Acute toxicity of potentially toxic elements on ciliated protozoa from Lake Maracaibo

toxicity

The potential of zooplanktonic organisms as bioindicators in toxicity tests has been well referenced (Vilas-Boas et al., 2020;Gomiero et al., 2012;Gomiero et al., 2013;Mortimer et al., 2010;Qing-Hua et al., 2008).The main advantage of using them in ecotoxicological research is to show toxic effects at individual level and its subsequent impact on higher levels of biological organization, naturais e de atuar em tempo hábil (sistemas de alerta precoce) em eventos de contaminação de EPT, bem como o desenvolvimento de métodos de biomonitoramento sensíveis e rápidos para a detecção desses elementos no meio ambiente.
elements were selected because to its appreciable magnitude in water, sediment and biota, as well as numerous anthropogenic sources of PTE in the hydrographic basin of this water body.The toxicity tests at short exposure times could serve as a practical and rapid tool to generate short-term results and, in this way, provide the opportunity to act on emergency situations or those that deserve immediate legal action.In this regard, certain investigations with exposure times of 1-h have been carried out, for example, to propose protozoan species as bioindicators of water quality (Nalecz-Jawecki et al., 1993) or to understand the ecological effects caused by stress in the presence of PTE (Blanck & Wängberg, 1988).

Study area
Lake Maracaibo is located in the West of Venezuela, Zulia state, between 70°30' and 73°24' W and 09°00' and 10°30' N (Figure 1).It is connected to the Gulf of Venezuela by a strait of 55 km long at North and fed by some 135 rivers, of which the most important are Catatumbo, Chama, Santa Ana and Escalante (South region).The lake has 13,210 km 2 of surface area, 95,923 km 2 of drainage, 1485.2 km of coastline and is one of the oldest on Earth.It has an altitude of 3 m and a maximum depth of 34 m (Rodríguez, 2000;Parra-Pardi, 1979).

Isolation and culture of ciliated protozoa
Small aliquots of water samples were observed under optical microscope (5X) to select the most abundant free-living ciliated protozoa using a capillary tube.The organisms were grown at an average temperature of 30.5 ± 1.5 °C in glass jars with pre-filtered lake water (0.22 μm) and oats to form oat liquor, and thereby promoting bacterial growth (Fried et al., 2002).The most abundant genera in the water samples were Urodema sp.(82.3%),Euplotes sp.(12.4%) and Loxodes sp.(5.3%), so they were used in the toxicity experiments.Microbial cultures were renewed every 48 h, centrifuged and observed under optical microscope to select organisms of the same species, using a capillary tube.These organisms were planted in fresh culture medium (per liter distilled water: 0.125 g K 2 HPO 4 , 0.025 g NH 4 Cl, 0.4 g NaCl, 0.2 g MgCl 2 .6H 2 O, 0.15 g KCl and 0.25 g CaCl 2 .2H 2 O), until a monospecific cultivation of ciliates (Narayanan et al., 2007).

Taxonomic identification of ciliated protozoa
Cultures were, initially, stained with lugol to detail the internal and external structures of the specimens, such as: location and number of cirrus in different regions, morphology of the individual, location of cytostome contractile vacuole and then stained with methyl green and safranin, to observe the morphology and location of the nuclei (Lynn, 2008;Foissner, 1999;Foissner & Berger, 1996).Taxonomic identifications were made under the light microscope through specific taxonomic keys.The dichotomous keys allowed to verify the following distinctive structural characteristics: i) Uronema sp.: oval to ovoid morphology, elongated, slightly flattened, narrow preoral groove leads from the frontal plate to mouth at middle, inconspicuous peristome, ciliated right border, cytostome on the ventral side near the left border in the anterior half, confused cytopharynx, central spherical macronucleus and terminal contractile vacuole; ii) Euplotes sp.: inflexible ovoid body with flattened ventral surface and convex dorsal surface, longitudinally wrinkled, wide and triangular peristome, absence of frontal cirrus, frontal part of the adoral area in a flat groove, nine or more ventral frontal cirri (five anal and four scattered caudal), band-shaped macronucleus, a micronucleus, and posterior contractile vacuole; and iii) Loxodes sp.: larger, anterior part with beak-shaped curved end, mouth at posterior end of a concave indentation near the front of the cell, surface covered with a membrane bound with granules containing a yellow-brown pigment, a large part of the cell interior with mineral inclusions located in specific positions on the dorsal border and absence of contractile vacuole (Paiva et al., 2013;Lynn, 2008).
Acute toxicity bioassays were conducted in Sedgewick-Rafter plates, in triplicate, according to APHA (2017).Each chamber had 1 mL of test solution and of 18 to 22 organisms.The exposure time to the PTE solutions was 1-h, during which counts and microscopic observations were performed every 5 min (Figure 2).After this time, the number of ciliated protozoa dead (non-moving) was determined (Girling et al., 2000).Toxic elements of ciliated protozoa… Acta Limnologica Brasiliensia, 2022, vol.34, e21

