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Selection of a bioassay battery to assess toxicity in the affluents and effluents of three water-treatment plants/Seleccion de una bateria de bioensayos para evaluar toxicidad en los afluentes y efluentes de tres plantas potabilizadoras/Selecao de uma bateria de bioensaios para avaliar a toxicidade em afluente e efluente de tres estacoes potabilizadoras.

Introduction

The discharge of wastewater into a water body involves a large number and diversity of chemicals, many of which are unknown. These substances can be mixed among them, increasing or decreasing the toxic effect and generating a negative impact on the structure and functioning of the natural ecosystem.

The tools commonly used to assess pollution in wastewater are based on physicochemical analyses such as pH, dissolved oxygen, Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Total Organic Carbon (TOC), Total Dissolved Solids (TDS) and Total Suspended Solids (TSS), (1-3), which do not reflect the biological effects that pollution can cause in animals, plants and humans. A good alternative to assess such effects are bioassays (4, 5).

To assess the toxicity of wastewater and drinking water, different types of bioassays have been used with fish, protozoa, bacteria, algae and others (6) Organisms to assess toxicity are diverse in their composition and their sensitivity to toxicants; therefore, a battery of bioassays is often used instead of a single species to cover a wide range of sensitivities (1, 7, 8). The test organisms included in a battery include representatives of the food chain at the level of consumers, producers and decomposers (9). The criteria for selection of the battery include autochthonous populations, in particular those that are environmentally attractive, with broad distribution and easy to maintain in the laboratory (10-12).

Keddy et al. (9) proposed a decision-making approach that consists in assessing whether organisms meet some essential criteria such as easy access to publications, standard test methods, acceptability, confidence intervals of 95%, and other desirable criteria like organisms identified by species, measurable endpoint, frequency of observation, environmental test conditions and statistical analysis, among others. Criteria are assigned a weight; if they are over 80% of acceptability they can be recommended as candidates to make part of a battery. Once organisms are selected, their sensitivity to polluted water is evaluated, and then those organisms that are most useful are chosen to make part of the battery of bioassays.

The selection of organisms that are part of the battery of bioassays can be performed by using multivariate analysis and/or by combining some of them such as non-linear mapping, principal component analysis, cluster analysis (CA) or matching factors analysis (13-14). The cluster analysis is a mathematical tool used to classify objects or variables into groups based on their similarities. The clustering procedure is often initiated by the conversion of raw data into a similarity matrix. Pandard et al. (15) mentioned that this mathematical technique can lead to various structures in the dendrogram given small errors in the distances calculated from the matrix of similarities. Bioassays to assess toxicity in Colombia were adopted after Decree 1575 of 2007, which states that any drinking water supply must have at the entrance to the treatment plant, and if possible in the water collection, an early warning system to detect the possible early toxic contamination in the water and to take precautionary measures and strategies for environmental management. Additionally, a risk map should be established for inspection, monitoring and control of risks associated with the conditions of the quality of the sources supplying water for human consumption.

To meet these requirements, the Bogota Water and Sewerage Company considered necessary to implement a battery of bioassays for analysing affluents and effluents of three drinking water treatment plants that supply the city of Bogota. To select this battery, we evaluated two animal models: Daphnia magna and Hydra attenuata; three model plants: Lactuca sativa, Allium cepa and Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum); and a bacterial model: Photobacterium leioghnathi (16-24).

Materials and methods

Test organisms

Animal models

Daphnia magna (25).

It is a static acute toxicity bioassay (48 h of exposure), in which 30 ml plastic containers are used with 25 ml of volume solution. As a positive control we used 0.13 mg [Cr.sup.+[sigma]]/L with confidence intervals between 0.05 and 0.21 mg [Cr.sup.+[sigma]]/L, and reconstituted hard water as a negative control. Three replicates were performed for each control and dilution. In each container 10 neonates 24 h-old were transferred. The neonates were observed after 24 h and 48 h of incubation at 21 [+ or -] 1[degrees]C, with a photoperiod of 16 h light/8h dark, and a light intensity of 800 lux, and the number of dead organisms was recorded. Based upon the dead counts, we calculated the lethal concentration 50 (L[C.sub.50]) at 48 h using the Probit method with a significance level of P<0.05.

Hydra attenuata (26).

It is a static test of acute toxicity (96 h of exposure), in which culture plates from 12 wells are used. As a positive control we used 0.78 mg [Cr.sup.+[sigma]]/L with confidence intervals between 0.73 and 0.83 mg [Cr.sup.+[sigma]]/L and reconstituted hard water as a negative control. Three replicates were performed for each control and dilution: in each well three hydras were transferred to a volume of 4 ml of the solution and incubated at a temperature of 20 [+ or -] 2[degrees]C, a light intensity of 800 lux and a photoperiod of 16 h light/8 h dark. The morphological changes of the test organisms were recorded at 24, 48, 72 and 96 h of exposure. Morphology includes a normal stage, two of sublethality (organisms with rounded and shortened tentacles), and two of lethality (tentacles tulip-shaped and disintegrated organisms). With this assay we determined the average concentration that produces an effect in the exposed population (sublethal E[C.sub.50] or lethal L[C.sub.50]) using the Probit method with a significance level of P<0.05.

Vegetable model

Lactuca sativa (27).

It is a static acute toxicity test (120 h of exposure) with Lactuca sativa variety Great Lake Batavia. In the test, 25 seeds of similar size, shape, and colour are placed on a Whatman No. 3 filter paper impregnated with 4 ml of sample in a Petri Dish and incubated at 22 [+ or -] 2[degrees]C in darkness for 5 days. As a positive control 18 mg [Zn.sup.+2]/L were used with confidence intervals between 6.8 and 30 mg [Zn.sup.+2]/L, and reconstituted hard water as a negative control. After incubation, the average length of roots per sample concentration is recorded and five outliers are discarded to reduce the coefficient of variation in the results. Finally, the concentration that produces 50% inhibition in root elongation (I[C.sub.50]) is estimated using the Probit method with a significance level of P<0.05.

Pseudokirchneriella subcapitata (28).

