potential environmental toxicity from hemodialysis effluent
TRANSCRIPT
Elsevier Editorial System(tm) for Ecotoxicology and Environmental Safety Manuscript Draft Manuscript Number: EES-13-1089R1 Title: EVALUATION OF POTENTIAL ENVIRONMENTAL TOXICOLOGY RESULTING FROM EFFLUENTS GENERATED BY HEMODIALYSIS TREATMENTS Article Type: Research Paper Section/Category: Ecotoxicology Keywords: dialysis effluent, Daphnia magna, Euglena gracilis, environmental toxicity. Corresponding Author: Dr. Gilmar Sidnei Erzinger, Ph.D. Corresponding Author's Institution: University of Joinville Region First Author: Carla K Machado, Msc Order of Authors: Carla K Machado, Msc; Gilmar Sidnei Erzinger, Ph.D.; Luciano Henrique Pinto H Pinto, Msc; Lineu F Del Ciampo, Msc; Luciano Lorenzi, Ph.D.; Cláudia Hack Gumz H Gumz Correia, graduate; Donat P Häder, Ph.D. Abstract: The knowledge of the toxicity of certain potentially toxic compounds on various aquatic organisms allows to assess the impact that these pollutants on the aquatic biota. The processes of sewage treatment is inefficient in inhibition and removal of pathogenic bacteria resistant to antibiotics by dialysis. 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 oxygen demand, and chemical oxygen demand, exceeding the thresholds defined in the CONAMA Resolution 430 from 2011. 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 4.59 (± 3.48). In tests with Daphnia magna, the acute toxicity EC50 was 7.74 (± 3.46) as the dilution, and a NOEC value of 3.70% and a LEOC value of 18.75%.
Dear editor Albert Allen of Ecotoxicology and Environmental Safety
Please find attached the paper of our research work "Evaluation Of Potential
Environmental Toxicology Resulting From Effluents Generated By Hemodialysis Treatments"
Although the effluents generated by processes of hemodialysis are, in their
composition, very similar to those generated by human urine excretion of water
volumes and quality used and its fresh form, together with untreated wastewater showed
through the results obtained in our work a strong environmental impact. The use of
bioassays with algae (Euglena gracilis) and micro crustaceans (Daphnia magna), try
testing in acute and chronic was able to indicate the toxicological potential of this
important environmentalist effluent. The study aimed to contribute to better understand
the potential of this effluent, mainly by the high volume of water that is necessary for
the performance of hemodialysis.
We believe that the information and data provided in this paper is very valuable for
researchers and environmental engineers working in the field of wastewater treatment
and reuse and sustainable use of water resources .We hope that our paper will be given
full consideration for publication in Ecotoxicology and Environmental Safety
With best regards
Gilmar Erzinger on behalf of the co-authors
Cover Letter
Reviewer Suggestions
First
Name
Middle
Initial
Last
Name
Academic
Degree Institution E-mail Address
Afonso
Celso Dias Bainy PhD
Universidade Federal de
Santa Catarina
or
Evaldo
Luiz Gaeta Espindola PhD [email protected]
Adilson
Pinheiro PhD
Fundação Universidade
Regional de Blumenau -
FURB
*Response to Reviewers
Graphical Abstract (for review)
Highlights
The effluent generated by hemodialysis in hospitals and clinics is moderately toxic and
causes environmental contamination risks when disposed of directly into the
environment, especially in cities without sewage treatment.
The value obtained by testing the acute EC50 for Daphnia magna was 7.72, and the
dilution factor generated an RQ of 0.129
A chronic test of Dapnhia magna was an effluent dilution factor of 7.7, classified as a
medium risk environment.
The results of this study demonstrated that EC50 for algae (Euglena gracilils) of 7.08 as
the dilution factor.
*Highlights (for review)
POTENTIAL ENVIRONMENTAL TOXICITY FROM 1
HEMODIALYSIS EFFLUENT 2
1 - Carla Keite Machado, E-mail: [email protected] Department of Biology Rua Paulo Malschitzki, 10 Campus - Industrial Zone PO Box 246 - CEP 89219-710 - Joinville SC, Brazil.
