211

Hormonal Parameters of Ecohydrology Laurence Shore, Ph.D, Kimron Veterinary Institute, Israel

Abstract The objective of this work is to demonstrate that hormone measurements, which can be done rapidly and simply, can be used to assess the “health” of an ecosystem. Initially we determined natural (testosterone, estradiol, estrone, estriol) and ethinylestradiol in a watershed to measure pathways of pollution from animal and human sources. It was found that testosterone and estrogen alone were characteristic of animal pollution while combined with ethinylestradiol and estriol (hormone of human pregnancy) were characteristic of human pollution. The source of the pollution could be determined 60 km downstream from the source. Unexpectedly it was found that testosterone was present in the topsoil and vadose zone and was eluted during rain events. The data suggested that there are three patterns of testosterone transport in the environment. (1) Testosterone associated with estrone, ethinylestradiol and estriol which is characteristic of sewage effluent; (2) Testosterone associated with estrone and estradiol which is characteristic of runoff from cattle pasture and manure fertilized fields; and (3) Testosterone alone, characteristic of leaching from soil and baseflow. Levels of ethinyl estradiol higher than 1 ng/L were found in 70% of the samples from polluted river stretches. This level was shown to affect the sex ratio in fish. In addition, preliminary evidence indicated that fecal corticosterone concentrations in wildlife and plant phytoestrogen concentrations are elevated when the ecosystem is stressed. We therefore propose that determining the hormonal parameters of an ecosystem would enable both short term and long term detection of disturbances in the system. This research was supported by a grant (GLOWA - Jordan River) from the Israeli Ministry of Science and Technology; and the German Bundesministerium fuer Bildung und Forschung (BMBF). Introduction Ecohydrology is a broadly defined field that studies the interface between hydrological and ecological sciences, specifically how do hydrological systems control ecological systems. To study such systems a large database of very many variables is necessary which is impractical without large modeling programs for monitoring the status of a watershed. The present paper proposes that simple, rapid measurement of hormone parameters can give water management real time information of the ecological status of the system. The main premise is a watershed is a like a vein bringing hormones from the source to the target. Measuring hormones can monitor the “health” of the ecosystem and river. The main discussion will be on the value of hormonal measurements in water. However, the value of non-invasive hormonal measure in wildlife and phytoestrogen measurement in plants will also be discussed. Steroid hormones produced by humans and animals are constantly excreted into the environment (see Drewes and Shore, 2001; Lintelmann et al., 2003; Shore and Shemesh, 2003 for reviews). The primary steroid hormones are estrone, estradiol, progesterone, testosterone, androstenedione, cortisol and corticosterone, all of which are lipophilic and poorly soluble in water (log Pow between 3 to 4, Lintelmann et al., 2003). The natural steroids of major concern are estrone and estradiol-17β since they exert their physiological effects at lower concentrations than other steroids and can be found in the environment in concentrations above their Lowest Observed Effect Level (LOEL) for fish (increased vitellogenin) and plants (increased growth) (10 ng/l) (Christiansen, 2002; Shore et al., 1992,1995b). In rivers and soil, estradiol is converted abiotically to estrone, so for environmental studies estradiol and estrone can be considered together as “estrogens” (Colucci et al., 2001; Jürgens et al., 2002). Effluent from human sources also contains estriol, a weak estrogen excreted in the urine of pregnant women, and synthetic estrogens such as mestranol and ethinylestradiol (Wenzel et al., 1998). These synthetic compounds are of particular concern as they have LOEL’s in the order of 1 ng/l (Lange et al., 2001). Progesterone and testosterone are also excreted in the free active form but at the maximal level found in rivers (about 200 ng/l, Kolpin et al. 2002), there are no documented effects in the environment. The testosterone metabolite androstenedione are also excreted in the free active form. It is a matter of controversy if the masculinization of fish seen in some rivers is due the high androstenedione (40 ng/L) level (Durham et al., 2002).

