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Trang 1Sensitivity of a tropical micro-crustacean (Daphnia lumholtzi) to trace
metals tested in natural water of the Mekong River
Thanh-Son Daoa,⁎ , Vu-Nam Leb, Ba-Trung Buic, Khuong V Dinhd,e, Claudia Wiegandf, Thanh-Son Nguyenc,
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
• 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
G R A P H I C A L A B S T R A C T
a b s t r a c t
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
Metal contamination is one of the major issues to the environment worldwide, yet it is poorly known how expo-sure 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
4μg L−1), Zn (50 and 56μg L−1), and Ni (six concentrations from 5 to 302μg L−1) for 21 days The concentrations
of 4μg Cu L−1and 6μg Ni L−1enhanced the body length of D lumholtzi but 46μg Ni L−1and 50μg Zn L−1resulted
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
Keywords:
Acute toxicity
Life history traits
Mekong River water
Sensitivity
Trace metals
Science of the Total Environment xxx (2016) xxx–xxx
⁎ Corresponding author.
E-mail address: dao.son@hcmut.edu.vn (T.-S Dao).
STOTEN-20661; No of Pages 11
http://dx.doi.org/10.1016/j.scitotenv.2016.08.049
0048-9697/© 2016 Elsevier B.V All rights reserved.
Contents lists available atScienceDirect
Science of the Total Environment
j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / s c i t o t e n v
Trang 2important 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
ag-ricultural activities have increased the metal concentrations in the
envi-ronment 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
in-destructible 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 andfishes,
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
micro-crustacean andfish 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
pulex-pulicaria 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
tem-perate regions (reviewed byGrosell et al., 2002; Tsui and Wang, 2007)
For examples, exposure to metals e.g., Cu, Ni, Zn, Cr, or Ag caused
im-pairments of life history traits such as growth rate, maturity age,
life-span, 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
knowl-edge of how tropical species deal with contaminants (Ghose et al.,
2014) Few studies have investigated the responses of tropical
zoo-plankton 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
dis-solved metals infield 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,
urbaniza-tion, transportation of goods, agriculture (Wilbers et al., 2014),
aquacul-ture (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
rel-atively low (b1.6 μg L−1;Ikemoto et al., 2008), a high level of
anthropo-genic 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
fresh-water 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 seeDao 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 re-gions such as the Mekong River For example, the Vietnamese regula-tions 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 tem-perate 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 hard-ness 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 spe-cies, especially D magna enabled comparisons and recommendations for ecological risk assessment programs in tropical countries like Viet-nam 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-relat-ed traits in D lumholtzi such as survival, growth rate, maturation and fe-cundity 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 ex-periments 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 Thefiltered 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) ac-cording toAPHA (2005) Total hardness was determined based on con-centrations 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)
Trang 32.1.2.1 Analysis of metals The pooledfiltered waters (from 10
sub-sam-ples per sampling site,Relyea and Diecks, 2012; Relyea, 2012; Dinh Van
et al., 2013) for metal characterization were acidified with concentrated
HNO3(Merck) to pHb 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
param-eters for metal analysis in samples are presented in the Supplementary
1 A multi-element tuning solution was used to check accuracy of
mea-surement (relative standard deviation, RSDb 5%, Agilent Technologies)
The calibration curve was prepared using single stock solutions for each
metal The concentrations of metals in mixed working standard
solu-tions were prepared based on their estimated concentrasolu-tions in water
samples from preliminary semi-quantitative analysis The weighted
cal-ibration curves for each element with R2N 0.999 were accepted for
con-centration calculation All samples and working standard solutions for
calibration were spiked with 10μg Scandium L−1as 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
sub-samplings from each storage tanks) for organochlorine pesticides
(OCPs) and organophosphate pesticides (OPPs) characterization were
taken and kept in dark glass bottles on ice in thefield until analysis in
the laboratory Water samples werefiltered (Sartorius, Germany) to
re-move 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
hex-ane (15/85, v/v; Merck & Labscan Inc.) The mixture was shaken for
15 min, followed by phase separation The organic phase was
trans-ferred 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
ex-traction (SPE) column (Silica Gel 100/200 mesh) (US EPA, 1996-
Meth-od 3630) The column was eluted with 40 mL of hexane and 30 mL of
DCM with theflow 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 mmfilm thickness) The
recov-eries 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 0.01μg L−1
and 0.1μg L−1, respectively OCPs standard mixture includes 13 com-pounds: 2,4,5,6 Tetrachloro-m-xylene,α-HCH, α-Chlordane, 4,4′-DDE, β-Endosulfan, Delta-HCH, Aldrin, Heptachlor epoxide, δ-Chlordane, En-drin aldehyde, Endosulfan sulfate, EnEn-drin 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 ana-lyzed 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−1Cu, 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 fil-tered 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 pa-rameters were favorable for D lumholtzi
2.2.2 Test organisms The tropical daphnid D lumholtzi was collected from Bac Ninh Prov-ince, Vietnam (Bui et al., 2016) and has been maintained in the Labora-tory 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 accord-ing 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 fol-lowing 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
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).
