 days at to μg Cu L−1 in our study In a 15-day test, N 50% of D magna and 80% of Moinadaphnia macleayi survived exposure to 25 and 40 μg Cu L−1 (in artificial medium, pH of 7.6–7.7; hardness of 160– 180 mg L−1 as CaCO3 (Regaldo et al., 2014)) These results indicate that temperate daphnid species seem to be more resistant to Cu than D lumholtzi Previous studies have shown that intraspecific populations at lower latitudes with faster life history (e.g., higher growth rate and shorter generation times) may be more vulnerable to contaminants (Dinh Van et al., 2014) as a result of energy allocation trade-off (Sibly and Calow, 1989; Congdon et al., 2001) It remains to be tested whether this is also the case at the species levels for the higher sensitivity of tropical daphnid species to metals compared to temperate one Muyssen et al (2006) reported chronic exposure to 80–250 μg L−1 Zn at pH of 7.6 and DOC of mg L−1 did not significantly decrease D magna survivorship while survival of D lumholtzi in our study was already decreased 46% at 50 μg Zn L−1 at a lower DOC, however (Fig 2d) Again, either the DOC mitigated toxicity for D magna by up to factor or D lumholtzi seems more susceptible The difference in Zn-induced mortality of D lumholtzi in waters from two different sites in Mekong River may also be partly attributed to the lower DOC content at site 2, possibly leaving more Zn bioavailable, but this speculation needs further investigations For Ni treatment, our results are in line with a study of Munzinger (1994), reporting reduced survival of chronically exposed D magna to Ni concentrations of 40–200 μg L− in natural water Similarly, D magna exposed to 85 μg Ni L−1 decreased up to 70% of its population (Pane and McGeer, 2004) Therefore, both D lumholtzi and D magna had a similar survival when exposed to Ni However, in a previous study, D lumholtzi survived up to 750 μg L−1 for 14 days but not higher concentration (Dao et al., 2015) It seems that Ni increased its toxicity in Mekong River water than in COMBO medium This should relate to some other organic chemicals/substances in Mekong river water when combined with spiked metals (Cu, Zn, Ni) might induce negative effects on life history traits of daphnids (e.g survival) Further investigations Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx Fig Survival of Daphnia lumholtzi exposed to metals spiked into filtered water from Mekong River (a), (c) and (e), field water collected at site – Vinh Loc; (b), (d) and (f), field water collected at site – Tan Chau with pure organic chemicals should be implemented for confirmation Besides, as Mekong River water already contained some trace metals (2–5 μg Al L−1, 1–3 μg As L−1, 3–4 μg Zn L−1, Table 2), there might be combined effects of mixed metals to D lumholtzi (in the chronic tests with metal addition solution) which needs further investigations with artificial medium 3.3.2 Effects on maturation In the Mekong River water controls, D lumholtzi reached their maturity at approximately 2.7 days (Fig 3) The development time of D lumholtzi in the current study was less than half compared to a previous documented one of days for this species This discrepancy is probably due to differences in food availability and quality, and the lower experimental temperature (25 °C), lowering growth rates, moreover with some contributions of clone variabilities (Acharya et al., 2006) It is well known that daphnids only mature when they reach a certain body size (Ebert, 1992; Chopelet et al., 2008) The same authors also reported that the age to maturity of D magna correlates inversely with temperature, e.g around 4.5 days at 25 °C compared to around 11.6 days at 15 °C Overall, exposure to metals extended the time to maturation of D lumholtzi that is in line with the pattern observed in previous investigations For example, D obtusa shortly exposed to Cr delayed the age to first reproduction (Coniglio and Baudo, 1989) In our study, the detailed patterns somewhat differed among three metals Firstly, exposure to Cu (at the concentration of μg Cu L−1 dissolved in water from site 2) only extended the time to maturation by ca day, but not at the concentration of μg Cu L−1 dissolved in water collected from site (Fig 3b) Exposure to 1.8 μg Cu L− in artificial medium did not cause a postponement on the maturation of D pulex-pulicaria (Taylor et al., 2016) It seems that the threshold of effects of Cu on maturity age for