It is a static acute toxicity test with P. subcapitata (96 h of exposure). In the test, 18 25-ml Erlenmeyer flasks are used with a 10 ml solution volume. As a positive control 0.25 mg [Cr.sup.+[sigma]]/L with confidence intervals between 0.05 and 0.46 mg [Cr.sup.+[sigma]]/L was used and culture medium as a negative control. For each control and dilution three replicates were performed. The volume calculated from the culture is inoculated in each Erlenmeyer flask to set an initial cell density of [10.sup.4] cell/ml. Subsequently, the cultures are incubated at 23 [+ or -] 2[degrees]C, light intensity of 4.300 [+ or -] 10 lux and at continuous agitation of 100 revolutions per minute. After the incubation period of 96 h the percentage of inhibition is determined for each concentration compared to the control turbidity at 750 nanometres and the concentration that produces 50% of inhibition in the growth of algal cells (I[C.sub.50]) is calculated with the Probit method with a significance level of P<0.05

Bacterial model

Photobacterium leioghnathi (29).

Bioluminescence test is used to determine the toxicity of compounds that interfere with the enzymatic system of bacteria causing a reduction in light output. Variations in light output are measured with a high sensitivity luminometer (1 femtomole) at a wavelength of 490 nanometers. ToxScreen II test (CheckLight[R] Ltda.) includes the use of two buffers, one that favours the detection of heavy metals (Pro-Metal Buffer) and another one (Pro-Organic Buffer) that favours the detection of organic pollution. Toxicity is determined by the average effective or inhibitory concentration (I[C.sub.50] (15-30 minutes) 30[degrees] C) in a given time and under controlled temperature. The C[I.sub.50] is calculated when the inhibitory effect is greater than or equal to 50%, otherwise it is reported as a percentage of volume/volume effect.

Selection criteria for organisms

The first step in selecting the organisms of the battery in the affluent and effluent from three treatment plants was to apply the approach of Keddy et al. (9) which states that the following requirements must be met:

Essential requirements

1. To have easy access to the publications reported as standard test methods.

2. To have toxic reference values and their actual or median lethal concentration.

3. To have acceptability criteria, ideally associated to confidence intervals of 95%.

4. To have controls to ensure the health of test organisms to carry out the bioassays and the interpretation of results.

Desirable requirements

There were 12 inclusion criteria to be met by the test organisms and each criterion was assigned a score. The scores for each criterion were assigned as follows:

1. Test organisms identified by species (1)

2. Measurable endpoints (1)

3. Morphological characteristics of the test organism (1)

4. Number of organisms per replicate (1)

5. Frequency of observation (1)

6. Volume of test solution (1)

7. Volume of test containers (1)

8. Preparation of the test substance and its addition to the test container (2)

9. Continued cultivation of the organisms (1)

10. Environmental test conditions (3)

11. Definition of culture media and dilution (2)

12. Statistical analysis (2)

When methods meet the four essential requirements, tests are considered as 'potentially useful'; then they are analysed to find whether they meet all the desirable requirements to be regarded on the long term as 'prototype tests', that is both inclusion criteria mentioned above must be complemented to become 'useful tests'. When the analysed organism meets the 12 desirable criteria, it gets 17 points equivalent to 100% of acceptability for desirable requirements. In this case all the indicators to be evaluated obtained 17 points, which are equivalent to 100% of acceptability for desirable requirements. In addition to the selection of organisms, relevant information was considered for the application of the tests, such as representing the trophic level, sensitivity, reproducibility (coefficient of variation in control charts < 30%) and ecological relevance, all criteria that complement the tests and make them more robust to be recommended in a battery of bioassays (9).

Water samples

Ten samples of raw water (affluent) and 10 samples of treated water (effluent) were taken from three drinking-water treatment plants that supply the city of Bogota, Colombia. The water samples from the three treatment plants comply with national legislation. Given the physicochemical characteristics of the three affluents, they were analysed as untreated wastewater.

The Tibitoc plant collects water from Bogota River to be treated by a conventional system, which consists of a presedimentation, coagulation, flocculation, sedimentation, downward flow filtration through a bed of anthracite, and gas chlorination. El Dorado plant collects water from La Regadera water reservoir and its treatment is a pre-treatment where the water is stabilized with hydrated lime, coagulation, flocculation, sedimentation, downflow filtration through a bed of anthracite and gas chlorination, and finally a dosing with lime to stabilize the pH of the water. The Francisco Wiesner plant collects water from two sources: the Chingaza Paramo and the San Rafael reservoir in which water is stored from the Chingaza Paramo and the Teusaca River. Its treatment is a direct filtration with sand and anthracite, and gas chlorination. The average flow treated in plants is 8.50 [m.sup.3]/s for Tibitoc, 11.75 [m.sup.3]/s for El Dorado, and 0.35 [m.sup.3]/s for Francisco Wiesner.

Two litres of water were collected from each affluent and effluent at different days of the week to get a better assessment of variation of input water and the operation of each plant. Water samples were refrigerated at 4[degrees]C during transportation to the laboratory and were analysed within 48h after collection.

We used as test organisms two animal models: D. magna and H. attenuate; three vegetable models: L. sativa, P. subcapitata and Allium cepa; and a bacterial model: Photobacterium leioghnathi. The results of A. cepa are not included in this study because of the difficulty in obtaining homogeneous onion bulbs, so we obtained a coefficient of variation of 59% in the control card.

In the bioassay with P. leioghnathi, the effluent samples were processed with chlorine and chlorine neutralizing with sodium thiosulfate pentahydrate 3% (60[micro]l/ 50 ml of treated water).

Data analysis

Calculation of LC/EC/I[C.sub.50]

To calculate the LC/EC/I[C.sub.50] and their 95% confidence limits, the Probit method was used (EPA, V). This is a parametric method to estimate the effective concentration or lethality (E[C.sub.50] or L[C.sub.50]) by adjusting mortality data with a technique or effect of probability. One of the restrictions of the method is that to calculate the E[C.sub.50] or L[C.sub.50] intermediate values should be obtained between 0 and 100% effect. When results in EC or L[C.sub.50] cannot be reported by the demands of the statistical program, they are reported as the percentage of effect in the lowest concentration at which the event is still present on the evaluated population. The effects may be inhibition, sub-lethality and lethality or volume/volume.

