Effects of copper on the survival, hatching, and reproduction of a pulmonate snail (Physa acuta)

Effects of copper on the survival, hatching, and reproduction of a pulmonate snail (Physa acuta)

Chemosphere 185 (2017) 1208e1216 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Effect...

1MB Sizes 8 Downloads 69 Views

Chemosphere 185 (2017) 1208e1216

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Effects of copper on the survival, hatching, and reproduction of a pulmonate snail (Physa acuta) Lei Gao a, b, *, Hai Doan b, Bhanu Nidumolu b, Anupama Kumar b, Debra Gonzago b a b

School of Geography and Planning, Sun Yat-Sen University, Guangzhou, 510275, China Commonwealth Scientific Industrial Research Organisation (CSIRO) Land and Water, PMB 2, Glen Osmond, S.A., 5064, Australia

h i g h l i g h t s  Physa acuta is sensitive to copper (Cu) exposure with a 96 h LC50 of 23.8 mg L1.  Cu exposure causes delay in embryonic development, deformity and lesion of embryos.  Snails exposed at 12.5 and 25 mg L1 Cu produce polynuclear eggs in one egg capsule.  Cu exhibits potential genetic toxicity effect from multigenerational exposure.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 May 2017 Received in revised form 13 July 2017 Accepted 18 July 2017 Available online 24 July 2017

Acute and chronic bioassays provide essential basis for establishment of environmental quality standards. The effects of Cu on a pulmonate snail, Physa acuta, were investigated at a number of sublethal and lethal endpoints. Cu exposure suppressed movement and triggered an escape response in P. acuta at low and high concentrations, respectively, exerting acute toxic effects on adult snails exposed to a 96 h LC50 of 23.8 mg L1. Following 16 d exposure of Cu to the egg masses, successful hatching decreased with increasing Cu concentration. High Cu concentrations (12.5 and 25 mg L1) resulted in inhibition of eye and shell development at the veliger stage, and a deformed shell, abnormal eyes, and different morphological shapes with lesions and hemorrhages were observed after 9 days of exposure. A large number of eggs exposed to 2.5e25 mg L1 Cu remained in the veliger and hippo stages for 2e7 days, with no further development. Results from reproduction tests showed that adult snails exposed to various Cu treatments produced more than three broods, with the total number of eggs ranging from 770 to 1,289, revealing little difference between the control and Cu-treated groups (p > 0.05). However, snails exposed to 12.5 and 25 mg L1 Cu produced polynuclear eggs in one egg capsule. The hatching success rate and shell length of the filial generation were significantly reduced in a dose-dependent manner (p < 0.05). The shell length of newly hatched snails was shorter in the reproduction test than in the hatching test, indicating inherent Cu toxicity in the filial generation from the exposed parent strain. The present study provides essential data regarding Cu toxicity in pulmonate snail P. acuta. © 2017 Elsevier Ltd. All rights reserved.

Handling Editor: Jim Lazorchak Keywords: Copper Physa acuta Egg masses Mortality Development Oviposition

1. Introduction Copper (Cu) is not only an important nutrient for human health but also an essential transition metal ion for organisms due to its vital function in mitochondrial enzyme activity (Uriu-Adams and Keen, 2005; Mehta et al., 2006). In natural water bodies with

* Corresponding author. School of Geography and Planning, Sun Yat-Sen University, Guangzhou, 510275, China. E-mail address: [email protected] (L. Gao). http://dx.doi.org/10.1016/j.chemosphere.2017.07.101 0045-6535/© 2017 Elsevier Ltd. All rights reserved.

minimal pollution, Cu concentrations are usually low, ranging only from about <0.1 to 4 mg L1 (Gaillardet et al., 2007; Wilbers et al., 2014; Mebane et al., 2015). Gaillardet et al. (2007) estimated a global average for large rivers of about 1.5 mg L1, excluding extensively urbanized or industrialized basins. Copper concentrations in some large rivers and estuaries in China may also be similarly low (Zhang, 1995). However, in water bodies affected by industrial or urban discharges, Cu concentrations may be much higher, reaching at least 90 mg L1 (Wong et al., 2007; Gao et al., 2015). In extremely polluted areas influenced by mine drainage, copper concentrations in streams may be much higher, reaching mg L1 levels, with an

L. Gao et al. / Chemosphere 185 (2017) 1208e1216

extreme value of 37,620 mg L1 being reported from the Odiel River, Spain (Olias et al., 2004; Mebane et al., 2015). Therefore, water pollution caused by Cu has attracted growing concerns due to its toxicity and ecological risk to aquatic organisms (Xia et al., 2012). Many studies on the toxic effects of Cu have been conducted on aquatic organisms, including crustaceans, mollusks, invertebrates, and vertebrates, revealing different levels of sensitivity to Cu among aquatic organisms (Flemming and Trevors, 1989; Hsieh et al., 2004; Kwok et al., 2008; Cooper et al., 2009). Freshwater snails, as a typical representative of gastropods, may experience more stress when exposed to accidental pollution in ambient water, because they are less mobile than many other aquatic organisms, especially vertebrates (e.g., fish), which can move fast to escape the pollution area. Furthermore, widespread concern has been raised regarding gastropods, because they can accumulate substantial levels of trace metals during growth as a result of their high Ca requirements, resulting in a higher metal concentration in soft tissues than in ambient water (Ray, 1984; Ng et al., 2011). In long-term community exposures to Cu, pulmonate snails such as Lymnaea spp. and Physa sp. were the most sensitive invertebrates (Joachim et al., 2017). However, non-pulmonate snails may be even more sensitive to copper than Physa sp. (Besser et al., 2016). Pyatt et al. (2003) found that the freshwater snail Lymnaea peregra accumulated marked levels of Cu from the aquatic environment, with low Cu concentrations of 4 mg L1. However, under such exposure conditions, Cu has been shown to cause mortality in the gastropods Pomacea paludosa and Lymnaea stagnalis (Rogevich et al., 2008; Brix et al., 2011). Physiological abnormalities were found after an extended exposure time; for example, Lymnaea luteola L. exposed to 56 mg L1 Cu for 7 weeks stopped feeding activity, leading to a significant decrease in reproductive capacity, and sublethal effects in terms of egg mass abnormalities were observed after exposure to 10 mg L1 Cu (Khangarot and Das, 2010; Das and Khangarot, 2011). Gastropod exposure to Cu assessed at different endpoints corresponding to various life stages has provided baseline data for assessing the toxic effects of Cu on ecosystem health. The pulmonate snail, Physa acuta, is regarded to be an early invader in Europe and is found in many regions of Asia, Australia, and South Africa (Appleton, 2003; Beckmann et al., 2006; Zalizniak et al., 2007a) due to its high adaptability to different environmental characteristics and a high reproductive capacity (Kefford and Nugegoda, 2005). Hence, a worldwide distribution of P. acuta could be achieved in the future, becoming a useful bioindicator of toxicants. In previous studies, P. acuta or its egg masses have been exposed to ionic solutions of different strengths (Kefford and Nugegoda, 2005; Zalizniak et al., 2007a, b), cadmium (Cd) (Piccinni et al., 1985; Cheung and Lam, 1998), organic compounds (Sanchez-Arguello et al., 2009; Ma et al., 2014), nanomaterials (Musee et al., 2010), and ionic liquids (Bernot et al., 2005; Li et al., 2014) in ambient water and the effects evaluated at various lethal, biochemical, developmental, and reproductive endpoints. However, little information regarding the toxic effects of Cu on movement, development, reproduction, and multigenerational exposure of P. acuta to Cu has been reported. To supplement essential data regarding Cu toxicity in gastropods, this study aimed to (1) assess the sensitivity of P. acuta to Cu at various endpoints and (2) to investigate the adverse impacts of multigenerational Cu exposure on populations of P. acuta. 2. Materials and methods 2.1. Snail culture The P. acuta specimens used in this study were collected from

