Environ Sci Pollut Res DOI 10.1007/s11356-015-4238-8 RESEARCH ARTICLE Behaviour of estrogenic endocrine-disrupting chemicals in permeable carbonate sands Benjamin O. Shepherd & Dirk V. Erler & Douglas R. Tait & Lukas van Zwieten & Stephen Kimber & Bradley D. Eyre Received: 16 September 2014 / Accepted: 13 February 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract The remediation of four estrogenic endocrinedisrupting compounds (EDCs), estrone (E1), estradiol (E2), ethinylestradiol (EE2) and estriol (E3), was measured in saturated and unsaturated carbonate sand-filled columns dosed with wastewater from a sewage treatment plant. The estrogen equivalency (EEQ) of inlet wastewater was 1.2 ng L−1 and was remediated to an EEQ of 0.5 ng L−1 through the unsaturated carbonate sand-filled columns. The high surface area of carbonate sand and associated high microbial activity may have assisted the degradation of these estrogens. The fully saturated sand columns showed an increase in total estrogenic potency with an EEQ of 2.4 ng L−1, which was double that of the inlet wastewater. There was a significant difference (P < 0.05) in total estrogenic potency between aerobic and anaerobic columns. The breakdown of conjugated estrogens to estrogenic EDCs formed under long residence time and reducing conditions may have been responsible for the increase in the fully saturated columns. This may also be explained by the desorption of previously sorbed estrogenic EDCs. The effect of additional filter materials, such as basalt sediment and coconut fibre, on estrogenic EDC reduction was also tested. None of these amendments provided improvements in estrogen remediation relative to the unamended unsaturated carbonate sand columns. Aerobic carbonate sand filters have good potential to be used as on-site wastewater treatment systems for the reduction of estrogenic EDCs. However, the use of fully saturated sand filters, which are used to promote denitrification, and the loss of nitrogen as N2 were shown to cause an increase in EEQ. The potential for the accumulation of estrogenic EDCs under anaerobic conditions needs to be considered when designing on-site sand filtration systems required to reduce nitrogen. Furthermore, the accumulation of estrogens under anaerobic conditions such as under soil absorption systems or leachate fields has the potential to contaminate groundwater especially when the water table levels fluctuate. Keywords Estrogenic EDCs . Saturation state . Carbonate sand . Remediation . Wastewater treatment systems . Groundwater Responsible editor: Hongwen Sun B. O. Shepherd (*) : D. V. Erler : D. R. Tait : B. D. Eyre Centre for Coastal Biogeochemistry, School of Environment, Science and Engineering, Southern Cross University, Lismore, NSW 2480, Australia e-mail: [email protected] L. van Zwieten : S. Kimber Department of Primary Industries, Bruxner Highway, Wollongbar, NSW 2477, Australia L. van Zwieten Southern Cross Plant Science, School of Environment, Science and Engineering, Southern Cross University, Lismore, NSW 2480, Australia Introduction A growing area of environmental and human health concern is contamination of groundwater with endocrine-disrupting chemicals (EDCs) (Jenssen 2006; Kookana et al. 2007). The United States Environmental Protection Agency (USEPA) defines an EDC as Ban exogenous agent that interferes with the synthesis, secretion, transport, binding, action or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development and/ Environ Sci Pollut Res or behaviour^. Chronic, low-level exposure to EDCs in both humans and animals has been linked to a number of reproductive disorders (Kookana et al. 2007). EDCs encompass a wide variety of chemical classes, including natural and synthetic hormones, plant constituents, pesticides, certain compounds in plastics, personal care products, pharmaceuticals, pollutants and by-products of industry (Damstra et al. 2002). EDCs are widely dispersed in the environment and have been found in rivers, estuaries, coastal samples and even in the arctic (Braga et al. 2005a; Jenssen 2006). Some of the most potent EDCs include steroid estrogens such as estrone (E1), 17β-estradiol (E2), estriol (E3) and 17αethinylestradiol (EE2). These estrogenic