Cluster analysis

CA was used for the selection of the battery of bioassays (13, 15). Cluster analysis is a mathematical tool that classifies objects or variables into groups. The procedure begins with the conversion of raw data into a similarity matrix. We used the method of classification by hierarchical clustering (linkage Intra-Group), whose graphical representation is a dendrogram (15). To calculate the distance matrix between the values of each bioassay, the results were consolidated at 100% effect, using the measure of the Chi-2. CA as a mathematical tool can lead to various structures in the dendrogram, providing small errors in the distances calculated from the similarity matrix.

Results

Selection criteria for organisms

The selection of organisms used to evaluate the affluent and effluent water of the treatment plants was conducted according to the scheme proposed by Keddy et al (9). Bioassays to identify whether they met this proposal took into account the four key requirements and the 12 desirable qualifications to determine if they are considered useful tests (Tables 1 and 2). The analysis found that all organisms are potentially useful to meet four key requirements and 17 points for the desirable qualifications, equivalent to 100% acceptability. P. leioghnathi does not meet two essential requirements: the C[I.sub.50] for the toxic reference and the confidence interval.

Battery of Bioassays

Regarding the analysis of toxicity in animal, plant and bacterial models, there were different levels of sensitivity to input and output of water treatment systems.

In the affluent of Francisco Wiesner plant (Table 3), H. attenuata presented sublethal effects in most samples with E[C.sub.50] values between 49.6 and 107.42 and case lethality rates between 11.1 and 100%. D. magna showed low sensitivity in mortality rates between 4 and 57% to 100%. In the plant model, a similar sensitivity was observed in bioassays P. subcapitata and L. sativa. The algae growth presented an inhibition in 70% of the samples and the rest of the growth stimulation assays. In weeks 6 and 8, L. sativa showed growth-stimulating effects while other samples observed inhibition of root growth between 1 and 20%. In the case of plant models, when the volume/ volume percentage is greater than 100% effect, it indicates that there has been an overgrowth of algal cells and/or root elongation compared to the negative control, so it is also seen as a sign of toxicity. In the bacterial model, P. leioghnathi; showed sensitivity only to organic in week 1, exceeding a 50% inhibition as suggested by the protocol.

At El Dorado plant (Table 4), H. attenuata showed sublethality rates in most trials with E[C.sub.50] values between 25.32 and 177.80. D. magna presented mortality rates in 70% of the processed samples, with values between 9 and 36%. The model plants (L. sativa and P. subcapitata) showed a similar behaviour, presenting percentages of inhibition and stimulation of growth. P. leioghnathi showed no toxicity in any sampling event.

In the affluent of the Tibitoc Plant (Table 5), the indicator H. attenuata presented sublethality effects of 22.2 and 55.6% in the undiluted sample (weeks 7 and 9), E[C.sub.50] values between 30.51 and 130.62 in two events and an E[C.sub.50] of 82.48 and 55.54. D. magna showed toxicity in 60% of the samples, with values between 23% and 100% in the second week, and E[C.sub.50] of 151.73. P. subcapitata presented growth inhibition between 5 and 11% at week 4 and an E[C.sub.50] value of 56.10. Other results show a stimulating effect, overcoming a 100% effect with respect to the negative control. L. sativa presented, in the same proportion, stimulation and inhibition. P. leioghnathi did not exhibit this kind of sensitivity to water.

In the effluent of Francisco Wiesner Plant (Table 6), Hydra attenuata exhibited sensitivity in all the effluent samples except for week 3. The sample 10 yielded a value of 100% sub-lethality and lethality in weeks 5, 6, 7 and 9 with values between 33.3 and 66.7%. Daphnia magna showed toxicity in the 10 samples tested indicating a high sensitivity of this organism in this type of water. In P. subcapitata we observed inhibition of cell growth in 70% of the cases and growth was stimulated only in the first three weeks. L. sativa in all samples showed inhibitory effects on root elongation, with values between 1 and 24% to 100%. P. leioghnathi did not provide sensitivity to possible toxicity by organic or inorganic in 10 samples of water with chlorine neutralization. In water samples without neutralization of chlorine, chlorine concentration was between 2 and 2.8 mg/l.

Table 7 presents the results of toxicity bioassays in the effluent from El Dorado Plant. H. attenuata showed toxicity in all samples tested, with E[C.sub.50] values between 19.89 and 66.15. D. magna showed high rates of mortality and L[C.sub.50-48h] between 6.44 and 24.53. P. subcapitata showed growth inhibition in 80% of the cases and stimulation of growth in two samples. L. sativa showed both inhibition and stimulation of growth. Finally, P. leioghnathi presented an I[C.sub.50-15min] in the chlorine samples neutralized with sodium thiosulfate pentahydrate only in the first week, indicating toxicity of inorganic origin. The chlorine concentration in El Dorado was between 2.1 and 2.4 mg/l. In the remaining samples no effect of inhibition of bioluminescence was detected.

In the Tibitoc effluent (Table 8), Hydra and Daphnia were sensitive in 100% of the samples tested, but the Daphnia had greater mortality rates in the higher dilutions of the sample. Plant models showed no significant difference in terms of response or inhibition of growth effect; however, P. subcapitata showed greater sensitivity to the present average I[C.sub.50] values of 48.43 in 70% of the samples. Only in the first two weeks showed a stimulating effect on cell growth. L. sativa showed inhibition values between 1 and 15%, and samples from week 3 and week 10 showed a stimulating effect on root elongation. The bacterial model P. leioghnathi did not show toxicity in chlorine neutralizing samples. The chlorine concentration in the effluents was between 3 and 7 mg/l.

Cluster analysis

For the CA we used data obtained at 100%, i.e. from the undiluted sample. Dendrograms are shown in Figure 1. Bacterial model results with P. leioghnathi were excluded from the analysis due to failure to report positive results above 50% as suggested by the protocol.