1209

creeks in Adelaide Hills, Australia and were acclimated in an inhouse static culture. Approximately 150 adult snails were cultured in 15 L aquariums containing at least 10 L modified FETAX solution (MFS) (75 mg MgSO4$7H2O, 96 mg NaHCO3, 15 mg CaCl2$2H2O, 400 mg NaCl, 30 mg KCl, and 60 mg CaSO4 per liter deionized water), with sufficient aeration, a constant temperature of 21 ± 0.5  C, and a photoperiod regime of 16 h light: 8 h dark. Snails were fed algae wafers (Hikari, Hayward, CA, USA) once a week after renewal of the culture medium. 2.2. Acute exposure of adult snails to Cu Acute Cu exposure was evaluated over a 96 h period in adult snails, with a shell length of 11.6 ± 1.54 mm and dry weight of 65.8 ± 19.0 mg. The adult snails were fasted for 2 days before exposure to avoid Cu loss due to absorption by feces. The test was conducted under semi-static conditions without a food supply and aeration. The test solution was aerated and renewed on day 2 (48 h). Following a range-finding test, six concentrations of Cu (0, 5, 10, 20, 40, and 80 mg L1) were prepared from a stock solution (50 mg L1 Cu as CuSO4$5H2O) by diluting with the MFS to 600 mL in a precleaned glass jar (1 L) and then left for 6 h to equilibrate. MFS was used as a negative control. Five snails were randomly collected and placed into a sealed polyethylene cage that was submerged in the solution to prevent the snails from escaping as a result of Cu stress. The test solutions were measured before and after renewal for pH, electrical conductivity (EC), and dissolved oxygen (DO), and approximately 10 mL of the water sample were filtered through a 0.45 mm syringe filter and stored at 4  C prior to measurement of the Cu concentration by inductively coupled plasma-mass spectrometry (Agilent 7700, Santa Clara, CA, USA). All measurements were performed in quadruplicate at 25 ± 0.5  C. Survival was evaluated at 24, 48, 72, and 96 h by gently touching the snail with a soft probe. Death was defined as a lack of response to stimulation, and dead snails were immediately removed from the jar. 2.3. Embryo toxicity test Fifty adult snails reaching sexual maturity were collected from the aquarium cultures, and equal numbers were placed into 10 beakers (1 L) containing 900 mL MFS, a food supply, and sufficient aeration. Generally, snails produce egg masses (egg jelly) after acclimation for 1 or 2 days under “new” culture conditions. Then, 24h-old egg masses, typically containing 30e100 egg capsules, were gently collected from the back of shells or the container walls. Eighteen egg masses with similar size were collected and placed into 18 polyethylene cups containing approximately 30 mL of the testing solution, where they sank to the bottom. The embryos were exposed to a range of Cu concentrations (0, 1, 2.5, 5, 12.5 and 25 mg L1) in triplicate under conditions identical to those described for the acute test. The exposure period was 16 days, and the test solutions were renewed every 2 days. Water samples were collected before and after each renewal for measurements of pH, EC, DO, and Cu concentration. Observations were made at various developmental stages of the egg embryos (morula, trochophore, veliger, and hippo stages at days 2, 5, 7, 9, and 12e16) using an inverted microscope (Olympus SZX9, Tokyo, Japan). Normal or abnormal development of embryogenesis at different stages was recorded and photographed. Coagulation of embryos (e.g., a whitish cloud) with no capsule rotation was defined as death, and both normal and abnormal egg embryonic development was regarded as survival. During the exposure period, the numbers of hatched snails per treatment were counted. The shell length of hatched snails was also determined using the inverted microscope.

1210

L. Gao et al. / Chemosphere 185 (2017) 1208e1216

2.4. Reproduction test of adult snails Thirty snails that had reached sexual maturity were collected from the aquarium cultures and exposed to six Cu concentrations (0, 1, 2.5, 5, 12.5, and 25 mg L1) in five replications. One snail was placed into each of the 30 glass jars with 600 mL test solution under the same conditions as those used in the acute test. However, the reproduction test was conducted with aeration and a food supply throughout the entire exposure period. The test solution was renewed at 2-day intervals. Each jar was checked every day for egg masses, which were then transferred to a polyethylene cup containing the same Cu concentration as that of the water to which the parents were exposed and under the same environmental conditions. The reproduction test was continued until each snail in the negative control condition (MFS) produced three egg masses, and death of exposed adult snails was defined as a lack of response to stimulation. An inverted microscope was used to determine the following: abnormal egg masses, number of eggs per egg mass, number of hatched snails at 16 days, and shell length of newly hatched snails. 2.5. Statistical analysis Data are expressed as means ± standard deviation. For the acute exposure test, a survival rate of more than 90% in the control was accepted, and for the hatching test, a hatching success rate of more than 80% of the embryos was accepted. The LC50 (lethal concentration of Cu causing 50% of the snails to die) and EC50 (effective concentration of Cu inducing a hatching success rate of 50% of embryos) with respective 95% confidence intervals (CIs) were calculated using Maximum Likelihood Probit analysis (Gissi et al., 2013). The data obtained from the survival, hatching, and reproduction tests at different Cu concentrations were compared with those of the control using Dunnett’s test. Significant differences were confirmed if p < 0.01 (**) or < 0.05 (*). All statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). All figures were produced using Excel 2007. 3. Results 3.1. Water quality The water quality in the survival, hatching, and reproduction tests was relatively stable. The temperature, DO concentration, pH, and EC of the test solution in all assays were 25.0 ± 0.50  C, 92.7 ± 10.5% saturation, 8.01 ± 0.25, and 1172 ± 28.9 ms cm1, respectively. It was assumed that the losses of all major ions due to absorption onto the experimental materials and uptake by exposed snails were negligible, thus, the cation and anion concentrations were 7.90 mM for Naþ, 0.403 mM for Kþ, 0.576 mM for Ca2þ, 0.305 mM for Mg2þ, 7.44 mM for Cl, 0.746 mM for SO2 4 , and 1.067 mM for HCO 3 , respectively, in all test solution, with a total charge concentration of 20.0 mEq$L1. And the calculated alkalinity and hardness were 107 mg L1 (as CaCO3) and 84.8 mg L1 (as CaCO3), respectively. The mean Cu concentrations measured in the acute tests were 0.58 (nominal concentration: 0 mg L1), 4.94 (5), 7.10 (10), 15.2 (20), 46.7 (40) and 81.4 (80) mg L1, with the corresponding figures for the hatching and reproduction tests presented in Tables 1 and 2, respectively. 3.2. Mortality of adult snails The behavioral characteristics of adult snails in the acute test were recorded. Adult snails in the control group moved freely during the exposure period, and no abnormal behavior was