EDCs have been shown to have adverse affects on the reproductive systems of marine and freshwater animals at less than 5 ng L−1 (Jobling et al. 1998, 2002a, b, 2006; van Aerle et al. 2001; Pelley 2003). Estrone is a metabolite of estradiol and is an important pregnancy estrogen. Estradiol is a reproductive hormone naturally excreted by female animals including humans and in much smaller quantities by males. Ethinylestradiol is the main ingredient in the female contraceptive pill and hormone replacement medication (Trussell 2001; Song et al. 2009). A major source of estrogenic EDCs to natural waterways is wastewater treatment plant effluent and leachate of sewage sludge either buried in landfill or applied as fertilizer (Jobling et al. 2006; Koyama et al. 2006; Thorpe et al. 2009; Karnjanapiboonwong et al. 2011; Marti and Batista 2014). Concentrations of estrogenic EDCs in wastewater can range from nanograms per litre (ng L−1) to micrograms per litre (μg L−1) (Ying and Kookana 2005). Other potential sources of estrogenic EDCs in developed and agricultural catchments include on-site wastewater management systems, effluent run-off following manure application or run-off of livestock wastes (Reneau and Degen 1989; Hanselman et al. 2003; Zhang et al. 2011). Water contamination by sewage is common in all island countries of the Pacific region with many streams and groundwater supplies currently considered unsafe for human consumption without treatment (UNESCAP 2000). In the porous carbonate sands of many Pacific islands, groundwater is particularly susceptible to contamination from poorly performing sanitation systems. Indeed ground and surface water pollution associated with septic tank and absorption trench disposal systems is common in many Pacific countries (Dakers and Evans 2007). Some soil absorption systems which are used as on-site treatment systems in these countries can operate successfully for years, whilst others can fail within weeks of installation (Beal et al. 2005). Studies by Karnjanapiboonwong (2011) showed that EDCs that were present in the effluent from a wastewater treatment plant were able to reach the groundwater after land application of the effluent. Given the porous nature of the soil in most Pacific islands, the steadily increasing human population and greater use of synthetic compounds, such as the contraceptive pill, it is quite likely that groundwater may contain elevated concentrations of estrogenic EDCs. However, it is also possible that carbonate sands may act to physically, chemically and/or biologically remove estrogenic EDCs from effluent water. There is some evidence that EDCs can be effectively degraded during transport through porous media (Nakada et al. 2007). Biodegradation of EDCs has been shown to be the most cost-effective way of removing EDCs relative to advanced removal mechanisms such as activated carbon, membrane filtration, chemical advanced oxidation and ozonation (Liu et al. 2009). Using quartz sand filtration systems, Nakada et al. (2007) showed a 50 % removal rate of estrogenic EDCs. Carbonate sands may be particularly effective for the treatment of EDCs (Rasheed et al. 2003). They may be an ideal candidate for incorporation into wastewater treatment systems in developing Pacific nations due to their ready availability. However, the behaviour of EDCs in carbonate sands has not been studied previously. In particular, it is not understood how the biodegradation of estrogens will be affected by the degree of saturation within carbonate sands. Other studies have shown that unsaturated aerobic soils are more likely to degrade EDCs relative to saturated anaerobic soils. Permeable carbonate sands however are unique in that they have extremely high surface areas capable of sustaining a large microbial biomass and contain greater adsorption capabilities and pH buffering relative to quartz sands (Rasheed et al. 2003; Schöttner et al. 2011). The behaviour and degradation of EDCs in permeable carbonate sands therefore may be enhanced relative to other sands and soils. To date, there have been no studies that have looked at the removal of EDCs in carbonate sands under saturated or unsaturated conditions. The aim of this study was to investigate the behaviour