212

Estrogen and testosterone in the environment are excreted from the same sources in comparable amounts (Shore et al., 1995a). The major sources that have been investigated are animal manures and sewage effluent. Studies of fields fertilized with chicken and pig manure, or laboratory studies of soils with exogenous steroid added, indicated that estrogen binds tightly to the soil and does not migrate to the ground water (Shore et al., 1995a, 1997). However, estrogen is found in surface runoff and in ground water where the soil is rocky or where the terrain is highly permeable karst (Shore et al., 1993; Peterson et al., 2000). In contrast, testosterone is loosely bound to the soil and is found in both surface and ground water (Shore et al, 1997) as well as in surface soil (Finley-Moore et al. 2000). Both estrogen and testosterone are rapidly metabolized in wild duck ponds (half life - 0.5 h, Shore, unpublished observations) but in irrigation ponds these contaminants may persist for several months (Shore et al., 1993, 1995a). In laboratory studies of river water, Jürgens et al. (2002) reported that the half time reduction for estrogen was between 2-6 days, while half time reduction for ethinylestradiol was 46 days. However in field studies, Williams et al (2003) reported that sewage effluent had little effect on the estradiol and ethinylestradiol content of the river but that estrone persisted for 10 km below the effluent input. In soil, both estrogen and ethinylestradiol absorb rapidly and cannot be extracted after 2 days. However mineralization of the hormones in the soil may take several months (Colucci et al, 2001, Colucci and Topp 2001). Transport of Hormones in a watershed The transport of testosterone, estrogen, ethinylestradiol and estriol was measured in fifteen sites in a watershed, the Upper Jordan River Catchment area (Figure 1) after major rain events. Precipitation occurs in this area occurs only the rainy season from Oct. to May. The area consists of small farms, cattle pasture, fishponds with some urban development. The rain season depicted was the first above average season after a three-year period of well below average rainfall. It was found in the rain season of 2001/2002, that following a rain sequence of 131 mm/wk there was an initial large increase in the concentration of testosterone (maximum 6 ng/l) accompanied by high estrogen (maximum 6 ng/l), which then gradually declined to non-detectable levels (<0.3 ng/l) over a period of three months. These peaks originated from runoff from cattle pasture and fishpond effluent (Figure 2). Later peaks consisted only of testosterone that was moderately associated with sulfate (r2=0.53, P<0.05) and somewhat associated with total phosphorus (r2=0.49, P<0.1) indicating that the origin was leaching from the sulfurous peat soil (Shore et al., 2004). Testosterone in a watershed was therefore due initially to surface runoff from cattle pasture and then as discharge from the soil. The groundwater in the Upper Jordan Catchment area is generally close to the surface. When a different area with a much deeper water table was examined, testosterone was found in soil from the surface to the groundwater to a depth of at least 15 meters. (Figure 3.) Estrone, estradiol and ethinylestradiol apparently do not penetrate beyond the topsoil and are not generally found in the soil beyond the initial layer (Lee et al., 2003). It is not clear if androstenedione penetrates the soil or reaches the groundwater as the presence of androstenedione could be do to conversion from testosterone (Casey et al., 2004).

213

Figure 1. Upper Catchment area of the Jordan River. Symbols indicate sampling sites. Inset shows the sites sampled on the South Jordan River from dam at the lower limit of the Kinneret to the Dead Sea.

214

0.01.02.03.04.05.06.0

39 40 41 73 76 87 151 155 157 163 177 225 254 284Days from season start

Figure 2. Testosterone concentration in the Upper Jordan River during the rainy season of 2001/2002. The Y axis is ng testosterone/l. The x axis is the days from the start of the hydrological season. Samples were taken after each major rain event. There was an initial high testosterone due to runoff from cattle pasture. At day 177 there was an unusual intense late season storm. Much of the testosterone peak seen at that time can be attributed to overflowing sewage treatment ponds, which were already filled in anticipation of the use for irrigation in the dry season.

Figure 3. Hormonal profile of runoff from cattle pasture. The Y axis is ng estrogen or testosterone/l. The x axis is days after the initial rainfall of the hydrological season. After the initial rainfall, both testosterone and estrogen are present. However since the estrogen strongly binds to soil, only testosterone is seen following later rain events.