Fig Maturity age of Daphnia lumholtzi (mean value ± SD of adult daphnids; n as indicated in the columns) exposed to metals spiked into filtered water from Mekong River (a) field water collected at site – Vinh Loc; (b), field water collected at site – Tan Chau Asterisks indicate significant difference between control and exposures by Kruskal-Wallis test (*, P b 0.05; **, P b 0.01; ***, P b 0.001) Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx D lumholtzi in natural waters is around μg Cu L−1 but this needs further verification More obviously, exposure to Zn (50 and 56 μg L−1) and the highest Ni concentrations (225 and 302 μg L−1) resulted in a significant postponement of the daphnids' maturation (Fig 3a, b) Similarly, Dao et al (2015) reported the maturity age of D lumholtzi raised in COMBO medium increased at higher Ni concentrations (500 and 750 μg L−1) within the 14 days of exposure Hence, after a longer time of incubation (21 days), it becomes evident that Mekong River contained some unfavorable elements interfering with Ni-toxicity for D lumholtzi, which requires further investigation However, daphnids in the incubations of and 59 μg Ni L−1 in river water from site (Fig 3a) delayed their maturation whereas those of and 46 μg Ni L−1 in river water from site (Fig 3b) did not show a significant change in their maturity age The reason for no Ni-induced postponement on the maturation of D lumholtzi in river water from site is unknown, as both other metals retarded maturity 3.3.3 Effect on growth At the day 21 (the end) of the exposure duration, the average body length of daphnids was 2304 μm (Fig 4a) and 2265 μm in the two control treatments (Fig 4b), without significant difference (P = 0.066, Kruskal-Wallis test) Exposure to Cu (3 or μg L−1) and the lowest Ni concentrations (5 or μg L−1) resulted in an increase in body length (Fig 4a, b) indicating that the concentrations of these essential metals Fig Body length of 21 days old Daphnia lumholtzi (mean value ± SD of n adult daphnids as indicated in the columns) exposed to metals spiked into filtered water from Mekong River (a) field water collected at site – Vinh Loc; (b), field water collected at site – Tan Chau Asterisks indicate significant difference between control and exposures by Kruskal-Wallis test (*, P b 0.05; **, P b 0.001) in Mekong River water did not fulfill the daphnid requirements or were within safe ranges for the daphnids (Biesinger and Christensen, 1972) Previous studies demonstrated that exposure to low concentrations of essential metals may stimulate the growth rate in Daphnia species (De Schamphelaere and Janssen, 2004) For example, Dave (1984) showed that D magna increased the body length when exposed to 0.026 μg Cu L− for and 21 days Therefore, low concentrations of trace metals in the test solutions in our study probably contribute to the enzyme activities regulating energetic resources available for growth or directly regulating the growth of D lumholtzi In contrast, exposure to high concentrations of metals typically reduces both growth rate and body length (Ghazy and Habashy, 2003) as these metals then become toxic Indeed, in exposures to Zn at concentrations of 50 and 56 μg L− and Ni at concentrations of 46 and 59 μg L− the body length of the daphnids was significantly shorter than of those in the control D magna however increased body length in exposures to 600 and 800 μg Zn L−1 (Muyssen and Janssen, 2001) Tsui and Wang (2007) report D magna to be the most tolerant daphnid species to Zn and another evidence for the higher sensitivity of D lumholtzi compared to D magna to trace metals Our results are also supported by the study of Pane and McGeer (2004) showing a strong decrease of D magna wet weight after 21 days exposure to 85 μg Ni L−1 Regaldo et al (2014) noted the decrease of molting of daphnids (D magna, C dubia, Moinadaphnia macleayi) when they were exposed to Cu (2.5–60 μg L−1), Cr (5–25 μg L−1) and Pb (30–270 μg L−1) during 15 days of exposure It was found that high concentrations of trace metals retard the molting of crustaceans (Weis et al., 1992), which also explains our observations with D lumholtzi As typically, Daphnia increases their body size after every molting, the lower number of molts may be associated with the shorter body length of animals at the end of the exposure periods In Ni treatments of 225 and 302 μg Ni L−1 no daphnids were alive at the end of experiment (21 