observed. Snails exposed to the low Cu concentrations (5, 10, and 20 mg L1 Cu) displayed similar behaviors during the first 48 h of exposure. A tendency for a decreased movement speed was observed after 72 h, especially at 20 mg L1 Cu. Snails exposed to the high Cu concentrations (40 and 80 mg L1) moved rapidly to the edge of the cage, and a stress response and attempt to escape were clearly observed at the beginning of the test. Some snails either died or remained static on the bottom of the cage, only responding to a gentle touch during the first 24 h. Most snails were dead after 48 h of exposure to 40 and 80 mg L1 Cu. The number of surviving snails decreased with increasing Cu concentration (Fig. 1). Snails exposed to 80 mg L1 Cu experienced a 100% mortality rate after 72 h of exposure, whereas no mortality was observed at 10 mg L1 or lower concentrations during the test period. The LC50 (CI) values for Cu exposure in adult snails at 24, 48, 72, and 96 h were 71.3 (55.1e117), 39.6 (28.2e47.9), 26.4 (20.6e33.4), and 23.8 (18.7e30.3) mg L1, respectively. 3.3. Hatching success of egg masses The numbers of egg embryos exposing in the control group and different Cu treatments were not significantly different (p > 0.05) (Table 1). Egg masses started hatching on day 12 and the hatching success rates at the end of exposure were 90.9 and 86.9% in the control group and the 1 mg L1 Cu treatment group, respectively. The numbers of hatched snails were much lower after exposure to 2.5, 5, and 12.5 mg L1 Cu compared with the control group. All egg masses exposed to 25 mg L1 Cu failed to hatch until day 16. Exposure to 5 mg L1 Cu and higher concentrations resulted in a significantly reduced hatchability (p < 0.05) compared with the 1 mg L1 Cu and control treatments. Interestingly, the number of hatched snails was higher in the 1 mg L1 Cu group than in the control group from days 12e14. The estimated EC50 in the hatching test at day 16 was 1.66 mg L1 Cu. 3.4. Egg embryonic mortality Egg masses were examined to determine the mortality rate during Cu exposure. All eggs that stopped rotating completely or that developed a cloudy appearance (Fig. 3b), were assumed to be dead. As shown in Table 1, the mortality rate of embryos in both the control and the Cu treatment groups was low during the 16-day exposure, except for the 25 mg L1 Cu treatment group, which had a significantly higher mortality rate of 17.1% compared with the control (p < 0.01). In general, mortality increased as the Cu concentration increased. Thus, a dose-dependent relationship was found between embryo mortality and the Cu concentration. 3.5. Development of egg masses 3.5.1. Normal embryonic development The normal embryonic development of freshwater pulmonate snails occurs in four stages (Fig. 2ae2e): the morula, trochophore, veliger, and hippo stages. These stages generally occur at days 2.5e3, 3e5, 5e8, and 8e10 after egg deposition, respectively, (Gomot, 1998; Khangarot and Das, 2010; Das and Khangarot, 2011). The characteristics of the embryo at different stages were recorded. During the morula stage, the embryo left the vitelline membrane and moved very slowly within the egg capsule by means of cilia (Fig. 2a). During the trochophore stage, the shell gland and prototroch developed, and the embryo became more transparent and rotated faster within the capsule (Fig. 2b). During the veliger stage, the development of eyes, foot, and shell was observed (Fig. 2c and 2d). During the hippo stage, the foot and viscera became well separated. The embryo occupied the whole egg capsule fully and

L. Gao et al. / Chemosphere 185 (2017) 1208e1216

1211

Table 1 The rates of egg masses hatching, mortality, and deformity. Nominal Cu conc.(mg$L1)

Measured Cu conc.(mg$L1)

Control 0.32 1.0 0.48 2.5 2.28 5.0 5.34 12.5 13.2 25 25.2 EC50 and 95% C.L. (mg$L1 of Cu)

Number of eggsa 43.4 47.8 31.5 35.9 27.7 33.2

± ± ± ± ± ±

11.5 12.7 5.03 1.53 2.65 3.61

Hatching rate of egg masses (%) Day 12a

Day 13a

Day 14a

Day 15a

Day 16a

13.3 ± 3.85 22.2 ± 7.11 2.86 ± 4.95* 0** 0** 0** N.A. N.A.

32.6 ± 2.90 51.3 ± 22.4 9.95 ± 8.64* 2.07 ± 2.04c 0** 0** 0.51 0.41e0.62

62.5 ± 6.34 66.4 ± 5.60 24.1 ± 23.0* 7.63 ± 6.65** 0** 0** 0.85 0.70e1.01

78.6 ± 8.70 70.9 ± 1.83 33.8 ± 32.9 9.73 ± 8.43* 1.23 ± 2.14** 0** 1.06 0.87e1.26

90.9 ± 5.39 86.9 ± 1.34 39.3 ± 40.0 13.2 ± 11.4** 2.47 ± 4.28** 0** 1.66 1.09e2.37

Mortality ratea (%) 0.51 4.86 0.74 1.45 1.71 17.1 N.A. N.A.

