Comparing two biological indexes using benthic macroinvertebrates : positive and negative aspects of water quality assessment

Aim: The aim of the present study, was to apply two biological indexes based on benthic macroinvertebrates (BMWP and ICB RES-P) using as model the Itupararanga Reservoir to know what the advantages and disadvantages of the application of each index in that environment. Methods: The methodology consisted on three stages: 1) macroinvertebrates sampling and determination of limnological variables in situ, and sorting of the organisms in laboratory, 2) taxonomic identification of organisms and 3) determination of the saprobic degree for the collection sites and effectiveness of the application of the BMWP (Biological Monitoring Working Party score system) index and the Benthic Community Index created by CETESB (Environmental Sanitary Company of State of São Paulo) for deep regions (ICB RES-P index). Results: The study was performed over two periods (spring/summer and autumn/winter), and 8,841 organisms were sampled and divided in 17 families. The Chaoboridae (genus Chaoborus), Chironomidae (subfamilies Chironominae and Tanypodinae) and Tubificidae families had the most abundant taxa. The saprobic degree of the Itupararanga reservoir region resulted in the general classification of the reservoir as β-α mesasaprobic (βmsαms), considered critical due to its organic charge content. The BMWP index was adapted to the sample points and the scores subsequently obtained ranked the reservoir as category V, representing very poor water quality. The ICB RES-P index classified the sample points in the spring/summer period as having regular water This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Rodrigues, V.M. et al. Acta Limnologica Brasiliensia, 2016, vol. 28, e25 benthic macroinvertebrate biota using the BMWP (Biological Monitoring Working Party score system) index can be highlighted. The BMWP index was created in England in 1976 (Hawkes, 1997) based on the premise of being easy to use and quick to operate. The index is based on the scores given to the families of benthic macroinvertebrates, with the sum of the scores of these organisms used to determine the quality of the ecosystems (Hawkes, 1997). The BMWP index has been widely used in the United Kingdom to characterize the quality of rivers, streams and creeks (Metcalfe, 1989), as well as in other countries (López et al., 2005) such as Brazil (Junqueira & Campos, 1998; Junqueira et al., 2000; Monteiro et al., 2008; Roche et al., 2010; Ruaro et al., 2010). Another biotic index that uses benthic macroinvertebrates as bioindicators of aquatic environments through scoring is the Benthic Community Index (ICB), which was created by CETESB (Environmental Sanitary Company of the State of São Paulo) (CETESB, 2012). This index is a multimetric with different and specific versions used for each type of system to be analyzed. It has already been used in some studies (Yoshida & Rolla, 2012;


Introduction
The increase in anthropogenic activities in recent decades has intensified pressure on water resources.Nowadays there are very few aquatic or land ecosystems that have not suffered the direct and/or indirect influence of human activities (Goulart & Callisto 2003;Valipour 2014aValipour , b, c, 2015;;Valipour et al., 2015).As water is a key natural resource for establishing and maintaining life, several methodologies have been developed in order to determine and diagnose the environmental impacts and disturbances that result from human activities (Buss et al. 2003).One of these is the biomonitoring of aquatic organisms, such as benthic macroinvertebrates (Rosenberg et al., 1999;Egler et al., 2012;Coe et al., 2013, Feio et al., 2014;Smetanová et al., 2014).
To improve and shorten the time needed to evaluate the quality of aquatic environments, biotic indexes have been created based on the conception of the sensitivity and tolerance of benthic macroinvertebrates in response to different impacts (Cairns Junior & Pratt, 2000).Among the indexes that are currently used to investigate the water quality of aquatic ecosystems, analysis of quality, and the autumn/winter period as having good water quality.Conclusions: From previous findings, it can be concluded that further study and refinement of the components of both indexes are required in order to effectively assess the quality of water in reservoirs.
The Itupararanga Reservoir is located in the southwest region of the state of São Paulo, Brazil, in an environmental protection area (Figure 1).This reservoir is important as it provides water supply to the population and power generation to industry.The rivers responsible for supplying the reservoir have suffered a degree of human impact due to the removal of riparian vegetation, the entry of untreated domestic sewage, and demographic pressure (Cunha & Calijuri, 2011).These impacts on the reservoir drainage basin can be classified as between minimum and high (Salles et al., 2008).
It is important to note that the Itupararanga Reservoir has been the subject of a number of studies with different focuses (Conceição et al., 2011;Bottino et al., 2013;Beghelli et al., 2014).All the studies conducted so far have shown that the quality of water of the reservoir has been declining (Salles et al., 2008;Beghelli et al., 2012;Beghelli et al., 2014;Beghelli et al., 2016).None of the earlier conducted studies using macroinvertebrates as bioindicators have focused on the tributaries regions of the Itupararanga Reservoir, which are more densely occupied, and neither to have focused on test which is the most effective and applicable biotic index to determine the water quality.To enlighten this gaps the aim of this study was therefore to apply two biological indexes based on benthic macroinvertebrates (BMWP and ICB RES-P ) to answer the following question: What are the advantages and disadvantages of application of each index in a reservoir?The answers to this question will improve the use and applicability of these indexes for future biomonitoring of similar lentic environments.