0

1

2

3

4

5

6

7

20 40 76 87Days from start of rain

testosteroneestrogen

215

Figure 4. Testosterone in soil. The Y axis is ng testosterone/ 5 g soil. The x axis is the probe depth. Samples were taken every 0.5 meters. Testosterone was present in the soil throughout the vadose zone until the water table. Transport of Hormones in a polluted river The transport of hormones in a polluted river differs from that in an unpolluted watershed in that the base flow values are very high which means the compounds persist many km from their source. The polluted river is characterized by high BOD, TOC, ammonia and bacterial counts. The polluted river studied was the Lower or South Jordan. The Upper Jordan River Catchment area described above empties into Lake Kinneret. However the outlet from the lake is blocked by a dam and water flows directly into the Jordan only if the Lake overflows which has not happened since 1991. As opposed to the Upper River, which is a drinking water source, the lower river below the dam receives sewage effluent, some from bypass channels around the Kinneret and some from small settlements, which only partially treat their sewage (Figure 1 inset). The principal sources of hormonal pollution are sewage and fishpond effluents. The flow rate of the river was about 20 cm3/sec. The hormone concentrations from Spring 2002 sampling are depicted graphically in Figure 5. Estriol (not shown) was the most rapidly destroyed followed by ethinylestradiol and testosterone. On the other hand estrogen, presumably mostly estrone, appears to persist the whole stretch of the river.

Shefaim V

01234567

0 10 20 30 40meters

ng te

stos

tero

ne

sand

Water Table

clay

216

00.5

11.5

22.5

33.5

44.5

1.3 2.3 6.5 7.3 23.7 28.7 67.4 92.6 101.1

Testosterone Estrogen EE

Figure 5. Transport of hormones in a polluted river. The Y axis is the hormonal value in ng/l. The y axis is the distance in km from the start of the river. The South River originates in agricultural runoff and sewage effluent. Along its initial course, some freshwater sources enter the river. At 23.7 km it received effluent from from fishponds. From 28.7 to the 101.1 km mark, there were no significant influxes of water. Use of hormones to define pollution sources Hormone measurements can be used to identify non-point sources of pollution. If the organic pollution originates from sewage effluent, the contraceptive drug, ethinylestradiol will be present. If it a rural area with a relative large number of pregnant women, the level of a hormone produced during pregnancy, estriol, will be present. In the absence of ethinylestradiol or estriol, estrogen together with testosterone is characteristic of runoff from cattle early in the rainy season (Figure 6). Testosterone alone is characteristic of soil and groundwater contamination or after the initial rainfall when the estrogen, which binds tightly to the soil is no longer present. However, another testosterone metabolite, androstenedione, can be used to characterize testosterone originating in runoff. Androstenedione Androstenedione and testosterone are present in cattle manure in comparable quantitative. After the initial rainfalls, both compounds are present in equal amounts in the runoff. Preliminary evidence indicates as the rainy season progresses, testosterone enters the soil while androstenedione remains on the surface, therefore later in the rainy season only androstenedione is present in the runoff from cattle pasture.

217

Figure 6. Use of hormone ratios to determine source of pollution. The y axis represent the testosterone/ethinylestradiol or testosterone/estriol (last bar) ratio. A high ratio indicates that the hormone source is not anthropogenic. The presence of ethinylestradiol is typical of urbanized sewage while high estriol, a hormone produced by pregnant women, is typical of a non-contraceptive using population. Hormone measurement as a measure of effectiveness of water quality management Hormone measurements to determine the effectiveness of sewage water treatment plant Hormone measurements can be used to determine the efficacy of upgrading sewage treatment (Figure 7). The reduction of anthropogenic hormones (ethinylestradiol and estriol) indicates that the treatment of the urban sewage component was effective. On the other hand the much smaller reduction of testosterone and natural estrogen (estradiol and estrone combined), indicates that the hormones from non-anthropogenic origin, e.g. cattle pasture or fishponds, was still entering the stream from upstream sources.

0.0

1.0

2.0

3.0

4.0

5.0

6.0

sewagesewagemixedmixedcattlepasture

cattlepasture

218

0

10

20

30

40

testosterone estrogen ethinylestradiol estriol

ng h

orm

one

beforeafter

Figure 7. Hormone levels in a stream before and after upgrading of sewage facilities. The y axis shows the hormone concentrations in ng/l. Hormone measurement to determine overall effectiveness of management Testosterone measurements can also be used to determine if efforts to reduce pollution from animal husbandry is effective. In the area measured, substantial efforts were made to reduce the pollution from dairy cattle waste. As can be seen in Table 1, this apparently resulted in a 50% reduction in testosterone while the input from human sources as represented by ethinylestradiol remained the same. Table 1. Comparison of mean testosterone and ethinylestradiol concentrations at 14 sites in the UPJR during two rainy seasons.