± ± ± ± ± ±

0.87 8.42 1.28 2.51 2.96 2.86**

Deformity ratea (%) 0 0.67 ± 1.15 3.39 ± 3.00 9.33 ± 8.17 19.2 ± 15.9 59.2 ± 5.91** 25.1 12.7eN.A.

*,** Significant differences compared with control p < 0.05, p < 0.01. N.A. Data were not available. a Mean ± standard deviation of six observations.

Table 2 Effects of different Cu concentrations on reproduction of snails and hatching success.

**

Nominal Cu conc. (mg$L1)

Measured Cu conc. (mg$L1)

The time of first brooda (d)

Control 1 2.5 5 12.5 25

0.95 1.02 3.08 5.22 12.0 28.3

3.2 3.6 4.4 3.2 3.6 3.4

± ± ± ± ± ±

1.1 1.7 1.3 1.1 2.3 1.3

Number of egg masses per snaila 3.80 2.80 3.40 4.20 4.40 4.40

± ± ± ± ± ±

1.30 0.45 0.89 1.10 2.07 2.30

Number of eggs

Hatched snails

Hatching rate (%)

1243 1289 928 1086 971 770

1007 1166 665 543 432 86**

81.0 90.5 71.7 50.0 44.5 11.2**

Significant differences compared with control p < 0.01. Mean ± standard deviation of six observations.

a

moved freely. The shell, foot, eyes, and tentacle could be identified (Fig. 2e). The larval structures gradually degraded within the following 2e3 days (at days 12e13), and then the young snails hatched (Fig. 2f). 3.5.2. Abnormal embryonic development Almost all surviving egg embryos exposed to each Cu solution reached the veliger stage. There were differences between the control and Cu treatment groups following development of the egg embryos. A normal veliger stage was observed at 5 mg L1 or lower Cu concentration, while the development of the eyes and shell was inhibited at 12.5 and 25 mg L1 Cu, respectively; these concentrations resulted in a deformed shell, abnormal eyes, and differences

Percentage survival (%)

120 24 h 48 h 72 h 96 h

100 80

* 60

**

40

** 20 0 0

20

40

60

80

Nominal Cu conc. ( g·L 1) Fig. 1. Percentage mortality in adult snails exposed to various Cu concentrations at different experimental periods (* and ** significantly different from the survival rate of the control, p < 0.05 or p < 0.01).

in morphology during further development. All egg embryos exposed to 5 mg L1 Cu reached the veliger stage, but the size of the egg embryos was abnormal. Furthermore, formation of the shell gland, shell, eyes, and foot was not clear at the high Cu concentrations (12.5 and 25 mg L1), and egg embryos were smaller than those in the control group. At day 9, separation between the foot and viscera was observed in normal egg capsules. Some egg embryos exposed to 12.5 and 25 mg L1 Cu reached the hippo stage, although the mantle ridge and shell edge disappeared, and lesions and hemorrhaging of the larval body were observed (Fig. 3d). At 25 mg L1 Cu, 59.2% of egg embryos were identified as deformed. A more distinct dose-dependent relationship was observed with regard to deformity than mortality. The EC50 for egg embryo deformity was estimated to be 25.1 mg L1 Cu (Table 1). 3.5.3. Delay in egg embryonic development Egg embryos in the control group developed normally and started hatching on day 12 after egg deposition, with more than 90% of eggs hatching by the end of the experiment. In contrast, not only was the number of hatched snails less after Cu solution treatment than after the control treatment, but the development of egg embryos after the various Cu treatments was significantly delayed compared with the control group (p < 0.05) (Fig. 4). In summary, all eggs in the control and Cu treatment groups reached the trochophore stage at day 5, and more than 95% of eggs in both the control and 1 mg L1Cu treatment groups reached the early veliger stage. An obvious delay in egg development was found at the high Cu concentrations (nominal: 2.5, 5, 12.5, and 25 mg L1), especially at 12.5 mg L1, in which almost 50% of eggs remained in the trochophore stage (Fig. 4a). As shown in Fig. 4b, almost half of the eggs exposed to 1 mg L1 Cu were in the hippo stage after 7 days of exposure. Notably, approximately 10% of eggs exposed to 12.5 mg L1 Cu remained in the trochophore stage. The percentage of eggs in the hippo stage decreased with increasing Cu concentration. At day 9, the eggs were mainly in the veliger and hippo

1212

L. Gao et al. / Chemosphere 185 (2017) 1208e1216

a

b

c

F Sh

P

Shg

EC d

f

e

Sh

F

Sh

HS E E

Te

Fig. 2. Normal morphological features of snail (Physa acuta) egg embryos exposed to control at different developmental stages: (a) 2-day-old morula stage has a round shape, (b) 4day old trochophore stage, shell formation begins, (c) 5e6 day early veliger stage, shell and foot formation are observed, (d) 7-day old late veliger, shows well developed foot, shell and eyes, (e) 8e10-day old hippo stage, development of tentacle is completed and larvae occupies the whole space in the egg capsule, (f) newly hatched snail. (P, Prototroch; E, eye; F, foot; Ec, egg capsule; Sh, shell; Shg, shell gland; Te, tentacle; HS, hatched snail).

stages. The percentage of eggs reaching the hippo stage was 98.2% for the control and 96.5% for the 1 mg L1 Cu treatment (Fig. 4c). Some eggs in the 1 mg L1 Cu treatment group hatched, and eggs in all treatment groups survived. There was a tendency for the number of eggs in the hippo stage to decrease with increasing Cu concentration from days 7e9. After 12 days of exposure, eggs in the control group and the 1 and 2.5 mg L1 Cu treatment groups

a

b

CA

NE c

d DSh

E AL NL Te

LL

Fig. 3. Morphological features of snail (Physa acuta) egg embryos exposed to control and Cu solution at morula and hippo stage: (a) normal egg embryo, (b) dead embryo egg with cloudy appearance, (c) normal larvae, and (d) deformed larvae. (NE, normal egg; CA, cloudy appearance; NL, normal larvae; Te, tentacle; E, eye; LL, lesion larvae; AL, abnormal larvae; DSh, deformed shell).