Statistical analysis
For descriptive statistical calculations (arithmetic mean, range, and standard deviation), the program Microsoft Excel version 10 was used.LC 10 , LC 50 and LC 90 calculations (including confidence limits 95%) for PTE were performed with the program PriProbit version 1.63.Two-way analysis of variance (ANOVA) with Tukey test a posteriori was used to determine statistical significance of the differences between LC 50 values considering the element and the protozoa (genera), whereas the level of significance was accepted at p<0.05.These analyses were performed using the program SPSS version 20.0.
The 1-h LC 50 values for ciliated protozoa from Lake Maracaibo ranged between 0.14 mg.L -1 (Cd(II), Loxodes sp.) and 213.59 mg.L -1 (Cr(VI), Uronema sp.) (Table 1).The LC 10 and LC 90 values have been included to relate them to the concentrations that could cause minor effects on the population and those that would have a greater effect on it, respectively.
The two-way ANOVA showed highly significant differences (p<0.001) of the LC 50 values with respect to the genera of ciliated protozoa (p<0.001,F value = 98.00), to the PTE (p<0.001,F value = 1271.70)and for the interaction element*protozoan (p<0.001,F value = 119.86).The Tukey test revealed three independent groups (p<0.05) with the three genera of ciliated protozoa (Table 2).Thus, the toxic effect of the elements on ciliated protozoa was Cd(II) = V(V) > Cr(III) > Cr(VI) (Table 1).Loxodes sp. was the protozoan most sensitive to Cd(II) and V(V), while Uronema sp. was the most tolerant to Cr(VI), however, Euplotes sp.showed the highest sensitivity to the set of PTEs tested (1-h mean LC 50 30.77mg.L -1 ) (Figure 6).

Discussion
In this work, the adverse toxic effects of the PTEs tested on four genera of protozoa isolated from surface water samples from Lake Maracaibo were confirmed, allowing the estimation of the LC 50 for an exposure time of 1-h and with the following pattern: Cd(II) = V(V) > Cr(III) > Cr(VI).