3 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx
Trang 4tropical species Neonates of D lumholtzi (age≤ 24 h) were used for
test-ing 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
ex-posure (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
ter-minated, 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
tox-icity test Based on the 48 h-LC50values 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
4μg Cu L−1, 50 and 56μg Zn L−1, and from 5 to 302μg 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 4μg Cu L−1;
57μg Zn L−1, Dao et al., manuscript in preparation; 151 Niμg L−1;Bui
et al., 2016) Chronic tests were performed according to theAPHA
(2005)andDao et al (2010)with minor modifications (see 2.2.3)
Brief-ly, neonates (15 individuals per treatment) of D lumholtzib24 h-age
from 2nd to 3rd clutch were individually and independently incubated
for each treatment in 50-mL polypropylene cups containing 20 mL
con-trol 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,
approxi-mately 140,000 cells mL−1) and YTC (~20μL) just after the exposure
so-lutions 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
Re-production 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
moth-er daphnid The chronic tests lasted for 21 days At test tmoth-ermination,
liv-ing mother daphnids were immediately fixed with Lugol solution
(Sournia, 1978) and body length was measured to the nearest 1μm,
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
USA) Kruskal-Wallis test (Sigma Plot, version 12) was applied for calcu-lation 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 solu-tions made from waters collected at each sampling site
3 Results and discussion 3.1 Physical and chemical characteristics offield water from Mekong River All analyzed organic pesticides infiltered 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, Beta-Endosulfan, Delta-HCH, Aldrin, Heptachlor epoxide, Gamma-Chlordane, Endrin aldehyde, Endosulfan sulfate, Endrin ketone, Decachlordiphenyl, Diazinon, Ethion, Malathion, Pazathion and Trithion Overall, concentra-tions of trace metals infiltered water from both sampling sites of the Mekong River were very low They ranged from 2 to 5μg L−1of Al, 1
to 3μg L−1of As, 25 to 30μg L−1of Ba, 2 to 5μg L−1of Fe and 3 to
4μg L−1of 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,
1μg L−1(Table 2) The concentrations of trace metals and pesticides
infiltered 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 (3μg L−1) was ca 1000 times lower than the lowest concentration inducing acute negative effects
on other daphnid species e.g D magna (3000μg 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
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 BDL a , 0.1 μg L −1 for BDL b , and 0.01
μg L −1 for BDL c
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 5 2 Tetrachloro-m-xylene BDL b BDL b
BDL b
Ba 25 30 Alpha-Chlordane BDL b
BDL b
BDL b
Zn 4 3 Beta-Endosulfan BDL b
BDL b
Delta-HCH BDL b
BDL b
Co BDL a BDL a Aldrin BDL b BDL b
Cr BDL a BDL a Heptachlor epoxide BDL b BDL b
Mn BDL a
BDL a
Gamma-Chlordane BDL b
BDL b
Ni BDL a
BDL a
Endrin aldehyde BDL b
BDL b
Se BDL a
BDL a
Endosulfan sulfate BDL b
BDL b
Mo BDL a
BDL a
Endrin ketone BDL b
BDL b
Ag BDL a
BDL a
Decachlordiphenyl BDL b
BDL b
Cd BDL a BDL a Diazinon BDL c BDL c
Pb BDL a BDL a Ethion BDL c BDL c
Malathion BDL c
BDL c
Pazathion BDL c
BDL c
Trithion BDL c
BDL c
Trang 52007) and D pulex (2500–3900 μg L−1;Shaw et al., 2007) Similarly,
dis-solved Zn (4μg L−1) and aluminum (5μg L−1) in the test water were
considerably lower 48 h-LC50 values (61.8–130 μg Zn L−1; 742–
1900μg 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 CaCO3L−1, respectively (Table 3) Both pH and DO in the
test water were within the favorable range for the growth and
develop-ment 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
concentra-tions 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 CaCO3L−1) and hardness (79–87 mg
CaCO3L−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
con-centrations, 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-LC50values for D lumholtzi incubated in Mekong River
water ranged from 11.57 to 16.67μg L−1of Cu, 179.3 to 280.9μg L−1
of Zn and 1026 to 1516μg L−1of Ni (Table 4, Supplementary 2) The
48 h-LC50values were lower in the test with river water from site 2
than that from site 1 probably associated with the lower DOC
concentra-tion 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 CuN Zn N Ni (Table 4) which is in line with previous
in-vestigations (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–
8.61μg L−1, and 5.77–7.23 μg L−1in two tropical micro-crustaceans,
D lumholtzi and Ceriodaphnia cornuta, respectively, exposed to Cu
spiked into Mekong River water These 48 h-LC50values are two times
lower than those from our study (Table 4) It seems that the higher
alka-linity and hardness in the water used in the current study contributed to
the lower toxicity of Cu compared toBui 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
hard-ness 180 and 200 mg CaCO3mg L−1), the 48 h-LC50value of 54.6μg 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 ofVardia et al (1988), may have contributed to
the lower sensitivity.Chishty et al (2012)used several daphnid species