The choice of battery for each affluent and effluent of the three treatment plants was based on the comparison of the sensitivity of the test organisms by the CA. We obtained homogeneous groups of organisms, with the same potential for toxicity detection and the same range of sensitivity, with distances below 5 standard units with the Chi-2 method. In the affluents of Francisco Wiesner and El Dorado, the animal model H. attenuata showed a greater homogeneity in the results. For Tibitoc, it was D. magna the organism with the greatest homogeneity. Regarding the plant model, P. subcapitata showed a greater homogeneity in the three treatment plants, although L. sativa was also highly homogeneous for El Dorado. In the three effluents it can be seen that D. magna and P. subcapitata represent greater homogeneity in their behavior, although H. attenuata is also below 5% in El Dorado. Based on these results the battery to the affluents of the treatment plants includes the following organisms: Francisco Wiesner: H. attenuata, P. subcapitata and P. leioghnathi; El Dorado: H. attenuata, L. sativa and P. leioghnathi; and Tibitoc: D. magna, P. subcapitata and P. leioghnathi. In the case of the effluents, Francisco Wiesner: D. magna, P. subcapitata and P. leioghnathi; El Dorado: H. attenuata, P. subcapitata and P. leioghnathi; and Tibitoc: D. magna, P. subcapitata and P. leioghnathi. This selection included representatives of the food chain for animals, plants and bacteria.

[FIGURE 1 OMITTED]

Discussion

The results of bioassays with H. attenuata demonstrate an increased sensitivity of this organism for affluent or raw water from the three water treatment plants, a finding that coincides with the results obtained by Castillo et al. (30) who assessed wastewater with H. attenuata and D. magna and found that H. attenuata shows a greater sensitivity to this type of water. On the other hand, Pardos (24) reported that in 35.7% of the wastewater studied, mortality was observed for H. attenuata and 71.4% sublethal responses. In subsequent studies, Pardos et al. (31) compared the sensitivity of H. attenuata and Microtox (Vibrio fischeri) in wastewater samples and higher sensitivity was observed by H. attenuata, attributing the observed toxicity for this organism to ammonia levels.

Slabbert and Venter (32) evaluated domestic sewage effluent and industrial wastewater with D. magna and S. capricornutum and toxic activity was detected between 20 and 100% for both indicators. In our study we observed in D. magna as in P. subcapitata toxicity levels above 20% in a single sampling event in affluents of Wiesner and El Dorado, while in Tibitoc toxicity levels were lower. By contrast, Kontana et al. (33) found mortality rates of 50% of D. magna in most wastewater samples. The toxicity values found in the affluents of this study showed that P. subcapitata presents both inhibition and overgrowth in all the events analysed in the three treatment plants. By contrast D. magna has little sensitivity to this type of water. Similar results reported Ra et al. (34) in assessing wastewater with S. capricornutum and D. magna, who found that 33% of the samples showed acute toxicity to D. magna compared to 92% with S. capricornutum.

In assessing the effect of wastewater toxicity on H. attenuata, Bacillus cereus, Panagrellus redivivus, D. magna, L. sativa, and Oncorhynchus mykiss, Castillo et al. (30) found that H. attenuata showed the highest sensitivity in this type of water, while L. sativa had lower sensitivity even compared to P. subcapitata. Pica-Granados et al. (35) and Arkhipchuk et al. (36) reported inhibitory effects against organic substances, but Bohorquez and Campos (37) showed growth-stimulating effects of this alga.

In the case of effluent or potable water, D. magna showed higher sensitivity compared to other organisms evaluated. Cao et al. (18) reported similar results with D. magna when assessed town's secondary effluents before and after disinfection with chlorine, noting that this organism was more sensitive in samples treated with chlorine. Garzon (38) evaluated the toxicity of drinkable water from the Bogota River and found the highest sensitivity with H. attenuata, showing E[C.sub.50] of 21.1 and L[C.sub.50] of 30.2. L. sativa and S. capricornutum showed moderate sensitivity, whereas in D. magna mortality was not observed, probably because chlorine was inactivated after purification.

In the vegetable models, although there was a similar inhibition effect between Pseudokirchneriella and Lactuca, microalgae showed signs of toxicity reflected in the cell overgrowth. On the other hand, the bacterial model P. leioghnathi showed sensitivity only against inorganic compounds in a sample of the effluent of El Dorado plant.

The results obtained in the CA do not coincide entirely with those obtained in bioassays of toxicity in relation to the animal model in the affluent of The Tibitoc plant, since the results suggest the use of D. magna, but in the affluent of the three water treatment plants H. attenuata appears to be more sensitive. In the effluent of El Dorado plant the cluster analysis suggests the use of H. attenuata, but in the results of the three water treatment plants D. magna shows greater sensitivity. This could be explained by the number of samples and/or the fact of using only the results in the concentration of 100%. In many cases, positive results are obtained at lower concentrations, but these data are lost when entering into the analysis only the concentration of 100%. In these cases it is suggested to analyse a larger sample before making a decision and to take into account the initial results of the toxicity organisms tested. Such information is appropriate for decision making.

For the selection of organisms that are part of the battery of bioassays other tools can be used as suggested by Pandard et al. (15) who used CA as well as Principal Component Analysis (PCA) to select a battery of bioassays as part of the classification of hazardous waste of the Directive 91/689-CEE (39). In this case, they included L. sativa and P. subcapitata as vegetable models, E. foetida, D. magna and C. dubia as animal models and V. fischeri as a model for assessing bacterial toxicity in 40 residues. The authors note that the multivariate analysis can reduce the number of tests without changing the characteristics of the waste and that the combination of CA with PCA provides more robustness to the hierarchy of groups. Similarly, RojickovaPadrtova (14) used only PCA to select a battery of bioassays including 6 microarrays and three standard acute toxicity tests in environmental samples. The analysis showed three main components that explain 60% of the variance of the variables as follows: the first component (P. subcapitata, T. platyurus, D. magna and B. calyciflorus) explains 26%, the second component (C. dubia, S. ambiguum) explains 20.6% and the third component (V. fischeri) explains 13.5%. Results indicate that such selection is possible with this tool, allowing to conclude that the battery may contain P. subcapitata, B. calyciflorus, T. platyurus and V. fischeri.

Devillers (13) and Pandard (15) suggest a combination of multivariate analysis such as nonlinear mapping and principal component analysis among others, to provide more information about the analysed matrix for optimal selection. However, the structure and amount of data obtained in the three treatment plants did not meet the requirements of these tools, the reason why they were not implemented.