4 - Luciano Lorenzi, E-mail: [email protected] Department of Biology Rua Paulo Malschitzki, 10 Campus - Industrial Zone PO Box 246 - CEP 89219-710 - Joinville SC, Brazil.
2 - Luciano Henrique Pinto, E-mail: [email protected] Department of Pharmacy Rua Paulo Malschitzki, 10 Campus - Industrial Zone PO Box 246 - CEP 89219-710 - Joinville SC, Brazil.
5 - Cláudia Hack Gumz Correia, E-mail: [email protected] Laboratory of Ecotoxicology Rua Paulo Malschitzki, 10 Campus - Industrial Zone PO Box 246 - CEP 89219-710 – Joinville SC, Brazil.
3 - Lineu Fernando Del Ciampo, E-mail: [email protected] Inovaparq Rua Paulo Malschitzki, 10 Campus - Industrial Zone PO Box 246 - CEP 89219-710 – Joinville. SC, Brazil.
6 - Donat Peter Häder, E-mail: [email protected] Neue Str. 9. 91096. Möhrendorf Germany
7 - Gilmar Sidnei Erzinge E-mail: [email protected] 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.
3
*ManuscriptClick here to view linked References
ABSTRACT 4
Examining the toxicity of compounds such as hemodialysis effluent to aquatic 5
organisms can help assess the impact that these pollutants have on aquatic biota. The 6
limitations of sewage treatment processes make removing microorganisms that are 7
resistant to antibiotics, such as pathogenic bacteria, from dialysis effluent difficult. In 8
many emerging countries such as Brazil, sewage and effluent from hospitals are 9
discharged directly into waterways without any treatment (Emmanuel, 2005). The 10
objective of this study was to characterize the effluents generated by hemodialysis and 11
to assess their acute and chronic environmental toxicities. Hemodialysis effluent 12
showed high concentrations of nitrites, phosphates, sulfates, ammonia and total 13
nitrogen, and it also had elevated conductivity, turbidity, salinity, biochemical oxygen 14
demand and chemical oxygen demand. Few of the measured values fell within the 15
standards that were put forth in National Council for the Environment (CONAMA) 16
Resolution 430 in 2011. The sample showed acute toxicity to the algae Euglena gracilis 17
in four physiological parameters (motility, precision of gravitational orientation, 18
compactness, and upward movement and alignment) and a mean EC50 of 4.59 (± 3.48). 19
For tests with Daphnia magna, the acute toxicity EC50 was a dilution factor of 7.74 (± 20
3.46), and the chronic toxicity was measured by the NOEC (3.70%) and the LEOC 21
(18.75%). 22
23
Keywords: dialysis effluent, Daphnia magna, Euglena gracilis, environmental toxicity. 24
25
INTRODUCTION 26
27
Tarrass et al (2010) reported that water resources are dwindling due to global warming, 28
climate change and recurring droughts; in fact, water is too valuable to waste. 29
Hemodialysis uses large volumes of water. For a patient undergoing 3 weekly dialysis 30
treatments of 4 h each, approximately 18,000 liters of dialysis fluid is used. In addition, 31
for each liter of water used to prepare the dialysis fluid. 32
As our planet‘s population continues to grow, so does the population of dialysis 33
patients. The annual growth rate of the population requiring dialysis is now estimated to 34
be 6%, which will result in approximately 4 million patients by 2025. As the number of 35
patients on dialysis continues to grow, so does the consumption of natural resources and 36
the production of waste by dialysis facilities (Connor et al 2010). 37
In the United States, 5,000 dialysis clinics, which served 325,000 patients and 38
represented 26% of the global dialysis market in 2007, perform over 50 million dialysis 39
treatments per year, consuming over 5 trillion liters of fresh water. In Australia, an 40
estimated 400 million liters (0.4 gigaliters) or 400 olympic-sized swimming pools of 41
water is discarded annually (Tarrass et al, 2010). In Brazil, more than 105,000 patients 42
were on hemodialysis in 2010, consuming more than 17 million liters of fresh water per 43
year at hemodialysis facilities. Due to this high water consumption, dialysis centers 44
should be a focus for water conservation (Machado, 2013). 45