Materials and Methods
The present study consisted of three distinct stages: 1) macroinvertebrate sampling and determination of water variables in situ, followed by sorting of the organisms in the laboratory, 2) taxonomic identification of the organisms, and, 3) determination of the saprobic degree of the collection sites and the adequacy of the BMWP index and the CETESB benthic community index for reservoirs (ICB RES-P ).Samplings were carried out in the Itupararanga Reservoir over two seasons: spring/summer (October to December of 2013 and January to February of 2014) using nine stations, and autumn/winter (June to September of 2014) using eight stations (Figure 2).The sampling stations were preferably located in the tributaries of the left bank of the reservoir, which are more densely occupied areas.These stations were georeferenced, and their geographic coordinates are listed in Table 1.The reservoir was divided into three zones: riverine, near the tributaries; transitional, in the middle of the reservoir; and lacustrine, close to the dam.According to Thornton et al. (1982), in a reservoir these zones occur due to differences in the limnological characteristics of each region.The division between the zones of the Itupararanga Reservoir was proposed by Beghelli et al. (2012).
Benthic macroinvertebrates were sampled using a modified Peterson dredge.Cumulative samples were taken at several times, until 10 L of sediment had been collected.The sediment was washed in the laboratory with running water with sieves with a 250 µm mesh screen to remove excess material, and was transferred to translucent plastic trays in a box with fluorescent lighting to facilitate the visualization of the organisms.After sorting, the specimens were preserved in 70% alcohol, counted and identified through specialized literature (Brinkhurst & Gelder, 2001;Mungnai, 2010;Trivinho-Strixino, 2011).In general, identification was carried out to family level.Individuals of the Chironomidae family (Diptera) were identified to genus level, molluscs to species level and Oligochaeta individuals were separated into two morphotypes.The organisms were deposited in the Benthic Macroinvertebrates Collection of the Biology Department of the Human and Biological Science Center of the Federal University of São Carlos, Sorocaba Campus, Brazil.
The regional adaptation of the BMWP model involved the calculation of a new score for each family of macroinvertebrates present in the reservoir.The saprobiotic valences of the macroinvertebrate families were determined by correlating the frequencies of occurrence with the different levels of saprobity of each sampling station.The saprobiotic levels for the sampling stations were obtained  using the reference values provided by Junqueira & Campos (1998) (Table 2), which, in turn, were adapted from the saprobiotic degrees proposed in the Richtlinie für die Ermittlung der Gewässergüte-Klasse ("Guidelines for Determining Water Quality Class") manual, created by Kolkwitz & Marsson (1909).
The parameters dissolved oxygen (OD), biochemical oxygen demand (BOD5) and chemical oxygen demand (COD) were selected as a basis for this study.The dissolved oxygen was determined in situ using an YSI 556 multi-probe.A HACH kit, which is based on the oxidation of the organic matter present in the samples by reaction with a chemical agent (potassium dichromate), was used to measure COD.After the digestion of the solution in a dry block, COD measurement was performed using the colorimetric method by spectrophotometry (model DL 5000, HACH).BOD5 was determined by the 5-day BOD protocol, which employs the technique of incubation with dilution, according to the technical standards established by the Brazilian Association of Technical Standards (ABNT, 1992).
To perform the determination and adequacy of the biotic indexes, the sampling points were organized in two ways: 1) according to temporal variation (spring/summer and autumn/winter) and 2) according to the spatial variation of the sampling points along the dam, considering the zonation of the reservoir.After the identification of the organisms and tabulation of their sampling station, the frequency of occurrence of each family was correlated with saprobiotic level, and new scores were obtained using the following Equation 1: In this equation S i is the score for the family i, n ij is the family abundance for i in location with saprobic degree j, V j is the value weighted to this saprobiotic degree and n i is the total family abundance.The weighting values used were: 10 for Os (Oligosaprobic), 5 for Os-βms (Oligo-β-mesosaprobic), 4 for βms (β-mesosaprobic), successively decreasing to 0 for Ps (Polisaprobic).From the BMWP index obtained in this study, score ranges to determine the classes of water quality (excellent, good, satisfactory, poor, and very poor) for each sampling station were proposed (Alba-Tercedor & Jiménez-Millá, 1987).This classification facilitates the visualization of biomonitoring results, as it allows graphical representation (Junqueira et al., 2000).
There are several versions of the Benthic Community Index (ICB), with each version related to a specific type of water environment.
In the present study, considering the characteristics presented by the sampled community, the ICB RES-P (Benthic Community index for Deep Regions of Reservoirs) was applied.To calculate the index, the richness of the benthic macroinvertebrates community (S), Shannon-Wiener diversity (H'), Tanytarsini/Chironomidae ratio (Tt/Chi) from community density, and tolerant group dominance (T/DT, where: T = tolerance density and DT = total density of the sample organisms) were applied.The individuals from the Chironomus Table 2. Reference values to saprobiotic levels, based on physical and chemical parameters (Junqueira & Campos, 1998).Sat Def.(Saturation defiance), Super Sat.(Super Saturation), COD (Chemical oxygen demand) and BOD (Biochemical oxygen demand).and Tubificidae family without capillary bristles are considered tolerant in these reservoirs (CETESB, 2012).The final score was achieved from the arithmetic average value obtained from the sum of each metric of the sampling stations, and this score was used to classify the water quality.The scores obtained for each sampling station were classified according to the CETESB protocol (Table 3) (CETESB, 2012).

