STOTEN-20661; No of Pages 11

Science of the Total Environment xxx (2016) xxx–xxx

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Science of the Total Environment

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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
b
University of Science, Vietnam National University – Hochiminh City, 227 Nguyen Van Cu Street, District 5, Hochiminh City, Vietnam
c
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

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 in-
crease environmental realistic exposure
scenarios.
• D. lumholtzi showed higher sensitivity
to metals than temperate Daphnia spe-
cies.
• D. lumholtzi is recommended for
assessing toxicity of metals in tropical
environments.

a r t i c l e i n f o a b s t r a c t

Article history: Metal contamination is one of the major issues to the environment worldwide, yet it is poorly known how expo-
Received 14 June 2016 sure to metals affects tropical species. We assessed the sensitivity of a tropical micro-crustacean Daphnia
Received in revised form 2 August 2016 lumholtzi to three trace metals: copper (Cu), zinc (Zn) and nickel (Ni). Both, acute and chronic toxicity tests
Accepted 6 August 2016
were conducted with metals dissolved in in situ water collected from two sites in the lower part of the Mekong
Available online xxxx
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
Keywords:
(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,
Acute toxicity 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
Life history traits 4 μg L−1), Zn (50 and 56 μg L−1), and Ni (six concentrations from 5 to 302 μg L−1) for 21 days. The concentrations
Mekong River water of 4 μg Cu L−1 and 6 μg Ni L−1 enhanced the body length of D. lumholtzi but 46 μg Ni L−1 and 50 μg Zn L−1 resulted
Sensitivity in a strong mortality, reduced the body length, postponed the maturation, and lowered the fecundity. The results
Trace metals 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
2 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.

1. Introduction upon chronic exposure (but see Dao et al., 2015). The direct application
of ecological risk assessments based on toxicity tests of temperate
Anthropogenic emissions from mining operations, industrial and ag- model species such as D. magna (Dave, 1984; De Schamphelaere et al.,
ricultural activities have increased the metal concentrations in the envi- 2004, 2007) may not be relevant to extrapolate the risk in tropical re-
ronment so that they have become the common contaminants in gions such as the Mekong River. For example, the Vietnamese regula-
aquatic ecosystems and a challenge to control (Tomasik and Warren, tions on surface water quality regarding trace metals for protection on
1996; Schwarzenbach et al., 2010; Lanctot et al., 2016). Several metals aquatic life (QCVN-38, 2011) are not based on the toxicity tests with
are essential, while others do not have a function in organisms, but all local species. This may be problematic as tropical animals differ in key
become toxic at a certain concentration (Wetzel, 2001). Metals are in- important life history traits such as faster life history comparing to tem-
destructible contaminants with high potential for bioaccumulation, in perate species thereby differing in the sensitivity to contaminants
particular in their organic-metal form (e.g., Lau et al., 1998; Waykar (Kwok et al., 2009; Dinh Van et al., 2014). Given that toxicity of metals
and Shinde, 2011) and can be transferred to higher trophic levels of depends on the presence of the dissolved organic matter, water hard-
the food chain (Ikemoto et al., 2008). As exposure to metals impairs ness and alkalinity, these parameters should be taken into account in
aquatic organisms such as aquatic crustaceans, insects and fishes, ecotoxicological studies (Ryan et al., 2009; Jo et al., 2010).
metal contamination has been identified as one of the major threat to To address these issues, we aim to test the sensitivity of a tropical
freshwater biodiversity (Millennium Ecosystem Assessment, 2005; crustacean species Daphnia lumholtzi to three essential metals: Cu, Ni
Dinh Van et al., 2013; Moldovan et al., 2013; Lanctot et al., 2016). and Zn at ecologically relevant concentrations (Jing et al., 2013;
Toxicity of dissolved metals to aquatic organisms such as micro- Onojake et al., 2015) in in situ water collected from two sites in Mekong
crustacean and fish is regulated by several environmental parameters River. Daphnia lumholtzi was chosen as the study species as it is a key
such as pH, alkalinity, dissolved organic carbon (DOC) and hardness species in freshwater ecosystems in the lower basin of the Mekong
(De Schamphelaere and Janssen, 2002; Hoang et al., 2004; Linbo et al., River. Cu, Zn and Ni were chosen to test their acute and chronic toxicity
2009; Ryan et al., 2009; Jo et al., 2010). The increase of pH and humic to Daphnia lumholtzi because of (i) these metals are among the most
acid concentration in the test medium decreased bioavailability of Zn, common metal contaminants in the Mekong River (Cenci and Martin,
thus reducing its toxicity to Daphnia magna (Paulauskis and Winner, 2004; Bui et al., 2016; Dao et al., manuscript in preparation), and (ii)
1988). Similarly, toxicity of metals decreased with increasing water the availability of toxicity data of Cu, Zn and Ni on other daphnid spe-
hardness in daphnid species, e.g. Ceriodaphnia dubia or Daphnia pulex- cies, especially D. magna enabled comparisons and recommendations
pulicaria testing with Cu, Ni, Zn, and Cd (Naddy et al., 2015; Taylor et for ecological risk assessment programs in tropical countries like Viet-
al., 2016). nam. The water samples collected from two sites were comprehensively
Considerable progress has been made on understanding the effects analyzed for the environmental parameters and metal and pesticide
of trace metals on aquatic organisms, including daphnid species in tem- contamination before using them for the acute and chronic toxicity
perate regions (reviewed by Grosell et al., 2002; Tsui and Wang, 2007). tests. We documented how exposures to metals affect key fitness-relat-
For examples, exposure to metals e.g., Cu, Ni, Zn, Cr, or Ag caused im- ed traits in D. lumholtzi such as survival, growth rate, maturation and fe-
pairments of life history traits such as growth rate, maturity age, life- cundity. Finally, recommendations for ecological risk assessment in
span, reproduction, and survival in many temperate Daphnia species tropical ecosystem are provided.
such as D. magna, D. pulex, D. parvula, D. ambigua and D. obtusa
(Winner and Farrell, 1976; Coniglio and Baudo, 1989; Munzinger, 2. Materials and methods
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 knowl- 2.1. Test solutions
edge of how tropical species deal with contaminants (Ghose et al.,
2014). Few studies have investigated the responses of tropical zoo- 2.1.1. Water samples collection
plankton such as Daphnia species to metals (Vardia et al., 1988; Surface water was collected at 2 sampling sites in Mekong River: site
Chishty et al., 2012; Dao et al., 2015; Bui et al., 2016). As mentioned 1 at Vinh Loc ferry-port, An Phu district and site 2 at Tan Chau ferry-port,
above, among the trace metals, Cu, Zn and Ni, were commonly used to Tan Chau district, An Giang Province (Fig. 1). The water samples were
evaluate the chronically negative effects on zooplankton, e.g. temperate transferred to the Environmental Toxicology Laboratory, Institute for
daphnids. However, the chronic effects of these metals, especially dis- Environment and Resources in Hochiminh City and prepared for the ex-
solved metals in field water, on tropical Daphnia lumholtzi have not periments at the same day. In the laboratory, the water samples were
been reported. filtered through 0.45 μm syringe filter (Sartorius, Germany) and stored
The Mekong River is one of the biggest rivers in the world with high in pre-cleaned low density polyethylene plastic containers at 4 °C prior
level of anthropogenic activities such as hydropower plants, urbaniza- to the tests.
tion, transportation of goods, agriculture (Wilbers et al., 2014), aquacul-
ture (Marcussen et al., 2014), and industrialization (Quyen et al., 1995). 2.1.2. Water samples characteristics
While the concentrations of most trace metals (e.g. Ag, As, Cr, Co, Cu, Cd, The filtered waters from each sampling site were analyzed for water
Pb, Se, Sn, Zn) in water in the lower part of the Mekong River were rel- quality parameters that may affect the bioavailability of dissolved
atively low (b 1.6 μg L−1; Ikemoto et al., 2008), a high level of anthropo- metals and the survival and growth of Daphnia such as DOC, alkalinity
genic activities in this region may pose a risk of metal contamination. In and hardness, pH, trace metals and pesticides. The DOC was analyzed
fact, metal contaminations have been occurring locally in several places with a total organic carbon (TOC) analyzer (TOC-5000, Shimadzu) ac-
in the lower part of the Mekong River and its basin (e.g., Cenci and cording to APHA (2005). Total hardness was determined based on con-
Martin, 2004). Despite this, the assessment of metal impacts on fresh- centrations of Ca2 + and Mg2+ and the alkalinity was determined by
water and tropical daphnids (e.g. D. lumholtzi) is neglected (but see titration method (APHA, 2005). The pH of water was measured with a
Vardia et al., 1988; Chishty et al., 2012; Bui et al., 2016), especially 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 3

