Assessment of wastewater effluent quality in Thessaly region, Greece, for determining its irrigation reuse potential

Ecotoxicology and Environmental Safety 74 (2011) 188–194

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Assessment of wastewater effluent quality in Thessaly region, Greece, for determining its irrigation reuse potential S. Bakopoulou n, C. Emmanouil, A. Kungolos 1 Department of Planning and Regional Development, School of Engineering, University of Thessaly, Pedion Areos, 38334 Volos, Greece

a r t i c l e in f o

a b s t r a c t

Available online 16 August 2010

The objective of the present study is to assess wastewater effluent quality in Thessaly region, Greece, in relation to its physicochemical and microbiological burden as well as its toxic potential on a number of organisms. Wastewater may be used for agricultural as well as for landscape irrigation purposes; therefore, its toxicity potential is quite important. Thessaly region has been chosen since this region suffers from a distinct water shortage in summer period necessitating alternative water resources. During our research, treated effluents from four wastewater treatment plants operating in the region (Larissa, Volos, Karditsa, and Tirnavos) were tested for specific physicochemical and microbiological parameters [biochemical oxygen demand (BOD5), chemical oxygen demand (COD), total suspended solids (TSS), pH, electrical conductivity, selected metals presence (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn, As), and fecal coliforms’ (FC) number]. The effluents were also tested for their toxicity using two different bioassays (Daphnia magna immobilization test and Phytotoxkit microbiotest). The findings were compared to relative regulations and guidelines regarding wastewater reuse for irrigation. The results overall show that secondary effluents in Thessaly region are generally acceptable for reuse for irrigation purposes according to limits set by legislation, if effective advanced treatment methods are applied prior to reuse. However, their potential toxicity should be closely monitored, since it was found that it may vary significantly in relation to season and location, when indicator plant and zooplankton organisms are used. & 2010 Elsevier Inc. All rights reserved.

Keywords: Daphnia magna Phytotoxkit Physicochemical analyses Microbiological analyses Wastewater reuse Thessaly region

1. Introduction The use of alternative water resources has been recognized in recent years as a valid solution to problems such as water scarcity and water quality deterioration that have been noticed in many countries all over the world (Bouwer, 2000). Popular alternative water resources include desalination of seawater as well as reclamation and reuse of municipal wastewater (Brenner et al., 2000). Till date, the most common application for treated municipal wastewater has been agricultural irrigation while landscape irrigation is gaining interest in recent years. In the case of agricultural irrigation, wastewater can serve as a source of water and nutrients, reducing fertilization costs as well (Metcalf and Eddy, 2003). Wastewater reuse applications, especially for agricultural irrigation purposes, have already been developed and applied in many countries in the Mediterranean basin (Haruvy

n

Corresponding author. Fax: + 302421074380 E-mail addresses: [email protected], [email protected] (S. Bakopoulou), [email protected] (A. Kungolos). 1 Tel.: + 302421074480; fax: + 302421074380. 0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.06.022

et al., 1999; Papaiacovou, 2001; Angelakis et al., 2003; Bixio et al., 2006). In Greece, water imbalance is often experienced. This is due to temporal and regional variations of rain precipitation, increased water demand during summer months (due to tourism and cultivation irrigation), and the difficulty of transporting water due to the mountainous terrain. Since more than 83% of the Greek treated wastewater effluents are produced in regions with a deficient water balance, reclaimed wastewater in these areas may satisfy this demand (Angelakis et al., 2003). The region of our present research, Thessaly, is a Greek region which also suffers from a deficient water balance especially in summer months because of increased agricultural activity and high ambient temperatures. Total irrigated area in the region reaches up to 240,000 ha. This area is now partly irrigated with approximately 750 Mm3 of water, while full irrigation of the area demands approximately 1600 Mm3 of water. The use of water from a different water region (Western Greece water region, Acheloos River) is currently examined as a valid solution to the problem but water shortage may still exist even after this work is executed (Goumas, 2006). Furthermore, the intense and extensive agriculture of water-demanding crops in Thessaly (cotton and maize) may lead to over-exploitation of groundwater and

S. Bakopoulou et al. / Ecotoxicology and Environmental Safety 74 (2011) 188–194

subsequently to water resources deterioration (Loukas et al., 2007). Taking all these into account, the possibility of wastewater reclamation and reuse in the region for agricultural purposes is an appealing solution. In our research, treated effluents from four wastewater treatment plants operating in the region were tested for their quality, aiming at determining whether this kind of water could be reused for agricultural and urban irrigation purposes. Two aspects of research have been followed: (a) the adherence of wastewater quality to set physicochemical and microbiological criteria based on international or national guidelines proposed for the case of Greece, (b) the toxicity of reclaimed wastewater to model plant and animal organisms. The analyses have been repeated seasonally (autumn, winter, and summer) encompassing the seasonal changes in water abundance/consumption equilibrium and in small industrial scale/ domestic activities observed in this region. Some studies aiming at testing the quality of treated wastewater from specific treatment plants in Thessaly have already been performed (SakellariouMakrantonaki and Angelaki, 2007), but no study yet has taken into account the majority of high performance plants operating in the region, so that more generalized conclusions regarding wastewater reuse potential in Thessaly area can be drawn.

2. Materials and methods Quality of the effluents from Larissa, Volos, Karditsa, and Tirnavos treatment plants was checked. Three samples were taken from each examined plant (except Volos plant from which two samples were taken) during July 2008–January 2009, corresponding to three different periods (summer, autumn, and winter). The third sampling (winter period) was not possible for Volos treatment plant. The above plants have been chosen according to their treating capacity, since they produce approximately 75% of the total treated effluents in the region (Thessaly region, 2005). All the plants are centralized, operating as activated sludge ones. Larissa, Volos, and Karditsa plants disinfect the produced secondary effluent by use of chlorination method while Tirnavos plant uses UV irradiation method for its effluent disinfection. Furthermore, a sand filtration technique is applied for the advanced treatment of secondary effluents in Tirnavos plant prior to their disinfection (by use of UV). The effluents were tested for specific physicochemical and microbiological parameters, including BOD5, COD, TSS, pH, electrical conductivity, and FC number for summer, autumn, and winter and selected metals presence (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn, As) for summer and autumn. The physicochemical and microbiological parameters were measured according to Standard Methods for the Examination of Water and Wastewater (APHA–AWWA–WEF, 1995). For dissolved metals determination, the procedure was essentially as described in the study by Mitsios et al. (2005): a 200 mL sample was filtered through a 0.45 mm pore-size membrane filter and acidified with 0.6 mL of 1:1 HNO3 solution. Fe, Mn, Zn, Ni and Cu were determined by Flame Atomic Absorption Spectroscopy (AAS), while Cd, Co, Cr, Pb and As were determined by Graphite Furnace AAS, using a Perkin Elmer instrument, model 3300. Metal concentrations presented were estimated as a mean of two replicates and the accuracy of the determinations were verified through the standard addition technique. Differences between sites were evaluated by a one-way ANOVA for all groups of two or three replicates followed by Tukey’s HSD test, after verification of equal variances (Levene’s test), via the statistical package SPSS 16.0 for Windows. All the above parameters have been chosen for testing based on several studies or laws determining or aiming at determining specific criteria regarding wastewater reuse procedures in Greece and worldwide (Tsagarakis et al., 2004;

189

Andreadakis et al., 2003; Common Ministerial Decree, 133551/2008; WHO, 1989, 2006; EPA, 2004; FAO, 1992). The above studies propose different quality criteria for different uses of treated wastewater (agricultural, landscape irrigation, aquifer recharge, potable uses, etc.). The criteria of concern for the present study are those referring to agricultural and landscape irrigation uses. Besides physicochemical and microbiological analyses, the toxicity of the effluents was tested using two different bioassays (Daphnia magna immobilization test and Phytotoxkit microbiotest). The toxicity tests on D. magna were carried out using Daphtoxkit F magna (Microbiotest Inc., Belgium), which follows ISO 6341 (1996) standard method. Experiments with Daphtoxkit F are based on the immobilization of the crustacean D. magna due to the action of toxicants. D. magna were hatched from dormant eggs (ephippia) in 3 days under continuous illumination (6000 lx) at 20 1C. Neonates (younger than 24 h) were exposed to the undiluted samples for 24 h at a temperature of 20 1C in darkness. Twenty neonates were used for each sample in a series of four wells; each well was containing 10 mL sample and 5 neonates. The toxicity of each sample was evaluated by the estimation of D. magna mortality (immobilization) rates. In agreement to ISO 6341, control mortality was less than 10% in all replicates. The phytotoxicity of the wastewater samples was evaluated using the Phytotoxkit microbiotest, provided by Microbiotests Inc., Belgium. This microbiotest measures the rate of both seed germination and root growth of three higher plant species: the monocotyl sorghum Sorghum saccharatum, the dicotyl garden cress Lepidium sativum, and mustard Sinapis alba. The tests were performed essentially as described in Phytotoxkit protocol (Biohidrica Ltd., Chile). Ten seeds of each plant were placed at equal distance near the middle ridge of the tests plate on a black filter paper placed on reference soil that was hydrated with wastewater samples. One series of three test plates (containing reference soil) of each plant species was hydrated by distilled water and it was used as control. About 30 mL of wastewater sample, or deionized water for the control plates, was added for the hydration of the reference soil. Three replicates were used for each plant species. The plates were placed vertically in a holder and incubated for 3 days at 25 1C, in darkness. The pictures of the test plates at the end of the exposure period were taken by a digital camera and the length of the root of each plant was measured using the Image Tool 3.0 software (UTHSCSA, USA). The percent inhibition of root growth was calculated using the formula AB  100 A adapted from Phytotoxkit protocol (Biohidrica Ltd., Chile) where A is the mean root length in the reference soil hydrated with deionized water and B is the mean root length in the reference soil hydrated with wastewater samples. Differences between sites and differences between seasons were evaluated by a two-way ANOVA for all groups of three replicates followed by Tukey’s HSD test, after verification of equal variances (Levene’s test), via the statistical package SPSS 16.0 for Windows.

3. Results 3.1. Physicochemical and microbiological analyses The majority of physicochemical and microbiological analyses results are presented in Table 1 where average values as well as standard deviation values from the three different sampling periods (summer, autumn, and winter period, respectively) are shown. Furthermore, the fluctuation of the physicochemical and microbiological parameters’ values during the three different samplings for each wastewater treatment plant is depicted in Fig. 1. It should be noted that a third sampling (winter period) was not possible for Volos treatment plant. Additionally, the metal analyses (average values for summer and autumn period) are shown in Table 2. The comparison of the physicochemical and

Table 1 Results of physicochemical and microbiological analyses of treated effluents in Thessaly region.

Larissa tr. plant Volos tr. plant Karditsa tr. plant Tirnavos tr. plant

BOD5 (mg/L)

COD (mg/L)

TSS (mg/L)

pH

Electrical conductivity (mS/cm)

FC (MPN/100 mL)

7.33 74.16 9.50 72.12 6.00 72.00 6.00 73.46

34.67 7 17.67 427 15.56 18.67 7 6.66 29.33 7 21.94

7.25 7 0.83 9.50 7 0.99 7.3 7 0.66 2.5 7 1.32a

9.67 0.36 8.957 0.07b 9.47 0.36 7.917 0.17a

11.13 7 1.53 10.92 7 0.11 9.45 7 1.14 0.97 0.17a

115 7161.17 – 136.67 7151.7 absence

Note 1: FC number for Volos treatment plant was not calculated because the effluent sample taken was not disinfected. Note 2: Average values 7 standard deviation values. a b

Significantly different from Volos, Karditsa, Larissa. Significantly different from Karditsa, Larissa.

190

S. Bakopoulou et al. / Ecotoxicology and Environmental Safety 74 (2011) 188–194

100 Volos

10 8

Karditsa

6

Larissa

4

Tirnavos

80 COD (mg/L)

BOD5 (mg/L)

14 12

Volos

60

Karditsa

40

Larissa Tirnavos

20

2 0

0 Autumn

Summer

Winter

Summer

12

10 Volos Karditsa

6

Larissa 4

Volos

8 pH

8

TSS (mg/L)

Winter

12

10

Karditsa

6

Larissa 4

Tirnavos

2

Tirnavos

2 0

0 Summer

Autumn

Winter

Summer

Sampling

Autumn

Winter

Sampling

350

14 Volos

8 6

Karditsa

4 2

Tirnavos

Larissa

FC/100mL

300

12 10

Conductivity (mS/cm)

Autumn Sampling

Sampling

250

Karditsa

200 150

Larissa Tirnavos

100 50

0

0

0

0

0

0 Summer

Autumn

Winter

Summer

Sampling

Autumn

Winter

Sampling

Fig. 1. Fluctuation of physicochemical and microbiological parameters during the three different samplings for each of the examined effluents. A: BOD5 values, B: COD values, C: TSS values, D: pH values, E: electrical conductivity values, and F: FC number

Table 2 Seasonal metal range concentrations (mg/L) in the effluents of the wastewater treatment plants. Metal

As Cd Co Cr (tot) Cu Fe Mn Ni Pb Zn

Volos

ND2 2  10  3 7 1  10  3 4.6 7 0.64 0.85 7 0.23 1.14 7 0.35 16.15 7 0.85 4.95 7 0.48 3.05 7 0.85 0.037 0.01 2.2 7 0.71

Karditsa

ND 3  10  3 7 1  10  3 2.97 0.57 2.357 0.28a 0.957 0.42 16.55 7 0.92 6.507 0.53 1.957 0.42 0.027 0.01 1.17 0.57

Larissa

ND 2  10  3 71  10  3 2.6 70.66 1.00 70.21 0.90 70.71 25.7 70.85b 7.35 70.35 1.00 70.49 0.03 70.01 1.85 70.28

Tirnavos

ND 2  10  3 7 1  10  3 4.8 7 0.99 1.75 7 0.17 1.6 7 0.43 18.60 7 0.78 2.80 7 0.51c 1.20 7 0.85 0.027 0.01 0.89 7 0.42

Detection Limit

0.005 0.5  10  3 0.005 0.002 0.05 0.1 0.05 0.1 0.002 0.02

Maximum concentration1 Long-term

Short-term

0.1 10  10  3 0.05 0.1 0.2 5 0.2 0.2 5 2

2 50  10  3 5 1 5 20 10 2 10 10

1

Source: EPA, 2004; FAO, 1992. ND: not detected. a Significantly different from Volos, Larissa. b Significantly different from Volos, Karditsa, Tirnavos. c Significantly different from Volos, Karditsa, Larissa. 2

microbiological effluent quality from the four different wastewater treatment plants indicates that Tirnavos plant produces effluents which are characterized in general by better physicochemical and microbiological parameter values than the other three plants. BOD5 and COD values are the only exceptions

since they are not different enough between the four plants. FC numbers were high in Karditsa and Larissa samples but their considerable seasonal variation led to annual non-significant differences between the three sites. In reference to metal analyses, Tirnavos effluents are not different from the rest of

S. Bakopoulou et al. / Ecotoxicology and Environmental Safety 74 (2011) 188–194

191

Table 3 Immobilization rates for Daphnia magna after exposure to the wastewater effluents. Sampling

Volos treatment plant

Karditsa treatment plant

Larissa treatment plant

Tirnavos treatment plant

Summer Autumn Winter

0% 100% –

0% 0% 0%

0% 100% 0%

0% 100% 0%

Root growth inhibition level/summer period

A

100

%root growth inhibition

80

a

a

b

A c

a

b

A c

a

b

a

b

d

Volos

60

Karditsa Larissa

40

T irnavos

20 0 S alba

-20

L sativum

S saccharatum

-40 -60

Root growth inhibition level/autumn period

B

% root growth inhibition

100 80

a

a

A

b

B

c a

b

c

d

a

b

a

b

Volos Karditsa

60

Larissa T irnavos

40 20 0 S alba

L sativum

S saccharatum

-20

Root growth inhibition level/winter period C A C 100

% root growth inhibition

80 60

b a

b

c

Karditsa

c b

a

40

b

Larissa T irnavos

20

d

0 -20

S alba

L sativum

S saccharatum

-40

since chlorine is a toxic substance for such organisms (EPA, 1984; Cao et al., 2009). In the majority of cases, mortality of D. magna was 0% (summer and winter period) (Table 3). The only exceptions were observed in autumn period where the toxicity rates were 100% for all the plants except the Karditsa one. These high toxicity rates observed in autumn were in accordance with chemical analyses noticed in the corresponding effluents, which showed high organic burden for the same period (see Fig. 1B). Regarding the application of the Phytotoxkit microbiotest, Fig. 2 shows the effects of the examined treated effluents (decrease or increase of root growth for each of the tested plants). As it can be observed in the aforementioned figure, the relative results varied significantly in relation to site and to season. Furthermore, there was a significant interaction between site and season (two-way ANOVA, Po0.05). Regarding S. alba, there was a significant difference between all three seasons with autumn samples being collectively more toxic than the summer or the winter ones. The winter samples were the least toxic. Volos and Karditsa effluent toxicities did not differ significantly from each other for S. alba, whereas Larisa and Tirnavos effluent toxicities differed significantly from each other. They were also significantly different from the Volos and Karditsa ones. Tirnavos samples were collectively the least toxic. Regarding L. sativum, there were no significant differences between all three seasons. Each site however showed different toxicities to L. sativum within the same season. In brief, Volos effluent toxicities were different from Karditsa, Larissa, and Tirnavos ones. Karditsa and Larissa effluent toxicities were comparable to each other but overall significantly different from each other. Tirnavos effluent toxicities were collectively the lowest and they were significantly different from the Volos, Karditsa, and Larissa ones. Regarding S. saccharatum, again there was a significant difference between all three seasons with autumn samples being collectively more toxic than the summer or the winter ones. Volos and Larissa effluent toxicities did not differ significantly from each other for S. saccharatum and Karditsa and Tirnavos effluents did not differ significantly from each other for S. saccharatum. Karditsa and Tirnavos effluents were less toxic than the Volos and Larissa ones.

-60 -80 -100

Fig. 2. Root growth inhibition level for the three different species of plants and the three different wastewater treatment plants. A: summer period, B: autumn period, and C: winter period. Capital letters (A, B, C) indicate differences between seasons among sites while small letters (a, b, c, d) indicate differences between sites among seasons.

effluents. The majority of metals in the examined effluents do not exceed the proposed limits for short-time use of reclaimed wastewater for irrigation purposes (see also Section 4). 3.2. Toxicity analyses Regarding toxicity of treated effluents on D. magna, nonchlorinated samples were used in all cases for toxicity testing,

4. Discussion 4.1. Physicochemical and microbiological analyses A comparison of the physicochemical and microbiological analyses values (Table 1) to corresponding reference values resulting from studies or laws proposing wastewater reuse criteria for the case of Greece (Tsagarakis et al., 2004; Andreadakis et al, 2003; Common Ministerial Decree, 133551/2008) indicates that the majority of BOD5 values as well as the TSS ones of all samples (Fig. 1A and C, respectively) are lower than the proposed limit values for the cases of unrestricted irrigation (10 mg/L for both BOD5 and TSS parameters). It should be mentioned that the unrestricted irrigation includes irrigation of vegetables that are eaten raw, irrigation of residential areas with high public access,

192

S. Bakopoulou et al. / Ecotoxicology and Environmental Safety 74 (2011) 188–194

etc. Thus, the secondary treatment of wastewater for the examined plants produces effluents characterized by BOD5 and TSS values suitable for use in unrestricted irrigation. This is generally true for the case of BOD5, however for the case of TSS the above conclusion is not always reliable. Relative studies assessing the secondary effluent quality of the majority of treatment plants operating in Greece (Andreadakis et al., 2003) have shown that besides low values of TSS, which characterize in general the secondary effluents from Greek plants, the corresponding values of turbidity are higher than the limit value of 2 NTU. In order to reduce such values, the application of an effective advanced treatment method (i.e. sand filtration) is suggested as essential prior to the disinfection process. Furthermore, the greater reduction of TSS values generally results in better removal of total coliforms (TC) and FC through the disinfection unit. This fact was also verified by the results of the present analyses. It is observed (Fig. 1C, F) that Tirnavos effluent, which is characterized by very low values of TSS (significantly lower from the rest of the plants), is also characterized by FC absence. Such results have also arisen from studies aiming at investigating advanced treatment technologies operation for wastewater reclamation and reuse purposes (Pollice et al., 2004; Petala et al., 2006). Regarding the COD parameter, the corresponding values of all samples fluctuate between the three different samplings (see Fig. 1B). In general, high values are observed in autumn period while in the other time periods the corresponding values are reduced. This could be attributed to increased agricultural domestic activity observed in the region (i.e. alcohol distilling). However, even if the highest COD values of all samples are compared to the criteria set by the Greek Ministry for the case of using reclaimed wastewater for agricultural irrigation in the greater area of Thessaloniki city (COD¼80 mg/L) (Ministerial Decree Ref No123805, 2004), the above values are still within the limits (except Larissa ones), implying that the secondary treatment could ensure in general the production of effluent suitable for reusing, regarding COD values. Concerning the pH and electrical conductivity parameters, by comparing the measured values (see Table 1 and Fig. 1D, E, respectively) to reference values proposed for the case of Greece (Andreadakis et al., 2003), it could be concluded that the effluents of Larissa, Volos, and Karditsa treatment plants did not fulfill the corresponding criteria. Such criteria impose that the highest electrical conductivity value of irrigation water should not exceed the 3 mS/cm, while an optimum value reaches up to 0.7 mS/cm. For the case of pH value, the optimum range is between 6.5 and 8. The above limit values are only fulfilled by the effluent of Tirnavos treatment plant and this can be attributed to the use of UV irradiation in this plant whereas the other plants use chlorination for their effluent disinfection. The most common disinfectant used for the chlorination in Thessaly plants is NaOCl, a strongly alkaline solution (pH 13.5–14) which in addition dissociates in water to NaCl and OH  . This alkaline behavior of NaOCl explains the high pH values of effluent in Larissa, Volos, and Karditsa treatment plants. Regarding the microbiological analysis of FC number, the calculated average numbers of such coliforms arising from the three samplings are much higher than the proposed limit values for unrestricted irrigation for Larissa and Karditsa samples while in Tirnavos sample there were no such coliforms. Volos effluent was not tested for FC number since it was not possible to take a disinfected sample. It is worth mentioning that the average FC number of Larissa and Karditsa sample still fulfill the proposed criteria for restricted irrigation in Greece (200 FC/100 mL) (Andreadakis et al., 2003). However, it should be mentioned that the above microbiological criteria proposed for the case of Greece are strict, since they are mainly based on California criteria. World

Health Organization guidelines for wastewater reuse, which were published in 1989, were less strict in FC number criterion (1000/ 100 mL as average value and for uses such as unrestricted irrigation) (WHO, 1989); however, WHO edited in 2006 the above guidelines by proposing stricter criteria (WHO, 2006). Comparison of the effluent quality from the four different wastewater plants operating in Thessaly region indicates that Tirnavos plant’s effluent was characterized in general by better physicochemical and microbiological quality characteristics than the other three ones, allowing, in all examined cases, the use of this effluent for beneficial uses such as unrestricted irrigation. This fact could be mainly attributed to the use of the sand filtration technique in Tirnavos plant in conjugation with use of UV irradiation for its effluent disinfection. Finally and in reference to metal analyses, comparison of the actual values to the limit values proposed by EPA (2004) and FAO (1992) (Table 2) shows that the use of Thessaly wastewater effluents for irrigation purposes is, in most cases, possible, but for a short period of time. The only exceptions observed were for Cr (Karditsa and Tirnavos plants), for Fe (Larissa plant), and finally for Ni (Volos plant). Furthermore, according to EPA (2004), the term ‘‘short time’’ refers to 20 years use maximum and for finetextured neutral and alkaline soils with high capacities to remove the different pollutant elements. After that time, a program aiming at alternation in use of fresh and recycled water should be implemented or, otherwise, advanced treatment methods aiming at metals removal (i.e. reverse osmosis) should be applied. However, such treatment methods are expensive and a cost– benefit analysis should be implemented. The only metals which fulfill the ‘‘long term’’ and ‘‘without any precautions’’ criteria for irrigation are Cd and Pb for all plants and Zn for Karditsa, Larissa, and Tirnavos plants. Furthermore, it is worth mentioning that As was not detected in any of the tested effluents. This is an important result since human risks arising from As-containing wastewater used for irrigation purposes are significant (Chiou, 2008).

4.2. Toxicity analyses Toxicity indices on living organisms have not been included in wastewater reuse criteria implemented worldwide (EPA, FAO, WHO guidelines); however, toxicity analyses are important since they can provide direct evidence regarding the overall adverse effects of reclaimed wastewater on environment. A meaningful battery of bioassays for quality evaluation of effluents and superficial waters should ideally include toxicity on producers (like algae and higher plants), consumers (like crustaceans, bacteria, and rotifers) and decomposers (Mankiewicz-Boczek et al., 2008). In our case, since the reclaimed wastewater was specifically examined for possible irrigation purposes, testing focused on higher plants. Toxicity on freshwater crustaceans was complementary and was done on the grounds of possible leaching of irrigation waters towards superficial aquifers (Okamura et al., 1999). D. magna is an excellent model organism for aquatic toxicology and its sensitivity is comparable to that of higher aquatic organisms. In some cases of pollutants the acute LD50 Daphnia test has been proven to be more sensitive than the acute LD50 fish test (i.e. Danio rerio or Pimephales promelas) (Martins et al., 2007; Marchini et al., 1993 respectively). Regarding toxicity towards D. magna test, all samples were characterized by 0% mortality of the neonates except for autumn samples for Volos, Larissa, and Tirnavos plants where mortalities were 100%. The high mortalities observed in autumn could probably be attributed to distilling activities and they are directly linked to the increased (in relation to winter and summer) COD

S. Bakopoulou et al. / Ecotoxicology and Environmental Safety 74 (2011) 188–194

values (Fig. 1B). Thessaly area is characterized by the presence of numerous small-scale and usually home-based alcohol distilleries which utilize exclusively grape marcs. Ethanol itself is not toxic for Daphnia as proved by relevant research (i.e. Hermens et al., 1984; Calleja et al., 1994; Lilius et al., 1995); however, the distillation byproducts are rich in phenolic compounds [i.e. 14.99–20.30 mg gallic acid equivalent/g dry weight in grape skin according to Yilmaz and Toledo (2006)]. These non-biodegradable compounds which significantly increase COD (Guerra, 2001; Negro et al., 2003) but not BOD, might have contributed to the high organic burden observed here for autumn period and they are inherently toxic for D. magna (Guerra, 2001; Kamaya et al., 2005). The root growth feature was used for assessing the phytotoxicity of the tested effluents since, according to results of relative studies (Oleszczuk, 2008a), seed germination proved to be a weaker indicator of phytotoxicity assessment relatively to root growth. It should be noted that the results varied significantly between the three different species of plants: the dicotyl L. sativum and S. alba and the monocotyl S. saccharatum. In brief, the tests with S. alba showed significant differences in effluent toxicities between seasons but the toxicities were not always distinguishable between sites (Fig. 2). The same principle applied more or less for S. saccharatum: there were significant differences in toxicities between summer, autumn, and winter, but the differences were not always significant between sites. In contrast, it was quite surprising that effluent toxicities for L. sativum as a whole did not differ significantly between seasons. These results show the limitations of using only one kind of toxicity testing and highlight the need for a battery of relevant bioassays. It was therefore rather complex to draw universal conclusions from the sum of high density data on plant toxicities. A significant interaction between site and season was evident for all tested plants. Furthermore, each plant species showed different toxicity behavior that was not always in agreement with the other two of the plant species tested. Some of the most meaningful deductions that can be made are (a) autumn effluents produced in general higher toxicity effects for S. alba and S. saccharatum and (b) Tirnavos plant generally produced effluents characterized by lower toxicity than the other plants. It is characteristic that the winter effluent sample of Tirnavos plant increased the root growth of all tested plant species (Fig. 2C). The high phytotoxicity rates observed in autumn period in the majority of plant species and the tested plants are corroborated by higher toxicities to D. magna as well as by worse chemical analyses results. This fact could also be attributed to higher non-biodegradable organic burden observed in this period in all the wastewater treatment plants. Indeed, a relative study (Ellouze et al., 2009) showed that one of the above plant species (L. sativum) is sensitive to the presence of non-biodegradable organic as well as mineral pollutants. Furthermore, CzerniawskaKusza et al. (2006) showed that sediment organic matter content increase has a significant impact on increasing the corresponding sediment phytotoxicity and Oleszczuk (2008a, 2008b) found that the composting of sewage sludge resulted in the corresponding decrease of the majority of its ingredients causing phytotoxicity. The metals present probably contributed to the observed phytotoxicity to a limited extent; phytotoxic metals like Cu, Mn, and Zn (EPA, 2004) were within the limits for short time use. These metals however were above limits for long-term irrigation. Cr and Fe, which exceeded the limits for short time use, are of limited toxicological value for plants (EPA, 2004). Overall toxicity comparison between the treatment plants showed that Tirnavos plant generally produced effluents characterized by lower toxicity than the other plants. This fact may be justified by the operation of the sand filtration unit in comparison

193

with a disinfection unit using UV irradiation in this plant. Therefore, such units operation generally results in producing effluents with not only better physicochemical quality characteristics (i.e. TSS, turbidity, electrical conductivity, pH values) but also with lower phytotoxicity.

5. Conclusions Application of physicochemical and microbiological analyses showed that secondary effluents produced in Thessaly region are generally suitable for reuse in irrigation purposes and especially in crops which are not used raw by humans (restricted irrigation case), i.e. cotton, a prominent cultivation in the region. Use of treated wastewater for irrigation of agricultural products that are eaten raw (unrestricted irrigation case) is also possible but only under certain conditions and not for all plants’ effluents (use of an effective advanced treatment method and no chlorination is essential). Furthermore, in such cases additional precautions should be taken in order to minimize the potential risks arising when humans come in contact with such water resources. Regardless the fulfillment of physicochemical and microbiological criteria, toxicity analyses showed that the examined effluents should still be used with caution. It is worth mentioning that some of the samples were highly toxic for D. magna while still fulfilling set criteria. As a result, potential leaching of these effluents towards aquatic reservoirs could cause detrimental effects to their zooplankton and possibly to other aquatic species. Regarding terrestrial plants, which would be the ultimate recipient of these irrigation waters, laboratory toxicity testing on dicotyls and monocotyls is by no means representative for infield situations. However, the season- and site-specific inhibition of growth noted throughout our experiments shows some of the problems which may arise through this irrigation in the field. Extended toxicity analyses should always accompany the physicochemical and microbiological analyses of reclaimed wastewater especially because of fluctuations in effluent quality and composition between the different seasons of the year.

Acknowledgments This paper is part of the 03ED375 research project, implemented within the framework of the ‘‘Reinforcement Program of Human Research Manpower’’ (PENED) and co-financed by National and Community Funds (25% from the Greek Ministry of Development, General Secretariat of Research and Technology and 75% from EU European Social Fund). References Andreadakis, A., Gavalaki, A., Mamais, D., Noutsopoulos, K., Tzimas, A., 2003. Proposal of determination of qualitative parameters and prerequisites for wastewater reuse in Greece. In: Proceedings of LIFE 99/ENV/GR/000590 Meeting: Aqcuisition and Reuse of Wastewater, Thessaloniki, pp. 19–75 (in Greek). Angelakis, A.N., Bontoux, L., Lazarova, V., 2003. Challenges and prospectives for water recycling and reuse in EU countries. Water Sci. Technol.: Water Supply 3 (4), 59–68. APHA–AWWA–WEF, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed., Washington, DC. Biohidrica Ltd: Phytotoxkit Bench Protocol /http://www.biohidrica.cl/pdfs/phyto toxkit-protocol.pdfS. Bixio, D., Thoeye, C., De Koning, J., Joksimovic, D., Savic, D., Wintgens, T., Melin, T., 2006. Wastewater reuse in Europe. Desalination 187, 89–101. Bouwer, H., 2000. Integrated water management: emerging issues and challenges. Agric. Water Manage. 45, 217–228. Brenner, A., Shandalov, S., Messalem, R., Yakirevich, A., Oron, G., Rebhun, M., 2000. Wastewater reclamation for agricultural reuse in Israel: trends and experimental results. Water Air Soil Pollut. 123, 167–182.

194

S. Bakopoulou et al. / Ecotoxicology and Environmental Safety 74 (2011) 188–194

Calleja, M.C., Personne, G., Geladi, P., 1994. Comparative toxicity of the first 50 multicentre evaluation of in vitro cytotoxicity chemicals to aquatic nonvertebrates. Arch. Environ. Contam. Toxicol. 26, 69–78. Cao, N., Yang, M., Zhang, Y., Hu, J., Ike, M., Hirotsuji, J., Matsui, H., Inoue, D., Sei, K., 2009. Evaluation of wastewater reclamation technologies based on in vitro and in vivo bioassays. Sci. Total Environ. 407, 1588–1597. Chiou, R-J., 2008. Risk assessment and loading capacity of reclaimed wastewater to be reused for agricultural irrigation. Environ. Monit. Assess. 142, 255–262. Common Ministerial Decree, 133551/2008: /http://www.elinyae.gr/el/lib_file_up load/2089b08.1225273735875.pdfS (in Greek). Czerniawska-Kusza, I., Ciesielczuk, T., Kusza, G., Cichon, A., 2006. Comparison of the phytotoxkit microbiotest and chemical variables for toxicity evaluation of sediments. Environ. Toxicol. 21, 367–372. Ellouze, M., Saddoud, A., Dhouib, A., Sayadi, S., 2009. Assessment of the impact of excessive chemical additions to municipal wastewaters and comparison of three technologies in the removal performance of pathogens and toxicity. Microbiol. Res. 164, 138–148. EPA, 2004. Guidelines for water reuse. EPA/625/R-04/108, Washington, p. 450. EPA, 1984. Ambient water quality criteria for chlorine. Office of Water Regulations and Standards Criteria and Standards Division, Washington, pp. 5–6. FAO, 1992. Wastewater treatment and use in agriculture. Irrigation and Drainage Paper 47, M.B. Pescod, Rome, p. 125. Goumas, K., 2006. Irrigation in Thessaly plain: consequences in water and groundwater. In: Hellenic Hydraulic Union (Eds.), Proceedings of Water Resources and Agriculture, Thessaloniki, 2/2/2006, Greece, pp. 39–53 (in Greek). Guerra, R., 2001. Ecotoxicological and chemical evaluation of phenolic compounds in industrial effluents. Chemosphere 44, 1737–1747. Haruvy, N., Offer, R., Hadas, A., Ravina, I., 1999. Wastewater irrigation—economic concerns regarding beneficiary and hazardous effects of nutrients. Water Resour. Manage. 13, 303–314. Hermens, J., Canton, H., Janssen, P., De Jong, R., 1984. Quantitative structure activity relationship and mixture toxicity studies of chemicals with anesthetic potency: acute lethal and sublethal toxicity to Daphnia magna. Aquat. Toxicol. 5, 143–154. ISO, 1996. Water quality determination of the inhibition of the mobility of Daphnia magna strauss (Cladocera, Crustacea) acute toxicity test. ISO 6341. Kamaya, Y., Fukaya, Y., Suzuki, K., 2005. Acute toxicity of benzoic acids to the crustacean Daphnia magna. Chemosphere 59, 255–261. ¨ Lilius, H., Hastabacka, T., Isomaa, B., 1995. A comparison of the toxicity of 30 reference chemicals to Daphnia magna and Daphnia pulex. Environ. Toxicol. Chem. 14, 2085–2088. Loukas, A., Mylopoulos, N., Vasiliades, L., 2007. A modeling system for the evaluation of water resources management strategies in Thessaly, Greece. Water Resour. Manage. 21, 1673–1702. Mankiewicz-Boczek, J., Na"˛ecz-Jawecki, G., Drobniewska, A., Kaza, M., Sumorok, B., Izydorczyk, K., Zalewski, M., Sawicki, J., 2008. Application of a microbiotests battery for complete toxicity assessment of rivers. Ecotoxicol. Environ. Saf. 71, 830–836.

Marchini, S., Hoglund, M.D., Broderius, S.J., Tosato, M.L., 1993. Comparison of the susceptibility of daphnids and fish to benzene derivatives. Sci. Total Environ. 134, 799–808. Martins, J., Oliva Teles, L., Vasconcelos, V., 2007. Assays with Daphnia magna and Danio rerio as alert systems in aquatic toxicology. Environ. Int. 33, 414–425. Metcalf and Eddy, 2003. Wastewater Engineering, Treatment and Reuse 4th ed. McGraw-Hill International Editions, New York. Ministerial Decree Ref no. 123805, , 2004. Ministry of Environment Physical Planning and Public Works, Ministry of Health and Social Solidarity, Ministry of Rural Development and Food. Approval of environmental terms and conditions on reuse of processed municipal wastewater for irrigation purposes in Thessaloniki (in Greek). Mitsios, I.K., Golia, E.E., Tsadilas, C.D., 2005. Heavy metal concentrations in soils and irrigation waters in Thessaly region, Central Greece. Commun. Soil Sci. Plant Anal. 36 (4–6), 487–501. Negro, C., Tommasi, L., Miceli, A., 2003. Phenolic compounds and antioxidant activity from red grape marc extracts. Bioresour. Technol. 87, 41–44. Okamura, H., Omori, M., Luo, R., Aoyama, I., Liu, D., 1999. Application of short-term bioassay guided chemical analysis for water quality of agricultural land runoff. Sci. Total Environ. 234, 223–231. Oleszczuk, P., 2008a. Phytotoxicity of municipal sewage sludge composts related to physico-chemical properties, PAHs and heavy metals. Ecotoxicol. Environ. Saf. 69, 496–505. Oleszczuk, P., 2008b. The toxicity of composts from sewage sludges evaluated by the direct contact tests phytotoxkit and ostracodtoxkit. Waste Manage. 28, 1645–1653. Papaiacovou, I., 2001. Case study—wastewater reuse in Limassol as an alternative water source. Desalination 138, 55–59. Petala, M., Tsiridis, V., Samaras, P., Zouboulis, A., Sakellaropoulos, G.P., 2006. Wastewater reclamation by advanced treatment of secondary effluents. Desalination 195, 109–118. Pollice, A., Lopez, A., Laera, G., Rubino, P., Lonigro, A., 2004. Tertiary filtered municipal wastewater as alternative water source in agriculture: a field investigation in Southern Italy. Sci. Total Environ. 324, 201–210. Sakellariou-Makrantonaki, M., Angelaki, A., 2007. Toxicity tests on reclaimed municipal wastewaters. In: Kungolos, A., Aravossis, K., Karagiannidis, A., Samaras, P. (Eds.), Proceedings of First International Conference on Environmental Management, Engineering, Planning and Economics (CEMEPE), June 24–28, Skiathos island, Greece, pp. 265–270. Tsagarakis, K.P., Dialynas, G.E., Angelakis, A.N., 2004. Water resources management in Crete (Greece), including water recycling and reuse and proposed quality criteria. Agric. Water Manage. 66, 35–47. Yilmaz, Y., Toledo, R.T., 2006. Oxygen radical absorbance capacities of grape/wine industry byproducts and effect of solvent type on extraction of grape seed polyphenols. J. Food Compos. Anal. 19, 41–48. WHO, 1989. Health guidelines for the use of wastewater in agriculture and aquaculture. Report of a WHO Scientific Group, WHO Technical Report Series 778, Geneva. WHO, 2006. WHO Guidelines for the Safe Use of Wastewater, Excreta and Greywater. Volume II: Wastewater Use in Agriculture, WHO publications, Geneva, pp.196.