of estrogenic compounds in permeable carbonate sands under saturated and unsaturated conditions. A broader goal of this research was to assist in the development of low-cost on-site wastewater treatment technologies for EDC removal in developing Pacific countries. Materials and methods Equipment and experimental design In this study, the main objective was to test the reduction of EDCs within large columns containing permeable carbonate sands. A secondary objective was to test whether estrogenic EDC removal would be affected by the addition of other soils/ materials (e.g. basalt soil, coconut fibre) to some of the sand columns. Basalt sediment and coconut fibre were used as amendments as these are readily available and commonly used with carbonate sand in wastewater treatment systems throughout the Pacific. Environ Sci Pollut Res The columns were operated under a range of saturation states, and some were amended with alluvial basalt soil and coconut husk fibre. Different saturation states and media types were used to increase retention times, adsorption capacity and degree of aeration and/or availability of microbial habitat. Basalt and carbonate sand sediments were shipped from Rarotonga in the Cook Islands (21°14′05″ S, 159°46′30″ W) and sterilized (Australian customs requirement) with gamma irradiation (Steritech Pty Ltd). Sediments were loosely packed into vertical flow PVC columns (diameter 150 mm, length 1.8 m). Columns were kept in a dark temperature-controlled room at the South Lismore sewage treatment plant (STP), Lismore, NSW, Australia. In all, there were seven carbonate sand treatments, with each treatment containing two columns. All columns contained 100 mm of 20-mm blue metal gravel above the carbonate sand to distribute the effluent evenly to the underlying media. Treatment 1 (T1) was a fully unsaturated carbonate sand column, treatment 2 (T2) was similar to T1 except that it received a double dosing of influent and treatment 3 (T3) was fully unsaturated sand with a 100-mm layer of coconut fibre at the top of the column. Treatment 4 (T4) contained an unsaturated homogenous mixture of sand and basalt sediment. Treatment 5 (T5) contained unsaturated carbonate sand with a 100-mm basalt sediment layer 900 mm from the top of the column. Treatment 6 (T6) contained carbonate sand with a saturated zone of 800 mm, created by lifting the outlet tubing to the desired height, and treatment 7 (T7) contained carbonate sand with a 1500-mm saturated zone. Grain size for the carbonate sand was ~81 % greater than 0.5 mm. Basalt soil was with ~49 % of grains less than 50 μm. Chemical composition of the sediments showed that trace metals were higher in the basalt soils. The carbonate sands contained 12 % carbon, and the basalt had 3 % carbon; both sediments had <0.5 % nitrogen. Grain size and soil analysis results were taken from unpublished studies by Tait et al. (manuscript submitted for publication). Columns were dosed regularly with effluent from the STP’s primary sedimentation tank. Primary treated effluent was transferred via a 25-mm pipe from the sedimentation tank to a control valve attached to a 200-μm filter (Arkal). The filter was intended to mimic septic tank discharge effluent and remove larger particulate matter that had potential to clog dosing lines. The primary treated influent was connected to a 240-L PVC barrel with a 15-mm ball float valve to maintain a constant volume. Primary treated influent was pumped from the barrel via a 12-V submersible pump (Amazon) to a capped 450×90 mm PVC header tank above the columns. The header tank allowed for even distribution of effluent to columns. The cap on the bottom of the header tank had 5 mm evenly distributed outlets inserted. The outlets were connected to 1.5 m of 6-mm PVC tubing. The header tank would fill and feed effluent through the tubes to distribution bars in the top 50 mm of the columns. The distribution bar consisted of a modified 50-mL plastic syringe with a series of 10-mm holes drilled along the bottom side. The distribution bar allowed for less restriction on outlet tube flow and created an even dispersal of influent over the media. The submersible pump was connected to a 12 V timer that was set to dose six times daily (7:00 am, 9:00 am, 11:00 am, 3:00 pm, 6:00 pm and 9:00 pm). The pump was set to deliver approximately 140 mL per column, which is equivalent to a hydraulic loading rate of 48 mm day−1. The double-dose treatment (T2) had two inlet tubes and was given 280 mL per dose equivalent to a hydraulic loading rate of 96 mm day−1. Figure 1 shows a picture of the experimental design. Flow rates were monitored regularly to maintain a consistent effluent flow to columns. Dosing rates were based on the maximum daily loading rates used for carbonate sand sediments in the Lagoon Protection Zone for the Cook Islands (CIMH 2009). Initially there was an equilibration period where columns were dosed with 2 L of primary treated effluent weekly for 6.5 weeks (20 April to 6 June 2011). The equilibration period allowed sediments to settle within columns and a biofilm to develop on amendments and previously sterilized sediments. Following this period, the columns were dosed daily, as described above, from 6 June 2011 to the end of the experiment. The experimental assay ran for 7 weeks (1 July 2011 and ended 21 August 2011). Sampling and analysis In the first 2 weeks of the experiment, a relative measure of column residence time was obtained using a pulsed addition Fig. 1 Experimental column design for T1 (unsaturated), T2 (unsaturated, double dosed), T3 (unsaturated, coconut), T4 (unsaturated basalt mix), T5 (unsaturated basalt layer), T6 (partially saturated), T7 (fully saturated) Environ Sci Pollut Res of a conservative tracer (NaBr) added to the inlet barrel (1680 μg L−1). The barrel was then disconnected from the incoming STP effluent to prevent the dilution of the NaBr. Samples of Br− were collected daily from the inlet barrel and every second day from the outlet of the columns. Following the tracer addition, the collection and analysis of influent and column outlet water for the determination of EDC concentrations commenced. Influent samples were collected three times a week, whilst column outlet sampling occurred once a week. The sampling was performed for four consecutive weeks, and samples were collected in 500-mm plastic PVC bottles. After collection, samples were stored on ice and processed within 3 h. Processing of water samples for EDC concentrations involved extraction using 4 mL (200 mg) C18 solid-phase extraction (SPE) cartridges (Grace Scientific). These cartridges were pre-conditioned by running 10 mL of acetonitrile through the cartridge under vacuum, followed by 10 mL of 50 % v/v methanol/milli Q water, 10 mL of methanol and then 20 mL of dilute phosphoric acid (pH<3). Extraction of EDCs from the wastewater involved filtering 200 mL of each effluent sample through a 47-mm glass microfiber filter (GF/F nominal pore size 0.7 μm). The filtered sample was then passed through a conditioned SPE cartridge under vacuum. The cartridge was then rinsed with 10 mL of the phosphoric acid under vacuum. Cartridges were oven-dried for 2 days at 50 °C. To extract the EDCs from the dry SPE cartridges, 10 mL of 50 % acetonitrile/ethanol was passed under vacuum. The eluate was collected in 15-mL centrifuge tubes, capped and refrigerated. The eluted samples were placed in a dry block heater (Ratek) at 60 °C, evaporated down to 0.5–1 mL under a stream of dry N2 gas and then transferred to 20-mL auto-sampler vials with a glass pipette. Centrifuge tubes were washed with 0.5–1 mL of hexane to remove any residue, and contents were put into the sampler vials. The vials were then dried at 70 °C whilst under a stream of dry N2 gas and derivatised with 100 μL of N,O-bis(trimethyl-silyl) trifluoroacetamide with 1 % trimethylchlorosilane (BSTFA +1 % TMCS) solution+ 100 μL of pyridine (see method by Li et al. (2007)). Vials were recapped with a lid containing a teflon insert and placed in the dry block heater for 30 min at 70 °C. Vial caps were then removed, and excess reagents were evaporated with a stream of dry N2. Hexane (0.5 mL) was then added to vials, caps were replaced and vials were shaken ready for analysis via gas chromatography/mass spectrometry (GCMS). An automated Agilent 7890 GCMS in EI (SIM) mode controlled by Chemstation software measured the EDC analyte concentrations. The 5975C Mass Spectrometer had a triple axis