Fig. 1. 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 2 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-sam- and 0.1 μg L−1, respectively. OCPs standard mixture includes 13 com-
ples per sampling site, Relyea and Diecks, 2012; Relyea, 2012; Dinh Van pounds: 2,4,5,6 Tetrachloro-m-xylene, α-HCH, α-Chlordane, 4,4′-DDE,
et al., 2013) for metal characterization were acidified with concentrated β-Endosulfan, Delta-HCH, Aldrin, Heptachlor epoxide, δ-Chlordane, En-
HNO3 (Merck) to pH b 2 and used for dissolved metal characterization drin aldehyde, Endosulfan sulfate, Endrin ketone, Decachlordiphenyl
(APHA, 2005) with an inductively coupled plasma/mass spectrometry and OPPs standard mix includes 5 compounds: Diazinon, Malathion,
(ICP/MS - Agilent 7500, USA). ICP-MS operating conditions and param- Parathion, Ethion, Trithion that were purchased from Sigma-Aldrich
eters for metal analysis in samples are presented in the Supplementary Co. Laboratory blanks consisted of milipore water extracted and ana-
1. A multi-element tuning solution was used to check accuracy of mea- lyzed in the same way as samples and did not contain OCPs and OPPs.
surement (relative standard deviation, RSD b 5%, Agilent Technologies).
The calibration curve was prepared using single stock solutions for each 2.2. Toxicity test
metal. The concentrations of metals in mixed working standard solu-
tions were prepared based on their estimated concentrations in water 2.2.1. Exposure solutions
samples from preliminary semi-quantitative analysis. The weighted cal- The Cu, Zn, Ni stocks were 1000 mg L− 1 Cu, Zn, Ni in Nitric acid
ibration curves for each element with R2 N 0.999 were accepted for con- (HNO3 ~ 2–3%, Merck). From these stock solutions, exposure solutions
centration calculation. All samples and working standard solutions for with different concentrations of each metal were prepared using the fil-
calibration were spiked with 10 μg Scandium L−1 as internal standard tered river water and exposure concentrations in one of the replicates of
to correct for instrument drift and physical interferences. The percent acute or chronic tests were determined when the tests terminated (see
recovery of the initial calibration verification standard should be 90– Table 2). During the toxicity tests, water temperature (WTW Oxi197i
110% for each element being determined. multi-detector), dissolved oxygen (DO, WTW 350i), and pH (Metrohm
744) were measured at the beginning and at the termination (for all
2.1.2.2. Analysis of pesticides. Pooled water samples (based on 10 sub- tests) and also at the time of medium renewal (chronic tests). These
samplings from each storage tanks) for organochlorine pesticides physical and chemical characteristics were used to confirm if these pa-
(OCPs) and organophosphate pesticides (OPPs) characterization were rameters were favorable for D. lumholtzi.
taken and kept in dark glass bottles on ice in the field until analysis in
the laboratory. Water samples were filtered (Sartorius, Germany) to re- 2.2.2. Test organisms
move residual suspended particulates prior to liquid – liquid extraction The tropical daphnid D. lumholtzi was collected from Bac Ninh Prov-
(AOAC, 1996). OSPs in water samples were extracted with methylene ince, Vietnam (Bui et al., 2016) and has been maintained in the Labora-
chloride (DCM) and OPPs were extracted with mixture of DCM and hex- tory of Environmental Toxicology, Institute for Environment and
ane (15/85, v/v; Merck & Labscan. Inc.). The mixture was shaken for Resources, Vietnam National University – Hochiminh City, for
15 min, followed by phase separation. The organic phase was trans- N2 years. The Daphnia was raised in COMBO medium (Kilham et al.,
ferred into a dry vial. The extraction process was repeated 3 times 1998), at 27 ± 1 °C with a photoperiod of 12 h: 12 h light: dark cycle
(AOAC, 1996; US EPA, 2008). The pooled extracts were concentrated and the light intensity of around 1000 Lux. The Daphnia was fed with
by rotary evaporation then cleaned on a neutral silica solid phase ex- a mixture of green alga (Chlorella sp.) cultured in COMBO medium
traction (SPE) column (Silica Gel 100/200 mesh) (US EPA, 1996 - Meth- and YCT (yeast, cerrophyl and trout chow digestion) prepared accord-
od 3630). The column was eluted with 40 mL of hexane and 30 mL of ing to the U.S. Environmental Protection Agency Method (US EPA,
DCM with the flow rate of 5 mL min−1. SPE extracts were concentrated 2002).
by rotary evaporation and with a gentle stream of nitrogen and
redissolved into 1 mL hexane for injection to GC-ECD. GC–ECD analysis 2.2.3. Acute toxicity tests
was carried out on an Agilent 7890 (USA) with a DB – 5.625 capillary The 48-h static nonrenewal acute toxicity tests were conducted fol-
column (30 m length 0.25 mm i.d., 0.25 mm film thickness). The recov- lowing the guidelines of the US EPA methods (US EPA, 2002) with two
eries of OCPs and OPPs were 80–91% (SD b 5%) and 103–109% (SD b 5%), adjustments of: i) light regime (a photoperiod of 12 h:12 h light:dark at
respectively. The detection limits of OCPs and OPPs were 0.01 μg L−1 a light intensity of ca. 1000 Lux) and ii) temperature (27 ± 1 °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
4 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 test- Table 2
ing. Each treatment had four replicates and each replicate consists of 10 Metal and pesticide concentrations in filtered field water from Mekong River. BDL, below
detection limits of the analytical methods, 1 μg L−1 for BDLa, 0.1 μg L−1 for BDLb, and 0.01
neonates in 40 mL of exposure solution in a 50-mL polypropylene cup.
μg L−1 for BDLc.
Five to seven concentrations of metals were prepared for each metal ex-
posure (Table 2). Controls were prepared by transferring the neonates Dissolved Site 1 – Site 2 – Pesticides (μg L−1) Site 1 – Site 2 –
into Mekong River water without metal addition. The neonates were metals Vinh Loc Tan Chau Vinh Loc Tan Chau
(μg L−1)
fed during the pre-exposure duration but starved during the tests (US
EPA, 2002). We checked daily for dead organisms and removed them Al 5 2 Tetrachloro-m-xylene BDLb BDLb
As 1 3 Alpha-HCH BDLb BDLb
from the cups. Dead of the animals was confirmed by observing the
Ba 25 30 Alpha-Chlordane BDLb BDLb
stop of the heart beat under a microscope. Mortality data were used to Fe 5 2 4,4′-DDE BDLb BDLb
determine median lethal concentrations (48 h-LC50). When the test ter- Zn 4 3 Beta-Endosulfan BDLb BDLb
minated, we randomly took test solution in one of the four replicates (in Cu 1 BDLa Delta-HCH BDLb BDLb
each metal concentration) for the metal analysis by ICP/MS. Co BDLa BDLa Aldrin BDLb BDLb
Cr BDLa BDLa Heptachlor epoxide BDLb BDLb
Mn BDLa BDLa Gamma-Chlordane BDLb BDLb
2.2.4. Chronic tests Ni BDLa BDLa Endrin aldehyde BDLb BDLb
Chronic tests were performed at the same condition as in acute tox- Se BDLa BDLa Endosulfan sulfate BDLb BDLb
icity test. Based on the 48 h-LC50 values and previous investigation (Dao Mo BDLa BDLa Endrin ketone BDLb BDLb
Ag BDLa BDLa Decachlordiphenyl BDLb BDLb
et al., 2015), the concentrations of metals (Cu, Zn, Ni) for chronic tests
Cd BDLa BDLa Diazinon BDLc BDLc
were chosen. The metal concentrations in chronic tests were 3 and Pb BDLa BDLa Ethion BDLc BDLc
4 μg Cu L− 1, 50 and 56 μg Zn L−1, and from 5 to 302 μg Ni L− 1 Malathion BDLc BDLc
(Table 1). Also, the chosen concentrations of Cu, Zn and Ni have been Pazathion BDLc BDLc
BDLc BDLc
found in natural water of the lower Mekong River (e.g. 4 μg Cu L−1; Trithion