Conclusions

Based on the results obtained, we suggest the use of H. attenuata, P. subcapitata and P. leioghnathi to evaluate the affluents of the three water treatment plants and D. magna, P. subcapitata and P. leioghnathi for effluents. This decision takes into account the variability in the response of organisms, the type of water analysed, the taxonomic group within the food chain and the cost-benefit. Similarly, it would be more convenient for the laboratories responsible of the management of treatment plants to use the same battery in all the three cases. Multivariate analysis and cluster analysis proved to be useful tools for selecting a battery of bioassays. The results for the effluents are useful as early warning systems for drinking-water treatment plants, but they do not determine by themselves the toxicity effects on the consumer. To rule out effects on human health other tests for an extended period of time are needed.

Financial support

The Empresa de Acueducto y Alcantarillado de Bogota-EAAB (Bogota Water and Sewerage Company) provided technical and financial support for the implementation of this project.

Conflict of interest

The authors have no conflict of interest.

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(23.) Cho CW, Phuong TTP, Jeon YC, Vijayaraghavan K, Choe WS, Yun YS. Toxicity of imidazolium salt with anion bromide to a phytoplankton Selenastrum capricornutum: Effect of alkyl-chain length. Chemosphere 2007; 69: 1003-1007.

(24.) Pardos M, Benninghoff C. Acute toxicity of polish (waste) water with a microplate based Hydra attenuate assay: A comparation with the Microtox test. Science of the Total Environment 1999; 243/244: 141-148.

(25.) Dutka, B. Methods for microbiological and toxicological analysis of water, wastewaters and sediments: National Water Research Institute (NWRI), Environment Canada; Burlington, Ontario, Canada. 1989, 140p.

(26.) Trottier S, Blasie C, Kusui T, Jhonson EM. Acute toxicity assessment of aqueous samples using a microplatebased Hydra attenuata assay: technical methodology. Environment Toxicology Water Quality 1997; 12: 265-271.

(27.) Dutka, B. Short-Term root elongation toxicity bioassay. Methods for toxicological analysis of waters, wastewaters and sediments: National Water Research Institute (NWRI), Environment Canada., Canada. 1989, 320p.

(28.) Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms., 3th ed. US EPA, EPA/600/4-91/002. Philip Lewis AL, Klem DJ, Lazorchak JM. 1994.

(29.) Standard Methods for the examination of water and wastewater, 21th ed., American Public Health Association, Ap. 8010G., Washington, D.C., APHA, 2005, 8-20, 8-23p.

(30.) Castillo GC, Vila IC, Neild E. Ecotoxiciy assessment of metals and wastewater using multitrophic assays. Environmental Toxicology 2000; 15: 370-375.

(31.) Pardos M, Benninghoff C, Gueguen C, Thomas R, Dobrowolski J, Dominik J. Water ecotoxicity studies in Cracow (Poland) using Hydra attenuata, Selenastrum capricornutum y Microtox toxicity test. Lakes & Reservoirs. Research and Management 2000; 5: 75-81.

(32.) Slabbert JL, Venter EA. Biological assay for aquatic toxicity testing. Water Science and Technology 1999; 39 (10-11): 367-373.

(33.) Kontana C, Papadimitriou A, Samaras P, Zdragas A, Yiangou M. Bioassays and biomarkers for ecotoxicological assessment of reclaimed municipal wastewater. Water Science and Technology 2008; 57 (6): 947-953.

(34.) Ra JS, Kim HK, Chang NI, Kim SD. Whole Effluent Toxicity (WET) Test on wastewater treatment plants with Daphnia magna and Selenastrum capricornutum. Environment Monitoring Assessment 2007; 129: 107-113.

(35.) Pica-Granados Y, Trujillo GD, Hernandez HS. Bioassay Standardization for Water Quality Monitoring in Mexico. Environmental Toxicology 2000; 15 (4): 322-330.

(36.) Arkhipchuk VV, Romanenko VD, Malinovskaya MV, Kipnis LS, Solomatina VD, Krot YG. Toxicity Assessment of Water Samples with a set of Animal and Plant Bioassays: Experience of the Ukrainian Participation in the WaterTox program. Environmental Toxicology 2000; 15 (4): 277-286.

(37.) Bohorquez PY, Campos MC. Evaluacion de Lactuca sativa y Selenastrum capricornutum como indicadores de toxicidad en aguas. Universitas Scientiarum 2007; 12 (2): 83-98.

(38.) Garzon C. Evaluacion de la calidad toxicologica y microbiologica de la fuente de abastecimiento del municipio de Agua de Dios en Cundinamarca. Tesis de Maestria. Universidad Nacional de Colombia, Bogota, 2002, 310p.

(39.) EC, 1991. Council Directive 91/689/ECC. Of 12 December 1991 on hazardous waste. Official Publications of the European Communities 1991; 377; 0020-7 [28/03/2011].

Paola Bohorquez-Echeverry [1], Marcela Duarte-Castaneda [1], Nubia Leon-Lopez [2], Fabian Caicedo-Carrascal [1], Myriam Vasquez-Vasquez, Claudia Campos-Pinilla [1] *

[1] Grupo de Biotecnologia Ambiental e Industrial (GBAI). Departamento de Microbiologia. Facultad de Ciencias. Pontificia Universidad Javeriana. Bogota, D.C. Colombia.

[2] Empresa de Acueducto y Alcantarillado de Bogota. Bogota, D.C. Colombia.

* campos@javeriana.edu.co

Received; 08-05-2012; Accepted: 10-07-2012
Table 1. Results of the essential requirements application for the
organism selection.

                   Essential requirements

Test organism       Test Method         Toxic reference
                                            endpoint
                                         I[C.sub.50]/
                                          L[C.sub.50]

H. attenuata        Trottier et      0.80 mg [Cr.sup.+6]/L
                      al. 1997          L[C.sub.50-96]h

D. magna            McInnis 1989     0.15 mg [Cr.sup.+6]/L
                                        L[C.sub.50-48]h

L. sativa           McInnis 1989      20 mg [Zn.sup.+2]/L
                                        I[C.sub.50-120]h

P. subcapitata        EPA 1994       0.30 mg [Cr.sup.+6]/L
                                        I[C.sub.50-96]h

P. leioghnathi        Standard
                      methods                 N.A
                       8050B

                   Essential requirements

Test organism       Confidence               Controls
                     Intervals

H. attenuata            95%                 (+) Chrome
                                        (-) Reconstituted
                                            hard water

D. magna                95%                 (+) Chrome
                                        (-) Reconstituted
                                            hard water

L. sativa               95%                  (+) Zinc
                                       (-) Distilled water

P. subcapitata          95%                 (+) Chrome
                                        (-) Culture medium

P. leioghnathi                       (+) Sodium chloroacetate
                        N.A            (pro-organic Buffer)
                                         Cupric chloride
                                      (pro-metallic Buffer)
                                       (-) Deionized water

N.A: Not Applicable. (+): Positive control. (-): Negative control

Table 2. Results of the desirable requirements application for
organism selection.

