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

STOTEN-20661; No of Pages 11
Science of the Total Environment xxx (2016) xxx–xxx

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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
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 2 August 2016
Accepted 6 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
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
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
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: (T.-S. Dao).

/>0048-9697/© 2016 Elsevier B.V. All rights reserved.

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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
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 do 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.
2. Materials and methods
2.1. Test solutions
2.1.1. Water samples collection
Surface water was collected at 2 sampling sites in Mekong River: site
1 at Vinh Loc ferry-port, An Phu district and site 2 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 4 °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).

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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-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 2 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
1. 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 3 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 5 mL min−1. SPE extracts were concentrated

by rotary evaporation and with a gentle stream of nitrogen and
redissolved into 1 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 5 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− 1 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 ± 1 °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 ± 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
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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 3 and
4 μg Cu L− 1, 50 and 56 μg Zn L−1, and from 5 to 302 μg Ni L− 1
(Table 1). Also, the chosen concentrations of Cu, Zn and Ni have been
found in natural water of the lower Mekong River (e.g. 4 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 (~ 1 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 1 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 2
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
μg L−1 for BDLc.
Dissolved
metals
(μg L−1)

Site 1 –
Vinh Loc

Site 2 –
Tan Chau

Pesticides (μg L−1)

Site 1 –
Vinh Loc

Site 2 –
Tan Chau

Al
As
Ba
Fe
Zn
Cu
Co
Cr
Mn
Ni
Se
Mo
Ag
Cd
Pb

5
1
25
5
4
1
BDLa
BDLa
BDLa
BDLa
BDLa
BDLa
BDLa
BDLa
BDLa

2
3
30
2
3
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.
3. 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 2 to 5 μg L−1 of Al, 1
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
4 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,
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
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 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)
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)

3
56
6, 59, 302

4
50
5, 46, 225

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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 2
than that from site 1 probably associated with the lower DOC concentration in water at site 2 (1.89 mg L−1) compared to site 1 (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 3
Physical and chemical characteristics of the field water from Mekong River and the exposure solutions during the experiments.
Parameters

Site 1 – Vinh Loc Site 2 – 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

5

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 2 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 3 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

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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
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 3 μg L−1 and 4 μg L−1 (Fig. 2a, b). The concentration of 56 μg
Zn L−1 in river water from site 1 resulted in 16% mortality of daphnids
whereas 50 μg Zn L−1 in river water from site 2 caused 54% mortality
(Fig. 2c, d). Ni in water from sites 1 and 2 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 1 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 3 weeks of incubation (Winner and Farrell,

1976), whereas D. lumholtzi suffered already 20% mortality during
21 days at 3 to 4 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 4 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
5 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− 1 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

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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 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 7 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 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 concentration of 3 μg Cu L−1 dissolved in water collected from site 1 (Fig. 3b). Exposure to 1.8 μg Cu L− 1 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. 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
River. (a) field water collected at site 1 – Vinh Loc; (b), field water collected at site 2 –
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).

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D. lumholtzi in natural waters is around 4 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 6
and 59 μg Ni L−1 in river water from site 1 (Fig. 3a) delayed their maturation whereas those of 5 and 46 μg Ni L−1 in river water from site 2

(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 2 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 4 μg L−1) and the lowest Ni
concentrations (5 or 6 μg L−1) resulted in an increase in body length
(Fig. 4a, b) indicating that the concentrations of these essential metals

Fig. 4. 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 1 – Vinh Loc; (b), field water collected at site 2 –
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− 1 for 7 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− 1 and Ni at concentrations of 46 and
59 μg L− 1 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 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) 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 2 °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 3 μg Cu L−1 in river water from site 1 (19.8 neonates per clutch; Fig. 5a, and 3058 offspring in total, Table 5) and reduced 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) 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 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 neonates per clutch, respectively) (Fig. 5b).
The Zn exposures decreased the total neonates of daphnids, to 746–
2081. Exposures to low Ni concentrations, from 5 to 59 μg L−1), the accumulative neonates were from 2277 to 2796, which were in range with

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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 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.
4. Conclusions

Fig. 5. 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 1 – Vinh Loc; (b), field water collected at site 2 – 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 3 μg Cu L−1 enhanced brood
size (Fig. 5a), whereas already at 4 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 5 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 5
Total accumulated offspring Daphnia lumholtzi in the exposures during 21 days of
experiment.
Sampling sites

Metal concentrations (μg L−1)

Site 1 – Vinh Loc

Accumulative
offspring

Control Cu = 3 Zn = 56 Ni = 6 Ni = 59 Ni = 302
2793
3058
2081
2796
2619
264

Site 2 – Tan Chau
Accumulative
offspring

Control Cu = 4 Zn = 50 Ni = 5 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.
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