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). Brief- USA). Kruskal-Wallis test (Sigma Plot, version 12) was applied for calcu-
ly, neonates (15 individuals per treatment) of D. lumholtzi b24 h-age lation the significant difference of the maturation, fecundity and body
from 2nd to 3rd clutch were individually and independently incubated length of D. lumholtzi between control and metal exposure solutions.
for each treatment in 50-mL polypropylene cups containing 20 mL con- To provide full overview of the sensitivity of the D. lumholtzi to metals,
trol solution or exposure solutions. Each treatment had 15 replicates we analyzed and documented the results separately for exposure solu-
(n = 15). Exposure solutions were renewed every second day. The tions made from waters collected at each sampling site.
Daphnia were fed with a mixture of Chlorella (~ 1 mg C L−1, approxi-
mately 140,000 cells mL−1) and YTC (~20 μL) just after the exposure so- 3. Results and discussion
lutions were renewed. Life history traits of the Daphnia including
mortality, maturation, and reproduction were scored daily. Maturity 3.1. Physical and chemical characteristics of field water from Mekong River
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 All analyzed organic pesticides in filtered Mekong River water were
each mother daphnid were checked daily, removed from the cup with below the detection levels of the equipment (Agilent 7890, USA;
a glass pipet and counted for clutch size to evaluate the fecundity. Re- Table 2), including Tetrachloro-m-xylene, Alpha-HCH, 4,4′-DDE, Beta-
production was calculated as total accumulated offspring reproduced Endosulfan, Delta-HCH, Aldrin, Heptachlor epoxide, Gamma-Chlordane,
by all mother daphnids in each treatment. Fecundity was defined as Endrin aldehyde, Endosulfan sulfate, Endrin ketone, Decachlordiphenyl,
the average number of offspring in one clutch reproduced by one moth- Diazinon, Ethion, Malathion, Pazathion and Trithion. Overall, concentra-
er daphnid. The chronic tests lasted for 21 days. At test termination, liv- tions of trace metals in filtered water from both sampling sites of the
ing mother daphnids were immediately fixed with Lugol solution Mekong River were very low. They ranged from 2 to 5 μg L−1 of Al, 1
(Sournia, 1978) and body length was measured to the nearest 1 μm, to 3 μg L−1 of As, 25 to 30 μg L− 1 of Ba, 2 to 5 μg L− 1 of Fe and 3 to
on a microscope (Olympus BX 51) coupled with a digital camera (DP 4 μg L−1 of Zn. Concentrations of other metals: Cu, Co, Cr, Mn, Ni, Se,
71). The body length was measured from the eye to the base of tail Mo, Ag, Cd and Pb were below the detection levels of the ICP/MS,
spine of the mothers. 1 μg L−1 (Table 2). The concentrations of trace metals and pesticides
in filtered Mekong River water in the current study were similar to
2.3. Data analyses 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
Median lethal concentrations with 95% confidence intervals (95% lower than the lowest concentration inducing acute negative effects
CIs) were calculated by Toxcalc Program (Tidepool Scientific LLC. on other daphnid species e.g. D. magna (3000 μg L− 1; Hoang et al.,

Table 1
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) 13, 15, 18, 19, 20 3, 7, 8, 10,11,13,15
Zn (μg L−1) 56, 156, 247, 343, 539 50, 87, 139, 192, 226, 476, 688
Ni (μg L−1) 1087, 1403, 1659, 1985, 2090 481, 766, 968, 1369, 1602, 1807

Chronic tests
Cu (μg L−1) 3 4
Zn (μg L−1) 56 50
Ni (μg L−1) 6, 59, 302 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 5

2007) and D. pulex (2500–3900 μg L−1; Shaw et al., 2007). Similarly, dis- such as D. lumholtzi, Moina, and Ceriodaphnia to test the acute toxicity of
solved Zn (4 μg L−1) and aluminum (5 μg L−1) in the test water were Zn, Pb and Cd dissolved in a natural water sample originating from a
considerably lower 48 h-LC50 values (61.8–130 μg Zn L− 1; 742– well (pH of 7.9, alkalinity and hardness of 512 and 582 mg CaCO3 L−1,
1900 μg Al L−1) to the micro-crustacean, Ceriodaphnia dubia, reported respectively). In their studies, the 48 h-LC50 was 2300 μg Zn L−1 (to D.
elsewhere (Gostomski, 1990; Naddy et al., 2015). lumholtzi), which is by a factor of 10 higher than that in our experiment
The pH of Mekong River water was 7.8 at both sampling sites (Table 4). Higher water hardness, pH and alkalinity as well as the use of
(Table 3). However, the pH decreased to 6.8 after metals (Cu, Ni, Zn) adult daphnids may have contributed to this higher value. However,
were spiked into the test water. During the toxicity tests the DO varied lacking experimental details (age of the animals and rearing conditions)
from 6.3 to 6.6 mg L−1 (ca. 78–80% of saturated oxygen concentration; impede the comparison.
Wetzel, 2001). The DOC concentrations in the water from Vinh Loc (site In acute toxicity tests of Cu in moderately hard water and similar
1) and Tan Chau (site 2) were 2.99 and 1.89 mg L−1, and hardness was range of DOC (1–3 mg L− 1), and pH (7–8) similar to our study, the
79 and 87 mg CaCO3 L−1, respectively (Table 3). Both pH and DO in the values of 48 h-LC50 of D. magna, D. obtusa, and D. pulex ranged from
test water were within the favorable range for the growth and develop- 60.3 to 156.1, 41.1 to 100.1 and 19.5 to 26 μg Cu L− 1, respectively,
ment of daphnids such as Daphnia magna, Daphnia pulex and which are higher than in our study with D. lumholtzi (Villavicencio et
Ceriodaphnia dubia (APHA, 2005; Ebert, 2005). However, lower pH al., 2005; Traudt et al., 2016). In addition, Rodriguez and Arbildua
could increase bioavailability and consequently toxicity of metals to (2012) found D. magna with the 48 h-EC50 of 16.5 μg Cu L−1, under
daphnids, thus contribute as confounding factor. The DOC concentra- the test conditions of 2 mL−1 of DOC, pH of 6.3 and hardness of
tions of the river water (1.89–2.99 mg L−1, Table 3) were considerably 169 mg L−1 as CaCO3. Though the same authors reported similar 48 h-
lower than that in a previous study (DOC = 14.4–14.7 mg L− 1) in LC50/EC50 value to our record, but the double hardness and lower pH
which samples were collected from a nearby location (Bui et al., in their study compared to ours revealed that D. lumholtzi (from our
2016). The alkalinity (64–68 mg CaCO3 L−1) and hardness (79–87 mg study) appeared to be more sensitive to Cu than the other three temper-
CaCO3 L− 1) were only slightly different between the two sampling ate Daphnia species, D. magna, D. obtusa, and D. pulex.
sites, and the water could be classified as moderately hard water In COMBO medium (0.67 mg L−1 of DOC, hardness and alkalinity of
(Villavicencio et al., 2005; Naddy et al., 2015). Probably, the DOC con- 44 and 10 mg L−1 as CaCO3, respectively) the 48 h-LC50 of 1775 μg Ni
centrations, alkalinity and hardness of water from Mekong River varied L−1 for D. lumholtzi (Dao et al., 2015) was a little higher than the
depending on the preceding meterological conditions but are in range 48 h-LC50 values of the current study (1026–1516 μg Ni L−1; Table 4).
with other tropical rivers (Villavicencio et al., 2005; Bui et al., 2016). 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
3.2. Acute effects of metals on Daphnia lumholtzi 3.6 mg L−1 which was in range with the 48 h-LC50 from our study. In
moderately hard water and 3 mg L−1 DOC, a 48 h-LC50 of 1633 μg Ni
The 48 h-LC50 values for D. lumholtzi incubated in Mekong River L−1 was attained for D. magna (Traudt et al., 2016). Therefore, D.
water ranged from 11.57 to 16.67 μg L−1 of Cu, 179.3 to 280.9 μg L−1 lumholtzi and D. magna seem to have a similar sensitivity regarding
of Zn and 1026 to 1516 μg L−1 of Ni (Table 4, Supplementary 2). The acute toxicity to Ni.
48 h-LC50 values were lower in the test with river water from site 2 Vardia et al. (1988) reported the 48 h-LC50 of D. lumholtzi of 2290 μg
than that from site 1 probably associated with the lower DOC concentra- Zn L−1, which is far higher than the 48 h-LC50 value in our study (Table
tion in water at site 2 (1.89 mg L−1) compared to site 1 (2.99 mg L−1). 4). Again, this difference could be the consequence of higher hardness
Overall, the toxicity order of the three metals to daphnids in our study and the age tolerance to metal of the daphnids as mentioned above.
decreased from Cu N Zn N Ni (Table 4) which is in line with previous in- Comparing D. lumholtzi 48 h-LC50 values for Zn of our study (179–
vestigations (e.g., Biesinger and Christensen, 1972; Wong, 1992; Vardia 280 μg Zn L− 1, in moderately hard water, Table 4) to those of D.
et al., 1988; Traudt et al., 2016). magna (928 μg Zn L−1 in moderately hard water) and C. dubia (102–
Bui et al. (2016) reported 48 h-LC50 values for Cu of 6.15– 130 μg Zn L−1 in hard water) reveals an increase of sensitivity from D.
8.61 μg L−1, and 5.77–7.23 μg L−1 in two tropical micro-crustaceans, magna to D. lumholtzi to C. dubia despite the possible mitigating effect
D. lumholtzi and Ceriodaphnia cornuta, respectively, exposed to Cu of water hardness (Naddy et al., 2015; Traudt et al., 2016). Notably,
spiked into Mekong River water. These 48 h-LC50 values are two times the Cu concentration of 200 μg L−1 is used as the safety level for protec-
lower than those from our study (Table 4). It seems that the higher alka- tion of aquatic life (QCVN-38, 2011), but this Cu concentration is even
linity and hardness in the water used in the current study contributed to 13 times higher than the 48-LC50 value of D. lumholtzi exposed to Cu
the lower toxicity of Cu compared to Bui et al. (2016), despite their in this study. Taking more safety factors into consideration (e.g. 10 for
higher DOC. In acute toxicity test with D. lumholtzi exposed to Cu in intra species differences and 10 for the chronic exposure scenario) the
dechlorinated tap water (pH 7–9, DOC 2–4 mg L−1, alkalinity and hard- QCVN-38 (2011) should be re-considered and adjusted for aquatic eco-
ness 180 and 200 mg CaCO3 mg L−1), the 48 h-LC50 value of 54.6 μg Cu system protection. To our knowledge this is the first report on the acute
L−1 (Vardia et al., 1988) was higher than that in our study (Table 4). The test of Ni and Zn spiked into field water to D. lumholtzi, which together
higher alkalinity and hardness together with the possibly older age of D. with previous results of Bui et al. (2016), may be used for the developing
lumholtzi in the study of Vardia et al. (1988), may have contributed to of the metal Biotic Ligand Model (Di Toro et al., 2001; Villavicencio et al.,
the lower sensitivity. Chishty et al. (2012) used several daphnid species 2005) with tropical micro-crustaceans.

