Ecotoxicology and Environmental Safety 102 (2014) 42–47

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Potential environmental toxicity from hemodialysis effluent Carla Keite Machado a, Luciano Henrique Pinto b, Lineu Fernando Del Ciampo c, Luciano Lorenzi a, Cláudia Hack Gumz Correia d, Donat Peter Häder e, Gilmar Sidnei Erzinger f,n a

Department of Biology, Rua Paulo Malschitzki, 10, Campus—Industrial Zone, PO Box 246, CEP 89219-710, Joinville, SC, Brazil Department of Pharmacy, Rua Paulo Malschitzki, 10, Campus—Industrial Zone, PO Box 246, CEP 89219-710, Joinville, SC, Brazil c Inovaparq, Rua Paulo Malschitzki, 10, Campus—Industrial Zone, PO Box 246, CEP 89219-710, Joinville, SC, Brazil d Laboratory of Ecotoxicology, Rua Paulo Malschitzki, 10, Campus—Industrial Zone, PO Box 246, CEP 89219-710, Joinville, SC, Brazil e Neue Str. 9., 91096. Möhrendorf, Germany f Department of Medicine and Pharmacy, Master's and PhD Program in Health and Environment, Rua Paulo Malschitzki, 10, Campus—Industrial Zone, PO Box 246, CEP 89219-710, Joinville, SC, Brazil b

art ic l e i nf o

a b s t r a c t

Article history: Received 27 October 2013 Received in revised form 6 January 2014 Accepted 8 January 2014 Available online 1 February 2014

Understanding the toxicity of certain potentially toxic compounds on various aquatic organisms allows to assess the impact that these pollutants on the aquatic biota. One source of pollution is the wastewater from hemodialysis. The process of sewage treatment is inefficient in inhibition and removal of pathogenic bacteria resistant to antibiotics in this wastewater. In many countries, such as Brazil, during emergencies, sewage and effluents from hospitals are often dumped directly into waterways without any previous treatment. The objective of this study was to characterize the effluents generated by hemodialysis and to assess the degree of acute and chronic environmental toxicity. The effluents of hemodialysis showed high concentrations of nitrites, phosphates, sulfates, ammonia, and total nitrogen, as well as elevated conductivity, turbidity, salinity, biochemical and chemical oxygen demand, exceeding the thresholds defined in the CONAMA Resolution 430. The samples showed acute toxicity to the green flagellate Euglena gracilis affecting different physiological parameters used as endpoints in an automatic bioassay such as motility, precision of gravitational orientation (r-value), compactness, upward movement, and alignment, with mean EC50 values of recalculate as 76.90 percent (74.68 percent) of the undiluted effluents. In tests with Daphnia magna, the acute toxicity EC50 was 86.91 percent (70.39 percent) and a NOEC value of 72.97 percent and a LEOC value 94.66 percent. & 2014 Elsevier Inc. All rights reserved.

Keywords: Bioassay Hemodialysis effluent Daphnia magna Euglena gracilis Environmental toxicity

1. Introduction Tarrass et al. (2010) reported that water is becoming a dwindling natural resource due to global warming, climate change, and frequent droughts; in fact, it is too valuable to be wasted. Hemodialysis uses large volumes of water. In a patient undergoing dialysis treatment three times per week for 4 h, about 18,000 L of dialysis fluid are used. Up to 25 percent of the water used for the treatment are discarded as waste. As our planet's population continues to grow the number of dialysis patients increases. The annual increase is expected to be 6 percent, which will result in approximately 4 million patients by 2025. As the number of dialysis patients continues to grow, the

