Influence of copper on Euplotes sp . and associated bacterial population

The influence of copper on the ciliate Euplotes sp. and associated bacteria isolated from sediment samples of Guanabara Bay were investigated in bioassays. This region is highly affected by heavy metals such as copper, from solid waste constantly dumped in the bay and other sources such as industrial effluents, antifouling paints, atmospheric deposition and urban drainage, and even today there are few data on the metal toxicity to the ecosystem of the Bay of Guanabara. Bioassays were conducted to estimate the LC50– 24 h of copper, in order to determine the concentration of metal bearing 50% of the population mortality. The results indicated that the concentrations of 0.05 and 0.009 mg L presented no toxicity to Euplotes sp. The associated bacteria are tolerant to copper concentrations used in bioassays, and suggest that they could be used as a potential agent in the bioremediation of areas affected by copper.

The microbial loop is mainly composed by bacteria, microflagelates and ciliates that have been studied primarily in oligotrophic environments.The main roles of microorganisms are recycling organic matter and, transforming dissolved organic matter (DOM) in particulate organic matter (POM) or vice versa (Paves & González, 2008).The DOM released by autotrophs is consumed by heterotrophic bacteria and transformed in growth.This DOM is rapidly converted into POM by bacteria, which is readily preyed by protists.So, nanoflagellates can control this process through pasture, converting the heterotrophic bacterial produc-tion in POM greater than 2.0 µm which is accessible to larger protists, as ciliates and dinoflagellates, and metazoans (Huppert et al., 2004).Thus, protists such as ciliates and nanoflagellates act as an efficient link in the microbial loop and the classic food chain (Fenchel, 2008).
Pollution due to chemicals, including heavy metals, is a problem that may have negative consequences on the biosphere.Heavy metals as copper, particularly from industrial effluents, are constantly polluting our environments (Rehman et al., 2006).These metals, depending on their concentration, may be considered harmful to exposed organisms, since they influence their growth processes, cell morphology, metabolism and function of plasma membrane and enzymes.Copper is considered a toxic element which may affect most of freshwater and marine invertebrates.However, it is also an essential micronutrient for bacterial metabolism, participating in energy production (Mayor et al., 2013).
The ciliate Euplotes sp. is a dorsoventrally flattened organism placed in the Spirotrichea, with prominent ventral cirri and less conspicuous dorsal bristle cilia in the Order Hypotrichida (Lynn, 2007).The genus is broadly distributed as both symbionts and free-living forms that occupy a wide range of habitats from freshwater to brackish and marine, in sands and soils, and edaphic habitats.They are typically benthic substrate-oriented, and have also been recorded in tropical litoral habitats (Dragesco & Dragesco-Kernéis, 1986).Presently, we have few data about the copper effects on Euplotes sp. and the associated microbiota in eutrophic environments.This paper aims to study the influence of copper on Euplotes sp. and associated population of bacteria isolated from sediment samples in Guanabara Bay.

Study area and sampling
Guanabara Bay is a 384 km 2 eutrophic coastal bay in the southeast of Brazil, which receives domestic untreated sewage from at least 10 million people, most of the discharge occurring directly into the bay (Kjerfve et al., 1997).Samples of Euplotes sp. were collected in sediment close to Ilha do Fundão (22°51'31''S, 43°13'14''W), in Guanabara Bay, and maintained in the laboratory to run toxicity tests in liquid medium containing natural seawater and rice grains as the carbon source (Dragesco & Dragesco-Kernéis, 1986).This culture medium was also used for maintenance and growth of associated bacterial microbiota.