Results
During the two study periods 8,841 individuals were sampled, which were classified into 17 taxa, with the Chaoborus (Chaoboridae), Chironomidae (subfamilies Chirominae and Tanypodinae) and Tubificidae the most abundant.The saprobic level of each sampling station was calculated from chemical parameters (Table 4).The reservoir was, therefore, in general classified as the β-α mesasaprobic (βms-αms), which is considered critical due to its high organic charge content.
The BMWP index was adapted for the Itupararanga Reservoir, and new scores were established for each sampling station based on the saprobiotic level measured (Table 5), as well as the frequency of occurrence of the identified organisms (Figures 3 and 4).Although scores are usually determined through taxonomic identification only to family level, in case the genus level was established for many taxa, mainly Chironomidae.After obtaining the scores for the identified taxa, the sum of scores was calculated and the rank for each sampling station in Itupararanga Reservoir was obtained (Table 6).The water quality was classified as category V, or in other words very poor, irrespective of the zone.
The ICB RES-P index classified the study sites in the spring/summer period as having regular water quality.In the autumn/winter period, however, an improvement in the water quality of most sample points was observed, and the water of the reservoir was considered to be of good quality (Table 7).

Discussion
The saprobiotic degrees measured demonstrated that these environments are undergoing a process of degradation, a finding which corroborates the expected results for the riverine zone (βms-αms and α ms degrees), but which is perhaps surprising for the lacustrine zone.This suggests that the L1, L2, L3 and L4 sampling stations with respectively of α ms, βms-αms, βms-αms and α ms saprobic degrees are receiving diffuse organic matter from domestic and/or agricultural sewage, consistent with the holiday homes and farms near the reservoir.The use of biological indicators of water quality to monitor watersheds is widely used in European countries, and has been included in national technical standards in several countries (Junqueira et al., 2000).As the saprobity (pollution levels) of rivers can be used as a measure of contamination resulting from the reduction of dissolved oxygen in the water, and as it directly affects the biota, different biological indexes for the assessment of water quality have been created (Monteiro et al., 2008).Acta Limnologica Brasiliensia, 2016, vol.28, e25 These indexes assign increasing values to organisms according to their tolerance to pollution.In an attempt to unify the methodology employed in each country, the BMWP (Biological Monitoring Working Party score system) was developed, which uses macroinvertebrates identified to the family taxonomic level, notably insects, which are the most numerous of such macroinvertebrates (Monteiro et al., 2008).In Brazil, the BMWP index has been adapted and used to determine the water quality of the watershed of the Rio das Velhas river in the state of Minas Gerais (Junqueira et al., 2000), and also the watershed of the Rio Meia Ponte river in the state of Goiás (Monteiro et al., 2008).In these watersheds, the BMWP has proved to be a highly effective method of monitoring water resources.However, Monteiro et al. (2008) emphasize that there is a need to adapt the biotic values of BMWP to local and regional conditions, as the BMWP tool was created for environments in temperate zones.Indeed, environmental change processes vary according to the history of human occupation around the watershed to be monitored.
The BMWP index was adapted to the Itupararanga Reservoir by establishing new scores based on the saprobic degrees obtained for the sampling stations, and the frequencies of occurrence, and abundance of the collected benthic macroinvertebrates.Despite the adjustments being performed to family levels, genus and species levels were also employed for hexapods and molluscs, and the oligochaetes were classified into two morphotypes.These adjustments were performed due to the fact that the values originally assigned to families in the BMWP list, as well as other studies, are calculated for lotic environments and, therefore, do not reflect the conditions of tropical reservoirs.The richness and diversity of organisms in reservoirs is known to be lower than those observed in rivers and streams, and the species that occur in this environment are, naturally, more tolerant to pollutants (Goulart & Callisto, 2003;Tundisi & Tundisi, 2008).