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Corresponding author. E-mail addresses: [email protected] (C.K. Machado), [email protected] (L.H. Pinto), [email protected] (L.F.D. Ciampo), [email protected] (L. Lorenzi), [email protected] (C.H.G. Correia), [email protected] (D.P. Häder), [email protected], [email protected] (G.S. Erzinger). 0147-6513/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2014.01.009

amount of natural resources consumed and wastewater produced by dialysis facilities increases accordingly (Connor et al., 2010). In the United States, 5000 dialysis clinics (which represent 26 percent of the global dialysis market) perform over 50 million dialysis treatments per year, consuming over 5 trillion liters of freshwater, based on its 325,000 patients in 2007. In Australia, an estimated 400 million liters or 400 olympic-sized swimming pools of water are used each year (Tarrass et al., 2010). In Brazil, in 2010, more than 105,000 patients were on hemodialysis, consuming more than 17 million liters of freshwater per year at hemodialysis facilities. Therefore, due to this huge water consumption, dialysis centers should focus on water conservation (Machado, 2013). Agar (2012) conducted an extrapolation of data for the dialysis population currently estimated at
2 million patients worldwide and concluded that a “world dialysis service” uses
156 billion liters of water and discard around two thirds of that during reverse osmosis and one third at the end of the hemodialysis process. Wastewater generated by hemodialysis may have a significant impact on the environment due to its high conductivity and salinity. However, the risk resulting from its discharge into bodies

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of water remains under-explored. It is evident that pollution can be reduced substantially through recycling of water. Moreover, limiting discharge can indirectly help in maintaining water quality (Tarrass et al., 2008, 2010; Tarrass and Benjelloun, 2010). In addition to the discharge of mineral salts into the water, the lack of sufficient sewage treatment poses another risk, since microorganisms such as pathogenic bacteria, that are resistant to antibiotics, are released to the environment. In many countries, such as Brazil, during emergencies, sewage and effluents of hospitals are often discharged without any treatment (Emmanuel et al., 2005). In 2008, in Brazil only 28.5 percent of the municipalities had wastewater treatment, and in the State of Santa Catarina, only 16.7 percent of the municipalities treated wastewater (IBGE, 2010). Given these facts, it is necessary to assess the potential environmental toxicology of effluents produced during hemodialysis. In order to quantify the toxicity of the pollutants several short-term and long-term test were conducted with two classical bioassays using the green flagellate Euglena gracilis and the cladoceran Daphnia magna.

2. Material and methods 2.1. Sample collection Out of the four dialysis centers in Joinville, Brazil, one was chosen at random. Samples were collected on four different days in the period from August 2012 to January 2013. Samples were collected 01 L using a peristaltic pump connected to the output of the dialysis machine. In total, samples were collected simultaneously from 14 hemodialysis machines in parallel, corresponding to 14 different patients. 2.2. Sample characterization Chemical analyses were performed by colorimetric methods, using a Smart 3 instrument (Poly Control Analytical Instruments, ISO 9001 certified, in accordance with the U.S. Environmental Protection Agency), measuring nitrite, nitrate, phosphate, silica, and sulfate. Chemical oxygen demand (COD) was analyzed according to the methodology described in Standard Methods (1998) by spectrophotometry (HACH Instruments, Model DR 4000 and biochemical oxygen demand BOD 5.20) was quantified according to standard methods. 2.3. Culture of Euglena gracilis Tests were conducted with the F1 strain of E. gracilis, obtained from the collection of the Friedrich-Alexander University, Erlangen, Germany. The cultures were grown in mineral medium (Checcucci et al., 1976) in an incubator under fluorescence light at an irradiance of 20 W m  2 for 12 h and 12 h darkness, at a temperature of 18 1C. 2.4. Motility and orientation analysis of Euglena gracilis For the experiments performed with E. gracilis we used the New Generation Ecotox (NGTOX) (Erzinger et al., 2010). This equipment is an evolution of an instrument called ECOTOX developed by Tahedl and Häder (2001). It is an automated bioassay in which a peristaltic pump driven by a stepper motor, transfers a cell suspension of the flagellate E. gracilis to an observation chamber after being automatically mixed with the pollutant. The images of the moving cells are detected and recorded in infrared by a CCD (charge-coupled device) camera connected to a microscope. The video images are displayed on a computer monitor. The software ImagingTox (Ciampo et al., 2012) determines the motion parameters and analyzes motility (percentage of motile cells), precision of gravitational orientation (r-value), swimming velocity and shape of the cells, and stores all this information in a database. Five different toxin concentrations produced in automatic serial dilutions (1:2, 1:4, 1:8, 1:16, 1:32) were evaluated in sequence. The system operates in real time and tracks a virtually unlimited number of cells in parallel. The software uses the vectors of the tracks to calculate various parameters. The motility parameter gives the percentage of cells moving at a velocity equal to or faster than the minimum velocity set in the program. The parameter velocity gives the swimming speed of the cells in mm s  1. The cell compactness (form factor) describes the shape of the cell and has the lowest value of 1 when the cell has an absolutely round shape and increases as the cell increases in length. The parameter “upward” gives the percentage of cells that are moving upward ( 7 901 around the vertical direction). The r-value is a statistical parameter