exhibited hatching rates of 13.3, 22.2, and 2.86%, respectively (Fig. 4d). Egg embryos exposed to Cu concentrations of 5 mg L1 and higher were significantly delayed in hatching (p < 0.05). Many of the eggs exposed to 2.5e25 mg L1 Cu remained in the veliger and hippo stages for 2e7 days, with no further development. 3.6. Reproduction of adult snails Adult snails were exposed to different Cu concentrations to determine the toxic effects of Cu on reproduction and hatching after egg deposition. All adult snails used in the reproduction test survived after 9 days of Cu exposure. As shown in Table 2, the timing of the first brood for adult snails exposed to Cu varied from 3.2 to 4.4 days, and the differences compared with that of the control group (3.2 days) were not significant (p > 0.05). Most adult snails produced more than three broods of egg masses within 9 days, with the exception of those exposed to nominal 1 mg L1 Cu. Although the cumulative number of egg masses produced by snails exposed to Cu was not significantly different from that of the control group (p > 0.05), the number of embryos contained within the egg masses decreased in a dose-dependent manner. Surprisingly, the 12.5 and 25 mg L1 Cu treatments resulted in polynuclear (three or more) eggs in one egg capsule (Fig. 5b). The hatching success rate was 81% in the control group after 16 days, which was less than that in the 1 mg L1 Cu treatment group (90.5%). The number of hatched snails decreased sharply as the Cu concentration increased, especially when the Cu concentration reached 25 mg L1. 3.7. Shell length of the hatched snails in the hatching and reproduction tests In the hatching test, the mean shell length was 871 mm (Fig. 6) in the control group and 832 and 934 mm in the 1 and 2.5 mg L1 Cu treatments, respectively, although the difference compared with

100%

*

a

*

80%

60%

40%

20%

0% Control

1

2.5

5

12.5

% of different stages of development

% of different stages of development

L. Gao et al. / Chemosphere 185 (2017) 1208e1216

100%

c

**

80%

60%

40%

20%

0% Control

1

2.5

5

Nominal Cu conc. (

12.5

25

g·L-1)

*

*

12.5

25

60%

40%

20%

0% Control

25

1

2.5

5

Nominal Cu conc. ( g·L-1) trochophore veliger hippo hatched

% of different stages of development

% of different stages of development

*

b

80%

Nominal Cu conc. ( g·L-1) 100%

1213

100%

d

dead

*

*

12.5

25

80%

60%

40%

20%

0% Control

1

2.5

5

Nominal Cu conc. (

g·L-1)

Fig. 4. Inhibition of different Cu concentrations on different developmental stages of Physa Acuta embryogenesis at days 5(a), 7(b), 9(c), and 12(d) (*,** indicate that developmental delay of embryogenesis in Cu treatments were significantly different from that in control, p < 0.05, p < 0.01).

the control group was not significant (p > 0.05). In contrast, there was an obvious decrease in the shell lengths (757 and 666 mm) of hatched snails exposed to the high Cu concentrations (5 and 12.5 mg L1, respectively) compared with the control (p < 0.01). In the reproduction test, shell length also decreased with increasing Cu concentration. The mean shell length was 854 mm in the control group compared with 793, 727, and 747 mm in the 1, 2.5, and 5 mg L1 Cu treatment groups, respectively. A significant reduction in shell length (633 and 446 mm) was observed at 12.5 and 25 mg L1 Cu, respectively (p < 0.01). A similar decreasing trend in shell length with increasing Cu concentration was found in both the hatching and reproduction tests. Remarkably, the shell length of hatched snails exposed to the same Cu concentration was slightly longer in the hatching test than in the reproduction test. 4. Discussion A high Cu concentration in ambient water can have serious adverse effects on aquatic organisms. In this study, nonlinear doseresponse relationships between the aquatic Cu concentration and behavioral characteristics of P. acuta were observed. At low concentrations (e.g., 20 mg L1), Cu exposure suppressed snail movement. It has been reported that concentrations exceeding a certain threshold trigger an escape response, with snails searching for refuge from the stress (Bernot et al., 2005). A previous study reported that the LC50 value varied with the water chemistry, especially the hardness and pH (Rogevich et al., 2008). The hardness of a test solution is an important chemical parameter that affects the

acute and chronic toxicities of Cu toward aquatic organisms. High levels of hardness and pH could result in a lower solubility and bioavailability of Cu in aqueous solution, reducing the toxicity of Cu. The 96 h LC50 of Cu in P. Acuta was 23.8 mg L1 in alkaline water (pH ¼ 8.01 ± 0.25), with a hardness of 84.8 mg L1 as CaCO3 (Ca ¼ 543 mM, Mg ¼ 305 mM). The 96 h LC50 of Cu in the adult Florida apple snail (P. paludosa) was 44.9 mg L1 at similar water chemistry conditions (hardness: 68 mg L1, pH > 7) (Rogevich et al., 2008). Brix et al. (2011) reported a 96 h LC50 of 31 mg L1 Cu in L. stagnalis in an acute toxicity test, which was in accordance with Ng et al. (2011), who reported a 96 h LC50 of 24.9 mg L1 Cu in water with a hardness of 123 mg L1 as CaCO3. P. acuta is very sensitive to Cu in moderately hard water but is well protected under the Canadian Water Quality Guidelines of a Cu concentration of 2 mg L1 and water hardness of <82 mg L1as CaCO3 (Canadian Council of Ministers of the Environment, 2007). In this study, the acute toxicity test showed a sharp increase in the mortality rate within the first 48 h, indicating that high Cu concentrations (40 and 80 mg L1) had significantly adverse effects on the snails. The high toxicity of Cu to snails could be attributed to the relatively greater accumulation rate of Cu, with gills and respiratory organs being the main target organs (Cheung et al., 2002). The toxicity mechanism of Cu in aquatic organisms is associated with the production of radical oxygen species or non-free radical oxygenated molecules, such as hydrogen peroxide, superoxide, singlet oxygen, and the hydroxyl radical (Pisoschi and Pop, 2015), which influence cellular processes, especially membrane system function, and may lead to membrane permeability and cell death (Pinto et al., 2003; Ali et al., 2012;

1214

L. Gao et al. / Chemosphere 185 (2017) 1208e1216

a

b

Fig. 5. Normal egg masses produced by snails exposed to the control group (a) and abnormal egg masses produced by snails exposed at 12.5 and 25 mg L1 Cu (b).

1200

Reproduction test

Hatching test

Shell length ( m)

1000

** 800

** **

600

** 400 200 0 Control

1

2.5

5

Nominal Cu conc. (

12.5

25

g·L-1)

Fig. 6. Effect of different Cu concentrations on shell length of hatched snails in hatching and reproduction tests (**Significant differences compared with control, p < 0.01).