Toxicity of potentially toxic elements on ciliated protozoa
In aquatic organisms, in general, Cr(III) is less toxic than Cr(VI), mainly due to: weak permeability in the cell membrane, use as a trace Table 1.Mean lethal concentration (LC) and confidence limits 95% (in parentheses) of potentially toxic elements (mg.L -1 ) to ciliated protozoa of Lake Maracaibo (exposure time 1-h, n= 3).*: the difference in means is significant at the level 0.05.element in metabolic processes, greater stability and low oxidizing power, between other factors; while Cr(VI) is more active in penetration through passages for isoelectric and isostructural anions (Hong et al., 2020;Jaishankar et al., 2014;WHO, 2009;CCME, 1999).Inside the cell, reactions between Cr(VI) and biological reducers such as thiols and ascorbate, result in the production of reactive oxygen species such as superoxide ion, hydrogen peroxide and hydroxyl radical, which in ultimately leads to oxidative stress in the cell that damages DNA and proteins (Jaishankar et al., 2014;Rico et al., 2009).However, in the isolated protozoa from Lake Maracaibo, the trivalent form of chromium showed a higher degree of toxicity in laboratory tests carried out (Figures 1, 2 and 3), possibly due to: solubility and mobility of chemical species (WHO, 2009;Panda & Choudhury, 2005), water hardness (USEPA, 1985; Pickering & Henderson, 1966), water pH (Aslam & Yousafzai, 2017), age of organisms (Guertin, 2005), use of non-specific salts (Vignati et al., 2010), direct effects on DNA (Fang et al., 2014;Plaper et al., 2002), genetic variations (Martín-González et al., 2006), among others.
Vignatiet al. ( 2010) reported acute toxicities of 0.65-0.81mgCr(III).L -1 and 1.58-2.24mgCr(VI).L -1 for the microalgae Pseudokirchneriella subcapitata, emphasizing that Cr(III) can be more toxic to certain aquatic microorganisms, even at concentrations below current environmental quality criteria and within the concentration range typically observed in aquatic systems impacted by industrial discharges.Also, it has eventually been observed that fish may be more sensitive to Cr(III) than to Cr(VI) (Aslam & Yousafzai, 2017;WHO, 2009), being able to generate tissue damage, including hyperplasia, toothpick formation in the gill lamellae and necrosis, as well as impaired ability to osmoregulate and breathe (Moore, 1991).
Several authors have shown the toxic effect of Cr(III) on various aquatic organisms, for example: the microcrustacean Daphnia magna with an acute toxicity of 22 mg.L -1 (Kuhn et al., 1989), the protozoan Tetrahymena pyriformis with 50-62 mg.L -1 (Sauvant et al., 1995), the microalgae Chlorella kessleri with 0.08-0.12mg.L -1 (Vignati et al., 2010), the fish Cyprinus carpio with 97.7 mg.L -1 (Wong et al., 1982), and the amphipod Crangonyx pseudogracilis with 291 mg.L -1 (Martin & Holdich, 1986).Similarly, in heterotrophic bacteria from Lake Maracaibo, a greater ecotoxic effect of Cr(III) was found compared to Cr(VI) in the sampling site of the present study (Lake Park Vereda), possibly due to spatial variability in terms of PTE sources' contribution in the basin and its effect on local microbial populations (Castro & Marín, 2018).
Regarding cadmium, it has been reported that protozoa can bioaccumulate between 0.05 and 332.75 mgCd.kg - dry weight (Fernandez-Leborans & Olalla Herrero, 2000), with percentages between 13.5 and 86.4% of the Cd available in their environment (Martín-González et al., 2006).In aquatic environments, Cd(II) shows relative mobility, which depends on pH, alkalinity, temperature, presence of organic molecules and water hardness.Thus, marine organisms are more resistant to Cd poisoning than those living in a freshwater environment (Borsari, 2014;CCME, 2014).Cd indirectly generates reactive oxygen species and ultimately increases oxidation of proteins, lipids, and DNA in cells, leading to DNA damage and tumor growth (Liu et al., 2009;Wang et al., 2004).It can also block the calcium absorption in water (CCME, 2014), also exhibiting embryotoxic, teratogenic and mutagenic properties (Sanders, 1986).