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that describes the precision of gravitactic orientation of the cells and ranges from 0 (when the cells are moving in random directions) to 1 (when all the cells are moving in a the same direction). For hardware and more details about ECOTOX see Tahedl and Häder (1999). The filling time of the observation chamber was set to 100 s and the rinsing time was 45 s. The duration of all tracking was 3 min. The minimum area for objects to be included in the vector analysis was set to 400 mm2 and the maximum area to 2000 mm2. Minimum speed at which the cells were considered motile was set to 15 mm s  1. In order to avoid any effects of light (which could induce e.g. phototaxis), the cells of E. gracilis were incubated in darkness for 30 min before making measurements and the recordings were done using an infrared monitoring light. 2.4.1. Daphnia magna The cultivation of the cladoceran D. magna was performed according to ISO 6342 (2012). Containers with a capacity of 500 mL culture medium (M4 medium) were used for growth of the organisms. Organisms were fed daily with an algal culture of Scenedesmus subspicatus, grown according to ISO 8692 (2012). The methodology for acute (short-term) tests with the test organism D. magna followed the standard NBR 12713 protocol (ABNT, 2003; EPA, 2002). The samples collected were tested based on the exposure of neonates of D. magna, 2–26 h old, in dilutions of the sample for a period of 48 h (Flohr et al., 2005). The chronic (long-term) toxicity tests were performed in accordance with ISO10706 (2000) with modifications according to Bianchini and Wood, 2002, and Knops et al. (2001) for 21 days. 2.5. Measurement procedures The size of the female Daphnias was estimated at the beginning and at the end of the test to evaluate the relationship between EL and BL (see below), using a stereomicroscope (Nikon SMZ 1500) with magnifications of 32  and 57  . The precision of the measurements was 15 mm for 32  , and 8.5 mm for 57  . BL is defined as the distance from the top of the head until the base of the carapace spine, and EL as the distance on the central axis from the base to the top of the first exopodite of the second antenna (Pereira et al., 2004). Measurements were made in a total of 350 animals. Somatic growth of females was calculated using Eq. (1): g¼

ln ðlf Þ  ln ðlo Þ Δtðdays

1

Þ

ð1Þ

where lf is the length of the body (mm), lo the initial length (mm), and Δt the time interval (days). 2.6. Statistical analysis Statistical analysis of data was performed by repeated measures one-way ANOVA. The significance level was set at 5 percent and 95 percent confidence. Statistical analysis was performed using SPSS v14.0 statistical software (SPSS, Chicago, IL). To determine the lethal concentration for E. gracilis, the EC50 was used in Eq. (2) interpreting the experimental data (Tahedl and Häder, 1999): y¼

y0 1þ ðc=EC50 Þb

ð2Þ

where y is the response variable (percentage of dead organisms), c is the concentration of the substance, y0 is the response when the concentration tends to infinity, and b is a scaling factor. The data were processed using the program SigmaPlot v12 (Systat Software Inc). This model corresponds to Eq. (3), proposed by Emmens (Tahedl and Häder, 1999) to interpret the concentration-effect relationships: n