Abdel-Halim et al., 2013). The LC50 values at 72 (26.4 mg L1 Cu) and 96 h (23.8 mg L1Cu) were comparable, suggesting that P. acuta exhibited some degree of resistance to Cu exposure after acclimation, which lasted for a considerable period. The embryos of aquatic organisms, especially pond snails, are generally more sensitive to toxic substances than adults (Leung et al., 2007; Khangarot and Das, 2010). For example, the 96 h LC50 of adult Lymnaea luteola and the 14 d EC50 for embryonic hatching success were estimated to be 27 and 3.58 mg L1 Cu, respectively (Khangarot and Ray, 1988; Khangarot and Das, 2010). The toxic effects of Cu on P. acuta and egg masses observed in this study were comparable, with an LC50 of 23.8 and EC50 of 1.66 mg L1 Cu. Cu is a vital component of hemocyanin, which is an oxygen-carrying molecule in snails and arthropods. Excess Cu accumulation in egg masses will probably result in dysfunctional egg embryos by influencing the neuroendocrine regulation of developing snails (Khangarot and Das, 2010), thus inhibiting hatching. Gomot (1998) suggested that the egg cells in egg masses can be protected by “two lines of defense” upon exposure to pollution. Trace metals such as Cd could penetrate the gelatinous matter and form a diffusion gradient in the egg masses surrounding the actual eggs, resulting in a direct pathway for Cd to enter the eggs. This would inhibit the development or cause mortality of eggs. Some studies have shown that toxicants can cause a delay in embryonic development and the hatching of aquatic organisms (Zha and Wang, 2006; Sawasdee and

Kohler, 2009; Bandow and Weltje, 2012). Das and Khangarot (2011) reported abnormal development of L. luteola embryos after exposure to a low concentration of Cu (32 mg L1). Furthermore, the number of deformed eggs increased significantly with an increase in the Cu concentration from 3.2 to 10 mg L1, at which abnormalities in morphological features and formation of the foot, shell, and shell gland were observed after 10 days. Embryonic development was previously shown to delay the veliger stage significantly (Khangarot and Das, 2010), which was in good agreement with the results of our study. Therefore, the rate of deformed eggs was greater than the mortality rate, indicating the significance of sublethal effects of Cu on eggs. The time to hatching of P. acuta in culture medium can be at least 8.16 days (Woodard, 2005), and it was 12 days in our study, which could be attributed to differences in exposure conditions and laboratory environments among studies. The development and hatching of egg masses exposed in the control group and the lowest Cu treatment (nominal: 1 mg L1), corresponding to measured concentrations of 0.32 and 0.48 mg L1, respectively, were similar. As an important trace element for aerobic organisms, Cu serves as a catalytic and structural cofactor for enzymes, playing roles in energy generation, iron acquisition, oxygen transport, cellular metabolism, and a host of other biochemical processes (Olias et al., 2004). However, Cu concentration exceeding 5 and 12.5 mg L1, respectively, significantly inhibited the hatching and development of egg masses. Salinity, affected by  essential elements (e.g., Ca2þ, Mg2þ, Kþ, Naþ, Cl,SO2 4 , HCO3 , and CO2 ), presents an inverted U-shaped concentration-response 3 curve for the survival, growth, and reproduction of P. acuta (Kefford and Nugegoda, 2005; Zalizniak et al., 2007a). Thus, chemical (e.g., copper) inputs at certain ranges can significantly promote or inhibit the metabolism of aquatic organisms at different stages of life. A more detailed range-finding test regarding to inverted U-shaped concentration-response curve of copper for the development and hatching of egg masses is necessitated. Reproduction, as one of the most important stages of the life cycle, contributes to the maintenance of organism populations in aquatic ecosystems. In this study, exposed snails survived in the reproduction test, which was attributed to the food supply. There were two possible reasons for this: (1) food might increase the concentration of dissolved organic matter in the test solution, which could then trap cations via the formation of stable chelates or complexes (Gao et al., 2016), resulting in reduced Cu bioavailability and toxicity to the snails; (2) food provides the necessary ions and energy to maintain a normal metabolism and develop internal detoxification mechanisms, such as metallothionein binding to trace metals, leading to a reduction in thiobarbituric acid-reactive substances and subsequently detoxification of poisonous substances (Ng et al., 2011). The second explanation was

L. Gao et al. / Chemosphere 185 (2017) 1208e1216

considered to be plausible due to the finding that only approximately 10% of adult snails survived after exposing to 40 mg L1 Cu treatment in the acute test, whereas 100% survived in the reproduction test with no obvious effects of Cu observed after 9 days. Furthermore, the soft tissues of snails contain various metal-rich granules, which could have an important role in sequestering trace metals in biologically inactive forms (Uriu-Adams and Keen, 2005), resulting in accumulation, storage, and excretion of trace metals and ultimately detoxification (Das and Khangarot, 2011). However, excessive accumulation of trace metals impacts breeding activities. It was reported that an increase in the Cd concentration significantly reduced the numbers of eggs and embryos produced, because Cd2þ can have adverse effects on the cerebral ganglia of freshwater snails, thus disrupting egg laying (Gomot, 1998). It was notable that snails exposed to 12.5 and 25 mg L1 Cu produced abnormal egg masses, with polynuclear eggs in one capsule (see Fig. 5b), which implies that Cu interferes with oogenesis and the formation of eggs in the genital tract. An inability to control the Cu balance, such as excessive uptake of Cu, will lead to genetic diseases and neurodegenerative disorders (Olias et al., 2004). It was interesting that the hatching success of egg masses under all Cu treatments was higher in the reproduction test than in the hatching test, indicating that the egg masses produced in the reproduction test were more resistant to Cu toxicity. This was likely attributed to acclimation of the egg masses to Cu, which was induced by detoxification of Cu in adult snails. Our hypothesis could be further supported by Reategui-Zirena et al. (2017) who found that hatchings from freshwater snail Lymnaea stagnalis exposed to Cd showed more tolerance than their parents, indicating a potentially important maternal effect. Snail shells consist mainly of CaCO3 and provide a shield to protect soft tissues from the physical impairments or predation by large aquatic animals. The formation of freshwater snail shells relies on Ca2þ uptake from ambient water and can be inhibited by trace metals such as lead, cobalt, and Cd, which often act as Ca antagonists (Real et al., 2003; Mehta et al., 2006; Wilbers et al., 2014). Brix et al. (2011) reported that adult snails exposed to 48 mg L1 Cu significantly reduced their Ca2þ uptake, leading to inhibited growth of adult snails. The inhibition of Ca2þ uptake was related to either inhibition of proteins found on the apical membrane or an indirect effect of Cu on another protein or system in snails. Therefore, Ca2þ uptake by egg masses was the main factor in the formation of shells from the veliger stage to hatching. Khangarot and Das (2010) found that the shell length of hatched snails showed a marked decline with increasing Cu concentration, suggesting that Cu acts as a Ca antagonist (Brix et al., 2011). Compared with the hatching test in the present study, the hatched snails in the reproduction test had an obvious reduction in shell length, which resulted mainly from inefficient Ca2þ uptake from the test solution as a result of long-term Cu exposure. 5. Conclusion The pulmonate snail P. acuta displayed a strong sensitivity to Cu, which induced an escape response to the stress in laboratory experiments. Cu had a significant acute toxic effect on P. acuta, resulting in a low LC50 value of 23.8 mg L1. Embryonic development was more vulnerable to chronic exposure. Different morphological shapes (e.g., abnormal eyes and a deformed shell), lesions and hemorrhaging in larvae, and a distinct delay in embryonic development were apparent when the egg masses were exposed to low Cu levels. In the reproduction test, adult snails exposed to more realistic conditions (i.e., provision of food and aeration) showed resistance to the toxic effects of Cu, but oviposition was significantly impacted,