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 CaCO3L−1, respectively) In their studies, the 48 h-LC50was 2300μg 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-LC50of D magna, D obtusa, and D pulex ranged from 60.3 to 156.1, 41.1 to 100.1 and 19.5 to 26μg 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-EC50of 16.5μg Cu L−1, under the test conditions of 2 mL−1 of DOC, pH of 6.3 and hardness of
169 mg L−1as CaCO3 Though the same authors reported similar 48
h-LC50/EC50value 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 temper-ate Daphnia species, D magna, D obtusa, and D pulex
In COMBO medium (0.67 mg L−1of DOC, hardness and alkalinity of
44 and 10 mg L−1as CaCO3, respectively) the 48 h-LC50of 1775μg Ni
L−1for D lumholtzi (Dao et al., 2015) was a little higher than the
48 h-LC50values of the current study (1026–1516 μg Ni L−1;Table 4)
Pane et al (2003)reported a 48 h-LC50of 1068μg Ni L−1for D magna
in (soft) tap water, pH of 7.3–7.6 and total organic carbon (TOC) of 3.6 mg L−1which was in range with the 48 h-LC50from our study In moderately hard water and 3 mg L−1DOC, a 48 h-LC50of 1633μg Ni
L−1was 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-LC50of D lumholtzi of 2290μg
Zn L−1, which is far higher than the 48 h-LC50value 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-LC50values for Zn of our study (179–
280μg Zn L−1, in moderately hard water,Table 4) to those of D magna (928μg Zn L−1in moderately hard water) and C dubia (102–
130μg Zn L−1in 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 200μg L−1is used as the safety level for protec-tion of aquatic life (QCVN-38, 2011), but this Cu concentration is even
13 times higher than the 48-LC50value 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 eco-system protection To our knowledge this is thefirst report on the acute test of Ni and Zn spiked intofield water to D lumholtzi, which together with previous results ofBui 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 thefitness (health, growth and reproduction) in an-imals However, increasing metal concentrations at some point impair physiological functions, reducefitness or even become lethal to organ-isms (Pane et al., 2003) Several nonexclusive mechanisms may under-lie the metal-induced reduction of thefitness-related traits in exposed aquatic animals such as growth, age to maturation and fecundity: the
Table 3
Physical and chemical characteristics of the field water from Mekong River and the
expo-sure solutions during the experiments.
Parameters Site 1 – Vinh Loc Site 2 – Tan Chau
pH (in the field water) 7.8 7.8
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
Dissolved organic carbon (mg L −1 ) 2.99 1.89
Hardness (mg CaCO 3 L−1) 79 87
Alkalinity of the field water (mg CaCO 3 L−1) 68 68
Alkalinity of the test water (mg CaCO 3 L−1) 64–68 64–68
5 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx
Trang 6impairment 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
anti-oxidant mechanisms (Amiard et al., 2006; Dinh Van et al., 2013)
There-fore, the maintenance cost is increased Furthermore, exposure to
metals may also reduce the foraging activity, hence lowering energy
in-take (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 3μg L−1and 4μg L−1(Fig 2a, b) The concentration of 56μg
Zn L−1in river water from site 1 resulted in 16% mortality of daphnids
whereas 50μg Zn L−1in 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 225μg Ni L−1for Ni dissolved in water from site 1 and 2,
respective-ly;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 CaCO3L−1; hardness of 130–160 mg CaCO3L−1; and pH of
8.2–9.5) survival of the four Daphnia species slightly decreased at
20μg 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 4μg Cu L−1in our study In a 15-day test,N50% of D
magna and 80% of Moinadaphnia macleayi survived exposure to 25 and
40μg Cu L−1(in artificial medium, pH of 7.6–7.7; hardness of 160–
180 mg L−1as 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 trop-ical 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−1did not significantly decrease D magna survivorship while survival of D lumholtzi in our study was al-ready decreased 46% at 50μg Zn L−1at 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 ofMunzinger (1994), reporting reduced survival of chronically exposed D magna to
Ni concentrations of 40–200 μg L−1in natural water Similarly, D magna exposed to 85μg Ni L−1decreased 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 750μg L−1for 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
Table 4
The values of 48 h median lethal concentrations (48 h-LC 50 ) of Cu, Zn and Ni for daphnid species (without *); *, values of 48 h-EC 50 (immobilization); **, values of 72 h-LC 50
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 ) 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
Trang 7with 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 4μg Cu L−1dissolved in water from site 2) only
extended the time to maturation by ca 1 day, but not at the
concentra-tion of 3μg Cu L−1dissolved in water collected from site 1 (Fig 3b)
Ex-posure 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 2 Survival of Daphnia lumholtzi exposed to metals spiked intofiltered 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.