Heavy metal exposure analysis
For the bioassay, 15 batches of 30 specimens of Euplotes sp. were prior maintained for 72 h in Petri dishes, with sterile seawater, without the addition of carbon source to reduce their microbiota and without food supply.After this period, healthy and motile free living individuals of Euplotes sp. were transferred to wells of in a sterile 24-well polystyrene plate.Each well was filled with 2.0 mL of seawater.The different treatments were prepared adding 0.001, 0.009, 0.05, 0.01 and 1 mg L -1 of a copper solution, which was taken from a stock solution of 1.0 M CuSO 4 .No copper was added to the control, which had seawater only.The bioassay occurred in triplicate with 30 Euplotes sp. in each well, and the plates were incubated at 25ºC for 24 h, without supplementary nutrients.
To determine the LC 50 three replicates were performed.Previously, 1.0 mL of each well was taken and 0.05 mL Bouin's fluid was added to fix cells (Foissner, 1991).Fixed and non damaged specimens of Euplotes sp. were quantified using a Sedgwick-Rafter counting chamber at 0 and 24 h, performed according to the standard CETESB (2005).The bacterial carbon was quantified in 1.0 mL of each well under an epifluorescent microscope, according to Kepner & Pratt (1994), and Carlucci et al. (1986).
Soluble metal concentration was measured using an inductive coupled plasma emission spectrometer (ICP-AES) sequential-multichannel Varian Ultra Mass, calibrated by direct comparison with standard solution of copper, considered as certified reference materials (Rauret et al., 1999).The survival data was treated by Probit analysis in order to estimate LC 50 -24 h of copper for Euplotes sp.(Diaz et al., 2006).After 24 h, the growth of Euplotes sp.occurred at 0.05 and 0.009 mg L -1 of copper, and had no growth with 0.1 and 1.0 mg L -1 of copper (Fig. 1a).The control group showed a slight decrease of Euplotes sp., with an increase in carbon of associated bacteria.The bacterial carbon ranged between 1.0x10 -4 and 4.5x10 -4 µg C cm -3 (Fig. 1b).The increase of Euplotes sp.number at 0.009 and 0.05 mg L -1 of copper favored the reduction of associated bacteria and there was no increase of bacterial carbon in presence of 0.1 and 1.0 mg L -1 of copper.The estimated rate of the Probit LC 50 was 0.04 mg L -1 to the Euplotes sp. cells counting.
In the control group, predator and prey functioned as expected in a microbial loop (Fenchel, 2008).Despite the increasing number of Euplotes sp. in the concentrations of 0.009 and 0.05 mg Cu L -1 , bacterial carbon also increased, since the associated microbiota may use defense mechanisms under stressed environments, thus explaining the increase of the bacterial population in this case (Matz & Kjelleberg, 2005).Bacterial self-defense includes production of biofilm and changes in size proportions.
Biofilms enhance the bacterial proportions in a way that the aggregates may be larger than the Euplotes peristome.This mechanism/strategy protects bacteria from predators and enables them to increase their biomass (Matz & Kjelleberg, 2005).At concentrations of 0.1 and 1.0 mg L -1 of copper, the associated bacteria showed no increase of carbon, even in the absence of grazing and nutritional supplements.Therefore, it can be inferred that the microbiota associated with Euplotes sp. may be tolerant to the higher concentrations of copper used in bioassays.
According to Harrison et al. (2007), the ability of microorganisms to survive but not grow in the presence of metals, metalloids cations or oxyanions, either alone or in combination, defines tolerance.Bacteria may be able to grow vigorously and rapidly in the medium with copper (Okafor, 2007), using nitrogenous excreta from protists (Kirchman, 2000;Hahn & Höfle, 2001).This ability enhances the relative proportion of bacterial biomass and it influences the metabolic activity.The increased biomass improves the enzymatic contact and exchanges with the medium in a more intense way, making bacteria prone to absorb Cu quickly.Bacteria tend to invest in the maintenance of their structures to face a stressful environment.As a consequence, they increase their metabolism, but maintain their biomass (Harrison et al., 2007).
After the addition of copper at concentrations of 0.05 and 0.009 mg L -1 a cell division occurred in Euplotes sp., since it seems to act as a micronutrient in lower concentrations, possibly contributing to maintain the predator-prey relationship.However, in higher concentrations, toxic effects may have occurred in Euplotes sp.The estimate LC 50 Probit test indicates that the concentration would lead to a fatality of 50% of the population would be 0.04 mg L -1 .This concentration was below that stipulated by the National Council of the Environment to saline waters which may be intended for primary contact recreation, as CONAMA Resolution 274/2000 (CONAMA, 2011), 1.0 mg Cu L -1 for marine environments.
Analyzing other studies, we observed that the tolerated concentrations of metals depend on the study site and species examined.For example, throughout 1991-1992, 29 ciliate species have been isolated from an activated-sludge plant in Manchester, U.K., and showed resistance to 60 ppb of copper (Abraham et al., 1997).In bioassays performed with heavy metals, the population of Colpoda steinii, a soil ciliated, was reduced by 50% in the presence of 0.10, 0.22, 0.25, and 0.85 mg L -1 of Ni, Cd, Cu and Zn respectively (Forge et al., 1993).The copper concentration 1.58 mg L -1 inhibited 50% growth of the Euplotes crassus population in bioassays during 48 h, as well the resistance to Euplotes vannus with 0.2 mg L -1 (Kim et al., 2011).According to Rehman et al. (2006), protozoa, specifically Euplotes mutabilis and Tachysoma pellionella used in media containing copper, lead, mercury and chromium were resistant to small amounts added daily (1.0 µg mL -1 ).These results compared to our bioassays suggest that Euplotes sp. is resistant to 0.001 to 0.05 mg L -1 copper, because the ciliates continued to grow in the presence of the metal (Harrison et al., 2007).
In the same way that copper is an important factor in the cells, it may also be harmful, promoting various deleterious effects such as changes in membrane due to depolarization and damage on receptor or carriers molecules, functional damage by their binding to macromolecules such as DNA and enzymes, forming protein damage or oxidative alterations of DNA and thereby causing multiple functional alterations (Bremner, 1998).In addition, it may cause cell damage due to the production of free radicals by Fenton reaction leading to a loss of cell integrity, decreased ATP synthesis, oxidation and lipid peroxidation, DNA damage and damage to vital organelles such as mitochondria and lysosomes (Bremner, 1998).A major concern is that environments such as Guanabara Bay are being threatened by the constant release of effluents containing heavy metals.In some sampling sites of Guanabara Bay concentrations of copper as high as 60 mg kg -1 have already been recorded, which undermine the self-sustaining of the ecosystem (Fonseca et al., 2013).According to Meyer-Reil & Köster (2000), when a eutrophic ecosystem suffers a disturbance, it goes through a period of resilience that is the time (speed) that an ecosystem needs after disturbance to return to the initial status.However, in the case of the Guanabara Bay and other ecosystems, the resilience capacity seems to decrease in response to long-term disturbances, which can lead to an impairment of the natural environmental self-sustaining.

CONCLUSIONS
The Cu concentrations of 0.05 and 0.009 mg L -1 were not harmful to Euplotes sp., and according to Harrison et al. (2007) they might also confer resistance to copper, i.e., the ability of a microorganism to continue growing in the presence of metals like copper.However, at concentrations higher than 0.05 mg L -1 this metal becomes toxic to Euplotes sp.The associated bacteria are tolerant to all copper concentrations used in bioassays, but keeping the bacterial carbon constant in the concentrations tested for the bacterial population of the Guanabara Bay sediments.

Figure 1 .
Figure 1.a) Number of individuals of Euplotes sp.under different Cu concentrations at the beginning (0 h) and the end (24 h) of the experiment, and b) bacterial carbon variation in function of different Cu concentrations at beginning 0 h and end 24 h of the experiment.Error bars denote standard deviation.