Agar (2012) extrapolated that the dialysis population, currently estimated at ~2 million 46
patients worldwide, uses ~156 billion liters of water, of which ~2/3 is discarded during 47
reverse osmosis and 1/3 at the end of the hemodialysis process. 48
The wastewater generated by hemodialysis may have a significant impact on the 49
environment due to its high conductivity and salinity. However, the risks resulting from 50
its discharge to bodies of water remain under-explored. As an alternative to releasing 51
untreated hemodialysis wastewater into the ocean, recycling the hemodialysis effluents 52
can substantially reduce pollution. Moreover, limiting discharge can indirectly help 53
maintain water quality (Tarrass et al, 2008, 2010; Tarrass and Benjelloun, 2010). 54
The limitations of sewage treatment processes also make removing and inhibiting 55
microorganisms that are resistant to antibiotics, such as pathogenic bacteria, from the 56
dialysis effluent difficult. In many emerging countries such as Brazil, sewage and 57
effluents from hospitals are often discharged directly into waterways without any 58
treatment (Emmanuel, 2005). 59
According to IBGE (2010), only 28.5% of Brazilian municipalities had wastewater 60
treatment as of 2008, and the state of Santa Catarina State currently has treatments in 61
only 16.7% of its municipalities. 62
Given these facts, it is necessary to assess the potential environmental toxicology of 63
environmental effluents generated by hemodialysis activities. 64
65
MATERIALS AND METHODS 66
Sample Collection 67
One dialysis center from the four in Joinville was chosen at random. Samples were 68
collected on four different days by placing peristaltic pump tubing in the effluent outlet 69
that was connected to the dialysis machine. This wastewater collection system was 70
connected to 14 simultaneous hemodialysis machines, corresponding to 14 different 71
patients. 72
Sample characterization 73
Chemical analyses were performed by the colorimetric method using Smart 3 (, São 74
Paulo, Brazilwhich is ISO 9001 certified by the Environmental Protection Agency of 75
the United States (2002). This method measures nitrite, nitrate, phosphate, silica, and 76
sulfate. An analysis of chemical oxygen demand (COD) was performed according to the 77
methodology developed in the Standard Methods of 1998 and using a 78
spectrophotometer (HACH Instruments, Model DR 4000). –and biochemical oxigen 79
demand (BOD5) was performed according to the standard methods issued in 2013. 80
Maintenance of Euglena gracilis 81
Tests were conducted with strains of Euglena gracilis that were obtained from the 82
collection of the University of FAU, Germany. These strains were maintained in a 83
mineral and organic culture medium as described by Checcucci et al (1976). The culture 84
was maintained in an incubator under a 12 hour light:dark cycle of 20 W/m2 at a 85
temperature of 18°C. 86
87
Motility and orientation analysis in Euglena gracilis 88
89
For the experiments performed with Euglenas gracilis, we used the New Generation 90
Ecotox (NGTOX) (Erzinger et al, 2009). This equipment is a modification of the 91
ECOTOX equipment developed by Tahedl and Häder (2001). NGTOX is an automated 92
bioassay system in which a cell suspension containing the algae is automatically 93
transferred by a peristaltic pump driven by stepper motor to an observation vessel via 94
darkroom mixing. The images of the movement of these cells are detected and recorded 95
by a CCD (Charge Coupled Device) that is well-lit and connected to a microscope. 96
These images are digitized and displayed on a computer monitor. ImagingTox software 97
(Ciampo et al, 2012) determines the motion parameters and analyzes motility 98
(percentage of dead cells), the precision of the gravitational orientation and the speed 99
and shape of the cells, and it stores all of this information in a database. These 100
parameters are subsequently measured under five different concentrations of toxins 101
produced in automatic serial dilutions (1:2, 1:4, 1:8, 1:16, 1:32). 102