days) so body length of adult daphnids in these treatments was not available 3.3.4 Effects on fecundity and reproduction During the exposure duration, one adult D lumholtzi raised in Mekong River water from site or site (controls) produced around 18 or 15.6 offspring per clutch, respectively (Fig 5a, b) The accumulative neonates from the two controls were 2793 (in river water from site 1) and 2375 (in river water from site 2, Table 5) Acharya et al (2006) recorded a lower average clutch size of b 12 neonates from adult D lumholtzi raised in Ohio River water, compared to our study The better food quality used in our study, green alga Chlorella and YTC, a very rich nutrient, compared to green alga Scenedesmus added with a phosphorus source in the study by Acharya et al (2006) in addition to the °C higher culture temperature in our study plus clone differences may have resulted in the different fecundities of the daphnids Exposure to Cu resulted in two opposite outcomes of the fecundity: increased neonates in μg Cu L−1 in river water from site (19.8 neonates per clutch; Fig 5a, and 3058 offspring in total, Table 5) and reduced neonates in μg Cu L− in river water from site (14.1 neonates per clutch; Fig 5b, and 1511 offspring in total, Table 5) which could be another evidence for a threshold for toxic effects of Cu around μg L−1 Exposure to Zn at the concentration of 50 and 56 μg L−1 and Ni at high concentrations (302 μg L−1 and 225 μg L−1) reduced the daphnids' fecundity (10.8 and 15.8 neonates per clutch, respectively for Zn, and 10.1 and 5.7 neonates per clutch, respectively for Ni At low Ni concentrations, effects were inconsistent for different waters from sites and 2: in the river water from site 1, Ni did not have any effect on fecundity at concentration of and 59 μg L−1 but in the river water from site 2, Ni at the concentrations of and 46 μg L−1 even stimulated daphnids reproduction (17.7 and 17.6 neonates per clutch, respectively) (Fig 5b) The Zn exposures decreased the total neonates of daphnids, to 746– 2081 Exposures to low Ni concentrations, from to 59 μg L−1), the accumulative neonates were from 2277 to 2796, which were in range with Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong River, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx lower concentration (65–750 μg Ni L−1) This further suggested that the some unknown chemicals in river water may have influenced on Ni toxicity on the growth of D lumholtzi However, brood size of D magna exposed to Ni at 42–85 μg L− (Pane and McGeer, 2004) or 40–200 μg L−1 (Munzinger, 1994) decreased concentration dependently which is supported by the result of our study in which D lumholtzi exposed to high Ni concentrations (225 and 302 μg L−1) reproduced 10– 20 times lower total neonates than the control (Table 5) Zinc at low concentrations is an essential trace element for the growth of daphnids (Kilham et al., 1998), but as any metal at high concentration could reduce the daphnid fecundity This might explain the strong reduction on fecundity and reproduction in the exposure to 50 and 56 μg Zn L−1 (Fig 5; Table 5) Daphnia magna in treated with 80– 170 μg Zn L−1 in the tap water containing 2–3 mg L−1 of DOC, pH of 7.6 and hardness of 180–200 mg as CaCO3, did not significantly reduce its fecundity compared to the control (Muyssen et al., 2006) This observation is another evidence suggesting a higher sensitivity of D lumholtzi to Zn than the temperate D magna Conclusions Fig Fecundity of Daphnia lumholtzi (number of offspring per clutch per individual adult, mean value ± SD) exposed to metals spiked into filtered water from Mekong River (a) field water collected at site – Vinh Loc; (b), field water collected at site – Tan Chau Asterisks indicate significant difference between control and exposures by KruskalWallis test (*, P b 0.05; **, P b 0.01; ***, P b 0.001) those from the controls, from 2375 to 2793 However, the higher Ni concentrations, 225 and 302 μg L−1 strongly reduced the accumulative offspring daphnids, to 103 and 264 neonates, respectively (Table 5) which is in line with the impaired survival and longer time to maturity at chronic exposure to higher Ni concentrations Daphnia magna fed on Cu and Zn burdened (algal) dietary strongly reduced its brood size and reproduction (De Schamphelaere et al., 2004, 2007) Interestingly, D magna chronically exposed to waterborne containing 10 mg L−1 of DOC, pH of 6.8, and Cu at