Table 3
3.3. Chronic effects of metals on life history traits of Daphnia lumholtzi
Physical and chemical characteristics of the field water from Mekong River and the expo-
sure solutions during the experiments. Several trace metals such as Zn and Cu are essential components of
more than hundred enzymes and various biological functions (Walker
Parameters Site 1 – Vinh Loc Site 2 – Tan Chau
et al., 1996) contributing to the function and regulation of many enzyme
pH (in the field water) 7.8 7.8
activities related to the fitness (health, growth and reproduction) in an-
pH (in the test water after metal addition) 6.8–7.8 6.6–7.8
Dissolved oxygen in the test water (mg L−1) 6.3–6.6 6.3–6.6 imals. However, increasing metal concentrations at some point impair
Dissolved organic carbon (mg L−1) 2.99 1.89 physiological functions, reduce fitness or even become lethal to organ-
Hardness (mg CaCO3 L−1) 79 87 isms (Pane et al., 2003). Several nonexclusive mechanisms may under-
Alkalinity of the field water (mg CaCO3 L−1) 68 68 lie the metal-induced reduction of the fitness-related traits in exposed
Alkalinity of the test water (mg CaCO3 L−1) 64–68 64–68
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
6 T.-S. Dao et al. / Science of the Total Environment xxx (2016) xxx–xxx

Table 4
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 Cu (μg L−1) 6.15–8.61 Mekong river Bui et al., 2016
Ceriodaphnia cornuta Cu (μg L−1) 5.77–7.23 Mekong river Bui et al., 2016
Daphnia lumholtzi Cu (μg L−1) 54.6 Tap water Vardia et al., 1988
Ceriodaphnia dubia Cu (μg L−1) 16.6 Artificial medium Naddy et al., 2015
Daphnia magna Cu (μg L−1) 60.3–156.1 Rivers and lakes Villavicencio et al., 2005
Daphnia magna Cu (μg L−1) 100 Artificial medium Traudt et al., 2016
Daphnia obtusa Cu (μg L−1) 41.1–100.1 Rivers and lakes Villavicencio et al., 2005
Daphnia pulex Cu (μg L−1) 19.5–26 Rivers and lakes Villavicencio et al., 2005
Ceriodaphnia reticulata Cu (μg L−1) 13.3–17.7* Artificial medium Bossuyt and Janssen, 2005
Ceriodaphnia pulchella Cu (μg L−1) 12.0–16.4* Artificial medium Bossuyt and Janssen, 2005
Daphnia magna Cu (μg L−1) 26.8–53.2* Artificial medium Bossuyt and Janssen, 2005
Daphnia galeata Cu (μg L−1) 22.6* Artificial medium Bossuyt and Janssen, 2005
Daphnia longispina Cu (μg L−1) 9.89–11.9* Artificial medium Bossuyt and Janssen, 2005
Daphnia magna Cu (μg L−1) 86.5** Pond water Winner and Farrell, 1976
Daphnia ambigua Cu (μg L−1) 67.7** Pond water Winner and Farrell, 1976
Daphnia pulex Cu (μg L−1) 86** Pond water Winner and Farrell, 1976
Daphnia parvula Cu (μg L−1) 72** Pond water Winner and Farrell, 1976
Daphnia lumholtzi Cu (μg L−1) 16.67 (15.92–17.38) Mekong river, site 1, Vinh Loc This study
Daphnia lumholtzi Cu (μg L−1) 11.57 (10.97–12.07) Mekong river, site 2, Tan Chau This study
Daphnia lumholtzi Zn (μg L−1) 2290 Tap water Vardia et al., 1988
Daphnia lumholtzi Zn (μg L−1) 2300 Water from a well Chishty et al., 2012
Ceriodaphnia Zn (μg L−1) 1400 Water from a well Chishty et al., 2012
Moina Zn (μg L−1) 1200 Water from a well Chishty et al., 2012
Daphnia magna Zn (μg L−1) 819 Artificial medium Shaw et al., 2006
Daphnia magna Zn (μg L−1) 928 Artificial medium Traudt et al., 2016
Daphnia pulex Zn (μg L−1) 273 Artificial medium Shaw et al., 2006
Daphnia ambigua Zn (μg L−1) 304 Artificial medium Shaw et al., 2006
Ceriodaphnia dubia Zn (μg L−1) 260 Artificial medium Shaw et al., 2006
Ceriodaphnia dubia Zn (μg L−1) 102–130 Artificial medium Naddy et al., 2015
Daphnia lumholtzi Zn (μg L−1) 280.9 (257–306.6) Mekong river, site 1, Vinh Loc This study
Daphnia lumholtzi Zn (μg L−1) 179.3 (162.4–198.2) Mekong river, site 2, Tan Chau This study
Daphnia lumholtzi Ni (μg L−1) 1775 Artificial medium Dao et al., 2015
Daphnia magna Ni (μg L−1) 1068 Tap water Pane et al., 2003
Daphnia magna Ni (μg L−1) 1633 Artificial medium Traudt et al., 2016
Daphnia lumholtzi Ni (μg L−1) 1516 (1398–1616) Mekong river, site 1, Vinh Loc This study
Daphnia lumholtzi Ni (μg L−1) 1026 (941.6–1114) Mekong river, site 2, Tan Chau This study