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).
7 T.-S Dao et al / Science of the Total Environment xxx (2016) xxx–xxx
Trang 8D lumholtzi in natural waters is around 4μg Cu L−1but this needs
fur-ther verification
More obviously, exposure to Zn (50 and 56μg L−1) and the highest
Ni concentrations (225 and 302μg L−1) resulted in a significant
post-ponement of the daphnids' maturation (Fig 3a, b) Similarly,Dao et al
(2015)reported the maturity age of D lumholtzi raised in COMBO
medi-um increased at higher Ni concentrations (500 and 750μg 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
unfa-vorable elements interfering with Ni-toxicity for D lumholtzi, which
re-quires further investigation However, daphnids in the incubations of 6
and 59μg Ni L−1in river water from site 1 (Fig 3a) delayed their
mat-uration whereas those of 5 and 46μg Ni L−1in 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
re-tarded maturity
3.3.3 Effect on growth
At the day 21 (the end) of the exposure duration, the average body
length of daphnids was 2304μm (Fig 4a) and 2265μm in the two
con-trol treatments (Fig 4b), without significant difference (P = 0.066,
Kruskal-Wallis test) Exposure to Cu (3 or 4μg L−1) and the lowest Ni
concentrations (5 or 6μg L−1) resulted in an increase in body length
(Fig 4a, b) indicating that the concentrations of these essential metals
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 concentra-tions of essential metals may stimulate the growth rate in Daphnia spe-cies (De Schamphelaere and Janssen, 2004) For example,Dave (1984)
showed that D magna increased the body length when exposed to 0.026μg Cu L−1for 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 re-duces both growth rate and body length (Ghazy and Habashy, 2003)
as these metals then become toxic Indeed, in exposures to Zn at con-centrations of 50 and 56μg L−1and Ni at concentrations of 46 and
59μg L−1the 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 800μg 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 sup-ported by the study ofPane and McGeer (2004)showing a strong de-crease of D magna wet weight after 21 days exposure to 85μg 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 302μg Ni
L−1no 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 ofb12 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 byAcharya 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 3μg Cu L−1in 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 4μg Cu L−1in 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 4μg L−1 Exposure to Zn at the concentration of 50 and
56μg L−1and Ni at high concentrations (302μg L−1and 225μg L−1) 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, rere-spectively 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 59μg L−1but
in the river water from site 2, Ni at the concentrations of 5 and
46μg L−1even stimulated daphnids reproduction (17.7 and 17.6 neo-nates 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 59μg L−1), the ac-cumulative neonates were from 2277 to 2796, which were in range with
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).
Trang 9those from the controls, from 2375 to 2793 However, the higher Ni
con-centrations, 225 and 302μg L−1strongly reduced the accumulative
off-spring 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−1of DOC, pH of 6.8, and Cu at concentrations of 35–
100μg L−1was 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 3μg Cu L−1enhanced brood
size (Fig 5a), whereas already at 4μg 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 46μg Ni L−1in which the exposed daphnids
increased brood size but decreased total newborns (Fig 5b;Table 5)
Similarly,Coniglio and Baudo (1989)observed afluctuation 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 1035μg L−1but not at the
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 302μg 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 56μg Zn L−1(Fig 5;Table 5) Daphnia magna in treated with 80–
170μg Zn L−1in the tap water containing 2–3 mg L−1of 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
CuN 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 con-centration 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 Me-kong River water in the Ni exposures At chronic exposure, some unde-termined 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 re-sults underscore the importance of including tropical species, e.g D lumholtzi, in ecological risk assessment in tropical regions such as Viet-nam to arrive at a better conservation and management plan to protect freshwater biodiversity from metal contaminants To the best of our knowledge, this is thefirst 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-crusta-ceans, e.g D lumholtzi
Supplementary data to this article can be found online athttp://dx doi.org/10.1016/j.scitotenv.2016.08.049
Acknowledgement
We would like to thank Prof Tham Hoang from Loyola University Chicago for his assistance on the calculation of median lethal concentra-tion (48 h-LC50) This study was funded by the Vietnam National Uni-versity– Hochiminh City under the granted project number B2014-48-01
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