Sensitivity of aquatic organisms to ethanol and its potential use as bioindicators

The aim of this research was to evaluate the feasibility for the use of the organisms Lemna minor, Azolla caroliniana, Hyphessobrycon eques, Pomacea canaliculata and Daphnia magna as exposure bioindicators for ethanol (lethal and effective concentration 50% LC50(I)/EC50(I)). Thus, the following concentrations were used 5.0; 10.0; 20.0; 30.0; 40.0 and 50.0 mg L ethanol on L. minor; 25.0; 50.0; 75.0; 100.0; 150.0 and 200.0 mg L on A. caroliniana; 0.3; 0.5; 1.0; 2.0 and 3.0 mg L on H. eques; 0.05; 0.10; 0.20; 0.40 and 0.80 mg L on P. canaliculata; and 40.0; 60.0; 80.0; 100.0; 120.0 and 140.0 mg L on D. magna. An untreated control was also kept for all organisms, with three repetitions. The increase in the ethanol concentration elevated the electrical conductivity and decreased the water dissolved oxygen and pH. The ethanol LC50 for L. minor and A. caroliniana were 12.78 and 73.11 mg L, respectively, and was classified as slightly toxic; 1.22 mg L for H. eques (moderately toxic); 0.39 mg L for P. canaliculata (highly toxic) and 98.85 mg L for D. magna (slightly toxic). Thus, H. eques and P. canaliculata have showed good potential for the use as ethanol exposure bioindicators on water bodies.


Introduction
The ethanol (CH 3 CH 2 OH) is used as fuel for combustion engines, alcoholic drinks (beer, wine and cachaça) and raw material for industry (perfume, cleaning products, paints, solvents, among others).The cited uses are related to both main ethanol properties: flammability and water solubility (Ferreira, Oliveira, & Duarte, 2004;Bastos, 2007).
At the harvest period of 2012 and 2013 in Brazil, more than 25.5 billion liters of ethanol were produced, with approximated consumption of 18 billion liters and exportation of more than 3.0 billion liters (EPE, 2014).Among the transportation methods, the river system stands out for being cheaper than roads and rails, for decreasing the trucks flow on the roads and the reduction of gas emission (CO 2 and CO) (Costa, 2004).
However, there are important concerns about the use of waterways for the ethanol transportation, especially about possible environmental impacts caused by direct ethanol contact on water bodies due to hull breaches on the ships, which may harm the aquatic environmental.

Nevertheless, little
information is available about the ethanol toxicity and its possible effects on non-target organisms.
In regard to the presence of ethanol in water bodies, some negative ethanol effects were reported, such as craniofacial abnormalities, cardiac and structural malformation to the Danio rerio fish (Reimers, Flockton, & Tanguay, 2004); alterations on motor coordination, sensorial perception, neuronal function (Esel, 2006) and on the activity of the acetylcholinesterase (AChE) (Rico, Rosemberg, Dias, Bogo, & Bonan, 2007), and changes on intoxication clinical signs, such as search for oxygen, agitation and increased opercular beating (Rosemberg et al., 2012).
The use of bioindicators as macrophytes duckweed (Lemna minor) and water fern (Azolla caroliniana); the fish mato grosso (Hyphessobrycon eques); snail (Pomacea canaliculata) and the microcrustacean water flea (Daphnia magna) are important for the understanding of the ethanol in water bodies.
The research about use of bioindicator on environmental impacts assessments for ethanol presence on the water bodies has fundamental importance, because the knowledge about ecotoxicological dynamics may assist at the decision making, if any amount of ethanol reaches the water bodies.
Thus, the aim of this research was to evaluate the use of thes test organisms L. minor, A. caroliniana, H. eques, P. canaliculata and D. magna

Material and methods
The tested chemical was the ethanol, common name for hydrated ethyl alcohol, a product composed by 6.2 -7.4% water and 92.6 -93.8% alcohol.

Macrophyte ecotoxicological assays
The acclimatization was performed under bioassay room condition, with air temperature of 25.0 ± 2.0°C and constant light with 1000 lux, for three days.After the acclimatization period, the plants were disinfected with water solution of sodium hypochlorite 2% (L.minor), 3% (A.caroliniana) and distilled water.Initially, the plants sensibility was evaluated through sodium chloride (NaCl) as the reference substance.The LC50(I); 7d was 6.67 g L -1 and 95% confidence intervals ranged from 5.48 to 6.85 g L -1 for L. minor and 2.14 g L -1 (1.97 -2.31 g L -1 ) for A. caroliniana.
To evaluate the macrophyte sensibility to ethanol, 4 L. minor colonies with three fronds each and five A. caroliniana individuals were selected and acclimated in a glass recipient (100 mL) containing 50 mL of culture medium (Hoagland's).The ethanol was applied in a static system, without aeration.The glass containers were covered in plastic film on both macrophyte, for more than 24 hours.
The mortality percentage evaluation was performed at 3, 5 and 7 days of ethanol exposure.The alterations observed on the L. minor fronds were the fronds number, chlorosis (natural chlorophyll pigmentation loss) and necrosis (plant's tissue death process) (OECD, 2002).The A. caroliniana evaluations were performed through the observations of chlorosis and necrosis, using a rank scale (E to A), according to Silva et al. (2012).