On the other hand, the ecotoxic effects of vanadium come mainly from the similar structure of vanadate (VO 4 3-, oxidation state +5) and o-phosphate and, therefore, from interference with the function of biomolecules containing P, turning out to be the most toxic, soluble and stable form of vanadium in the environment (Gustafsson, 2019;Mannazzu, 2001).Furthermore, vanadium appears to interfere with different enzymes such as ATPases, protein kinases, ribonucleases, and phosphatases (Mukherjee et al., 2013).Some examples of the acute toxicity of pentavalent vanadium on aquatic organisms are: Pseudomonas putida (bacteria) 180-200 mg.L -1 , Trachelophyllum sp.(protozoa) of 120-130 mg.L -1 (Kamika & Momba, 2014), D. magna (microcrustacean) of 1.2 mg.L -1 (Okamoto et al., 2015) and Salmo gairdneri (fish) of 2.0-13.2mg.L -1 (Stendahl & Sprague, 1982).In the case of Lake Maracaibo, an ecotoxicity of V(V) for P. solida of 401.96 mg.L -1 has been documented; this bivalve has been proposed as a bioindicator for PTE in this ecosystem, due to its ability to incorporate, tolerate and bioaccumulate elements in higher concentrations and proportional to exposure levels (Rojas, 2012).
The LC 50 values observed for the ciliated protozoa of Lake Maracaibo, in general, are relatively high and comparable to those of other water bodies (Maurya & Pandey, 2020;Kamika & Momba, 2014;Madoni & Romeo, 2006;Madoni et al., 1996).Madoni & Romeo (2006) reported toxic effects of 0.30, 0.89, 0.59 and 0.07 mgCd(II).L -1 , and of 110, 108, 0.1 and 0.1 mgCr(VI).L -1 for the ciliated protozoa Dexiotricha granulosa, Colpidium colpoda, Euplotes aediculatus and Halteria grandinella, respectively, showing great differences in tolerance, as observed in other species of ciliates.These variations make it difficult to define an absolute scale of PTE toxicity for aquatic organisms, but they establish a greater sensitivity of ciliated microorganisms compared to invertebrate metazoans.Kamika & Momba (2013), for their part, highlighted the synergistic effect of the combination of V(V) and Ni(II), increasing toxicity and impairing the microbial survival and its ability to eliminate PTE, due to presence resistant genes and microbial diversity in the environment.
Tolerance and adaptability processes are related to prolonged exposure to PTEs (Poirier et al., 2013;Gadd, 2010;Martín-González et al., 2006), which could be occurring in the Lake Maracaibo basin, as a result of constant discharge of large volumes of untreated industrial wastewaters (e.g.petrochemicals, gas processing, oil production and transportation, tanneries, metal-mechanics, among others), especially in the Strait of Maracaibo area, as well as the activity of approximately 10,000 oil drilling platforms located in the lake central area (CGR, 2010;Rodríguez, 2000;Parra-Pardi, 1979).In this sense, Martín-González et al. (2006) pointed to bioaccumulation as an important resistance mechanism for these xenobiotic substances in the environment, possibly being mediated by the presence of metallothioneins.Bioaccumulation in biota negatively affects the health of exposed organisms and their consumers.PTEs are transferred from the abiotic environment to living organisms (bioconcentration) and accumulate in biota, causing contamination of food chains with these elements.PTEs can enrich successive trophic levels of food webs, leading to their biomagnification.In this way, the bioconcentration, bioaccumulation and biomagnification of PTE in food chains have important implications for wildlife and human health (Ali & Khan, 2018).
Another mechanism that promotes tolerance to the PTE presence in aquatic environments is microbial reduction.When an element is microbially reduced to a lower redox state, its mobility and toxicity can be greatly decreased (Gadd, 2010).In this way, the bacteria-protozoan association could allow the survival of non-tolerant communities by reducing toxic forms of elements to more innocuous ones.In this regard, Kamika & Momba (2014) revealed that the V reduction capacity, adopted by Pseudomonas putida, can be an effective strategy to eliminate V from polluted environments.
The higher relative sensitivity of Euplotes sp. to the PTE tested (Table 1, Figure 4) makes it look like a possible bioindicator for PTE contamination in the Lake Maracaibo.The high sensitivity of ciliated protozoa to toxic compounds, coupled with their structural simplicity (unicellular models), ease of culture, physiological changes, behavioral activity, responses of cell organelles and short generational period (Maurya & Pandey, 2020;Gomiero et al., 2013;Mortimer et al., 2010;Qing-Hua et al., 2008;Martín-González et al., 2006) makes them good candidates for use as bioindicators, biosensors, and models for ecotoxicity studies.