concentrations of 35– 100 μg L−1 was not negatively impacted instead got beneficial effects of increasing reproduction and dry mass (De Schamphelaere and Janssen, 2004) In our study, D lumholtzi exposed to μg Cu L−1 enhanced brood size (Fig 5a), whereas already at μg Cu L−1, the animals decreased their brood size due to later maturation and lower survival compared to the daphnids in control (Figs 2b, 3b) Similar results were recorded in the treatments of and 46 μg Ni L−1 in which the exposed daphnids increased brood size but decreased total newborns (Fig 5b; Table 5) Similarly, Coniglio and Baudo (1989) observed a fluctuation of number of neonates produced by D obtusa after a short time (48 h) exposed to Cr at 20–100 μg L−1 Dissolved in COMBO medium, Ni impaired clutch size D lumholtzi at the concentration of 1035 μg L−1 but not at the Mekong River increased the environmental realistic exposure scenario without interfering in the acute toxicity tests using D lumholtzi The acute tests showed a high sensitivity of D lumholtzi to metals and toxicity order of the used metals to this micro-crustacean was Cu N Zn N Ni In river water from sampling site 2, dissolved metals displayed stronger effects compared to river water from site 1, probably due to the lower DOC despite little higher alkalinity Chronic low concentration exposures of the daphnids to Cu, Zn and Ni slightly decreased the daphnid survival but enhanced the body length of the surviving ones by the end of the incubation However, higher metal incubations caused high mortality rates, delayed maturation, reduced body length and fecundity thus consequently decreased reproduction For chronic exposures, however, we could not exclude interfering factors of the Mekong River water in the Ni exposures At chronic exposure, some undetermined chemicals other than the monitored metals and organic pesticides in river water enhanced the toxicity of spiked metals in our tests The responses of life history traits of D lumholtzi to Cu, Zn and Ni under chronic exposures tentatively suggested that this species has a higher sensitivity to metals than related temperate species These results underscore the importance of including tropical species, e.g D lumholtzi, in ecological risk assessment in tropical regions such as Vietnam to arrive at a better conservation and management plan to protect freshwater biodiversity from metal contaminants To the best of our knowledge, this is the first report on the chronic toxicity of Cu, Ni and Zn dissolved in river water on survivorship of the tropical daphnid D lumholtzi A direct comparative study of the sensitivity between tropical and temperate species of daphnids is highly recommended in future studies We also suggest investigating combined effects of a mixture of trace metals or metals with other pollutants on tropical micro-crustaceans, e.g D lumholtzi Supplementary data to this article can be found online at http://dx doi.org/10.1016/j.scitotenv.2016.08.049 Acknowledgement Table Total accumulated offspring Daphnia lumholtzi in the exposures during 21 days of experiment Sampling sites Metal concentrations (μg L−1) Site – Vinh Loc Accumulative offspring Control Cu = Zn = 56 Ni = Ni = 59 Ni = 302 2793 3058 2081 2796 2619 264 Site – Tan Chau Accumulative offspring Control Cu = Zn = 50 Ni = Ni = 46 Ni = 225 2375 1511 746 2277 2642 103 We would like to thank Prof Tham Hoang from Loyola University Chicago for his assistance on the calculation of median lethal concentration (48 h-LC50) This study was funded by the Vietnam National University – Hochiminh City under the granted project number B201448-01 References Acharya, K., Jack, J.D., Smith, A.S., 2006 Stoichiometry of Daphnia lumholtzi and their invasion success: are they linked? 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http://dx.doi.org/10.1016/j.scitotenv.2016.08.049 ... maturation and fecundity: the Please cite this article as: Dao, T.-S., et al., Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace metals tested in natural water of the Mekong. .. Daphnia galeata Daphnia longispina Daphnia magna Daphnia ambigua Daphnia pulex Daphnia parvula Daphnia lumholtzi Daphnia lumholtzi Daphnia lumholtzi Daphnia lumholtzi Ceriodaphnia Moina Daphnia magna... Artificial medium Pond water Pond water Pond water Pond water Mekong river, site 1, Vinh Loc Mekong river, site 2, Tan Chau Tap water Water from a well Water from a well Water from a well Artificial