detector installed with a 30 M J&W DB5-MS column. The carrier gas was helium set at a constant flow rate of 1 mL min−1. The temperature program starts at 80 °C, is ramped up to 180 °C at 20 °C min−1, is held for 1 min, then ramped up to 280 °C at 20 °C min−1 and then held for 18 min. Samples were analysed for estrone, estriol, estradiol and ethinylestradiol. Standards, blanks and control samples were prepared similarly. An eightpoint calibration curve was plotted from standards, which were injected in a descending order of 10, 8, 6, 4, 2, 1, 0.5 to 0.2 μg L−1. Linearity was obtained across the calibration range for all compounds. For the E1 and EE2, the RSD of the peak areas from 10, 8, 6, 4, 2 to 0.5 μg L−1 were in the range of 3–15 and 3–17 %, respectively; those for E2 and E3 calibrations were in the range of 3–15 % when the calibration were from 10 to 1 μg L−1. Chemical analyses were undertaken within an ISO9001:8000 certified facility. Standards, spikes and recoveries were run with each batch of samples. Recoveries ranged from 60 to 90 %, and results were adjusted for recoveries. Analytical method was as described by Li et al. (2007). Auxiliary measurements Measurements were taken using handheld probes (Hach HQ40D) in situ for pH, temperature and dissolved O2 (DO). Influent samples were collected three times a week, and outlet samples were tested once a week using the same equipment. Data analysis One-way analysis of variance tests were performed on treatment flow rate, pH and EEQ (estrogen equivalency) concentrations in all treatment outlets and the inlet barrel. Post hochonest significant difference tests were performed when P<0.05 or Fcalc > Fcrit to determine significant differences. Percentage reduction of EDCs was calculated by dividing outlet average by inlet average. All results are presented with the standard deviation of the measurement. Estrogen equivalency (equivalent to 17B estradiol) was defined by the sum of concentrations of individual estrogens (C) multiplied by their relative toxicity TEF, (EEQ = ∑ (Ci) × (TEFi). The TEF potency values used were taken from Metcalfe et al. (2001). These potency values were obtained by using yeast estrogenicity screening assays on estrogens in sewage treatment plants to determine TEF potency values relative to 17β-estradiol (E2). The TEFs used were 17β-estradiol (E2) = 1, 17α ethinylestradiol (EE2) = 0.38, estrone = 0.14 and estriol = 0.037. Results Flow and geochemistry The average flow rate from the inlet barrel to the columns was 143±19 mL dose−1. No significant difference (P>0.05) was found between the flow rates to the columns. The conservative Environ Sci Pollut Res tracer breakthrough curves for the seven treatments are shown in Fig. 2. The saturated treatments had the longest residence times followed by the mixed sand basalt columns (Fig. 2). The pH and DO of the column outlets and the inlet barrel are shown in Fig. 3b, c. Average inlet pH and DO concentrations were 7.93±0.33 and 1.27 mg L−1, respectively. The pH generally increased, and the DO generally decreased with increasing residence time (Fig. 3a–c). Treatment 2 (doubledosed unsaturated carbonate sand) had the highest DO with an average of 5.5 mg L−1. Treatment 7 had the lowest DO with an average of 2.0 mg L−1. Estrogens The average EEQ of the influent barrel over the 4-week experimental period was 1.2 ng L−1 (EEQ equivalent to 17β estradiol). Estrone was the dominant component of the inlet EDCs and constituted 58 % of the total EDC concentration (Fig. 4). Estradiol was the least abundant EDC in the inlet (6 %). Ethinylestradiol and estriol both contributed 18 % to the total estrogenicity of the inlet sample. The EEQs from the outlets of treatments 1–6 were lower than EEQ for inlet water (Fig. 5). T1 had the best reduction of EEQ with an EEQ of 0.5 ng L−1, followed by T5 which had an EEQ of 0.6 ng L−1. Both treatments T1 and T5 were significantly different (P<0.05) than treatment 7. In the fully saturated columns (T7), there was a 6 % increase of measured EDCs (ethinylestradiol and estradiol) to the outlet water. Treatment (T7) had the highest EEQ of 2.4 ng L−1, which was double that of the inlet wastewater which had an EEQ of 1.2 ng L−1. Discussion Surface and groundwater