impairment of the respiratory function (Pane et al., 2003), the inhibition 180 mg L−1 as CaCO3 (Regaldo et al., 2014)). These results indicate
of the sodium uptake, impairing the osmotic imbalance (Grosell et al., that temperate daphnid species seem to be more resistant to Cu than
2002) inducing oxidative stress and an increase in the energy expense D. lumholtzi. Previous studies have shown that intraspecific populations
for detoxification (e.g., upregulation of costly metallothioneins or anti- at lower latitudes with faster life history (e.g., higher growth rate and
oxidant mechanisms (Amiard et al., 2006; Dinh Van et al., 2013). There- shorter generation times) may be more vulnerable to contaminants
fore, the maintenance cost is increased. Furthermore, exposure to (Dinh Van et al., 2014) as a result of energy allocation trade-off (Sibly
metals may also reduce the foraging activity, hence lowering energy in- and Calow, 1989; Congdon et al., 2001). It remains to be tested whether
take (e.g., Janssens et al., 2014) Consequently, metal-exposed animals this is also the case at the species levels for the higher sensitivity of trop-
may suffer a lower growth and reproduction rate, or even mortality ical daphnid species to metals compared to temperate one.
(e.g., Winner and Farrell, 1976; Pane and McGeer, 2004; Muyssen et Muyssen et al. (2006) reported chronic exposure to 80–250 μg L−1
al., 2006). Zn at pH of 7.6 and DOC of 4 mg L−1 did not significantly decrease D.
magna survivorship while survival of D. lumholtzi in our study was al-
3.3.1. Effects on survival ready decreased 46% at 50 μg Zn L−1 at a lower DOC, however (Fig.
Mekong River water did not impair survival of D. lumholtzi during 2d). Again, either the DOC mitigated toxicity for D. magna by up to factor
three weeks of exposure (Fig. 2a, b). Exposure to Cu caused mortality 5 or D. lumholtzi seems more susceptible. The difference in Zn-induced
of 20% at 3 μg L−1 and 4 μg L−1 (Fig. 2a, b). The concentration of 56 μg mortality of D. lumholtzi in waters from two different sites in Mekong
Zn L−1 in river water from site 1 resulted in 16% mortality of daphnids River may also be partly attributed to the lower DOC content at site 2,
whereas 50 μg Zn L−1 in river water from site 2 caused 54% mortality possibly leaving more Zn bioavailable, but this speculation needs further
(Fig. 2c, d). Ni in water from sites 1 and 2 caused mortality of 14–27% investigations.
at 5–59 μg Ni L−1. This metal induced 100% mortality on day 10 at 302 For Ni treatment, our results are in line with a study of Munzinger
and 225 μg Ni L−1 for Ni dissolved in water from site 1 and 2, respective- (1994), reporting reduced survival of chronically exposed D. magna to
ly; Fig. 2e, f). Ni concentrations of 40–200 μg L− 1 in natural water. Similarly, D.
Comparing the vulnerability of four Daphnia species (D. magna, D. magna exposed to 85 μg Ni L−1 decreased up to 70% of its population
pulex, D. parvula, D. ambigua) to Cu in pond water (alkalinity of 110– (Pane and McGeer, 2004). Therefore, both D. lumholtzi and D. magna
119 mg CaCO3 L−1; hardness of 130–160 mg CaCO3 L−1; and pH of had a similar survival when exposed to Ni. However, in a previous
8.2–9.5) survival of the four Daphnia species slightly decreased at study, D. lumholtzi survived up to 750 μg L−1 for 14 days but not higher
20 μg Cu L−1 during 3 weeks of incubation (Winner and Farrell, concentration (Dao et al., 2015). It seems that Ni increased its toxicity in
1976), whereas D. lumholtzi suffered already 20% mortality during Mekong River water than in COMBO medium. This should relate to
21 days at 3 to 4 μg Cu L−1 in our study. In a 15-day test, N 50% of D. some other organic chemicals/substances in Mekong river water when
magna and 80% of Moinadaphnia macleayi survived exposure to 25 and combined with spiked metals (Cu, Zn, Ni) might induce negative effects
40 μg Cu L−1 (in artificial medium, pH of 7.6–7.7; hardness of 160– 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 7

Fig. 2. Survival of Daphnia lumholtzi exposed to metals spiked into filtered water from Mekong River. (a), (c) and (e), field water collected at site 1 – Vinh Loc; (b), (d) and (f), field water
collected at site 2 – 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 matu-
rity 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 7 days for this species. This discrepancy is probably
due to differences in food availability and quality, and the lower exper-
imental 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 re-
ported 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 investiga-
tions. 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 4 μg Cu L−1 dissolved in water from site 2) only
extended the time to maturation by ca. 1 day, but not at the concentra-
tion of 3 μg Cu L−1 dissolved in water collected from site 1 (Fig. 3b). Ex- Fig. 3. 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
posure to 1.8 μg Cu L− 1 in artificial medium did not cause a River. (a) field water collected at site 1 – Vinh Loc; (b), field water collected at site 2 –
postponement on the maturation of D. pulex-pulicaria (Taylor et al., Tan Chau. Asterisks indicate significant difference between control and exposures by
2016). It seems that the threshold of effects of Cu on maturity age for 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
8 T.-S. Dao et al. / Science of the Total Environment xxx (2016) xxx–xxx

D. lumholtzi in natural waters is around 4 μg Cu L−1 but this needs fur- in Mekong River water did not fulfill the daphnid requirements or
ther verification. were within safe ranges for the daphnids (Biesinger and Christensen,
More obviously, exposure to Zn (50 and 56 μg L−1) and the highest 1972). Previous studies demonstrated that exposure to low concentra-
Ni concentrations (225 and 302 μg L−1) resulted in a significant post- tions of essential metals may stimulate the growth rate in Daphnia spe-
ponement of the daphnids' maturation (Fig. 3a, b). Similarly, Dao et al. cies (De Schamphelaere and Janssen, 2004). For example, Dave (1984)
(2015) reported the maturity age of D. lumholtzi raised in COMBO medi- showed that D. magna increased the body length when exposed to
um increased at higher Ni concentrations (500 and 750 μg L−1) within 0.026 μg Cu L− 1 for 7 and 21 days. Therefore, low concentrations of
the 14 days of exposure. Hence, after a longer time of incubation trace metals in the test solutions in our study probably contribute to
(21 days), it becomes evident that Mekong River contained some unfa- the enzyme activities regulating energetic resources available for
vorable elements interfering with Ni-toxicity for D. lumholtzi, which re- growth or directly regulating the growth of D. lumholtzi.
quires further investigation. However, daphnids in the incubations of 6 In contrast, exposure to high concentrations of metals typically re-
and 59 μg Ni L−1 in river water from site 1 (Fig. 3a) delayed their mat- duces both growth rate and body length (Ghazy and Habashy, 2003)
uration whereas those of 5 and 46 μg Ni L−1 in river water from site 2 as these metals then become toxic. Indeed, in exposures to Zn at con-
(Fig. 3b) did not show a significant change in their maturity age. The centrations of 50 and 56 μg L− 1 and Ni at concentrations of 46 and
reason for no Ni-induced postponement on the maturation of D. 59 μg L− 1 the body length of the daphnids was significantly shorter
lumholtzi in river water from site 2 is unknown, as both other metals re- than of those in the control. D. magna however increased body length
tarded maturity. 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
3.3.3. Effect on growth species to Zn and another evidence for the higher sensitivity of D.
At the day 21 (the end) of the exposure duration, the average body lumholtzi compared to D. magna to trace metals. Our results are also sup-
length of daphnids was 2304 μm (Fig. 4a) and 2265 μm in the two con- ported by the study of Pane and McGeer (2004) showing a strong de-
trol treatments (Fig. 4b), without significant difference (P = 0.066, crease of D. magna wet weight after 21 days exposure to 85 μg Ni L−1.
Kruskal-Wallis test). Exposure to Cu (3 or 4 μg L−1) and the lowest Ni Regaldo et al. (2014) noted the decrease of molting of daphnids (D.
concentrations (5 or 6 μg L−1) resulted in an increase in body length magna, C. dubia, Moinadaphnia macleayi) when they were exposed to
(Fig. 4a, b) indicating that the concentrations of these essential metals 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 Me-
kong River water from site 1 or site 2 (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) re-
corded 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 2 °C higher
culture temperature in our study plus clone differences may have re-
sulted in the different fecundities of the daphnids.
Exposure to Cu resulted in two opposite outcomes of the fecundity:
increased neonates in 3 μg Cu L−1 in river water from site 1 (19.8 neo-
nates per clutch; Fig. 5a, and 3058 offspring in total, Table 5) and re-
duced neonates in 4 μg Cu L− 1 in river water from site 2 (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 4 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) re-
duced the daphnids' fecundity (10.8 and 15.8 neonates per clutch, re-
spectively 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 1 and 2: in the river water from site 1, Ni did not
have any effect on fecundity at concentration of 6 and 59 μg L−1 but
in the river water from site 2, Ni at the concentrations of 5 and
46 μg L−1 even stimulated daphnids reproduction (17.7 and 17.6 neo-
Fig. 4. Body length of 21 days old Daphnia lumholtzi (mean value ± SD of n adult daphnids nates per clutch, respectively) (Fig. 5b).
as indicated in the columns) exposed to metals spiked into filtered water from Mekong
River. (a) field water collected at site 1 – Vinh Loc; (b), field water collected at site 2 –
The Zn exposures decreased the total neonates of daphnids, to 746–
Tan Chau. Asterisks indicate significant difference between control and exposures by 2081. Exposures to low Ni concentrations, from 5 to 59 μg L−1), the ac-
Kruskal-Wallis test (*, P b 0.05; **, P b 0.001). cumulative 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 9