SS ¼ ∑ ðyi  yi Þ2 i¼1

ð3Þ

The software calculates the values of a nonlinear regression. The program Sigma-Plot was used to determine the sigmoidal curve, using the LevenbergMarquardt algorithm (Eq. (2)) and to calculate the parameters of the independent variables that give the best fit between the equation and the data. This algorithm determines the parameters iteratively so that the sum of the squared differences between the observed values and predicted values of the dependent variable are minimized. EC50, b and y0 (see Eq. (3)) are optimized. The confidence intervals of the set of optimized parameters are calculated from the covariance matrix with an error level of 5 percent. To determine the values of 48-h EC50 in Daphnia the statistical Probit Method (Weber, 1993) was used for parametric data and the Trimmed Spearman–Karber Method (Hamilton et al., 1977) for nonparametric data. For the determination of the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for different treatments were compared by the Kruskal–Wallis nonparametric test. When significant differences were found, the Mann–Whitney U test was used, with the significance level set for the

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number of comparisons made according to the Bonferroni technique (Sokal and Rohlf, 1995).

and requires that the effluent contains no floating materials. Other components that exceed those recommended for Class 3 are ammonia, which was approximately 8 times higher, and total nitrogen which was 9.5 percent above the allowed limit.

3. Results and discussion 3.1. Acute toxicity The results presented here include the period from August 2012 to January 2013. Table 1 shows the physical and chemical characteristics of the samples. Taking the CONAMA Resolution 430 as a reference, it can be seen that the samples do not meet the minimum standards established by law. The results of the physico-chemical characterization show that the samples had a conductivity 177 times higher than the limit defined by CONAMA and in terms of salinity, the samples are classified as brackish water. Likewise, the turbidity is 45 units higher; the main component responsible for the turbidity was silica (98.28 792.07 mg L  1). The biological oxygen demand (BOD) was found to be approximately 38 times the defined limit. The chemical oxygen demand (COD) was around 6.6 times higher than the maximum recommended. According to Piveli and Kato (2006), carbonate hardness is a measure of the alkalinity of water caused by the presence of anions such as carbonate or bicarbonate. It is expressed either as parts per million (ppm or mg L  1), or in degrees KH. One degree of carbonate hardness (dKH) corresponds to the carbonate and bicarbonate ions found in a solution of approximately 17.848 mg of calcium carbonate (CaCO3) per liter of water. The corresponding cations are mainly calcium and magnesium and others such as iron, manganese, strontium, zinc, aluminum, and hydrogen, associated with the anions carbonate and bicarbonate (which is more soluble), sulfate and mainly, among other anions, nitrate, chloride, and silicate. The four major compounds that impart hardness in water are calcium bicarbonate, magnesium bicarbonate, calcium sulfate, and magnesium sulfate. The results obtained are within the normal standards required. The samples had nitrite concentrations about 11 times higher than required by CONAMA (2011); however, nitrate and pH were within the required limits. The determination of the phosphate concentration, though not defined in CONAMA 430, is a major nutrient for the growth of cyanobacteria. High concentrations of silica (the baseline in drinking water is 1–30 mg L  1) occur as suspended particulate components. The resolution described above defines the conditions and standards of effluent release

In the State of Santa Catarina the environmental quality control is carried out by the Environmental Foundation (FATMA). FATMA (2002) established the maximum toxicity limits for domestic and hospital sewage (017/2002). Normally the concentrations (e.g. in mg L  1) are calculated for EC50 or NOEC value and LEOC values. Since no single concentration can be stated for the undiluted mixture of pollutants in the effluent the values are given as percent fraction after dilution. The value EC50 for D. magna obtained from the acute test was 86.91 percent (7 0.39 percent) of the undiluted control (Fig. 1). According to Goktepe et al. (2004), this value classifies these effluents as a medium-risk pollutant. The data presented in Table 2 and Fig. 2 show that the less diluted samples of the dialysis effluents affect various motility parameters in E. gracilis. The mean EC50 obtained for five physiological parameters showed a concentration of 76.90 percent, which is similar to that obtained in the acute tests with D. magna. In a healthy culture of E. gracilis about 70 to 90 percent of the cells are motile (Lebert and Häder, 1999). Motility was not affected by a 20-fold dilution of the dialysis effluent; but with higher concentrations, starting at 86 percent of the undiluted control, there was a sharp decrease in motility and only 30 percent of the cells were motile, compared to
93 percent in the control. Among the motion parameters, the r-value was found to be the most sensitive. The gravitactic orientation of the cells helps Euglena to find a place in the water column suitable for growth and reproduction (Lebert and Häder, 1999; Häder et al., 1999).