The system operates in real time and tracks a virtually unlimited number of cells in 103
parallel. The software uses the vectors of the tracks to calculate various parameters, 104
such as percent motility, percentage of cells moving upwards, mean velocity, cell 105
compactness and the precision of gravitational orientation. The motility parameter gives 106
the percentage of cells moving at a speed equal to or faster than a minimum velocity set 107
in the program. The velocity parameter gives the speed (swimming velocity) of the cells 108
in µms-1
. The cell compactness (form factor) describes the shape of the cell and has a 109
low value of 1 when the cell is perfectly round, and this value increases as the cell 110
increases in length. The parameter ―upward‖ provides the percentage of cells that are 111
moving towards the upper part of the cuvette (±90 around the vertical direction). This 112
parameter is a statistical parameter that describes the precision of the gravitactic 113
orientation of the cells and ranges from 0 (when the cells are moving randomly) to 1 114
(when all the cells are moving in a single direction). For a description of the hardware 115
and more details about ECOTOX, see Tahedl and Hader (1999). 116
The filling time of the cuvette was 100 s, and the rinsing time was 45 s. The cells were 117
tracked for 3 min. The minimum area of the objects included in the vector analysis was 118
set to 400 µm2, and the maximum was set to 2000 µm
2. The minimum speed at which 119
the cells were considered motile was set at 15 µms-1
. To avoid the effect of light, the E. 120
gracilis cells were incubated in darkness for 30 min before performing measurements. 121
122
Daphnia magna 123
Cultivation of the test organism was performed according to ISO 6342 (2012). 124
Containers with a capacity of 500 ml and the culture medium M4 were used for growing 125
the organisms. They were fed daily with algal culture of Scenedesmus subspicatus, 126
which was produced according to ISO 8692 (2012). 127
The methodology for the acute test with the test organism Daphnia magna was as 128
described in the standard NBR 12713 (ABNT, 2003). The samples were tested by 129
exposing neonates of Daphnia magna, 2-26 hours old, to dilutions of the sample for a 130
period of 48 hours (Flohr et al., 2005). 131
The Chronic Toxicity Test of toxicity at 21 days was performed in accordance with ISO 132
10706 (2000) with modifications according Knops et al. (2001). 133
134
Measurement procedures 135
The sizes of the females were estimated at the beginning and end of the test to evaluate 136
the relationship between the EL and BL using a Nikon SMZ 1500 stereomicroscope 137
with magnifications of 32x and 57x. 138
The precisions of the measurements were 15 µm for 32x and 8.5 µm for 57x. BL was 139
measured as the distance from the top of the head to the base of the carapace spine, and 140
EL was measured as the distance, on the central axis, from the base to the top of the first 141
exopodite of the second antennae. Measurements were performed on a total of 349 142
animals for D. magna (Pereira et al, 2004). The somatic growth of females was then 143
calculated by Equation 1: 144
145
(1)
146
147
where lf is the final length of the body (mm), lo is the initial length (mm), and Δt is the 148
time (days). 149
150
Statistical Analyses 151
The statistical analysis of the data was performed by a repeated measures one-way 152
ANOVA, using an average frequency under test. The significance level was set at 5% 153
and 95% confidence. The statistical analysis was performed using SPSS v14.0 statistical 154
software (SPSS, Chicago, IL). 155
To determine the lethal concentration of E. gracilis (EC50), we interpreted the 156
experimental data according to Equation 2 (Tahedl and Häder, 1999): 157
158
(2) 159
160
b
EC
c
yy
50
0
1
161
where y is the response variable (percentage of dead organisms), c is the concentration 162
of the substance, y0 is the response when the concentration tends to infinity, and b is a 163
scaling factor. 164