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− 1 (Pane and McGeer, 2004) or
40–200 μg L−1 (Munzinger, 1994) decreased concentration dependent-
ly which is supported by the result of our study in which D. lumholtzi ex-
posed 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 con-
centration 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 obser-
vation is another evidence suggesting a higher sensitivity of D. lumholtzi
to Zn than the temperate D. magna.

4. Conclusions

Mekong River increased the environmental realistic exposure sce-

nario 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
Fig. 5. Fecundity of Daphnia lumholtzi (number of offspring per clutch per individual adult, due to the lower DOC despite little higher alkalinity. Chronic low con-
mean value ± SD) exposed to metals spiked into filtered water from Mekong River. (a) centration exposures of the daphnids to Cu, Zn and Ni slightly decreased
field water collected at site 1 – Vinh Loc; (b), field water collected at site 2 – Tan Chau.
the daphnid survival but enhanced the body length of the surviving
Asterisks indicate significant difference between control and exposures by Kruskal-
Wallis test (*, P b 0.05; **, P b 0.01; ***, P b 0.001). 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
those from the controls, from 2375 to 2793. However, the higher Ni con- exposures, however, we could not exclude interfering factors of the Me-
centrations, 225 and 302 μg L−1 strongly reduced the accumulative off- kong River water in the Ni exposures. At chronic exposure, some unde-
spring daphnids, to 103 and 264 neonates, respectively (Table 5) which termined chemicals other than the monitored metals and organic
is in line with the impaired survival and longer time to maturity at pesticides in river water enhanced the toxicity of spiked metals in our
chronic exposure to higher Ni concentrations. tests. The responses of life history traits of D. lumholtzi to Cu, Zn and
Daphnia magna fed on Cu and Zn burdened (algal) dietary strongly Ni under chronic exposures tentatively suggested that this species has
reduced its brood size and reproduction (De Schamphelaere et al., a higher sensitivity to metals than related temperate species. These re-
2004, 2007). Interestingly, D. magna chronically exposed to waterborne sults underscore the importance of including tropical species, e.g. D.
containing 10 mg L−1 of DOC, pH of 6.8, and Cu at concentrations of 35– lumholtzi, in ecological risk assessment in tropical regions such as Viet-
100 μg L−1 was not negatively impacted instead got beneficial effects of nam to arrive at a better conservation and management plan to protect
increasing reproduction and dry mass (De Schamphelaere and Janssen, freshwater biodiversity from metal contaminants. To the best of our
2004). In our study, D. lumholtzi exposed to 3 μg Cu L−1 enhanced brood knowledge, this is the first report on the chronic toxicity of Cu, Ni and
size (Fig. 5a), whereas already at 4 μg Cu L−1, the animals decreased Zn dissolved in river water on survivorship of the tropical daphnid D.
their brood size due to later maturation and lower survival compared lumholtzi. A direct comparative study of the sensitivity between tropical
to the daphnids in control (Figs. 2b, 3b). Similar results were recorded and temperate species of daphnids is highly recommended in future
in the treatments of 5 and 46 μg Ni L−1 in which the exposed daphnids studies. We also suggest investigating combined effects of a mixture of
increased brood size but decreased total newborns (Fig. 5b; Table 5). trace metals or metals with other pollutants on tropical micro-crusta-
Similarly, Coniglio and Baudo (1989) observed a fluctuation of number ceans, e.g. D. lumholtzi.
of neonates produced by D. obtusa after a short time (48 h) exposed to Supplementary data to this article can be found online at http://dx.
Cr at 20–100 μg L−1. Dissolved in COMBO medium, Ni impaired clutch doi.org/10.1016/j.scitotenv.2016.08.049.
size D. lumholtzi at the concentration of 1035 μg L−1 but not at the
Acknowledgement
Table 5
Total accumulated offspring Daphnia lumholtzi in the exposures during 21 days of We would like to thank Prof. Tham Hoang from Loyola University
experiment. Chicago for his assistance on the calculation of median lethal concentra-
Sampling sites Metal concentrations (μg L−1) tion (48 h-LC50). This study was funded by the Vietnam National Uni-
versity – Hochiminh City under the granted project number B2014-
Site 1 – Vinh Loc Control Cu = 3 Zn = 56 Ni = 6 Ni = 59 Ni = 302
Accumulative 2793 3058 2081 2796 2619 264 48-01.
offspring

Site 2 – Tan Chau Control Cu = 4 Zn = 50 Ni = 5 Ni = 46 Ni = 225 References

Accumulative 2375 1511 746 2277 2642 103
Acharya, K., Jack, J.D., Smith, A.S., 2006. Stoichiometry of Daphnia lumholtzi and their inva-
offspring
sion success: are they linked? Arch. Hydrobiol. 165, 433–453.

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
10 T.-S. Dao et al. / Science of the Total Environment xxx (2016) xxx–xxx