Table 1 Results of physico-chemical characterization of effluents from hemodialysis. The average was obtained from four different samples obtained on different days.

n

Parameter

Mean7 SD

(CONAMA n1 430)n

OD (mg L  1) pH Salinity Conductivity (mS cm  1) Hardness of water (mg L  1 CaCO3) Turbidity (UNT)nn COB (mg O2 L  1) BOD 5 days (mg O2 L  1) Nitrite (mg L  1) Nitrate (mg L  1) Phosphate (mg L  1 P) Sulfate (mg L  1 SO4) Ammoniaa (mg L  1 N) Total Nitrogen (3 mg L  1 N, for pHr 7,5)

10.78 71.45 7.49 70.66 9.42 74.48 4080 7181 60.03 74.55 4513 7327 832 749 384 719 11.56 72.96 1.52 72.33 53.95 72.72 23.0 72.5 5.3571.49 126.7 75.8

Z4 6 to 9 23 to 0.36 r 500 r 100 125 10 1.0 10.0 0.15 250 0.70 13

Reference values for Class 3. 1 nephelometric turbidity unit (NTU) ¼ 7.5 ppm de SiO2.

nn

a Concentration limits for ammonia compounds according to CONAMA (2011) Resolution 357.

Fig. 1. Percentage inhibition of motility of Daphnia magna exposed to fraction (in percent) of the undiluted concentration of effluents of hemodialysis.

Table 2 Mean results of the percentage inhibitions and EC50 fraction (in percent) of the undiluted concentration of acute test obtained with the alga Euglena gracilis. Inhibition (%) Motility Ascending movement Precision of gravitational orientation Cell Compactness Alignment Mean

EC50 (%)

p

76.977 4.68 32.96 7 4.05

o 0.0001 o 0.0001

116.067 5.11 107.36 7 6.10 51.157 3.46 76.907 4.68

o 0.0001 o 0.0001 o 0.0001

Values given are means 7SD of three replicates. p is the one-way ANOVA with a significance level p o0.05 to 95 percent confidence.

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Fig. 2. Percentage inhibition of various physiological parameters of Euglena gracilis exposed to fraction (in percent) of the undiluted concentration of effluents of hemodialysis. A ¼ motility, B¼ Ascending movement, C¼ precision of gravitational orientation, D ¼Compactness and E¼ Alignment.

According to Azizullah et al. (2012), this response commonly is a good indicator for toxicity. The EC50 value of 116.06 percent demonstrated that the precision of gravitational orientation is the most sensitive parameter followed by cell compactness (107.36 percent). The parameters motility, ascending movement and alignment were less sensitive to the sample. Two other factors also evaluate inhibition of gravitactic orientation of E. gracilis, which are ascending and alignment movement (Hoda and Häder, 2010). In the present study, we used 15 days-old Euglena cultures which are considered to be in their static phase. These cultures showed a precise negative gravitactic orientation, i.e., most of the cells swim upwards (Häder, 1987). In the presence of a less diluted effluent 90 percent of the cells were non motile and therefore sedimenting compared with 89 percent actively moving cells in

the control. In their natural environment the cells would have difficulty swimming to the surface, so that they cannot harvest sufficient solar radiation for photosynthesis. Compactness of the cells decreased with an increase in concentration of the samples, indicating that the cells become rounder, compared with the control. Previous studies have reported that species of the genus Euglena change their shape in response to increasing concentrations of water pollutants and other physical or chemical stresses (Murray, 1981; Takenaka et al., 1997; Conforti, 1998). Many freshwater algae are known to change their form to a globular shape in response to osmotic stress, and a globular shape is considered to be a form of stress adaptation (Takenaka et al., 1997; Azizullah et al., 2012). E. gracilis was found to change its shape from a rod shape to a globular shape under salt stress (Takenaka et al., 1997; Azizullah et al., 2012). The salinity of 9.7 in the effluents may be