The data were processed using charts and visualized in the program SigmaPlot ver. 12 165
(Systat Software Inc). This model corresponds to Equation 3, which was proposed by 166
Emmens (Tahedl an Häder, 1999) to interpret the concentration-effect relationships 167
168
169
170
The software adjusts the values of the nonlinear regression. The program Sigma-Plot 171
was used to graph and obtain the sigmoidal curve with the Levenberg-Marquardt 172
algorithm (Equation 2) to determine the parameters of the independent variables that 173
give the best fit between the equation and the data. This algorithm determines the 174
parameter values iteratively such that the sum of the squared differences between the 175
observed and predicted values of the dependent variable is minimized, with the 176
observed value io and the expected value yi. The parameters EC50, b and y0 (see 177
Equation 1) are optimized to minimize the algorithm‘s SS. The confidence intervals of 178
the set of optimized parameters are calculated from the covariance matrix with an error 179
level of 5%. 180
To determine the value of the 48 h EC50 for Daphnia, we used the Probit Method 181
(Weber, 1993) for parametric data and a Trimmed Spearman-Karber Method (Hamilton 182
et al, 1977) for nonparametric data. 183
To compare the no observed effect concentrations (NOECs) and least observed effect 184
concentrations (LOECs) among the different treatments, we used a Kruskal-Wallis 185
nonparametric test. When significant differences were found, we also used the Mann-186
Whitney U test, with the significance level set for the number of comparisons made 187
according to Bonferroni techniques (Sokal and Rohlf, 1995). 188
189
RESULTS AND DISCUSSION 190
The presented results include the period from August 2012 to January 2013. The 191
physical and chemical characteristics of the samples are presented in Table 1. 192
193
n
i
ii yySS1
2)( (3)
Table 1. Results of physico-chemical characterization of effluents from hemodialysis. 194
The average was obtained from four different samples obtained on different days. 195
Parameter Mean ± SD mg l-1*
(CONAMA nº 430)
OD 10.78 ± 1.45 ≥ 4 mg l-1
pH 7.5 ± 0.6 6 - 9
Salinity (%) 9.42 ± 1.48
Conductivity 4080 ± 181 23 to 0.36 µS cm-1
Hard water 60.0 ± 4.5 ≤ 500 mg l-1
CaCO3
Turbidity 4513 ± 327 ≤ 100 UNT
COD 832 ± 49 125 mg O2 l-1
BOD5 384 ± 19 10 mg O2 l-1
Nitrite 11.56 ± 2.96 1.0 mg l-1
Nitrate 1.52 ± 2.33 10.0 mg l-1
Phosphate 53.95 ± 2.72 0.15 mg l-1
P
Sulfate 23.0 ± 2.5 250 mg l-1
SO4
Ammonia***
5.35 ± 1,49 0.70 mg l-1
N
Total Nitrogen 126.7 ± 5.8 13.3 mg l-1
N, for pH ≤ 7,5
* Reference values for Class 3 196
** 1 nephelometric turbidity unit (NTU) = 7.5 ppm de Si02 197
*** Concentration limits for ammonia compounds according to CONAMA Resolution 357. 198
199
The samples did not meet the minimum standards established by Brazilian law in 200
CONAMA Resolution 430. The samples had a conductivity 177 times greater than is 201
mandated by CONAMA, and in terms of salinity, the sample is classified as brackish 202
water. Likewise, the turbidity was 45 times higher than the guidelines, and the main 203
component of the turbidity was silica (98.28 ± 92.07 mg l-1
). BOD, the amount of 204
oxygen required to oxidize the biodegradable organic matter present in the water, was 205
approximately 38 times the current legislation. The chemical oxygen demand (COD) is 206
a parameter that measures the amount of organic matter capable of being oxidized by 207
chemical means in a liquid sample. The COD was approximately 6.6 times higher than 208
the recommended maximum. 209
According Piveli and Kato (2006), the hardness of water is a measure of its ability to 210
precipitate soap. Soap forms insoluble complexes rather than foam until the water‘s 211
cations are exhausted. Hard water is principally caused by the presence of calcium and 212
magnesium, as well as other cations, such as iron, manganese, strontium, zinc, 213
aluminum, and hydrogen, that are associated with carbonate anions (more specifically 214
bicarbonate, which is the most soluble). Sulfate and other anions, such as nitrate, 215