Test               Organism     Measurable       We know the
Organism           identi-      endpoints      characteristics
                   fied by                     of the organism
                   species

H. attenuata         Yes        Sublethal            Yes
                                lethality

D. magna             Yes        Mortality            Yes

L. sativa            Yes        Inhibition           Yes

P. subcapitata       Yes        Inhibition           Yes

P.                   Yes        Inhibition           Yes
  leioghnathi

Test                  Number         Observation      Test
Organism           og organisms       frequency     solution
                        per                          volume
                     replicate

H. attenuata             9             24, 48,        4 ml
                                       72, 96h

D. magna                10            24, 48 h        30 ml

L. sativa               25              120 h         4 ml

P. subcapitata           3             24, 96h        10 ml

P.                     N.A.          0, 15, 30        1 ml
  leioghnathi                       minutes

Test                 Test       Test substance      Cultures
Organism          container       preparation      are main-
                    volume       and addition        tained
                                   to vessel         in the
                                                    organism

H. attenuata         5 ml             Yes             Yes

D. magna            35 ml             Yes             Yes

L. sativa           55 ml             Yes              No

P. subcapitata      25 ml             Yes             Yes

P.                   3 ml             Yes              No
  leioghnathi

Test                    Test            Medium        Statistical
Organism             Conditions       definition       analysis

H. attenuata          800 lux            Yes          E[C.sub.50]
                    20 [+ or -]                           y/o
                     [degrees]C                       L[C.sub.50]
                   16 h light/8 h
                        dark

D. magna              800 lux            Yes          L[C.sub.50]
                   21 [+ or -] 2
                     [degrees]C
                   16 h light/8 h
                        dark

L. sativa          22 [+ or -] 2         Yes          I[C.sub.50]
                     [degrees]C
                        dark

P. subcapitata        4300 lux           Yes          I[C.sub.50]
                       21-25
                     [degrees]C

P.                 30 [degrees]C         Yes          I[C.sub.50]
  leioghnathi

Test              Total
Organism          score

H. attenuata        17

D. magna            17

L. sativa           17

P. subcapitata      17

P.                  17
  leioghnathi

N.A: Not Applicable

Table 3. Bioassay results from the Francisco Wiesner plant affluent.

          H. attenuata           D. magna          P. subcapitata
Week     E[C.sub.50-96]h     E[C.sub.50-48]h      I[C.sub.50-96]h
               or                   or                   or
         % (v/v) Effect       % (v/v)Effect        % (v/v) Effect

1           Lethality           Mortality            Overgrowth
          11.1% al 100%         0% al 100%          141% al 100%

2           EC 49.06            Mortality           Overgrowth
                                7% al 100%          201% al 100%

3           EC 107.42           Mortality           Overgrowth
                               17% al 100%          136% al 100%

4           EC 55.82            Mortality           Inhibition
                                4% al 100%          18% al 100%

5          Lethality            Mortality           Overgrowth
          100% al 100%         57% al 100%          125% al 100%

6           EC 86.06            Mortality           Inhibition
                                0% al 100%           6% al 100%

7          Lethality             LC 77.79           Inhibition
          100% al 100%                              14% al 100%

8           EC 90.19            Mortality           Inhibition
                                0% al 100%          10% al 100%

9           EC 88.17            Mortality           Inhibition
                                0% al 100%           8% al 100%

10        Sublethality          Mortality            Inhibition
          55.6% al 100%         0% al 100%          20% al 100%

             L. sativa             P. leioghnathi
Week     I[C.sub.50-120]h         I[C.sub.50-30]h
                or                       or
          % (v/v) Effect           % (v/v) Effect

1           Inhibition              Inhibition
            11% al 100%         Inorganic 0% al 100%
                                Organic 52% al 100%

2           Inhibition              Inhibition
            8% al 100%         Inorganic 20% al 100%
                                Organic 22% al 100%

3           Inhibition              Inhibition
            5% al 100%          Inorganic 0% al 100%
                                Organic 22% al 100%

4           Inhibition               Inhibition
            1% al 100%          Inorganic 0% al 100%
                               Organic 38.5% al 100%

5           Inhibition              Inhibition
            17% al 100%        Inorganic 27% al 100%
                               Organic 21.9% al 100%

6           Overgrowth              Inhibition
           115% al 100%         Inorganic 0% al 100%
                                 Organic 0% al 100%

7           Inhibition               Inhibition
            20% al 100%         Inorganic 6% al 100%
                                 Organic 0% al 100%

8           Overgrowth              Inhibition
           109% al 100%         Inorganic 0% al 100%
                                 Organic 0% al 100%

9           Inhibition               Inhibition
            8% al 100%          Inorganic 0% al 100%
                                Organic 28% al 100%

10          Inhibition               Inhibition
            1% al 100%          Inorganic 0% al 100%
                                 Organic 0% al 100%

Table 4. Bioassay results from the El Dorado plant affluent.