contamination from EDCs has the potential to significantly affect human and environmental health in developing countries that are increasingly using and disposing of EDCs. The behaviour of EDCs in carbonate sands under saturated and unsaturated conditions is poorly understood but is essential for developing low-tech on-site wastewater treatment systems for the removal of EDC-laden effluent in developing countries. The main objective of this study was to quantify the removal of EDCs within carbonate sand columns operated under different degrees of saturation. A secondary objective was to test whether estrogenic EDC removal would be affected by the addition of other soils/ materials (e.g. basalt soil, coconut fibre) to the sand columns. Retention time was an important factor on all of the measured parameters (pH, DO and EDC concentration). In the fully saturated columns, the DO concentration was the lowest, and pH was similar to the inlet water. However, in the unsaturated carbonate sand treatments with the shortest retention time, the pH was significantly lower, and the DO was significantly higher than the saturated columns. The decrease in pH is likely caused by nitrification in the unsaturated sediments through the release of H+ ions (Sharma and Ahlert 1977). The 8 Br-1 Concentrations (mg L-1) in column outlets Fig. 2 Concentration of Br (mg L−1) at the outlet of all seven treatments following addition to a common inlet wastewater tank. T1 (unsaturated), T2 (unsaturated, double dosed), T3 (unsaturated, coconut), T4 (unsaturated basalt mix), T5 (unsaturated basalt layer), T6 (partially saturated), T7 (fully saturated) All treatments reduced estrone below average influent levels. All treatments with the exception of T7 reduced estriol. Treatments 1 (unsaturated sand) and T6 (saturated sand 800 mm) reduced all estrogens (E1, E2, EE2, E3) below average inlet influent levels. Positive fluxes of estradiol were observed in all treatments except T1 and T6 (Fig. 4). T1 - Unsaturated T2 - Unsaturated double dose T3 - Unsaturated coconut T4 - Unsaturated basalt mixed T5 - Unsaturated basalt layer T6 - Partially saturated T7 - Fully saturated 6 4 2 0 0 5 10 15 Days after addition 20 25 Environ Sci Pollut Res Fig. 3 a Hydraulic residence time in days for each of the seven treatments, b mean pH for each treatment and the inlet wastewater tank and c mean dissolved oxygen concentration (DO mg L−1) for each treatment including the wastewater inlet tank. Error bars are standard deviations of multiple measurements (n=8) reduction in DO in the saturated columns is most likely due to the oxidation of wastewater organic carbon. Estrogen equivalency concentration in the inlet barrel was variable over the experimental period. EDC concentrations are known to be variable in STP inlet waters (Roda et al. 2006). Estrone was the dominant component of the measured EDCs in the inlet barrel, and estradiol was the least abundant. The influent used for this study came from the primary settling tank at the STP, and it has been shown by Dytczak et al. (2006) that estrone levels were higher leaving primary treatment than inlet levels largely due to the conversion of estradiol to estrone. The same study demonstrated that estrone was one of the least degraded in primary treatment and is added to by the cleavage of conjugated forms of estrone by microbial transformations in the activated sludge. The only treatments able to reduce all four measured estrogens and have an EEQ below the level of the inlet barrel were the fully unsaturated sand treatments T1 and T6. There is a considerable amount of literature on the increased removal of EDCs under aerobic conditions as opposed to anaerobic conditions (Kowalchuk and Stephen 2001; Braga et al. 2005b; Ying and Kookana 2005; Czajka and Londry 2006; Nakada et al. 2007; Ren et al. 2007; Koh et al. 2008; Cajthaml et al. 2009; Zhang et al. 2011). Studies have shown that chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification and use the conversion of ammonia to nitrate as their sole energy source (Kowalchuk and Stephen 2001). Ren et al. (2007) stated that estrogens are removed either by the direct use as electron donors for heterotrophs or by the co-metabolic degradation of ammonia-oxidizing bacterium. Shi et al. (2004) showed that Nitrosomonas europaea, an autotrophic nitrifying bacterium, was able to oxidize estrone, estriol, estradiol and ethinylestradiol in the presence of ammonia. Redox conditions play an important role in the removal efficiency of estrone and ethinylestradiol, with the highest removal occurring under aerobic conditions where estroneis reduced to estradiol (Joss et al. 2004). Bioreactor tests have shown a linear relationship between nitrification and ethinylestradiol (EE2) removal by enriched cultures of autotrophic ammonia oxidizers. These results suggested a cometabolic transformation of ethinylestradiol (EE2) by the aerobic organisms (Cajthaml et al. 2009). Therefore, there is a clear link between the activity of nitrifying bacteria and the degradation of EDCs. The treatment receiving double the loading rate of other treatments (T2) was able to reduce the EEQ of inlet water. Ren et al. 2007 stated that higher organic loading rates create higher rates of organic matter transformation, which causes less ammonia oxidation and less degradation of estrone, estradiol and ethinylestradiol. However, in this study, the removal of estrogenic EDCs in unsaturated carbonate sand filters was not affected by dosing rate. This may be due to the ability of carbonate sands to provide a much higher surface area for bacterial growth and therefore greater capacity to degrade EDCs. Most sand filters used for wastewater use either secondary treated effluent, are anaerobic or have other forms of treatment involved. There was a clear link between the degree of saturation and the removal of total EDCs. The variability in the inlet EDC concentrations could have been responsible for this result. Despite sampling the inlet EDCs 12 times over the study period, it is possible that large spikes in EDC concentration in the inlet water may have been missed. This would reduce the average inlet EDC concentration and would make treatment 7 appear as if it were a source of EDCs. The positive flux of total EDCs from treatment 7 could also be due to conjugated estrogens not detected as EDCs in the inlet. Studies by Koh et al. (2008) showed that conjugated Environ Sci Pollut Res 6 Fig. 4 Individual concentrations of estrone, estradiol, ethinylestradiol and estriol for each treatment (T1 to T7) and the common inlet wastewater. All concentrations given in ng L−1; error bars are the standard deviations of multiple measurements (n=8) 3.0 Estradiol 5 2.5 4 2.0 Estradiol Estrone (ng L-1) Estrone 3 2 1 0.0 In 3.0 2.0 Ethinylestradiol 2.5 2.0 1.5 1.0 Treatment estrogens make up to 50 % of estrogens found in wastewater. These conjugated estrogens can be partially degraded to their former compound within the treatment and therefore show up as an EDC flux. There is also the possibility that estrogens sorbed onto carbonate sand becoming desorbed and released in outlet water. There were occasional large spikes recorded in the outlets of the semi-saturated and saturated treatments. Studies by Lai et al. (2000) showed that both anoxic and anaerobic conditions favour the sorption process of EDCs when compared to aerobic. The majority of estrogens excreted from humans are conjugated (mainly glucuronides) and are broken down to their original compounds in the treatment works (Kumar et al. 3.5 3 EEQ ngL-1 2.5 2 1.5 1 0.5 0 T5 T6 Estriol 1.5 1.0 0.5 0.0 T1 T2 T3 T4 T5 T6 T7 T4 In 0.5 0.0 T3 T1 T2 T3 T4 T5 T6 T7 Estriol (ng L-1) Ethinylestradiol (ng L -1) T1 T2 T3 T4 T5 T6 T7 T2 1.0 0.5 0 T1 1.5 T7 Inlet Fig. 5 Estrogen equivalencies (EEQ) relative to 17β estradiol for each treatment (T1–T7) and common inlet wastewater. All concentrations given in ng L−1; error bars are the standard deviations of multiple measurements (n=8) In T1 T2 T3 T4 T5 T6 T7 In Treatment 2012; Griffith et al. 2014). This can lead to higher concentrations found during treatment than those recorded for inlet levels (Andersen et al. 2003). The estradiol fluxes observed within the treatments may be explained by the reversible interconversion of estradiol (E2) and estrone (E1) as observed by Andersen et al. (2003) and Czajka and Londry (2006). Ethinylestradiol (EE2) is the most resistant to degradation of all the estrogens. Our results confirm the work of Nakada et al. (2007) and show that there is limited removal of EDCs under anaerobic saturated conditions. There were also positive fluxes of EDCs reported by Nakada (2007); however, in the study, effluent was secondary treated with EDC concentrations measuring in the tens to hundreds of nanograms per litre. Furthermore, in the Nakada et al. (2007) study, the secondary treated effluent was introduced at the bottom of a sand column (2.5 m in outer diameter and 7.0 m in height) and flowed upwards through the sand bed. The flow rate and retention time through the sand were 110 m day−1 and 1 h, respectively. The differences in effluent levels and dilution, retention times, flow rates, filter size, compound levels and EDC analysis techniques make comparisons between this and the Nakada (2007) study difficult, although one main point seems consistent in that limited degradation of EDCs occurs under anaerobic conditions. The additional amendments of basalt sediments and coconut fibre (both readily available and commonly incorporated into sewage treatment systems in South Pacific islands) were put in to encourage microbial activity or increase retention Environ Sci Pollut Res time. Although the results vary, the amended treatments were not as efficient at estrogenic EDC reduction as carbonate sand treatments (T1). In the context of on-site wastewater treatment in the Pacific, the transformation of EDCs is an important consideration. Aerobic sand filters are known to nitrify ammonium to nitrate, which is then reduced to N2 gas via anaerobic denitrification. However, this study has shown that the anaerobic conditions that favour N removal can lead to EDC accumulation. Designing sand filtration systems specific to the constraints of South Pacific countries that contain both unsaturated aerobic environment and saturated anaerobic environment is essential (Tait et al. 2013). The environmental concern is that in groundwater where DO concentrations are low, e.g. under septic tanks and waste disposal fields, there is the potential for EDC accumulation. The results from this study show a clear pattern between aerobic treatments and estrogenic EDC reduction and anaerobic treatments and accumulation of estrogenic EDCs. Conclusion This study aimed to investigate the behaviour of estrogenic EDCs in permeable carbonate sands under saturated and unsaturated conditions. The broader goal of this research was to develop low-cost on-site wastewater treatment technologies for estrogenic EDC removal in developing countries. One of the main findings in this study was that aerobic conditions within carbonate sand treatments encouraged EDC reduction even at high dosing rates. Fully saturated conditions led to incomplete degradation of conjugated forms of estrogens or accumulation or transformation of inlet estrogens measured. Fully saturated systems had low EDC reduction with the tendency to accumulate EDCs. Semi-saturated systems that had an aerated section before a saturated section had high EDC reduction. All the amended aerobic treatments performed better than fully saturated treatments. The accumulation of EDCs in anaerobic treatments suggests that additional aerobic treatment, post-denitrificiation, would help to reduce accumulated EDCs. The results from this study show that aerobic environments greatly increase the reduction of estrogenic EDCs as opposed to anaerobic environments. Therefore, in order for estrogenic EDCs to be removed efficiently from wastewater, an aerobic stage or two is needed in the treatment process. The efficiency will depend on size, flow rate, and placement; these are parameters that need further investigation. Acknowledgments This project was supported by the School of Environmental Science and Management, Centre for Coastal Biogeochemistry and ARC Linkage project LP100200732. We would like to thank the technical staff at the Wollongbar Department of Primary Industries. We also thank the technical staff at the School of Environment, Science and Engineering at Southern Cross University. References Andersen H, Siegrist H, Halling-Sorensen B, Ternes TA (2003) Fate of estrogens in a municipal sewage treatment plant. 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