chloride, and silicate, may also play a role. There are four major compounds that impart 216
hardness to water: calcium bicarbonate, magnesium bicarbonate, calcium sulfate and 217
magnesium sulfate. The levels of these compounds fell within the required standards. 218
The samples had nitrite concentrations approximately 11 times above the standards, 219
though nitrate and pH fell within the required parameters. The phosphate concentration, 220
though not addressed in CONAMA‘s resolution 430, is a major nutrient for the growth 221
of cyanobacteria. The concentration of silica (the baseline in drinking water 1-30 mg l-1
) 222
is high enough to be considered a suspension component. Resolution 430 sets out the 223
Conditions and Standards for Effluent Release and requires that the effluent not have 224
floating materials. Other components that are higher than recommended for Class 3 225
were ammonia, at approximately 8 times higher, and total nitrogen, at 9.5 % above its 226
limit. 227
228
Acute toxicity 229
The legislation proposed by FATMA (2002) in Santa Catarina State for domestic 230
sewage and/or hospitals in accordance with the provisions of Decree 017/2002 on 231
Quality Standards states that the factor of dilution of the samples for microcrustacean 232
Daphnia magna tests should be 1:1. Assuming that this value is representative of the 233
Expected Environmental Concentration (CAE), we used the environmental risk 234
assessment method of risk quotients (RQ) described by Goktepe et al. (2004) and 235
applied by Fujimoto et al. (2012). The RQ is calculated by dividing the estimated value 236
of the CAE of each insecticide by the EC50 value calculated in acute toxicity tests. The 237
value of RQ, also called Q, is a pure number because the units of the parameters are 238
canceled out in the division. 239
In the present study, we used Daphnia cultures that were 15 days old and considered 240
mature. The value obtained by testing the acute EC50 for Daphnia magna was 7.72, and 241
the dilution factor (Figure 1) generated an RQ of 0.129. According Goktepe et al. 242
(2004), a value of RQ between 0.05 and 0.5 characterizes effluents posing a medium 243
risk to the environment. 244
245
Dilution
0 4 8 12 16 20 24 28 32 36 40 44 48
Inhi
biti
on (
%)
0
20
40
60
80
100
246
Figure 1. Percentage inhibition of motility of Daphnia magna exposed to different 247
concentrations of hemodialysis effluent. 248
249
The data presented in Table 2 and Figure 2 show that the less diluted samples of the 250
dialysis effluent affected various parameters of the E. gracilis. The mean EC50 obtained 251
for the algae through the five physiological parameters showed that the sensitivity was 252
7.03, and the dilution factor was similar to that obtained in tests for the acute micro 253
crustacean test with Daphnia magna. Approximately 10% to 30% non-motile cells 254
indicates an effect on the physiological state of the culture (Lebert and Hader 1999). In 255
the case of dialysis effluent, motility was not affected by the 20-fold dilution of the 256
sample; however, from the twelfth dilution, there was a sharp decrease in motility and 257
only 30% of the cells were mobile compared to ≈ 93% in control. 258
259
260
Dilution
0 4 8 12 16 20 24 28 32 36 40 44 48
Inh
ibit
ion
(%
)
0
20
40
60
80
100
Dilution
0 4 8 12 16 20 24 28 32 36 40 44 48
Inh
ibit
ion
(%
)
0
20
40
60
80
100
B
261
Dilution0 4 8 12 16 20 24 28 32 36 40 44 48
Inh
ibit
ion
(%
)
0
20
40
60
80
100
C
C
Dilution
0 4 8 12 16 20 24 28 32 36 40 44 48
Inh
ibit
ion
(%
)
0
20
40
60
80
100
D
262
Dilution
0 4 8 12 16 20 24 28 32 36 40 44 48
Inh
ibit
ion
(%
)
0
20
40
60
80
100
E
263
264
265
266
267
268
Table 2 Mean percentage inhibition and EC50 of acute test obtained with the alga 269
Figure 2. Percentage inhibition of the
various physiological parameters of
Euglena gracilis exposed to different
concentrations of hemodialysis
effluent. A = motility, B = Ascending
movement, C = precision of
gravitational orientation, D =
Compactness and E = Alignment.