Toxic effect of potentially toxic elements at different exposure times on ciliated protozoa
Acta Limnologica Brasiliensia, 2022, vol. 34, e21 Comparing the toxic effects of chemicals on ciliated protozoa is a complicated matter, due to the differences between experimental conditions used in studies (Amaro et al., 2011;Rico et al., 2009;Madoni & Romeo, 2006;Madoni et al., 1996;Sauvant et al., 1995;Simanov, 1987).While most research is conducted using a 24-hour exposure time; shorter periods offer the opportunity to better assess the existence of oxidative stress (Rico et al., 2009;Nalecz-Jawecki et al., 1993), demonstrate the early effects of toxicity on natural populations (Sauvant et al., 1995), act promptly (early warning systems) in contamination events by PTEs (Isibor et al., 2020), as well as development of sensitive and rapid biomonitoring methods for PTE detections in the environment (Amaro et al., 2011;Blanck & Wängberg, 1988).In this sense, Mortimer et al. (2010) reported that the toxic effects of copper compounds on the ciliated protozoan Tetrahymena thermophila did not depend on the exposure time (4 and 24 h), while the toxicity of zinc compounds was approximately 1.5 times lower after 24-h of exposure than after 4-h, probably due to adaptation.These findings support the need to further study the influence of exposure time in PTE toxicity tests on ciliated protozoa, in order to establish the most suitable periods of experimentation and achieve similar conditions to carry out the laboratory tests.Also, Nalecz-Jawecki et al. (1993) suggest that the tests with short exposure periods (1-h) allow to observe the adverse effects in a more rapid and sensitive way before the death of organism occurs, also pointing out that the effective concentration values (1-h EC 50 ) of the protozoan Spirostomum ambiguum for Hg(II), Ag(I), Cu(II) and Cd(II) were almost the same as the 24 and 48-h LC 50 .
The acute toxicity data (1-h LC 50 ) for Cr(III), Cr(VI), Cd(II) and V(V) of the ciliated protozoa of Lake Maracaibo are comparable to those reported for other water bodies at different exposure times (Table 3).Euplotes sp. and Loxodes sp. from Lake Maracaibo are the organisms most sensitive to Cr(III) and V(V), while Euplotes aediculatus (Garda Lake, Italy) (Madoni & Romeo, 2006) -Jawecki et al., 1993) for Cd(II) and Peranema sp.(wastewater, South Africa) (Kamika & Momba, 2014) for V(V).In general, these data suggest large differences in the levels of sensitivity and tolerance of ciliated protozoa to PTEs, regardless of the exposure time in laboratory tests.Finally, the need for further research on the influence of exposure time in both acute and chronic toxicity tests with ciliated protozoa is pointed out, considering that this variable is critical in the response of these organisms in the presence of chemical contaminants such as PTE.This response is also dependent on the natural condition to which the organism is accustomed in its ecosystem.

Conclusions
The PTE Cr(III), Cr(VI), Cd(II) and V(V) revealed a toxic effect on ciliated protozoa isolated from Lake Maracaibo.The performance of Euplotes sp.makes it a sensitive and convenient biomonitor for PTE contamination in this lake.
The short exposure time (1-h) and the simplicity of the laboratory procedure used in this work allowed obtaining LC 50 levels comparable to traditional studies with 24-h experiments.These minor periods facilitate the application of toxicity essays in the development of rapid and sensitive biomonitoring programs for the detection of PTEs in the aquatic environment, as well as in emergency and early warning situations that require immediate responses.
The results obtained reveal the importance of continuing to develop studies aimed at understanding and explaining the ecological risk related to the presence of PTE, particularly in tropical environments where native species are threatened by the constant dumping of untreated liquid wastes, limiting their survival and altering the functionality of food webs, as well as the integrity of ecosystems as a whole.

Figure 2 .
Figure 2. Scheme of the laboratory procedure to evaluate the acute ecotoxicological effects of potentially toxic elements on ciliated protozoa from Lake Maracaibo.

Table 2 .
Multiple comparisons of the mean 1-h LC 50 values of ciliated protozoa from Lake Maracaibo for the potentially toxic elements tested by means of the Tukey test.