Week       H. attenuata           D. magna          P. subcapitata
         E[C.sub.50-96]h      L[C.sub.50-48]h      I[C.sub.50-96]h
                or                   or                   or
             % (v/v)              % (v/v)               % (v/v)
              Effect               Effect               Effect

1          Sublethality          Mortality            Overgrowth
          22.2% al 100%         20% al 100%          138% al 100%

2          Sublethality           CL 76.44            Overgrowth
            0% al 100%                               207% al 100%

3            EC 68.54            Mortality            Inhibition
                                36% al 100%           22% al 100%

4            EC 73.80            Mortality            Overgrowth
                                13% al 100%          109% al 100%

5           EC 177.80            Mortality            Overgrowth
                                 0% al 100%          105% al 100%

6            EC 62.70            Mortality            Inhibition
                                 9% al 100%           17% al 100%

7           EC 125.82            Mortality            Inhibition
                                14% al 100%           9% al 100%

8            EC 95.53            Mortality            Inhibition
                                 0% al 100%           17% al 100%

9            EC 25.32            Mortality            Overgrowth
                                20% al 100%          103% al 100%

10          EC 130.62            Mortality            Inhibition
                                 0% al 100%           6% al 100%

Week         L. sativa             P. leioghnathi
         I[C.sub.50-120]h         I[C.sub.50-30]h
                or                       or
              % (v/v)                  % (v/v)
              Effect                   Effect

1           Inhibition               Inhibition
            9% al 100%          Inorganic 0% al 100%
                                 Organic 22% al 100%

2           Overgrowth               Inhibition
              110% al           Inorganic 20% al 100%
               100%              Organic 0% al 100%

3           Inhibition               Inhibition
            3% al 100%          Inorganic 0% al 100%
                                 Organic 30% al 100%

4           Inhibition               Inhibition
            4% al 100%          Inorganic 43% al 100%
                                Organic 6,2% al 100%

5           Overgrowth               Inhibition
              120% al           Inorganic 0% al 100%
               100%              Organic 0% al 100%

6           Overgrowth               Inhibition
              104% al           Inorganic 10% al 100%
               100%              Organic 0% al 100%

7           Overgrowth               Inhibition
              104% al           Inorganic 0% al 100%
               100%              Organic 0% al 100%

8           Inhibition               Inhibition
            1% al 100%          Inorganic 0% al 100%
                                 Organic 0% al 100%

9           Inhibition               Inhibition
            7% al 100%          Inorganic 0% al 100%
                                 Organic 41% al 100%

10          Overgrowth               Inhibition
              112% al           Inorganic 0% al 100%
               100%              Organic 0% al 100%

Table 5. Results of bioassays from the Tibitoc plant affluent.

Week        H. attenuata            D. magna         P. subcapitata
         EC/L[C.sub.50-63]h      L[C.sub.50-48]h     I[C.sub.50-96]h
                 or                    or                  or
           % (v/v) Effect        % (v/v) Effect      % (v/v) Effect

1             LC 82.48              Mortality          Overgrowth
                                   23% to100%          140% to100%

2             EC 30.51              LC 151.73          Overgrowth
                                                       273% to100%

3             EC 64.38              Mortality          Overgrowth
                                    7% to100%          121% to100%

4             LC 55.54             Mortality            IC 56.10
                                   13% to100%

5             EC 130.62             Mortality          Overgrowth
                                    3% to100%          124% to100%

6             EC 55.88              Mortality          Inhibition
                                    3% to100%           5% to100%

7           Sublethality            Mortality          Overgrowth
            22.2% to100%            0% to100%          105% to100%

8             EC 55.76              Mortality          Inhibition
                                    0% to100%           7% to100%

9           Sublethality            Mortality          Inhibition

            55.6% to100%            0% to100%           6% to100%

10            EC 66.72             Mortality           Inhibition
                                   0% to 100%          11% to100%

Week        L. sativa             P. leioghnathi
         I[C.sub.50-120]h        I[C.sub.50-30]h
                or                      or
          % (v/v) Effect          % (v/v) Effect

1           Inhibition             Inhibition
            8% to 100%        Inorganic 0% to 100%
                               Organic 22% to 100%

2          Inhibition               Inhibition
            8% to 100%         Inorganic 20 to 100%
                               Organic 11% to 100%

3           Overgrowth             Inhibition
           110% to 100%       Inorganic 0% to 100%
                               Organic 26% to 100%

4          Inhibition              Inhibition
            4% to 100%         Inorganic 0% to 100%
                              Organic 28.6% to 100%

5           Overgrowth             Inhibition
           101% to 100%       Inorganic 0% to 100%
                              Organic 6.5 % to 100%

6           Overgrowth             Inhibition
           110% to 100%       Inorganic 10% to 100%
                                Organic 0% to 100%

7           Inhibition             Inhibition
            1% to100%         Inorganic 0% to 100%
                                Organic 0% to 100%

8           Overgrowth             Inhibition
           111% to100%        Inorganic 0% to 100%
                                Organic 0% to 100%

9           Inhibition             Inhibition
                              Inorganic 0% to 100%
            9% to100%          Organic 32% to 100%

10         Overgrowth              Inhibition
           123% to100%        Inorganic 0% to 100%
                                Organic 0% to 100%

Table 6. Bioassay results from the Francisco Wiesner plant effluent.

Week       H. attenuata           D. magna           P. subcapitata
         E[C.sub.50-96]h       L[C.sub.50-48]h       I[C.sub.50-96]h
                or                   or                    or
          % (v/v) Effect       % (v/v) Effect        % (v/v) Effect

1            EC 26.02              LC 2.08             Overgrowth
                                                      176% al 100%

2            EC 84.10             LC 12.19             Overgrowth
                                                      215% al 100%

3          Sublethality           LC 22.29             Overgrowth
            0% al 100%                                121% al 100%

4            EC 14.64             LC 10.47             Inhibition
                                                       59% al 100%

5           Lethality             LC 10.32             Inhibition
          66.7% al 100%                                81% al 100%

6           Lethality              LC 7.33              IC 70.30
          44.4% al 100%

7           Lethality             Mortality             IC 80.32
          33.3% al 100%         100% al 12.5%

8            EC 31.53             Mortality             IC 28.78

                                 21% al 18%

9           Lethality             Mortality             IC 17.79
          55.6% al 100%
                                100% al 12.5%

10         Sublethality           LC 17.48             Inhibition
           100% al 100%                                98% al 100%

Week         L. sativa              P. leioghnathi
         I[C.sub.50-120]h          I[C.sub.50-30]min
                or                        or
          % (v/v) Effect            % (v/v) Effect

1           Inhibition                Inhibition
            9% al 100%           Inorganic 0% al 100%
                                  Organic 42% al 100%

2           Inhibition                Inhibition
            15% al 100%          Inorganic 18% al 100%
                                  Organic 14% al 100%

3           Inhibition                Inhibition
            13% al 100%          Inorganic 0% al 100%
                                  Organic 32% al 100%

4           Inhibition                Inhibition
            8% al 100%          Inorganic 10.6% al 100%
                                 Organic 43.4% al 100%

5           Inhibition                Inhibition
            19% al 100%          Inorganic 14% al 100%
                                 Organic 30.5% al 100%

6           Inhibition                Inhibition
            6% al 100%           Inorganic 0% al 100%
                                  Organic 0% al 100%

7           Inhibition                Inhibition
                                 Inorganic 0% al 100%
            2% al 100%           Organic 19.4% al 100%

8           Inhibition                Inhibition
                                 Inorganic 0% al 100%
            1% al 100%            Organic 0% al 100%

9           Inhibition                Inhibition
            24% al 100%          Inorganic 0% al 100%
                                  Organic 33% al 100%

10          Inhibition                Inhibition
            21% al 100%          Inorganic 0% al 100%
                                  Organic 0% al 100%

Table 7. Results of bioassays from El Dorado plant effluent.

