In order to determine the influence of the presence of one or more species in a system on Zn toxic response, acute (LC50 at 72 h) and sublethal toxicity (Zn uptake kinetics, and quantification of total protein content and lipid peroxidation levels) were evaluated in three benthic organisms (Hyallela azteca, Limnodrillus hoffmeisteri and Stagnicola attenuata). They were exposed either singly or together (single or multi-species test systems) to Zn-spiked sediment from Ignacio Ramírez Reservoir. Both assays showed that Zn-spiked sediments from Ignacio Ramírez Reservoir were toxic to S. attenuata, L. hoffmeisteri and H. azteca, and that toxic response was modified according to the number of species in the system. These differences may be due to multiple factors: such as benthic bioturbation which modifies physical and chemical characteristics of sediments and affects the fate and partitioning of sediment-bond contaminants, such as spiked Zn or other pollutants in sediments from Ignacio Ramírez Reservoir. In addition, the presence of more than one species in the system may lead to microenvironmental changes (pH, temperature, and organic matter and metabolic waste content), which can also contribute to toxic response differences. Also, particular characteristics of each of the species involved in the study became evident. The snail and amphipod are facultative benthic organisms, while the worm is obligate. Such way of life differences may have modified the bioavailability of contaminants, determining their toxicity.

Introduction

Zn is an essential element (Wu and Chen, 2004; Gioda et al., 2007), but it may also become toxic to some species at ecologically relevant concentrations (Elumalai et al., 2007). Thus, Vesela and Vijverberg (2007) showed this metal reduces body size and elicits shape alterations in neonates of various cladoceran. Also, Gheorghiu et al. (2007) found that 120 μ g Zn l−1 are toxic to Gyrodactylus turnbulli, eliciting morphological changes and reduced reproductive and survival rates.

Since this metal has a high affinity with sulfhydryl and hydroxyl groups as well as a great capacity to combine with amino acids, peptides, proteins and nucleic acids, it can elicit very diverse effects (Serafim and Bebianno, 2007). A major consequence of their presence is increased production of reactive oxygen species (ROS) due to disruption of the equilibrium between production of these species and antioxidant system activity; the end result being increased lipid peroxidation (Geret and Bebianno, 2004) with consequent damage to cell membranes, nucleic acids and chloroplastic pigments (Zhang et al., 2007).

In nature, one rarely has complete knowledge of all the factors that are known to affect toxicity of metals in the laboratory. Little is known about the possible confounding effects of other natural stressors not present under optimal laboratory conditions (Sverdrup et al., 2006). Most studies on toxicity have been carried out in single-species test systems; thus leaving largely unknown the possible changes in the bioavailability and toxicity of Zn in the presence of other organisms and limiting prediction of toxic response under real environmental conditions. The multi-species tests are an attempt to make some kind of connection between a precisely controlled laboratory experiment and an observational field study (Mothes-Wagner et al., 1992).

The purpose of this study was to evaluate the effect of Zn-spiked sediment from Ignacio Ramírez reservoir in terms of acute and sublethal toxicity to S. attenuata, L. hoffmeisteri and H. azteca in single and multi-species test systems.

Materials and methods

Study area

The study was conducted at Ignacio Ramírez Reservoir (IRR), located on the Río La Gavia, 22 km from the city of Toluca and 5 km from the Río Lerma (19°27′35″ N and 99°46′25″ W). Climate is temperate with summer rains; the warmest months being May and June. Mean annual temperature is 12.4°C. Deforestation, inefficient agricultural practices and steeply inclined soil are common problems that contribute notably to environmental degradation of the area (Galar-Martínez et al., 2006).

Sediment and test organisms sampling

Sediment sampling was conducted at the water gate in February 2005 (dry season). A stainless steel conical drag was used to obtain samples. The sediment was dried at 80°C and sieved through 0.02 mm stainless steel mesh.

The three test species, L. hoffmeisteri, H. azteca and S. attenuata, were collected at the same site and transported to laboratory in plastic bags with constant aeration.

Maintenance of test organisms

Organisms were taken to the laboratory and placed in aquariums holding a 1:3 ratio of sand (0.84 mm particle size) and reconstituted water (0.22 g MgSO4, 0.18 g NaHCO3, 0.08 g KCl and 0.13 g CaSO4.2H2O per liter) (Galar-Martínez et al., 2006). These were then maintained at room temperature (20 a 25°C), with natural light/dark cycles and constant aeration (filtered air, 0.31 l min−1) for 7 days prior to the beginning of the experiment. Ad libitum feeding of specimens during this stage was done as follows: worms were fed a 5% solution of sugarcane molasses in 0.9% sodium chloride; amphipods and snails were fed lettuce.