1215

with snails exposed to high concentrations of Cu producing polynuclear eggs in one egg capsule. Furthermore, the hatching success and shell length of the filial generation decreased with increasing Cu concentration, indicating a genetic toxicity effect by Cu. These findings suggest that long-term multigenerational exposure experiments are needed to investigate the potential effects of Cu on the abundance of freshwater snails in the natural environment. Acknowledgement The authors are very grateful to reviewers for providing invaluable suggestion and comment. This work was financially supported by the International Program of Project 985, Sun Yat-Sen University. References Abdel-Halim, K.Y., Abo El-Saad, A.M., Talha, M.M., Hussein, A.A., Bakry, N.M., 2013. Oxidative stress on land snail Helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals. Chemosphere 93, 1131e1138. Ali, D., Alarifi, S., Kumar, S., Ahamed, M., Siddiqui, M.A., 2012. Oxidative stress and genotoxic effect of zinc oxide nanoparticles in freshwater snail Lymnaea luteola L. Aquat. Toxicol. 124e125, 83e90. Appleton, C.C., 2003. Alien and invasive fresh water Gastropoda in South Africa. Afr. J. Aquat. Sci. 28, 69e81. Bandow, C., Weltje, L., 2012. Development of an embryo toxicity test with the pond snail Lymnaea stagnalis using the model substance tributyltin and common solvents. Sci. Total Environ. 435e436, 90e95. Beckmann, M.C., He, Q.Y., Yang, J., Xu, P., 2006. First report of Ferrissia wautieri and Physa acuta in Taihu Lake of China. South China Fish. Sci. 2, 63e65. Bernot, R.J., Kennedy, E.E., Lamberti, G.A., 2005. Effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail, Physa acuta. Environ. Toxicol. Chem. 24, 1759e1765. Besser, J.M., Dorman, R.A., Hardesty, D.L., Ingersoll, C.G., 2016. Survival and growth of freshwater pulmonate and nonpulmonate snails in 28-day exposures to copper, ammonia, and pentachlorophenol. Arch. Environ. Contam. Toxicol. 70, 321e331. Brix, K.V., Esbaugh, A.J., Grosell, M., 2011. The toxicity and physiological effects of copper on the freshwater pulmonate snail, Lymnaea stagnalis. Comparative biochemistry and physiology. Toxicol. Pharmacol. CBP 154, 261e267. Canadian Council of Ministers of the Environment, 2007. Canadian Water Quality Guidelines for the Protection of Aquatic Life. Summary Table. No. 1299, Winnipeg ISBN 1-896997-896934-896991. Cheung, C.C.C., Lam, P.K.S., 1998. Effect of cadmium on the embryos and juveniles of a tropical reshwater snail, Physa acuta (Draparnaud, 1805). Water Sci. Technol. 38, 263e270. Cheung, S.G., Tai, K.K., Leung, C.K., Siu, Y.M., 2002. Effects of heavy metals on the survival and feeding behaviour of the sandy shore scavenging gastropod Nassarius festivus (Powys). Mar. Pollut. Bull. 45, 107e113. Cooper, N.L., Bidwell, J.R., Kumar, A., 2009. Toxicity of copper, lead, and zinc mixtures to Ceriodaphnia dubia and Daphnia carinata. Ecotoxicol. Environ. Saf. 72, 1523e1528. Das, S., Khangarot, B.S., 2011. Bioaccumulation of copper and toxic effects on feeding, growth, fecundity and development of pond snail Lymnaea luteola L. J. Hazard. Mater. 185, 295e305. Flemming, C.A., Trevors, J.T., 1989. Copper toxicity and chemistry in the environment: a review. Water Air Soil Pollut. 44, 143e158. , B., 2007. Trace elements in river waters. Treatise Gaillardet, J., Viers, J., Dupre Geochem. 5, 225e272. Gao, L., Chen, J., Tang, C., Ke, Z., Wang, J., Shimizu, Y., Zhu, A., 2015. Distribution, migration and potential risk of heavy metals in the Shima River catchment area, South China. Environmental science. Process. Impacts 17, 1769e1782. Gao, L., Wang, Z., Shan, J., Chen, J., Tang, C., Yi, M., Zhao, X., 2016. Distribution characteristics and sources of trace metals in sediment cores from a transboundary watercourse: an example from the Shima River, Pearl River Delta. Ecotoxicol. Environ. Saf. 134P1, 186e195. Gissi, F., Binet, M.T., Adams, M.S., 2013. Acute toxicity testing with the tropical marine copepod Acartia sinjiensis: optimisation and application. Ecotoxicol. Environ. Saf. 97, 86e93. Gomot, A., 1998. Toxic effects of cadmium on reproduction, development, and hatching in the freshwater snail Lymnaea stagnalis for water quality monitoring. Ecotoxicol. Environ. Saf. 41, 288e297. Hsieh, C.-Y., Tsai, M.-H., Ryan, D.K., Pancorbo, O.C., 2004. Toxicity of the 13 priority pollutant metals to Vibrio fisheri in the Microtox® chronic toxicity test. Sci. Total Environ. 320, 37e50. Joachim, S., Roussel, H., Bonzom, J.M., Thybaud, E., Mebane, C.A., Van den Brink, P., Gauthier, L., 2017. A long-term copper exposure in a freshwater ecosystem using lotic mesocosms: invertebrate community responses. Environ. Toxicol. Chem. 9999, 1e17.