Amiard, J.C., Amiard-Triquet, C., Barka, S., Pellerin, J., Rainbow, P.S., 2006. Metallothioneins Jing, L., Fadong, L., Qiang, L., Shuai, S., Guangshuai, Z., 2013. Spatial distribution and
in aquatic invertebrates: their role in metal detoxification and their use as bio- sources of dissolved trace metals in surface water of the Wei River, China. Water
markers. Aquat. Toxicol. 76, 160–202. Sci. Technol. 67, 817–823.
AOAC, 1996. Official Methods of Analysis. 16th ed. Association of Official Analytical Chem- Jo, H.J., Son, J., Cho, K., Jung, J., 2010. Combined effects of water quality parameters on mix-
ists, Washington DC. ture toxicity of copper and chromium toward Daphnia magna. Chemosphere 81,
American Public Health Association (APHA), 2005. Standard Methods for the Examination 1301–1307.
of Water and Wastewater Washington DC. Kilham, S.S., Kreeger, D.A., Lynn, S.G., Goulden, C.E., Herrera, L., 1998. COMBO: a de-
Bianchini, A., Wood, C.M., 2002. Physiological effects of chronic silver exposure in Daphnia fined freshwater culture medium for algae and zooplankton. Hydrobiologia 377,
magna. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 133, 137–145. 147–159.
Biesinger, K.E., Christensen, G.M., 1972. Effects of various metals on survival, growth, re- Kwok, K.W.H., Leung, K.M.Y., Lui, G.S.G., Chu, V.K.H., Lam, P.K.S., Morritt, D., Maltby, L.,
production, and metabolism of Daphnia magna. J. Fish. Res. Board Can. 29 (12), Brock, T.C.M., Van den Brink, P.J., Warne, M.S.J., Crane, M., 2009. Comparison of trop-
1691–1700. ical and temperate freshwater animal species' acute sensitivities to chemicals: impli-
Bossuyt, B.T.A., Janssen, C.R., 2005. Copper toxicity to different field-collected cladoceran cations for deriving safe extrapolation factors. Integr. Environ. Assess. Manag. 3,
species: intra- and inter-species sensitivity. Environ. Pollut. 136, 145–154. 49–67.
Bui, T.K.L., Do-Hong, L.C., Dao, T.S., Hoang, T.C., 2016. Copper toxicity and the influence of Lanctot, C., Wilson, S.P., Fabbro, L., Leusch, F.D.L., Melvin, S.D., 2016. Comparative sensitiv-
water quality of Dongnai River and Mekong River waters on copper bioavailability ity of aquatic invertebrate and vertebrate species to wastewater from an operational
and toxicity to three tropical species. Chemosphere 144, 872–878. coal mine in central Queensland, Australia. Ecotoxicol. Environ. Saf. 129, 1–9.
Cenci, R.M., Martin, J.M., 2004. Concentration and fate of trace metals in Mekong river Lau, S., Mohamed, M., Tan Chi Yen, A., Su'ut, S., 1998. Accumulation of heavy metals in
delta. Sci. Total Environ. 332, 167–182. freshwater molluscs. Sci. Total Environ. 214, 113–121.
Chishty, N., Tripathi, A., Sharma, M., 2012. Evaluation of acute toxicity of zinc, lead and Linbo, T.F., Baldwin, D.H., McIntyre, J.K., Scholz, N.L., 2009. Effects of water hardness, alka-
cadmium to zooplanktonic community in upper Berach river system, Rajasthan, linity, and dissolved organic carbon on the toxicity of copper to the lateral line of de-
India. South Asian J. Exp. Biol. 2 (1), 20–25. veloping fish. Environ. Toxicol. Chem. 28 (7), 1455–1461.
Chopelet, J., Blier, P.U., Dufresne, F., 2008. Plasticity of growth rate and metabolism Marcussen, H., Lojmand, H., Dalsgaard, A., Hai, D.M., 2014. Copper use and accumulation
in Daphnia magna populations from different thermal habitats. J. Exp. Zool. 309A, in catfish culture in the Mekong Delta, Vietnam. J. Environ. Sci. Health, Part A: Tox.
1–10. Hazard. Subst. Environ. Eng. 49, 187–192.
Congdon, J.D., Dunham, A.E., Hopkins, W.A., Rowe, C.L., Hinton, T.G., 2001. Resource allo- Millennium Ecosystem Assessment, 2005. Ecosystems and Human Well-being: Biodiver-
cation-based life histories: a conceptual basis for studies of ecological toxicology. En- sity Synthesis. World Resources Institute, Washington, DC.
viron. Toxicol. Chem. 20, 1698–1703. Moldovan, O.T., Meleg, I.N., Levei, E., Terente, M., 2013. A simple method for assessing bi-
Coniglio, L., Baudo, R., 1989. Life-tables of Daphnia obtusa (Kutz.) surviving exposure to otic indicators and predicting biodiversity in the hyporheic zone of a river polluted
toxic concentrations of chromium. Hydrobiologia 188 (189), 407–410. with metals. Ecol. Indic. 24, 412–420.
Dao, T.S., Do-Hong, L.C., Wiegand, C., 2010. Chronic effects of cyanobacterial toxins on Munzinger, A., 1994. The influence of nickel on population dynamics and on some demo-
Daphnia magna and their offspring. Toxicon 55, 1244–1254. graphic parameters of Daphnia magna. Hydrobiologia 277, 107–120.
Dao, T.S., Le, V.N., Nguyen, T.S., Bui, B.T., Do-Hong, L.C., Hoang, T., 2015. Acute and chronic Muyssen, B.T.A., Janssen, C.R., 2001. Multigeneration zinc acclimation and tolerance in
effects of nickel on Daphnia lumholtzi. J. Sci. Technol. 53 (3 A), 271–276. Daphnia magna: implications for water-quality guidelines and ecological risk assess-
Dave, G., 1984. Effects of copper on growth, reproduction, survival and haemoglobin in ment. Environ. Toxicol. Chem. 20 (9), 2053–2060.
Daphnia magna. Comp. Biochem. Physiol. C: Comp. Pharmacol. 78C (2), 439–443. Muyssen, B.T.A., De Schamphelaere, K.A.C., Janssen, C.R., 2006. Mechanisms of chronic wa-
De Schamphelaere, K.A.C., Janssen, C.R., 2002. A biotic ligand model predicting acute cop- terborne Zn toxicity in Daphnia magna. Aquat. Toxicol. 77, 393–401.
per toxicity for Daphnia magna: the effects of calcium, magnesium, sodium, potassi- Naddy, R.B., Cohen, A.S., Stubblefield, W.A., 2015. The interactive toxicity of cadmium,
um and pH. Environ. Sci. Technol. 36, 48–54. copper, and zinc to Ceriodaphnia dubia and rainbow trout (Oncorhynchus mykiss). En-
De Schamphelaere, K.A.C., Janssen, C.R., 2004. Effects of chronic dietary copper exposure viron. Toxicol. Chem. 34, 809–815.
on growth and reproduction of Daphnia magna. Environ. Toxicol. Chem. 23, Onojake, M.C., Sikoki, F.D., Omokheyeke, O., Akpiri, R.U., 2015. Surface water characteris-
2038–2047. tics and trace metals level of the Bonny/New Calabar River estuary, Niger Delta, Nige-
De Schamphelaere, K.A.C., Canli, M., Van Lierde, V., Forrez, I., Vanhaecke, F., Janssen, C.R., ria. Appl Water Sci 1–9. http://dx.doi.org/10.1007/s13201-015-0306-y.
2004. Reproductive toxicity of dietary zinc to Daphnia magna. Aquat. Toxicol. 70, Pane, E.F., McGeer, J.C., Wood, C.M., 2004. Effect of chronic waterborne nickel exposure on
233–244. two successive generations of Daphnia magna. Environ. Toxicol. Chem. 23,
De Schamphelaere, K.A.C., Forrez, I., Dierckens, K., Sorgeloos, P., Janssen, C.R., 2007. Chron- 1051–1056.
ic toxicity of dietary copper to Daphnia magna. Aquat. Toxicol. 81, 409–418. Pane, E.F., Smith, C., McGeer, J.C., Wood, C.M., 2003. Mechanisms of acute and chronic wa-
Di Toro, D.M., Allen, H.E., Bergman, H.L., Meyer, J.S., Paquin, P.R., 2001. Biotic ligand model terborne nickel toxicity in the freshwater cladoceran, Daphnia magna. Environ. Sci.
of the acute toxicity of metals. Environ. Toxicol. Chem. 20, 2383–2396. Technol. 37, 4382–4389.
Dinh Van, K., Janssen, L., Debecker, S., De Jonge, M., Lambret, P., Nilsson-Ortman, V., Paulauskis, J.D., Winner, R.W., 1988. Effects of water hardness and humic acid on zinc tox-
Beroets, L., Stocks, R., 2013. Susceptibility of a metal under global warming is icity to Daphnia magna Straus. Aquat. Toxicol. 12, 273–290.
shaped by thermal adaptation along a latitudinal gradient. Glob. Chang. Biol. 19, QCVN-38, 2011. National technical regulation on surface water quality for protection of
2625–2633. aquatic lifes (in Vietnamese). BTNMT 7 pp.
Dinh Van, K., Janssen, L., Debecker, S., Stocks, R., 2014. Temperature- and latitude-specific Quyen, P.B., Nhan, D.D., San, N.V., 1995. Environmental pollution in Vietnam: analytical
individual growth rates shape the vulnerability of damselfly larvae to a widespread estimation and environmental priorities. Trends Anal. Chem. 14, 383–388.
pesticide. J. Appl. Ecol. 51, 919–928. Regaldo, L., Reno, U., Gervasio, S., Troiani, H., Gagneten, A.M., 2014. Effects of metals on
Ebert, D., 1992. A food-independent maturation threshold and size at maturity in Daphnia Daphnia magna and cladocerans representatives of the Argentinean fluvial littorale.
magna. Limnol. Oceanogr. 37, 878–881. J. Exp. Biol. 35, 689–697.
Ebert, D., 2005. Ecology, Epidemiology, and Evolution of Parasitism in Daphnia [Internet]. Relyea, R.A., 2012. New effects of roundup on amphibians: predators reduce herbicide
National Library of Medicine (US), National Center for Biotechnology Information, Be- mortality; herbicides induce antipredator morphology. Ecol. Appl. 22, 634–647.
thesda (MD) Available from http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db= Relyea, R.A., Diecks, N., 2012. An unforeseen chain of events: lethal effects of pesticides on
Books. frogs at sublethal concentrations. Ecol. Appl. 18, 1728–1742.
Ghazy, M.M., Habashy, M.M., 2003. Experimental toxicity of chromium to two freshwater Rodriguez, P.H., Arbildua, J.J., 2012. Copper acute and chronic toxicity to D. magna: sensi-
crustaceans, Daphnia magna and Macrobrachium rosenbergii. Egyptian Journal of tivity at three different hardness at pH 6.3 (MES buffered) in the presence of 2 mg/L
Aquatic Biology and Fisheries 7 (3), 49–70. DOC. Report Submitted to the International Copper Association (ICA) http://hdl.
Ghose, S.L., Donnelly, M.A., Kerby, J., Whitfield, S.M., 2014. Acute toxicity tests and meta- handle.net/1854/LU-5902885.
analysis identify gaps in tropical ecotoxicology for amphibians. Environ. Toxicol. Ryan, A.C., Tomasso, J.R., Klaine, S.J., 2009. Influence of pH, hardness, dissolved organic
Chem. 33 (9), 2114–2119. carbon concentration and dissolved organic matter source on the acute toxicity of
Gostomski, F., 1990. The toxicity of aluminum to aquatic species in the US. Environ. copper to Daphnia magna in soft waters: implications for the biotic ligand model. En-
Geochem. Health 12, 51–54. viron. Toxicol. Chem. 28, 1663–1670.
Grosell, M., Nielsen, C., Bianchini, 2002. Sodium turnover rate determines sensitivity to Schwarzenbach, R.P., Egli, T., Hofstetter, T.B., von Gunten, U., Wehrli, B., 2010. Global
acute copper and silver exposure in freshwater animals. Comp. Biochem. Physiol., water pollution and human health. Annu. Rev. Environ. Resour. 35, 109–136.
Part C: Toxicol. Pharmacol. 133, 287–303. Shaw, J.R., Dempsey, T.D., Chen, C.Y., Hamilton, W., Folt, C., 2006. Comparative toxicity of
Hoang, T.C., Tomasso, J.R., Klaine, S.J., 2004. Influence of water quality and age on nickel cadmium, zinc, and mixtures of cadmium and zinc to daphnids. Environ. Toxicol.
toxicity to fathead minnows (Pimephales promelas). Environ. Toxicol. Chem. 23, Chem. 25 (1), 182–189.
86–92. Shaw, J.R., Glaholt, S.P., Greenberg, N.S., Sierra-Alvarez, R., Folt, C., 2007. Acute toxicity of
Hoang, T.C., Gallagher, J.S., Klaine, S.J., 2007. Responses of Daphnia magna to pulsed expo- arsenic to Daphnia pulex: influence of organic functional groups and oxidation state.
sures of arsenic. Environ. Toxicol. 22, 308–317. Environ. Toxicol. Chem. 26, 1532–1537.
Ikemoto, T., Tu, N.P.C., Okuda, N., Iwata, A., Omori, K., Tanabe, S., Tuyen, B.C., Takeuchi, I., Sibly, R.M., Calow, P., 1989. A life-cycle theory of responses to stress. Biol. J. Linn. Soc. 37,
2008. Biomagnification of trace elements in the aquatic food web in the Mekong 101–116.
Delta, South Vietnam using stable carbon and nitrogen isotope analysis. Arch. Envi- Sournia, A., 1978. Phytoplankton Manual. UNESCO, UK, p. 77.
ron. Contam. Toxicol. 54, 504–515. Taylor, N.S., Kirwan, J.A., Johnson, C., Yan, N.D., Viant, M.R., Gunn, J.M., McGeer, J.C., 2016.
Janssens, L., Dinh Van, K., Debecker, S., Bervoets, L., Stoks, R., 2014. Local adaptation and Predicting chronic copper and nickel reproductive toxicity to Daphnia pulex-pulicaria
the potential effects of a contaminant on predator avoidance and antipredator re- from whole animal metabolic profiles. Environ. Pollut. 212, 325–329.
sponses under global warming: a space-for-time substitution approach. Evol. Appl. Tomasik, P., Warren, D.M., 1996. The use of Daphnia in studies of metal pollution of aquat-
7, 421–430. ic system. Environ. Rev. 4, 25–64.