Single and multi-species test systems

Test systems consisted of polyethylene containers (capacity of 150 mL for single and 300 mL for multi-species test system) with a 3:1 ratio of reconstituted water and IRR sediment (75 mL:25 g), using the same conditions of temperature, light/dark cycle and aeration employed for organism maintenance. Intoxication systems were static and specimens were not fed during exposure.

Single-species test system involved the separate placing of each of the species; while in multi-species test system all three species were placed together.

Acute toxicity assay

Five test systems for each species (single-species test system) and five for the multi-species test system (1 per each concentration tested) were set up and enriched with ZnSO4 (Sigma) at nominal concentrations of Zn2 + as follows: 0.625, 0.735, 1.842, 12.92 and 123.6 mg kg−1. The nominal concentration includes added zinc, as well as zinc present in IRR sediment (0.6129 mg kg−1). A control system with Zn-free sand as sediment, was used for each tested species (1 for single-species test system and 1 for multi-species test system). Systems were mechanically shaken for 2 h until equilibrium was reached (Galar-Martínez et al., 2006) and 10 organisms were placed in each single-species and 30 in each multi-species test system (10 H. azteca, 10 L. hoffmeisteri and 10 S. attenuata). Dead (motionless) specimens were counted after 72 h of exposure. The experiment was performed in quintuple replication. Mean lethal concentration of Zn2 + (LC50 at 72 h) and the 95% confidence limits for this value (p < 0.05) were estimated using computerized Probit analysis (DL50CS.B.I.-IRCT, Montpellier, 1987).

Sublethal toxicity assay

Five test systems for each species (single-species test system) and 5 for the multi-species test system (1 per each time tested) were set up and spiked with 0.2 mg ZnSO4 kg−1 (Sigma) up to a nominal concentration of 0.8129 mg Zn2 + kg−1. A control system with Zn-free sand as sediment was used for each tested species. Systems were mechanically shaken for 2 h until equilibrium was reached (Galar-Martínez et al., 2006) and 1 g organisms were placed in each single-species and 3 g in each multi-species test system (1 g H. azteca, 1 g L. hoffmeisteri and 1 g S. attenuata). The biomass content for each system was 1 g per each 100 mL of water:sediment. Specimens were exposed to the systems for 12, 24, 36, 48 and 72 h. Subsequently, 0.5 g biomass was taken and homogenized in 20 mL phosphate buffer (pH 7), then centrifuged at 12 500 rpm for 15 min at 5°C prior to biochemical analysis namely: total protein content (Bradford, 1976) and lipid peroxidation degree (Buege and Aüst, 1978). The remaining biomass was used for Zn quantification (Eaton et al., 1995).The experiment was performed in quintuple replication. A two-way analysis of variance (ANOVA) was performed on the data and the mean differences of each group were compared with the corresponding control using the Duncan method. Significance was set at p < 0.05.

Results

Acute toxicity assay

The acute toxicity assay showed Zn2 + is more toxic to H. azteca and L. hoffmeisteri exposed in single-species than in multi-species systems (Table 1), as LC50 values increased by 337 and 698 %, respectively. No significant changes were observed in S. attenuate. The sensitivity of H. azteca and L. hoffmeisteri was higher when exposed in single-species systems; while S. attenuata was equally sensitive in both exposure systems.

Table 1.

Acute toxicity (CL50) of Zn-spiked sediments from the IRR, on Hyallela azteca, Limnodrillus hoffmeisteri and Stagnicola attenuata, exposed to single and multi-species systems. CI: 95% confidence interval, p < 0.05.

OrganismSingle-species systemCIMulti-species systemCI
H. azteca 1.75 1.58 – 2.23 7.66 6.98 – 8.0 
L. hoffmeisteri 1.06 0.99 – 1.78 8.46 6.99 – 9.45 
S. attenuata 9.05 8.21 – 11.02 11.61 10.98 – 12.49 
OrganismSingle-species systemCIMulti-species systemCI
H. azteca 1.75 1.58 – 2.23 7.66 6.98 – 8.0 
L. hoffmeisteri 1.06 0.99 – 1.78 8.46 6.99 – 9.45 
S. attenuata 9.05 8.21 – 11.02 11.61 10.98 – 12.49 

Sublethal toxicity assay

Zn concentrations are shown in Figure 1 and Figure 2. At all exposure times for H. azteca, higher Zn concentrations with respect to controls occurred in single-species systems; while in multi-species systems a tendency towards a decrease was obtained, although these responses were not significant (p > 0.05). In L. hoffmeisteri and S. attenuata, Zn concentrations decreased significantly at all exposure times with respect to controls in both test systems (p < 0.05).