Fish and snails ecotoxicological tests
Homogenous groups of the test organisms with 2.32±0.22grams (fish) and 6.77±0.82grams (snail) were acclimated for seven days under bioassay room conditions, according to the test standard ABNT (2011).
The fish and snail feed went with commercial feed once a day, and the snail was also feed with macrophyte Hydrilla verticillata.Preliminary tests were also conducted to determine the range of concentration of ethanol for the H. eques and P. canalicualata.
The fish intoxication clinical signs were visually evaluated immediately after ethanol application and at four, 24 and 48 hours after application.The clinical signs observed for the fish were increased opercular beat, erratic swimming, inquietude, position along the water column, lethargy, muscle spasm (trembling), corrosion at the body surface (skin and fin) and skin and eyes color (Murty, 1998).
The water quality variables for the fish and snail, before the treatment, were as follows: pH between 6.5 -7.5; dissolved oxygen above 4.0 mg L -1 ; electrical conductivity between 170.0 -180.0 μS cm -1 ; water hardness from 10 to 60 mg CaCO 3 L -1 ; and alkalinity between 200.0 -210.0 mg CaCO 3 (ABNT, 2011).The tests were performed in a static system.The mortality evaluations for the fish and mobility for the snail were performed at 24 and 48 hours of ethanol exposure.

Daphnia magna ecotoxicological tests
The test organisms were grown under biological oxygen demand chamber (BOD) conditions, with constant temperature (20.0 ± 2.0ºC), in a culture medium composed by distilled water, reconstituted with nutrients (ABNT, 2009).The feeding was performed daily with algae culture (Scenedesmus subspicatus), containing 5x10 6 algae cells per D. magna individual, and a solution composed by fermented fish feed and yeast (Saccharomyces cerevisiae).The tests were performed by selecting neonates aging between six and 24 hours.
No acclimatization performed for D. magna tests.The organisms were transferred to plastic containers (Falcon type), with 10 mL capacity, which were filled with 9.0 mL of a mixture composed by culture medium along with the ethanol concentrations, and 1.0 mL culture medium containing five neonates, aging between six and 24 hours.
The following ethanol concentrations were used to perform the ecotoxicity tests: 40.0; 60.0; 80.0; 100.0; 120.0 and 140.0 mL L -1 (40.0; 60.0; 80.0; 100.0; 120.0 and 140.0 mg L -1 ).An untreated control was also kept, all with four repetitions, in a static system.The mobility evaluation (swimming capacity during 15 seconds after gentle agitation) was performed at 48 hours of ethanol exposure.