A B
A
C D
E
Euglena gracilis. 270
Inhibition (%) EC50 (dilution) p
Motility 5.83 ± 2.34 <0.0001
Upward swimming cells (%) 3.48 ± 3.00 <0.0001
precision of gravitational orientation 12.93 ± 5.83 <0.0001
Cell Compactness 8.63 ± 5.21 <0.0001
Alignment 4.52 ± 6.70 <0.0001
Mean 7.08 ± 3.40
Values given are the means ± SD of three replicates. (p* = one-way ANOVA, 271
significance level p <0.05 to 95% confidence) 272
273
Among the motion parameters, the precision of gravitational orientation was found to be 274
the most sensitive parameter. The orientation of gravitaxia cells is a physiological 275
phenomenon that helps Euglena actively find a place in the water column that is ideal 276
for growth and reproduction (Lebert and Hader, 1999; Hader et al., 1999). According to 277
Azizullah et al. (2012), this response is commonly found to respond to effluent toxicity. 278
The precision of gravitational orientation (r-value) was the most affected parameter and 279
showed more commitment to 54.91% with respect to the inhibition of motility, 280
compared to 73.09% inhibition of movement ascending, 33.26% higher than inhibition 281
of Compactness and 65.04 % greater than the inhibition of alignment. Two other factors 282
also evaluate the inhibition of the ability to gravitactically orient to a lesser degree. 283
These factors are ascending and alignment movement (Hoda and Hader, 2009). 284
Older cultures of E. gracilis generally have a negative gravitactic orientation, that is, 285
most of the cells swim up (Hader et al. 1998). The results of this study demonstrated 286
that positive gravitactic orientation was more pronounced in the less diluted effluent, 287
where 90% of the cells were in motion compared with 89% in the control. In a natural 288
environment, this phenomenon would cause the euglena to have difficulty swimming to 289
the surface and impair their photosynthetic capacity, perhaps leading to the loss of their 290
ability to generate biochemical energy. 291
The morphological data show that the compactness of the cells decreased in more 292
concentrated samples: the cells become rounder compared with the control. Previous 293
studies have reported that other species of the genus Euglena change their shape in 294
response to increasing concentrations of water pollutants and other physical or chemical 295
stresses (Murray, 1981; Takenaka et al, 1997; Conforti, 1998). Many freshwater algae 296
are known to change their form to a globular shape in response to osmotic stress, and a 297
globular shape is considered to be an adaptation to stress (Takenaka et al., 1997, 298
Azizullah et al., 2012). E. gracilis was found to change its shape from a rod shape to 299
globular under salt stress (Takenaka et al. 1997, Azizullah et al. 2012). The effluent‘s 300
relatively high salinity (9.7), rather than a specific toxicity, may be responsible for this 301
phenomenon. 302
303
Chronic Toxicity 304
A chronic test of Dapnhia magna was also established as a benchmark to compare the 305
data of the acute test, in which the lethal concentration (EC50) was an effluent dilution 306
factor of 7.7, classified as a medium risk environment. From these data, the tests were 307
performed in 10 replicates at concentrations of zero (control), 1, 5, 10, 25 and 50% 308
effluent. The results are described below and presented in Table 3. At concentrations 309
above 25%, there was no formation of pups due to the toxicity of the environment. 310
In the chronic tests, the highest concentration that did not cause a statistically significant 311
effect on fecundity (NOEC) was 3.70%, and the lowest sample concentration that 312
caused one was 18.75% (LOEC) (ABNT, 2003). 313
314
Table 3. Number of pups per day (mean) of Daphnia magna exposed to different 315
concentrations of hemodialysis effluent. Each point corresponds to ten Daphnias 316
magna. 317
Sample Number of pups per day p
Control 9.93 ± 4.88
1% 17.30 ± 3.34 0.4332
5% 31.80 ± 6.62 0.1152
10% 13.36 ± 1.00 0.4652
25% 0,0 ± 0.0 0.0268
50% 0.0 ± 0.0 0.0268
p* = one-way ANOVA, significance level p <0.05 to 95% confidence. 318
319
Ecotoxicological studies with living Daphnia often evaluate the growth of individual 320
organisms. According to Pereira et al. (2004), these measures on living organisms 321