Figure 1.

Effect of Zn-spiked IRR sediments on protein concentration in H. azteca, L. hoffmeisteri and S. attenuata exposed to single and multi-species systems. Zn single-species system (), protein single-species system (), Zn multi-species system (), protein multi-species system ().

Figure 1.

Effect of Zn-spiked IRR sediments on protein concentration in H. azteca, L. hoffmeisteri and S. attenuata exposed to single and multi-species systems. Zn single-species system (), protein single-species system (), Zn multi-species system (), protein multi-species system ().

Figure 2.

Effect of Zn-spiked IRR sediments on lipid peroxidation level in H. azteca, L. hoffmeisteri and S. attenuata exposed to single and multi-species systems. Zn single-species system (), protein single-species system (), Zn multi-species system (), protein multi-species system ().

Figure 2.

Effect of Zn-spiked IRR sediments on lipid peroxidation level in H. azteca, L. hoffmeisteri and S. attenuata exposed to single and multi-species systems. Zn single-species system (), protein single-species system (), Zn multi-species system (), protein multi-species system ().

When Zn concentrations in single and multi-species test systems were compared, no significant difference (p > 0.05) was found between the systems in the case of amphipods. In contrast, significant differences occurred with snails at all exposure times (p < 0.05); while significant increases of 67 and 45% were observed in worms at 48 and 72 h (p < 0.05), respectively.

Figure 1 shows the results of total protein content determinations. As can be seen, amphipods had a significant increase with respect to controls at all exposure times in both systems (p < 0.05). On the other hand, snail and worm protein levels decreased significantly in both single and multi-species test systems at all exposure times (p < 0.05).

When the protein content in single and multi-species test systems is compared, levels are seen to increase significantly in H. azteca up to 112 % at 48 h, and to decrease 18% at 72 h (p < 0.05). In L. hoffmeisteri this parameter rose 65 % at 12 h of exposure, while at subsequent exposure times it decreased by 10, 58, 26 and 44 % (p < 0.05). On the other hand, protein content decreased in S. attenuata at all exposure times (p < 0.05), except at 24 h when an increase of 12% was observed (p > 0.05).

LPX levels are shown in Figure 2. Amphipods exposed for 24, 48 and 72 h in single-species systems showed a significant decrease of 63, 97 and 97% respectively compared to controls (p < 0.05). In multi-species test systems, LPX levels increased significantly by 138% at 12 h of exposure, but decreased from 36 h on reaching a maximum decrease of 99% at 72 h (p < 0.05). In worms, a significant decrease with respect to controls occurred in single-species test systems at the three earliest exposure times, while at 48 and 72 h LPX levels increased 41 and 43% respectively (p < 0.05). In multi-species test systems, a significant decrease was found at all exposure times (p < 0.05). Snails exposed in single-species test systems for 12, 24 and 36 h showed a significant increase of 41, 72 and 59% respectively and a decrease of 48% at 72 h compared to controls (p < 0.05). In multi-species test systems, this biomarker increased significantly only at 48 and 72 h of exposure, by 55 and 226% respectively (p < 0.05).

When LPX levels in single and multi-species test systems are compared, this parameter is seen to have increased significantly in H. azteca after 12, 24, 48 and 72 h of exposure (p < 0.05), but to have declined 74% at 36 h. In L. hoffmeisteri, LPX was found to decrease significantly beginning at 36 h, up to 72 h, attaining a maximum decrease of 92% at 36 h (p < 0.05). S. attenuata showed significant decreases of 42, 40 and 39% at 12, 24 and 36 h respectively (p < 0.05). However, a significant increase was observed beginning at 48 h (p < 0.05).

Discussion

In the present study, the LC50 for H. azteca, L. hoffmeisteri and S. attenuata was higher in multi-species than in single-species test systems, i.e. toxicity of Zn-spiked IRR sediment decreases when all three organisms are present. The number of species in the system probably altered the bioavailability of Zn and other contaminants in IRR sediment, due to benthic bioturbation (Ciarelli et al., 2000). Earlier studies in this reservoir revealed the presence of Fe, Ni and Cu (Galar-Martínez et al., 2006), which can interact in synergy with Zn resulting in increased metal toxicity. Favari et al. (2002) also found significant concentrations of organophosphates such as malathion and methyl parathion, as well as chlorinated organic insecticides such as aldrin, dieldrin and endrin, which are very toxic to organisms in the reservoir due to their persistence and mechanisms of action.