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 11

Traudt, E.M., Ranville, J.F., Smith, S.A., Meyer, J.S., 2016. A test of the additivity of acute tox- daphnids in a range of natural waters in Chile. Environ. Toxicol. Chem. 24 (5),
icity of binary-metal mixtures of Ni with Cd, Cu and Zn to Daphnia magna, using the 1287–1299.
inflection point of the concentration-response curves. Environ. Toxicol. Chem. http:// Walker, C.H., Hopkin, S.P., Sibly, R.M., Peakall, D.B., 1996. Principles of Ecotoxicology. CRC
dx.doi.org/10.1002/ect.3342. Press, Taylor & Francis Group, U.S. 315 pp.
Tsui, M.T.K., Wang, W.X., 2007. Biokinetics and tolerance development of toxic metals in Waykar, B., Shinde, S.M., 2011. Assessment of the metal bioaccumulation in three species
Daphnia magna. Environ. Toxicol. Chem. 26 (5), 1023–1032. of freshwater bivalves. Bull. Environ. Contam. Toxicol. 87, 267–271.
US Environmental Protection Agency (US EPA), 1996. EPA Method 3630 “silica gel clean- Weis, J.S., Cristini, A., Rao, K.R., 1992. Effects of pollutants on molting and regeneration in
up” 15p. crustacea. Am. Zool. 32, 495–500.
US Environmental Protection Agency (US EPA), 2002. Methods for measuring the acute Wetzel, R.G., 2001. Limnology – Lake and River Ecosystems. Academic Press,
toxicity of effluents and receiving waters to freshwater and marine organisms. EPA- pp. 305–310.
821-R02-012, fifth ed. Office of Water, Washington, DC. Wilbers, G.J., Becker, M., Nga, L.T., Sebesvari, Z., Renaud, F.G., 2014. Spatial and temporal
US Environmental Protection Agency (US EPA), 2008. Method 8141B - Organophospho- variability of surface water pollution in the Mekong Delta, Vietnam. Sci. Total Environ.
rus Compounds by Gas Chromatography. 485, 653–665.
Vardia, H.K., Rao, P.S., Durve, V.S., 1988. Effects of copper, cadmium and zinc on fish-food Winner, R.W., Farrell, M.P., 1976. Acute and chronic toxicity of copper to four species of
organisms, Daphnia lumholtzi and Cypris subglobosa. Proc. Indian Acad. Sci. (Anim. Daphnia. J. Fish. Res. Board Can. 33, 1685–1691.
Sci.) 97 (2), 175–180. Wong, C.K., 1992. Effects of chromium, copper, nickel, and zinc on survival and feeding of
Villavicencio, G., Urrestarazu, P., Carvajal, C., De Schamphelaere, K.A.C., Janssen, C., Torres, the Cladocera Moina macrocopa. Bull. Environ. Contam. Toxicol. 49, 593–599.
J.C., Rodriguez, P.H., 2005. Biotic ligand model prediction of copper toxicity to

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