1216

L. Gao et al. / Chemosphere 185 (2017) 1208e1216

Kefford, B.J., Nugegoda, D., 2005. No evidence for a critical salinity threshold for growth and reproduction in the freshwater snail Physa acuta. Environ. Pollut. 134, 377e383. Khangarot, B.S., Das, S., 2010. Effects of copper on the egg development and hatching of a freshwater pulmonate snail Lymnaea luteola L. J. Hazard. Mater. 179, 665e675. Khangarot, B.S., Ray, P.K., 1988. Sensitivity of a freshwater pulmonate snails, Lymnaea luteolaL., to heavy metals. Bull. Environ. Contam. Toxicol. 41, 208e213. Kwok, K.W., Leung, K.M., Bao, V.W., Lee, J.S., 2008. Copper toxicity in the marine copepod Tigropus japonicus: low variability and high reproducibility of repeated acute and life-cycle tests. Mar. Pollut. Bull. 57, 632e636. Leung, K.M., Grist, E.P., Morley, N.J., Morritt, D., Crane, M., 2007. Chronic toxicity of tributyltin to development and reproduction of the European freshwater snail Lymnaea stagnalis (L.). Chemosphere 66, 1358e1366. Li, X.Y., Dong, X.Y., Bai, X., Liu, L., Wang, J.J., 2014. The embryonic and postembryonic developmental toxicity of imidazolium-based ionic liquids on Physa acuta. Environ. Toxicol. 29, 697e704. Ma, J., Zhou, C., Li, Y., Li, X., 2014. Biochemical responses to the toxicity of the biocide abamectin on the freshwater snail Physa acuta. Ecotoxicol. Environ. Saf. 101, 31e35. Mebane, C.A., Eakins, R.J., Fraser, B.G., Adams, W.J., 2015. Recovery of a miningdamaged stream ecosystem. Elem. Sci. Anthropocene 3, 000042. Mehta, R., Templeton, D.M., O’Brien, P.J., 2006. Mitochondrial involvement in genetically determined transition metal toxicity II. Copper toxicity. Chemicobiol. Interact. 163, 77e85. Musee, N., Oberholster, P.J., Sikhwivhilu, L., Botha, A.M., 2010. The effects of engineered nanoparticles on survival, reproduction, and behaviour of freshwater snail, Physa acuta (Draparnaud, 1805). Chemosphere 81, 1196e1203. Ng, T.Y., Pais, N.M., Wood, C.M., 2011. Mechanisms of waterborne Cu toxicity to the pond snail Lymnaea stagnalis: physiology and Cu bioavailability. Ecotoxicol. Environ. Saf. 74, 1471e1479. Olias, M., Nieto, J.M., Sarmiento, A.M., Ceron, J.C., Canovas, C.R., 2004. Seasonal water quality variations in a river affected by acid mine drainage: the Odiel River (South West Spain). Sci. Total Environ. 333, 267e281. Piccinni, E., Coppellotti, O., Giannoni, L., Ravera, O., 1985. Effects of Cu, Cd, V in Physa acuta (draparnaud) I: partial characterisation of chelating compounds. Environ. Technol. Lett. 6, 505e513. Pinto, E., Sigaud-Kutner, T.C.S., Leitao, M.A.S., Okamoto, O.K., Morse, D., Colepicolo, P., 2003. Heavy metals-induced oxidative stress in algae. J. Phycol. 39, 1008e1018. Pisoschi, A.M., Pop, A., 2015. The role of antioxidants in the chemistry of oxidative stress: a review. Eur. J. Med. Chem. 97, 55e74.

Pyatt, F.B., Metcalfe, M.R., Pyatt, A.J., 2003. Copper bioaccumulation by the freshwater snail Lymnaea peregra: a toxicological marker of environmental and human health? Environ. Toxicol. Chem. 22, 561e564. Ray, S., 1984. Bioaccumulation of cadmium in marine organisms. Experientia 40, 14e23. ~ oz, I., Guasch, H., Navarro, E., Sabater, S., 2003. The effect of copper Real, M., Mun exposure on a simple aquatic food chain. Aquat. Toxicol. 63, 283e291. Reategui-Zirena, E.G., Fidder, B.N., Olson, A.D., Dawson, D.E., Bilbo, T.R., Salice, C.J., 2017. Transgenerational endpoints provide increased sensitivity and insight into multigenerational responses of Lymnaea stagnalis exposed to cadmium. Environ. Pollut. 224, 572e580. Rogevich, E.C., Hoang, T.C., Rand, G.M., 2008. The effects of water quality and age on the acute toxicity of copper to the Florida apple snail, Pomacea paludosa. Arch. Environ. Contam. Toxicol. 54, 690e696. Sanchez-Arguello, P., Fernandez, C., Tarazona, J.V., 2009. Assessing the effects of fluoxetine on Physa acuta (Gastropoda, Pulmonata) and Chironomus riparius (Insecta, Diptera) using a two-species water-sediment test. Sci. Total Environ. 407, 1937e1946. Sawasdee, B., Kohler, H.R., 2009. Embryo toxicity of pesticides and heavy metals to the ramshorn snail, Marisa cornuarietis (Prosobranchia). Chemosphere 75, 1539e1547. Uriu-Adams, J.Y., Keen, C.L., 2005. Copper, oxidative stress, and human health. Mol. Asp. Med. 26, 268e298. Wilbers, G.J., Becker, M., Nga, T., Sebesvari, Z., Renaud, F.G., 2014. Spatial and temporal variability of surface water pollution in the Mekong Delta, Vietnam. Sci. Total Environ. 485e486, 653e665. Wong, C.S., Duzgoren-Aydin, N.S., Aydin, A., Wong, M.H., 2007. Evidence of excessive releases of metals from primitive e-waste processing in Guiyu, China. Environ. Pollut. 148, 62e72. Woodard, V.H., 2005. Feasibility for Utilization of a Freshwater Pulmonate Snail, Physa Acuta, as a Model Organism for Environmental Toxicity Testing, with Special Reference to Cadmium Toxicity. Ph.D. Dissertation. Xia, K., Zhao, H., Wu, M., Wang, H., 2012. Chronic toxicity of copper on embryo development in Chinese toad, Bufo gargarizans. Chemosphere 87, 1395e1402. Zalizniak, L., Kefford, B.J., Nugegoda, D., 2007a. Effects of different ionic compositions on survival and growth of Physa acuta. Aquat. Ecol. 43, 145e156. Zalizniak, L., Kefford, B.J., Nugegoda, D., 2007b. Effects of pH on salinity tolerance of selected freshwater invertebrates. Aquat. Ecol. 43, 135e144. Zha, J., Wang, Z., 2006. Acute and early life stage toxicity of industrial effluent on Japanese medaka (Oryzias latipes). Sci. Total Environ. 357, 112e119. Zhang, J., 1995. Geochemistry of trace metals from Chinese river/estuary systems: an overview. Estuar. Coast. Shelf Sci. 41, 631e658.