Anderson et al. (2001) evaluated the quality of bay sediment in Long Beach, California, showing that survival rates in the amphipods Rhepoxynius abronius and Eohaustorius estuarius changed depending on the total number of species present in the benthos of these substrates. Similarly, Sibley et al. (2001) found changes as to type and ratio of species in cladocerans, rotifers and copepods exposed to liquid creosote in multi-species systems.

In the acute toxicity assay, species sensitivity varied depending on the test system used. This test suggested that S. attenuata was equally sensitive in both exposure systems; while the sensitivity of L. hoffmeisteri and H. azteca was higher when exposed to the single-species test system. Probably, when all three organisms were present in the sediment, Zn as well as some other pollutants from the IRR sediment were removed, becoming part of the water column and decreasing their bioavailability in sediments. A second possibility is that pollutants were biotransformed into less toxic forms. A study by Fuma et al. (2000) showed that the alga Euglena gracilis exposed separately to Mn was more sensitive than either the bacteria Escherichia coli or the protozoan Tetrahymena thermopila. However, when these three species were exposed together the bacteria and protozoan showed higher sensitivity. The latter authors assume that the bacteria and protozoan are able to reduce the levels of bioavailable Mn by transforming it into a less toxic form, or by chelating it with metabolites or waste products of T. thermophila or E. coli.

Aquatic animals use two strategies to regulate essential metal body concentrations. One of these is active regulation, in which organisms (active regulators) are able to maintain relatively constant metal concentrations by coordinating their excretion and uptake rates. This means of regulation is probably limited to certain essential metals such as Zn and Cu (Muyssen and Janssen, 2002). Another strategy to regulate essential metal body concentrations is the process of storage/detoxification, in which organisms (net accumulators) have specific tissues that accumulate and detoxify these xenobiotics.

In the present study, Zn concentrations decreased with respect to controls in snails and worms exposed in both single and multi-species test systems. It is possible that these organisms are net accumulators and, in the presence of high concentrations, Zn excretion is favored via accumulation/detoxification or sequestering in the snail shell. Another possible explanation of this occurrence is, as mentioned earlier, that other metals present in IRR sediment, such as Fe, are able to modify Zn uptake and regulation. Prusod and Beck (1996) showed Fe at high concentrations decreases Zn intake in different organisms, thus favoring Fe uptake. Pawlik-Skowronska et al. (2007) state simultaneous exposure to diverse metals may significantly alter the uptake and accumulation of some of them, and these interactions between metals are species dependent.

In our study on the other hand, metal loss was higher when both organisms were in single-species test systems. A study by Ciarelli et al. (2000) showed that fluoranthene uptake in the polychaete Nereis virens decreased with introduction of the amphipod Corophium volutato.

In contrast to results with S. attenuata and L. hoffmeisteri, Zn concentrations in H. azteca remained constant in both systems and showed no significant differences with respect to controls. The latter is probably an active regulator. Rainbow and White (1989) found that the amphipod Echinogamarus pirloti showed few changes in net body Zn accumulation even with increased bioavailability of this metal.

With regards to total protein content, concentration increased with respect to controls in H. azteca at all exposure times in both single and multi-species test systems. Various studies have shown that stress protein synthesis and metallothionein synthesis increase with exposure to different metals, including Zn (Pawlik-Skowronska et al., 2007; Franco et al., 2006). In the present study, amphipod Zn concentrations were not modified in relation to exposure time in either of the test systems. However, previous studies found Fe, Ni and Cu to be present in IRR sediment (Galar-Martínez et al., 2006), thus making it likely that the synthesis of these biomolecules was induced. Zn is a cofactor of various enzymes involved in protein synthesis, such as DNA and RNA polymerase (Geret and Bebianno, 2004; Kendrick, 1992). In snails and worms exposed to Zn-spiked IRR sediment, total protein content decreased with respect to controls in both single and multi-species test systems at all exposure times, and Zn loss also occurred. Lack of the metal probably inhibited the synthesis of these biomolecules. On the other hand, presence of other contaminants such as organophosphates in IRR sediment must be taken into account. These compounds have been shown to be capable of reducing protein content in L. hoffmeisteri and S. attenuata (Martínez-Tabche et al., 2002; Martínez-Tabche et al., 2000).