           H. attenuata            D. magna          P. subcapitata
Week    EC/L[C.sub.50-96]h     L[C.sub.50-48]h      I[C.sub.50-96]h
          % (v/v) Effect        % (v/v) Effect       % (v/v) Effect

1            EC 38.01              Mortality           Overgrowth
                                 100% al 12.5%        138% al 100%

2           Lethality              LC 19.34           Overgrowth
           77.8 al 100%                               194% al 100%

3            Lethality             Mortality           Inhibition
            100 al 100%          100% al 12.5%       84.99% al 100%

4            EC 66.15               LC 9.50           Inhibition
                                                      39% al 100%

5            EC 64.54             Mortality             IC 46.29
                                 87% al 12.5%

6            Lethality              LC 6.44             IC 31.21
           55.6% al 100%

7           Lethality              LC 17.97             IC 33.03
           100% al 100%

8            EC 19.89              LC 24.53           Inhibition
                                                      79% al 100%

9            Lethality             Mortality           Inhibition
           77.8% al 100%          100% al 25%         74% al 100%

10         Sublethality            Mortality           Inhibition
           44.4% al 100%          100% al 18%         88% al 100%

            L. sativa             P. leioghnathi
Week    I[C.sub.50-120]h        I[C.sub.50-30]min
         % (v/v) Effect           % (v/v) Effect

                                    Inhibition
1          Inhibition          Inorganic 54% al 100%
           9% al 100%          C[I.sub.50-15min] 10
                                Organic 26% al 100%

2          Overgrowth               Inhibition
          116% al 100%         Inorganic 0% al 100%
                                Organic 29% al 100%

3          Inhibition               Inhibition
           53% al 100%         Inorganic 0% al 100%
                                Organic 42% al 100%

4          Inhibition               Inhibition
           2% al 100%         Inorganic 46.5% al 100%
                               Organic 43.9% al 100%

5          Inhibition               Inhibition
           14% al 100%         Inorganic 6% al 100%
                                Organic 26% al 100%

6          Overgrowth               Inhibition
          107% al 100%         Inorganic 6% al 100%
                                Organic 0% al 100%

7          Inhibition               Inhibition
           16% al 100%         Inorganic 8% al 100%
                               Organic 26.2% al 100%

8          Overgrowth               Inhibition
          129% al 100%         Inorganic 0% al 100%
                                Organic 0% al 100%

9          Inhibition               Inhibition
           25% al 100%         Inorganic 0% al 100%
                                Organic 48% al 100%

10         Overgrowth               Inhibition
          108% al 100%         Inorganic 0% al 100%
                                Organic 0% al 100%

Table 8. Results of bioassays from the Tibitoc plant effluent.

             H. attenuata              D. magna
         EC/L[C.sub.50-96]h        L[C.sub.50-48]h
Week              Or                      or
            % (v/v) Effect          % (v/v) Effect

1            Sublethality              LC 32.83
             33.3 al 100%

2              EC 74.52               Mortality
                                    100% al 12.5%

3              EC 45.38               Mortality
                                    100% al 6.25%

4              EC24.68                Mortality
                                     100% al 25%

5              EC 67.87               Mortality
                                     100% al 25%

6              EC 23.64               Mortality
                                    100% al 3.12%

7            Sublethality             Mortality
             55.6 al 100%            100% al 25%

8              EC 26.00                LC10.48

9              EC 66.30               Mortality
                                    100% al 12.5%

10             EC 32.46                LC 14.09

          P. subcapitata          L. sativa
         I[C.sub.50-96]h      I[C.sub.50-120]h
Week            or                    or
          % (v/v) Effect        % (v/v) Effect

1           Overgrowth            Inhibition
           108% al 100%          15% al 100%

2           Overgrowth            Inhibition
           243% al 100%           4% al 100%

3            IC 71.34             Overgrowth
                                 114% al 100%

4            IC 71.19            Inhibition
                                  1% al 100%

5            IC 62.32            Inhibition
                                  5% al 100%

6            IC 35.74            Inhibition
                                 105% al 100%

7            IC 40.63             Inhibition
                                  3% al 100%

8          Inhibition            Inhibition
           62% al 100%            9% al 100%

9            IC 28.93            Inhibition
                                 12% al 100%

10           IC 28.89            Overgrowth
                                 110% al 100%

             P. leioghnathi
          I[C.sub.50-30 min]
Week               or
             % (v/v) Effect

1             Inhibition
         Inorganic 0% al 100%
          Organic 29% al 100%

2             Inhibition
         Inorganic 0% al 100%
          Organic 29% al 100%

3             Inhibition
         Inorganic 0% al 100%
          Organic 30% al 100%

4             %Inhibition
          Inorganic 0% al 100%
         Organic 43.8% al 100%

5             Inhibition
          Inorganic 0% al 100%
         Organic 30.8% al 100%

6             Inhibition
         Inorganic 0% al 100%
          Organic 30% al 100%

7             Inhibition
         Inorganic 0% al 100%
          Organic 35% al 100%

8             Inhibition
          Inorganic 7% al 100%
           Organic 0% al 100%

9             Inhibition
          Inorganic 0% al 100%
          Organic 41% al 100%

10             Inhibition
          Inorganic 0% al 100%
           Organic 0% al 100%
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Title Annotation:Original paper
Author:Bohorquez-Echeverry, Paola; Duarte-Castaneda, Marcela; Leon-Lopez, Nubia; Caicedo-Carrascal, Fabian;
Publication:Revista Universitas Scientarum
Date:May 1, 2012
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