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responsible for this phenomenon and therefore not necessarily a response to toxicity of the sample. 3.2. Chronic toxicity In the acute tests it was determined that the lethal concentration (EC50) was 86.91 percent of the undiluted effluent, and therefore the effluent was classified as a medium-risk environment. For the chronic tests we used Daphnia to verify the data of the acute tests. The tests were performed in 10 replicates at concentrations ranging from zero (control), to 50 percent effluent (Table 3). At concentrations above 25 percent there was no survival due to the toxicity of the sample. In chronic tests, the highest concentration of the sample that did not cause a statistically significant effect on the organisms (NOEC) in terms of fecundity, when compared to the control, was 72.97 percent, and the lowest sample concentration that caused a statistically significant effect in comparison to the control organisms, also in terms of fertility was 94,66 percent (LOEC) according ABNT (2003). Ecotoxicological studies with Daphnia often evaluate the growth of individual organisms. The most widely used method to evaluate the growth of microorganism is to remove it from the culture medium for microscopic observation (ABNT, 2003). According to Pereira et al. (2004), these measures on living organisms should be avoided since it may cause death or change certain physiological functions by stress, distorting the results. Considering that the living Daphnia were used throughout their life cycle, we determined allometric relationships rather than the growth parameter. The ventral and dorsal lengths of D. magna were recorded in living organisms at the end of the chronic tests with all samples shown in Fig. 3. Regression analysis was used to Table 3 Number of offspring per day (mean) of Daphnia magna exposed to fraction (in percent) of the undiluted different concentrations of hemodialysis effluent. Each point corresponds to ten D. magna. Sample

Number of offspring per day

p

Control 1% 5% 10% 25% 50%

9.93 74.88 17.30 73.34 31.80 76.62 13.36 71.00 0.0 70.0 0.0 70.0

0.4332 0.1152 0.4652 0.0268 0.0268

p ¼One-way ANOVA, significance level p o 0.05 to 95 percent confidence.

Fig. 4. Life-history endpoints of D. magna exposed to fraction (in percent) of the undiluted concentration of effluents of hemodialysis. EC50 ¼ 110.87 7 6.45, po 0.0001. Each point corresponds to ten D. magna.

establish the relationship between the two allometric measurements of all Daphnia used at the beginning and the end of the process at all dilutions used. As high R-squared values were achieved, the equation obtained demonstrated that the effluent from hemodialysis caused no physiological changes up to 10 percent concentration. Concentrations above 25 percent caused the early death of females. Another technique used to check for toxicity on the ability of females to generate offspring was the somatic growth test described by Joaquim (2007). In Fig. 4 it can be seen that the straight line increases up to a concentration of 110.87 percent, which proves that the female Dapnhia showed no physiologic response to the effluent. Data from the LOEC (94.66 percent) statistically support this result. The data from this study demonstrate that the effluent resulting from hemodialysis, when discarded directly into the environment, and in conjunction with the urban wastewater present environmental risks, especially in cities without sewage treatment. The results indicate the importance of toxicity studies not only for hemodialysis effluents but for all household and industrial sewage to determine the risks of wastewater disposal.

Acknowledgments The authors thank Professors of Nephrology of the Department of Medicine of Univille: Prof. Dr. Anderson Gonçalves Ricardo Roman, Dr. Helbert Mascimento Lima and Dr. Marcos Scheidemantel for their assistance with technical information about the studied area. The authors also wish to thank the CNPq (National Counsel of Technological and Scientific Development) for partially supporting this study.

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Fig. 3. Allometric relations between body (BL) and exopodite (EL) lengths for D. magna, R-square values (R2 ¼ 0.9619) and equations for each regression analysis are presented (BL ¼  0.364243þ 0.695466EL) exposed to fraction (in percent) of the undiluted different concentrations of hemodialysis effluent.

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