In all three test organisms, behavior was similar in regard to protein content with both systems, although a more pronounced effect took place in multi-species test systems. This may be a result of the fact that when all three species are present together, microenvironmental changes are produced at pH, carbon, ammonium and organic matter concentrations, as well as potential production of toxic metabolites that can modify toxic response. Furthermore, Ciarelli et al. (2000) mentioned that benthic bioturbation has an effect on physical and chemical characteristics of sediments, pore water and overlaying water, affecting the fate and partitioning of sediment-bond contaminants in sediment profiles, pore water and water columns.

Reactive oxygen species (ROS) such as superoxide (O2), hydroxyl radicals (OH.) and hydrogen peroxide (H2O2) are produced in cells as a result of exposure to different contaminants including heavy metals (Li et al., 2006). ROS induce cell damage by promoting lipid peroxidation of membranes. To counteract their effects, the cell requires antioxidants such as glutathione peroxidase (GPX), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) (Sinha et al., 2007). It is important to point out that in addition to ROS generation heavy metals have a high affinity with thiol groups, so that they are able to link to these enzymes altering their structure and antioxidant function.

One of the most important roles of Zn is related to its antioxidant properties, and deficiency of the metal leads to oxidative damage (Geret and Bebianno, 2004). However, different studies have shown that increased LPX occurs in organisms such as the fish Leporinus obtusidens, the microalga Pavlova viridis and the clam Ruditapes decussatus exposed to Zn at high concentrations (Gioda et al., 2007; Li et al., 2006).

In our study, LPX behavior was similar in H. azteca exposed to Zn-spiked IRR sediment in both systems. This parameter increased at 12 h of exposure and decreased beginning at 24 h, with respect to controls. In multi-species test systems however, a significant increase was observed at 72 h. In this organism, Zn concentrations remained constant throughout the study in both systems, with respect to controls. However, as was already mentioned, IRR sediment contains other metals, such as Fe, Ni and Cu as well as organophosphate pesticides which may be responsible for the increase in LPX at the earliest exposure times (Li et al., 2006; Martínez-Tabche et al., 2002). Decrease of this biomarker from 24 h on may be ascribed to stimulation of production of antioxidant enzymes such as GPX, CAT and SOD, which can be modulated under stress conditions linked with exposure to heavy metals (Franco et al., 2006; Geret and Bebianno, 2004). In our study, total protein content increased, which allows us to postulate this assumption. Franco et al. (2006) found a similar effect in gill of Perna perna exposed to Zn, where the increase in LPX was followed by a rise in the activity of these enzymes, related to an adaptive-like response. The increase in multi-species test systems at 72 h of exposure may be due to benthic bioturbation affecting the fate and partitioning of sediment-bond contaminants, as well as potential production of toxic metabolites that can modify toxic response.

Worms showed a significant decrease in LPX levels at all exposure times in multi-species test systems. Singh and Khangarot (2002) state this organism has a high tolerance to heavy metals and other contaminants. It may therefore be that the antioxidant defense system of the worms went into action. In contrast, LPX levels decreased in single-species test systems up to 36 h of exposure, but from 48 h on a tendency to increase is seen, possibly due to reduced antioxidant enzyme activity, since both protein content and Zn levels declined.

In S. attenuata, this biomarker showed similar trends in both test systems. In single-species test systems a significant increase with respect to controls occurred until 48 h of exposure, while in multi-species test systems, although LPX decreased significantly until 36 h, a significant increase took place from 48 h on. In this organism, as was also the case in the worm, both protein content and Zn concentrations decreased. This is consistent with results obtained by Campana et al. (2003), who found increased LPX in blood, kidney and liver of Halobatrachus didactylus as a result of exposure to metals, as well as with data obtained by Geret and Bebianno (2004), who found an increase in this biomarker in R. decussatus exposed to Zn.

Conclusions

In conclusion, Zn-spiked sediments from IRR were toxic to S. attenuata, L. hoffmeisteri and H. azteca. This toxicity could be attributed to spiked Zn and other pollutants in the IRR sediments. Nevertheless, the toxic response (acute and sublethal) was modified depending on the test system used (single or multi-species). These differences may be due to multiple factors, such as benthic bioturbation produced by the presence of more than one species in the system, which may affect the fate and partitioning of sediment-bond contaminants, such as spiked Zn or other pollutants in IRR sediment. In addition, the presence of more than one species in the system may lead to microenvironmental changes (pH, temperature, and organic matter and metabolic waste content), which can also contribute to toxic response differences. Also, particular characteristics of each of the species involved in the study became evident. The snail and amphipod are facultative benthic organisms, while the worm is obligate. Such way of life differences may have modified the bioavailability of contaminants, determining their toxicity.

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