Statistical analysis
The mortality and mobility data from the bioindicators L. minor, A. caroliniana, H. eques, P. canaliculata and D. magna was submitted to linear regression and the lethal and/or effective concentration 50% (LC50(I)/EC50(I)) was estimated by the software "Trimmed Spearmam Karber" (Hamilton, Russo, & Thurston, 1977).The LC50(I)/EC50(I) was used to identify the ethanol toxicity, as proposed by Zucker (1985).
The lesser sensibility displayed in this research by the macrophyte in comparison to the fish may be due to wax presence at the leaves abaxial surface.Sheaths or cuticles may also be present at the root system, which diminishes the xenobiotics absorption by the organisms (Begon, Townsend, & Harper, 2006).
No mortality occurred on the exposure tests over H. eques with 0.25 and 0.50 mg L -1 .With 1.0 mg L -1 , occurred 22.0% mortality; and on 2.0 and 3.0 mg L -1 occurred 100% mortality (Figure 1c).All ethanol exposed animals displayed intoxication clinical signs.The observed changes were increased opercular beat, inquietude, pitch capacity loss and oxygen search at the water-air interface, within all assessments.
According to Rosemberg et al. (2012), Danio rerio fish exposed to ethanol (1%, v/v) for 20 and 60 minutes also displayed clinical signs changes, such as inquietude and oxygen search, similar as displayed on H. eques in the present research.
According to Rico et al. (2007), B. rerio fish exposed to 1.0% ethanol (v/v) presented 33% increase on the enzyme acetylcolinesterase activity, in comparison to the untreated control (25.5%).Esel (2006) and Pannia, Tran, Rampersad, and Gerlai (2014), had observed that ethanol induced clinical signs like changes in motor coordination, sensory perception and neuronal function, similar as observed for H. eques on this research.
The effects caused by ethanol can also occur directly or indirectly through oxidative stress metabolites (acetaldehyde and acetate) of ethanol that can promote craniofacial abnormalities, heart malformations, and delayed development of fish (B.rerio) (Bilotta, Saszik, Givin, Hardesty, & Sutherland, 2002;Reimers et al., 2004).
Regarding the toxicity test for the fish (H.eques), the dissolved oxygen ranged of 5.4 mg L -1 on the untreated control to 4.8 mg L -1 at the highest tested concentration (3.0 mg L -1 ethanol), immediately after the application.Thus, at 24 hours the dissolved oxygen was between 2.3 and 0.7 mg L -1 ; and after a 48 hours exposure, it ranged from 2.0 to 0.2 mg L -1 (Figure 2a).
The highest variation on electrical conductivity for the test with H. eques occurred between the untreated control (184.400μS cm -1 ) and the highest tested concentration (182.700μS cm -1 at 3.0 mg L -1 ethanol), immediately after the application (0 hour).After a 24 hours exposure, the electrical conductivity was 187.000 μS cm -1 on the untreated control and 185.600 μS cm -1 at the 3.0 mg L -1 rate; and on the last assessment (48 hours exposure), it ranged from 186.500 μS cm -1 the untreated control to 184.900 μS cm -1 the 3.0 mg L -1 rate (Figure 2b).
The highest pH variation at the H. eques toxicity test also occurred between the untreated control and the highest exposure rate (3.0 mg L -1 ethanol), in all assessments.The variation was from 8.2 to 8.0 (untreated control and highest exposure rate, respectively) at the beginning of the exposure (0 hour); 7.68 to 7.49 (24 hours) and 7.67 to 7.42 after 48 hours exposure (Figure 2c).
The lack of data in the Figure 2 regarding dissolved oxygen, electrical conductivity and pH at the rates of 2.0 and 3.0 mg L -1 ethanol occurred due to the mortality of all tested subjects exposed to those rates.
In regard to the toxicity test for the snail (P.canaliculata), immediately after the exposure beginning, the dissolved oxygen ranged from 4.5 to 4.1 mg L -1 (control and 0.8 mg L -1 ethanol exposure rate, respectively).In the following assessments, the highest variation on dissolved oxygen also occurred between the control and the highest tested exposure rate (0.8 mg L -1 ethanol); at 24 hours exposure, it was between 2.3 and 0.5 mg L -1 and at 48 hours exposure, it ranged from 2.6 to 0.7 mg L -1 (Figure 3a).
The highest variations on electrical conductivity and pH at the toxicity tests for the snail was similar to the previous variations, standing between the untreated control, and the highest tested rate (0.8 mg L -1 ethanol).The electrical conductivity was between 187.700 to 184.700 μS cm -1 at 0 hour.The increase in electrical conductivity and decrease in dissolved oxygen with ethanol increment provided for this study H. eques and P. canaliculata (Figures 2 and 3) were lower than that observed by Silva et al., (2015) with the effluent vinasse, originating from the ethanol distillation process, which was 0.189 to 0.890 μS cm -1 (electric conductivity) and 5.0 to 0.40 mg L -1 (dissolved oxygen) to the same biomarker (H.eques) According with Grady, Daigger, and Lim (1999), the ethanol biodegradation consists in an oxidation reaction performed along the microbial respiratory process.The ethanol biodegradation requires high oxygen concentrations (Castello, Moreira, & Braga, 2011), which decreased in the tests on H. eques and P. canaliculata in the present study (Figures 2a and  3a).
The increased electrical conductivity at 24 and 48 hours exposure in this study may be due the molecule bond breaking (CH 3 CH 2 OH) in aqueous solution (Delgado, Araújo, & Fernandes-JR, 2007).The lower pH values at 24 and 48 hours exposure occurred due to the ethanol aerobic degradation process, which uses the dissolved oxygen as oxidant agent, such as the respiration process of test organisms, which promoted a higher carbonic gas concentration (CO 2 ) in the experimental units (Swift, 2003).
The ethanol was classified as slightly toxic to L. minor, A. caroliniana and D. magna; moderately toxic to H. eques and highly toxic to P. canaliculata (Zucker, 1985).The Pomacea canaliculata snail and Hyphressobrycon eques fish were the most sensible bioindicators to ethanol exposure.These organisms are at the initial level of development for toxicity studies, with positive effects for being endemic to the tropical America (Huang, Liao, Chang, Kuo, & Wu, 2003).
Based on the present research, these organisms do display good potential as bioindicators of possible negative effects promoted by the ethanol presence in water bodies.If a chemical agent promotes toxic effects over a species, it may be toxic for several organisms along the food chain, being therefore capable of causing negative environmental impacts (Magalhães & Filho, 2008).
The results presented on this research may be used as monitoring tool for environmental survey, Acta Scientiarum.Biological Sciences Maringá, v. 38, n. 4, p. 377-385, Oct.-Dec., 2016 in order to detect the impacts caused by accidental release of the xenobiotic on the aquatic environment.

Conclusion
The organisms Lemna minor, Azolla caroliniana, Daphnia magna and Hyphessobrycon eques showed potential to be used as bioindicators of ethanol effetcs in water bodies, specially the Pomacea canaliculata snail, which had presented elevated sensibility to small ethanol concentrations.