Despite the use of lower taxonomic levels, the BMWP index ranked all the sampling stations as category V, representing a water quality of very poor.Furthermore, considering that the BMWP index was originally designed for rivers and streams with a naturally higher diversity and richness of organisms, quite different from those presented in reservoirs and lakes, the BMWP index should also be adapted to the context of tropical reservoirs, which are mainly composed of organisms known to be indicators of impacted environments, such as Chironomidae and Oligochaeta.These organisms groups have shown high frequency of occurrence in almost of the sample points with βms-αms and α ms saprobic degrees considered respectively as critical and problematic due to its organic charge content levels.But in these environments such organisms constitute a large part of the community and are not solely a sign of the depletion of water quality and the environment as observed in the T1 and T2 sampling stations (βms saprobic degree/moderate organic charge content level).However, this index has the advantage of combining abiotic and biological data, providing a wider analysis of the study environment and a realistic profile of the quality of the water.
In searching for an index that did not suffer too much interference from the changes caused by the low diversity of in the studied sites, which is expected for reservoirs, the benthic community index for deep regions of reservoirs (ICB RES-P ) was selected and compared to the BMWP index, in order to provide a comparative analysis between the results obtained for the quality and integrity of the study sites.Although both indexes are based on the premise of determining the water quality of aquatic environments from scores obtained from calculations, the ICB index, in contrast to the BMWP index, does not include the saprobic degree of the site in the analysis, being based only on the constitution of the benthic macroinvertebrate community.The ICB index classified the reservoir as having water of regular quality in spring/summer and good in the autumn/winter, indicating there was a noticeable improvement in the quality of water in the autumn/winter period.This index also demonstrated that the riverine zone had worse water quality than the lacustrine and transitional zones during the autumn/winter season.
In southeast Brazil, there are two conspicuous seasons, the rainy (spring/summer), and the dry (autumn/winter).The horizontal stratification in reservoirs is less evident in rainy seasons, when water quality was considered regular by the ICB index.During the dry season, considering that there is less sediment entering the reservoir from adjacent areas and also that the quantity of organic matter and resuspended sediment is lower (Tundisi et al., 1999), the diversity of the benthic macroinvertebrate community increases, which can explain the classification of the water quality in the reservoir by the ICB index as good in the autumn/winter.Furthermore, during dry seasons in tropical environments, macroinvertebrates, especially insects, are already in an advanced developmental stage, and are therefore easier to sample (Merrit & Cummins, 1996).Nevertheless, the ICB index, despite being created to characterize tropical reservoirs, was found to give a restrictive analysis of the study environment, as it only considers the benthic macroinvertebrate community, and does not employ abiotic data.As a consequence, if only the ICB index was employed, it would provide the impression that during the autumn/winter period the water quality was better than the rainy period, mainly because of the ease of collecting macroinvertebrates (insects) in an advanced developmental stage.It is expected that during the dry season there is deterioration in water quality due to the increased system stability of tropical reservoirs (Tundisi et al., 1999).Therefore, the ICB index must be applied restrictively to one season of the year.

Conclusions
In summary, it can be concluded that both indexes are different and that neither is truly perfect in its analysis.Further study and refinement of the components of the indexes are required in order to effectively assess the quality of water in reservoirs, especially when located in tropical regions.For the BMWP index is essential not only to adjust the score of the taxa for each site, but also to modify the weightings applied to saprobic degree in reservoirs.The ICB index, meanwhile, should be applied restrictively to one season per year, or also employ abiotic data in its calculations of water quality, in order to provide more accurate results.

Figure 1 .
Figure 1.Localization of Itupararanga Reservoir in an environmental protection area, State of São Paulo, Brazil.

Figure 3 .
Figure 3. Frequency of occurrence of identified organisms of benthic macroinvertebrates according to saprobiotic levels for spring/summer station.

Figure 4 .
Figure 4. Frequency of occurrence of identified organisms of benthic macroinvertebrates according to saprobiotic levels for autumn/winter station.

Table 1 .
Sampling stations and their geographic coordinates.

Table 4 .
Saprobic levels of the sampling stations studied in the Itupararanga Reservoir.

Table 5 .
Scores assigned to each taxon of benthic macroinvertebrates in Itupararanga Reservoir.

Table 6 .
Values of the adapted BMWP and water quality ranking system based on benthic macroinvertebrates to the sampling stations of Itupararanga.

Table 7 .
Water quality ranking system based on benthic macroinvertebrates for each sampling station, zone and seasonal period obtained with ICB RES-P index.