should be avoided because they can lead to impairment or death and therefore alter the 322
results. Therefore, because living Daphnia were used throughout the life cycle, average 323
measurements are preferred over individual changes in almost all studies. Thus, the 324
allometric relationships provide an estimated parameter for viable growth. The ventral 325
and dorsal lengths of Daphnia magna were recorded at the end of the chronic tests on 326
all of the samples shown in Figure 3. A regression analysis was used to establish the 327
relationship between the two allometric measurements of Daphnia at the beginning and 328
end of the experiment for each dilution. The high R-squared values indicate that the 329
effluent did not cause physiological changes below a 10% dilution. Concentrations 330
above 25% caused early death of females 331
EL (nm)
4,0 4,5 5,0 5,5 6,0 6,5 7,0
BL
(nm
)
2,0
2,5
3,0
3,5
4,0
4,5
Control
1 % dilution
5 % dilution
10 % dilution
332
Figure 3. Allometric relationships between body (BL) and exopodite (EL) lengths for 333
D. magna. R2 and the equations for each regression are presented. 334
335
Another technique that was used to check for toxic effects on fertility was measuring the 336
somatic growth as described by Joachim (2007). In Figure 4, the fact that the lines 337
remain straight at dilutions as high as 10% proves that the female Dapnhia showed no 338
physiological response because of the effluents‘ toxicity. The LOEC value (18.75%) 339
also confirms these data. 340
341
Dilution
0 4 8 12 16 20 24 28 32 36 40 44 48 52
Som
ati
c gro
wth
rate
(D
ay-1
)
0,0000
0,0050
0,0100
0,0150
0,0200
0,0250
0,0300
0,0350
0,0400
342
Figure 4. Life-history endpoints of Daphnia magna exposed to different % dilutions of 343
hemodialysis effluent. EC50 = 17.71 ± 0.08 dilution, p =<0.0001. Each point 344
corresponds to ten Daphnia magna. 345
346
Thus, it can be concluded that the effluent generated by hemodialysis in hospitals and 347
clinics is moderately toxic and causes environmental contamination risks when disposed 348
of directly into the environment, especially in cities without sewage treatment having an 349
EC50 for algae (Euglena gracilils) of 7.08 and 7.74 as the dilution factor for Daphnia. 350
The results demonstrate that the sewage effluent of this important urban source requires 351
further study of the toxicity and the public policies that regulate effluent disposal. 352
353
354
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Table 1. Results of physico-chemical characterization of effluents from hemodialysis.
The average was obtained from four different samples obtained on different days.
Parameter Mean ± SD mg l-1*
(CONAMA nº 430)
OD 10.78 ± 1.45 ≥ 4 mg l-1
pH 7.5 ± 0.6 6 - 9
Salinity (%) 9.42 ± 1.48
Conductivity 4080 ± 181 23 to 0.36 µS cm-1
Hard water 60.0 ± 4.5 ≤ 500 mg l-1
CaCO3
Turbidity 4513 ± 327 ≤ 100 UNT
COD 832 ± 49 125 mg O2 l-1
BOD5 384 ± 19 10 mg O2 l-1
Nitrite 11.56 ± 2.96 1.0 mg l-1
Nitrate 1.52 ± 2.33 10.0 mg l-1
Phosphate 53.95 ± 2.72 0.15 mg l-1
P
Sulfate 23.0 ± 2.5 250 mg l-1
SO4
Ammonia***
5.35 ± 1,49 0.70 mg l-1
N
Total Nitrogen 126.7 ± 5.8 13.3 mg l-1
N, for pH ≤ 7,5
* Reference values for Class 3
** 1 nephelometric turbidity unit (NTU) = 7.5 ppm de Si02
*** Concentration limits for ammonia compounds according to CONAMA Resolution 357.
Table
Table 2 Mean percentage inhibition and EC50 of acute test obtained with the alga
Euglena gracilis.
Inhibition (%) EC50 (dilution) p
Motility 5.83 ± 2.34 <0.0001
Upward swimming cells (%) 3.48 ± 3.00 <0.0001
r-vaule 12.93 ± 5.83 <0.0001
Cell Compactness 8.63 ± 5.21 <0.0001
Alignment 4.52 ± 6.70 <0.0001
Mean 7.08 ± 3.40
Values given are means ± SD of three replicates. (p* = one-way ANOVA, significance
level p <0.05 to 95% confidence)
Table
Table 3. Number of pups per day (mean) of Daphnia magna exposed to different
concentrations of hemodialysis effluent. Each point corresponds to ten Daphnias
magna.
Sample Number of pups per day p
Control 9.93 ± 4.88
1% 17.30 ± 3.34 0.4332
5% 31.80 ± 6.62 0.1152
10% 13.36 ± 1.00 0.4652
25% 0,0 ± 0.0 0.0268
50% 0.0 ± 0.0 0.0268
p* = one-way ANOVA, significance level p <0.05 to 95% confidence.
Table
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure