Desalination 281 (2011) 185–189

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Safety evaluation of an artificial groundwater recharge system for reclaimed waterreuse based on bioassays

Xue Zhang a, Xuan Zhao a,⁎, Meng Zhang a, Qian-yuan Wu b

a Laboratory of Environmental Technology, Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, Chinab Environmental Simulation and Pollution Control State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China

⁎ Corresponding author. Tel.: +86 10 62796428; fax:E-mail address: zhxinet@tsinghua.edu.cn (X. Zhao).

0011-9164/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.desal.2011.07.060

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 April 2011Received in revised form 23 July 2011Accepted 25 July 2011Available online 24 August 2011

Keywords:BiotoxicityOzonationReclaimed water reuseSafety evaluationSoil aquifer treatment

A set of bioassays (acute toxicity to Daphnia, genotoxicity, estrogenic and antiestrogenic toxicity) was used toevaluate the safety of reclaimed water reuse in a lab-scale artificial groundwater recharge system. Ozonationof 1 mg O3·(mg DOC)−1 was used as the pretreatment and was proved to be effective in removing the fourtoxicity, with removal ratios ranging from 56% to 99%. In the vadose layer, the responsible compounds fortoxicity were less efficiently removed than the bulk organic compounds. However, they were preferentiallyremoved during the saturated aquifer treatment, with all the toxic effects per carbon mass dropping by 20–50%. Therefore, more than onemonth traveling in the aquifer is suggested before water reuse. Since UV254 wassignificantly correlated with the toxicity data, it could serve as an indicator of the toxicity. With the bioassay-based safety evaluation, the combination of soil aquifer treatment and ozonation could provide new watersources with no higher toxic than the conventional natural drinking water source.

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Water crisis in both water quantity and quality has risen as a globalurgent issue and received more and more attention from government,industry and academe. Reclaimed water (e.g., municipal effluent fromthe Wastewater Treatment Plant (WWTP)) has been generallyacknowledged as an alternative water source and provides an approachto coping with the water crisis. Among all kinds of reuse strategies,artificial groundwater recharge (AGR) with reclaimed water is apromising choice due to its advantages of long time storage and furtherpurification by soil and aquifer [1].

The safety of reclaimedwater reuse is themajor concern, since thereare lots of complex organic compounds existing in the reclaimed water.Recently, many studies reported a number of problematic tracepollutants in WWTP effluents, such as endocrine disruptors, pesticides,pharmaceuticals and personal care products [2–5]. Such trace pollut-ants can cause various toxic effects, e.g. reproductive disorders, intersexinduction, DNA damages and mutations even in quite low concentra-tion (μg·L−1 or even ng·L−1). As a result, more andmore attention hasbeen paid to this area. A number of studies have investigated on the fateand removal of various individual or specific groups of trace pollutantsto evaluate the safety of water during AGR [6–8]. Many trace organicpollutants could be removed to some extent during AGR depending ona number of factors, such as climate and physiochemical conditions ofthe aquifer [9]. However, it is not enough or even impossible for the

safety evaluation of AGR only by the determination of individual tracechemicals considering the large variability and synergistic effects ofvarious chemicals. Additionally, trace chemical detections are time-consuming and expensive. Biological assays are considered to be amoreefficient method for direct measurement of the overall toxicity of thecomplex compounds and can provide useful information on potentialtoxicity risk assessments.

Many investigations have reported various toxic effects (e.g.,estrogen, genotoxicity, acute toxicity) in WWTP effluents and riversreceiving WWTP effluents [10–12]. However, so far as we know, littleis known on how the biotoxicity of the trace pollutants changesduring AGR. Some studies revealed that little attenuation of estrogenicactivity was apparent during percolation of effluent in the riverchannel to well withdrawal points [13], while genotoxicity (on a permass basis) increased during vadose transport [14]. Further studies onthe safety of AGR based on diverse bioassays are necessary.

In this study, a set of bioassays was used for the safety evaluation ofa lab-scale AGR system with ozonation as the pretreatment. Fourbioassays, i.e. acute toxicity toDaphnia, estrogen and antiestrogen usingyeast two-hybrid test and genotoxicity using umu test were selected,since they are widely used in the toxicity evaluation ofWWTP effluentsand various wastewater treatment technologies [10,11,15,16].

2. Materials and methods

2.1. The laboratory-scale AGR system

The AGR system was simulated in the lab with five soil columns inseries. The first column (C1)was used to simulate the vadose soil layer

Table 1Physiochemical parameters of water samples (data presented as the average of 5 weekrecords, standard deviations shown in brackets).

R0 R1 WS1 WS5

DOC(mg·L−1) 4.06±0.11 4.15±0.14 1.20±0.16 0.97±0.06UV254 (m−1) 10.85±0.57 3.58±0.21 1.95±0.37 2.33±0.15SUVA (L·mg−1·m−1) 2.67 0.86 1.63 2.40NH4–N (mg·L−1) 1.34±0.99 1.46±0.90 0.14±0.01 0.13±0.01NO3–N (mg·L−1) 19.08±1.50 19.44±1.46 21.38±0.88 21.37±0.91PO4–P (mg·L−1) 1.48±0.83 1.44±0.80 0. 85±0.06 0.08±0.02pH 7.27±0.01 7.34±0.02 7.64±0.02 8.03±0.01

186 X. Zhang et al. / Desalination 281 (2011) 185–189

(aerobic condition) and operated under unsaturated conditions bypumping reclaimed water to the top of the column, with the cycle of3-day flooding and 1-day drying. The other four columns (C2–C5)were operated under saturated conditions and used to representanoxic and anaerobic redox saturated aquifer treatment. Thecumulative hydraulic retention times (HRT) of C1–C5 columns were0.8, 7.8, 14.8, 21.8 and 28.8 days, respectively. To balance the waterflux in this system, the size of C1 was smaller than the other four. Theinternal diameters were 12 cm for C1 and 24 cm for the following fourcolumns (C2–C5). All five columns were 200 cm in height, with apacked-bed height of 180 cm. Theywere all filledwith sandy powderysoil (grain size ranging from 0.4 to 0.8 mm) collected from a 9 to 17 mdepth aquifer of in the south-east suburb of Beijing, with porosity of0.39. All columns were kept at room temperature of 20±2 °C in thedark.

The secondary effluent from Gaobeidian Wastewater TreatmentPlant (traditional activated sludge treatment process was used in thisWWTP) was supplied as the feed water of C1 after ozonation. Theozone dosage of 1 mg O3·(mg DOC)−1 had been optimized in theprevious study to achieve a higher ratio of BDOC/DOC. The effluent ofC1 was stored in a 10-L tank and part was pumped through C2–C5columns in sequence. The system was biologically adapted and readyfor sampling after acclimatization of around a year.

2.2. Water sample collection and physiochemical analysis

The secondary effluent from GaobeidianWWTP (R0), the effluentsafter ozonation (R1) and from bottom of C1 and C5 (identified asWS1and WS5, respectively) were sampled at least once a week. All watersamples were firstly filtered by 0.45 μm filters and then the followingparameters were measured: dissolved organic carbon (DOC), absor-bance at 254 nm (UV254), nitrate (NO3–N), ammonia (NH4–N) andphosphate (PO4–P). DOC was measured using a Shimadzu TotalOrganic Carbon Analyzer (TOC-VWP) (Shimadzu Corporation, Japan).UV254 was analyzed with a Shimadzu UV-3100 ultraviolet–visible(UV/VIS) spectrophotometer (Shimadzu Corporation, Japan) at254 nm. The nitrate (NO3–N), ammonia (NH4–N) and phosphate(PO4–P) were determined according to the Standard Methods ofWater and Wastewater Monitoring of China [17]. The average waterquality parameters of 5 weeks' records (two weeks before, two weeksafter and one week during the soil sampling) would be given anddiscussed.

2.3. Concentration of water sample

Certain volumes (0.5–1 L) of water samples (R0, R1, WS1 andWS5) were filtered through 0.7 μm GF/C membrane (Whatman, UK),and acidified to pH 2.0 with 2 M H2SO4. Then the water samples werepassed through OASIS HLB resin cartridges (6 cm3 200 mg, WatersCorporation, America), which were previously washed with 10 mL ofmethanol and 10 mL of ultrapure water. The cartridges were thendried under air flow. Retained organic compounds on the cartridgewere eluted with methanol (10 mL), dichloromethane (5 mL) andhexane (5 mL), successively. The eluents were dried under nitrogenflow. The dry residues were dissolved in 200 μL of dimethylsulfoxide(DMSO) for estrogenic/antiestrogenic/genotoxicity activity assays orin 200 μL of acetone for Daphnia acute toxicity test.

2.4. Biotoxicity assays

The genotoxicity of concentrated water samples was evaluatedwith the SOS/umu test based on Salmonella typhimurium TA1535/pSK1002 without S9 activation in triplicates according to ISO 13829[18]. The 4-nitroquinoline-N-oxide (4-NQO) was used as the positivecontrols for the dose–response curve. The genotoxicity of watersamples was standardized to a toxic equivalent concentration

(TEQ) of 4-NQO. More than 20% growth inhibition of S. typhimuriumindicates a cytotoxic effect.

The estrogenic and antiestrogenic activities of the concentratedwater samples were evaluated with the yeast two-hybrid assay basedon yeast cells (Saccharomyces cerevisiae Y190) which contained therat estrogen receptor ERα and the coactivator TIF2 according to [19].The estrogenic and antiestrogenic activities of water samples werestandardized to TEQs of estrogen 17β-estradiol (E2) and antiestrogentamoxifen (TAM), respectively. More than 20% growth inhibition ofthe yeast cells indicates a cytotoxic effect.

The acute toxicity to Daphnia magna was carried out according toOECD 202 [20]. The cladoceran species Daphnia magna Straus waskindly supplied by the Freshwater Biological Laboratory, University ofCopenhagen. The D. magna animals were cultured in ADaM mediumwith S. acutus as the sole food source. The animals for experimentswere prepared in the same way in [21]. Young daphnids, aged lessthan 24 h, were exposed to the test water samples at a range ofconcentrations for 48 h, which was expressed as the relativeenrichment factor (REF) of the water sample. The REF was calculatedas described in [22]. Both concentrated water samples in acetone andnon-concentrated water were diluted with the ADaM medium andtested in 25 mL beakers with 5 neonates in 20 mLwater samples. Fourparallel samples were prepared for each test. Meanwhile, blankcontrol of ADaM medium and solvent control of acetone were testedin the same way and no deaths were detected in 48 h.

2.5. Data analysis

Differences in toxicity between samples were determined statis-tically using Student's t-test.

3. Results and discussion

3.1. Physiochemical water quality

The average water parameters of 5 weeks' records were listed inTable 1. No obvious change in DOC was observed after ozonation,while more than 60% of UV254 was removed, which meant thatozonation at this dose could not completely oxide the organic mattersinto carbon dioxide, but could significantly destroy the UV-sensitivematters like unsaturated/aromatic organic compounds. The unsatu-rated vadose treatment (C1) removed 70% of DOC and 15% of UV254.No significant decrease of DOC was observed during the saturatedaquifer treatment (pN0.05, t-test), whereas a slight increase of UV254

was detected. The SUVA, representing the relative aromaticity of theorganic compounds, decreased significantly after ozonation, whereasit kept increasing during the soil treatment, indicating that aliphaticmatters were preferentially removed during the soil treatment.Around 90% of NH4–N was converted by C1 into almost equal amountof NO3–N. No further change of NO3–N was detected in the saturatedaquifer treatment. Most of PO4–P was removed by the saturatedaquifer treatment. All these physiochemical parameters were com-parably steady for further toxicity tests after one-year running.

Fig. 1. TEQ and removal ratio of estrogen activity.

187X. Zhang et al. / Desalination 281 (2011) 185–189

3.2. Acute toxicity to D. magna

Acute toxicity toD.magna of the fourwater sampleswas tested andcompared by the parameters of LL100, NOEC and LC50 (Table 2). D.magna deaths happened in 20% R0 and all neonates died in 50% R0. Thecalculated LC50 for R0 was 0.4 (representing as REF), which indicatedthe secondary effluent from WWTP was acutely toxic to D. magna. Asfor the other water samples (R1, WS1 andWS5), no death of D. magnawas observed in non-concentrated samples, whereas deaths werefound in various concentrations presented as REF. The LC50 of R1 was4.5, which was about 10 folds of R0's LC50 value. The LC50 values keptincreasing to 12.1 and 28.7 for WS1 and WS5, respectively. Theincrease of LC50 indicated the decrease of acute toxicity to D. magna.Therefore, it could be concluded that both ozonation and soil aquifertreatment could remove pollutants responsible for the acute toxicity.

3.3. Estrogen and antiestrogen activity

The TEQs and removal ratios of estrogen activity were presented inFig. 1. The estrogen activity was 1.51 ng-E2 L−1 in the secondaryeffluent R0, which was comparable with previous reports of estrogenin WWTP effluents [11,23–25]. Estrogen was removed by 57% afterozonation, and dropped to 0.66 ng-E2 L−1. Further removal ofestrogenwas observed during the soil aquifer treatment. The estrogenactivity decreased to 0.52 ng-E2 L−1 after the vadose layer treatmentand further decreased to 0.32 ng-E2 L−1 after 28 days traveling in thesaturated aquifer treatment. Considering that no observed effect(intersex or vitellogenin induction) concentration of E2 is 1 ng L−1

[26], AGRwith ozonation as pretreatment exhibited as a safe approachfor wastewater reclamation with respect to estrogen activity. Theinhibition ratios of yeast growth were all below 10% in the testedsamples, indicating no growth inhibition during the tests.

Antiestrogenic chemicals are another class of chemicals existing inthe wastewater effluents, which can affect reproduction and devel-opment of animals as the estrogenic chemicals [27]. However, theremoval characteristics were different during water treatments (i.e.,rapid infiltration) [28]. Therefore, it is important to pay attention toboth estrogenic and antiestrogenic chemicals. Here, the antiestrogenactivity was measured along the treatment processes and presentedin Fig. 2. The antiestrogen activity was 0.62 mg-TAM L−1 in R0, ofwhich 70% was removed by ozonation. The antiestrogen activityremained at 0.18 mg-TAM L−1 during the unsaturated vadosetreatment, whereas 15% more was further removed during the 28-day saturated aquifer treatment. The concentration dropped to0.09 mg-TAM L−1 in the terminal effluent. The inhibition ratios ofyeast growth were all below 20%, which meant there were nosignificant growth inhibitions. Compared with estrogen, antiestrogencould be more efficiently removed by both ozonation and saturatedaquifer.

3.4. Genotoxicity

Genotoxicity describes a deleterious action on a cell's geneticmaterials, which would be caused by mutagenic or carcinogenicchemicals. The TEQs and removal ratios of genotoxicity were

Table 2Acute toxicity to D. magna of different water samples (data present as REF).

Water sample LL100 NOEC LC50±SE

R0 0.5 0.2 0.4±1.3R1 8 2 4.5±1.1WS1 15 10 12.1±0.9WS5 36 15 28.7±1.0

LLC100: the lowest concentrated factor that caused 100% death of D. magna in 48 h;NOEC: no observed effect concentrations in 48 h; LC50: the calculated concentrationfactor that caused 50% death of D. magna in 48 h.

presented in Fig. 3. The genotoxicity in the secondary effluent was28.7 μg-4-NQO L−1, which was at the same level of the previousreports [29]. It was dramatically reduced to 0.07 μg-4-NQO L−1 byozonation, with a removal ratio of 99.8%. No significant further changewas detected in the following soil aquifer treatment (pN0.05, t-test).No cytotoxic effects were detected in the umu tests, since theinhibition ratios of bacteria growth were all below 20%. Ozonationseems to be the main contributor for the genotoxicity removal, whichagreed with previous studies [22,30].

The toxic effects per carbon mass, representing the relativetoxicity, were listed in Table 3. Ozonation played a significant rolein removing the toxic effects, since all the three toxic effects percarbon mass decreased significantly after ozonation. Therefore, aspretreatment of AGR, ozonation performed as an important barrier todestroy toxic pollutants. Considering no obvious change in DOC afterozonation (Table 2), it could be deduced that ozonation couldtransform the relevant toxic chemicals without complete mineraliza-tion, which agreed with previous studies in both lab and full scaleozonation systems [15,22]. However, all the relative toxic activitiesincreased during the unsaturated vadose zone, which meant thatresponsible compounds for toxicitywere removed less efficiently thanthe bulk organic compounds. After 28-day travelling in the saturatedaquifer, all the three toxic effects per carbonmass dropped by 20–50%,indicating that the toxic chemicals were preferentially removedduring the saturated aquifer. Further removal of toxic chemicals maytake place during the following travelling in the aquifer. Therefore, atravel time of more than one month in the aquifer was suggestedbefore extracting water from an AGR system. In summary, ozonation

Fig. 2. TEQ and removal ratio of antiestrogen activity.

Fig. 3. TEQ and removal ratio of genotoxicity.

Table 4Pearson's correlation between the toxicity and organic content parameter (Pearson'scorrelation coefficient, significant factor).

Estrogen Antiestrogen Genotoxicity Acute toxicitya

DOC 0.726, 0.274 0.632, 0.368 0.559, 0.441 0.613, 0.387UV254 0.980⁎, 0.020 0.980⁎, 0.020 0.986⁎, 0.014 0.994⁎, 0.006

⁎ Significantly correlated, pb0.05.a Acute toxicity to D. magna was represented as the reciprocal of LC50 in this

correlation calculation, since LC50 was represented using REF.

Table 5Water qualities of drinking water resource (DWR) and the final effluent of the AGR(WS5).

DWR WS5

188 X. Zhang et al. / Desalination 281 (2011) 185–189

and soil aquifer treatment could complement with each other inremoving toxic chemicals.

3.5. Correlation of the toxicity and the physiochemical parameters

Compared with regular physiochemical parameters, biotoxicitytests were complicated and difficult for real time detections.Therefore, it would be useful if some physiochemical parameterscould serve as indicators of the biotoxicity. Pearson's correlation wasused to find the correlation between the organic content (DOC, UV254)parameters and toxicity data. Significant correlations were foundbetween UV254 and all the four toxicity data (estrogen, antiestrogen,genotoxicity and acute toxicity to D. magna), with the correlationcoefficients all above 0.98 (Table 4). In another way, the removalefficiency of UV254 in this system was similar to those of the toxicactivities. It may be deduced that unsaturated/aromatic organiccompounds represented by UV254 are closely related to the toxicchemicals according to their similar fates during the treatments.

3.6. Safety assessment by comparing with a drinking water resource

Although the TEQs of several toxic activities were detected for theAGR system, it is still difficult to evaluate the water safety, since norelevant guideline is available. Therefore, a reference water sourcewas selected. Water samples from Jingmi canal were used as thereference, since the Jingmi canal was used for transporting drinkingwater source (DWR) from Miyun reservoir to Water Treatment Plantin urban area of Beijing and generally considered as a safe watersource. The physiochemical and toxic parameters of DWR and WS5were summarized in Table 5. After the pre-oxidation of ozone andadditional long term purification of soil aquifer treatment, thephysiochemical parameters of the AGR terminal effluent are lowerthan those of DWR. Although higher content of NO3–N was detectedin WS5 due to the lack of denitrification, it can be avoided byoptimizing the pretreatment of AGR. As for all the three toxicactivities, the TEQs of WS5 were all significantly lower than those ofDWR (Pb0.05, t-test). As a result, the wastewater reclamation by AGR

Table 3Biotoxicity per carbon mass for different water samples.

Estrogen(ng-E2·mg DOC−1)

Antiestrogen(mg-TAM·mg DOC−1)

Genotoxicity(μg-4-NQO·mg DOC−1)

R0 0.37 0.15 7.07R1 0.16 0.04 0.02WS1 0.43 0.15 0.09WS5 0.33 0.10 0.04

at least provides an additional water source, which is less toxic thanthe local natural drinking water resource, with respect to the toxicitydetected here.

Considering the whole system, ozonation reduced the toxicity ofreclaimed water (estrogen, antiestrogen and genotoxicity) to thesame level as the drinking water resource (DWR). However, the bulkorganic contents (DOC and UV254) were still significantly higher in theozonated water (R0) than the DWR. The soil treatment (especially forthe vadose layer) was effective in removing these compounds. Also,further removal of toxicity was achieved during the soil treatment,such as the acute toxicity to Daphnia (Table 2). Therefore, the soiltreatments and ozonation are synergistic with each other and arenecessary for this system.

4. Conclusions

This study focused on the evaluation of water quality in a lab-scaleAGR systembased on a set of bioassays and thewater safety assessmentby comparingwith a drinking water source. The secondary effluent hadacute toxicity to D. magna with LC50 of 0.4, estrogen of 1.5 ng-E2 L−1,antiestrogen of 0.62 mg-TAM L−1 and genotoxicity of 28.7 μg-4-NQO L−1, most of which were above the safety limits. Ozonation of1 mg O3·(mg DOC)−1 acted as an important barrier for AGR, withremoval ratios of the four toxic effects ranging from 56% to 99%. Duringthe unsaturated vadose zone, removals of acute toxicity and estrogenwere detected and the bulk organic compounds were more effectivelyremoved (DOC removed by 70%) than responsible compounds fortoxicity. In the saturated aquifer with HRT of 28 days, the toxicchemicals were preferentially removed, with decrease of the toxicityper carbon mass by 20–50% when compared with the effluent of thevadose treatment. Therefore, a long time travel in the aquifer isimportant to get rid of the toxic chemicals, and a travel time of morethan one month in the aquifer was suggested before extracting waterfrom an AGR system. UV254 was significantly correlated with thetoxicity data, and could be used as an indicator of biotoxicity to avoidthe complicated analysis under certain conditions. In well-managedAGR systems, new water sources can be supplied with no higher toxicthan the conventional natural drinking water source, with respect tothe toxicity detected here.

DOC (mg·L−1) 1.64±0.12 0.97±0.06UV254 (m−1) 2.30±0.16 2.33±0.15SUVA (L·m−1·mg−1) 1.40 2.40NH4–N (mg·L−1) 0.36±0.06 0.13±0.01NO3–N (mg·L−1) 1.30±0.06 21.37±0.91PO4–P (mg·L−1) – 0.08±0.02pH 8.27±0.02 8.03±0.01Estrogen (ng-E2·L−1) 0.51±0.01 0.32±0.01Antiestrogen (mg-TAM·L−1) 0.20±0.01 0.09±0.01Genotoxicity (μg-4-NQO·L−1) 0.14±0.06 0.04±0.02

–, Under the detection limit.

189X. Zhang et al. / Desalination 281 (2011) 185–189

Acknowledgements

This researchwas supported by National Natural Science Foundationof China (Grant No. 50878115 and 51078211). The authorswould like tothank Prof. Hu Hong-ying for his great support in the bioassays.

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