Testosterone ng/l

Ethinylestradiol ng/l

2001/02 2002/03 2001/02 2002/03 Mean±SD

(no. of samplings) 1.5±0.4 (125)

0.8±0.5 (109)

0.7 ±0.4 (47)

0.9±0.3 (112)

2001/02 vs. 2002/03 P<0.01 NS

Non-invasive determination of hormones as a means of monitoring wildlife Measurement of fecal testosterone and corticosterone can be done using the same techniques (column extraction; ELISA, RIA) as those for measurements in water. Although progesterone has been used for monitoring pregnancy in wildlife (see Shore and Shemesh, 2003 for review), little use has been made of testosterone and corticosterone levels. Our laboratory has found that fecal corticosterone is elevated by at least two kinds of stress, cold (oryxes) and removal of females (eland). Further information on male reproductive status can be obtained from the testosterone levels. Fecal corticosterone levels have also been shown to increase when both passerine and non-passerine birds are stressed. (Kotrschal et a., 2000, 2002). Phytoestrogen content of legumes as measurement of stress

219

Many plants, especially legumes, contain phytoestrogens. These plant estrogens can cause infertility and other reproductive disorders in domestic animals and wildlife. The phytoestrogens have two functions, one as a signal for rhibozobia attraction and secondly as phytoallexins (generalize plant response to trauma). A dramatic increase of phytoestrogens in alfalfa (medicago sativa) as the result of fungal infection or irrigation with treated sewage water has been reported (Shore et, al, 1992, 1995b). The effect on phytoestrogen content in alfalfa by increase in phytoestrogen can be mimicked by the addition of estrogen. The effect of other types of stress in other legumes is not known but preliminary evidence indicated that overgrazing decreases the phytoestrogen content. Although the techniques for measuring phytoestrogens (Radio-receptor assay, modified yeast cells) are not readily available, the modified yeast cells can also be used to measure estrogen and testosterone in water and soil and commercial biosensors to perform these tasks are being developed. Conclusions Hormone measurement can give information as the transport and source of the hormones in an ecosystem. It is possible to differentiate hormones from human sources due to the presence of ethinylestradiol, a contraceptive, or estriol, a hormone of human pregnancy from the testosterone and estrogen from animal sources. Measurements of the hormone are a good indicator to determine steps taken by managers are effective, e.g. closing off of barn effluent and upgrading sewage facilities. In a clean river, hormone pulses are seen after rain events, particularly in the early part of a hydrological season. Testosterone, androstenedione and estrogen pulses are observed only in the initial samples due to runoff from cattle grazed fields and effluent from fishponds. The initial testosterone and androstenedione pulses dissipate over a three and five month period respectively. The absence of an estrogen pulse is due to estrogen binds tightly to the soil. The longer androstenedione pulse is due to testosterone penetrating the soil while the androstenedione remains on the surface. The rest of the season is characterize by testosterone pulses which presumably come from soil washout as the pulses correlate with sulfate and phosphorus which are released from peat soils during the same rain events. This was in an area with a shallow water table. In drier areas, testosterone can be traced from the topsoil through the vadose zone to the groundwater to at least 15 m. In a polluted river, the hormones present are unrelated to the rain events. The pollution in the river can be seen by high ammonia, BOD, TOC and fecal coli counts which drop rapidly downstream after the influx of contaminated water. Ethinylestradiol and estriol concentrations parallel this decrease. On the other hand testosterone and estrogen (estradiol and estrone) could remain above 2 ng/l for the whole stretch of the river. This indicated that bacterial, adsorption or photolytic activity are more effective in reducing estriol and ethinylestradiol than testosterone, estradiol and estrone. Estrogen, presumably mostly estrone, was more persistent than testosterone concentrations. In the polluted river, ethinylestradiol was above the LOEL level (1 ng/l) in 70% of the samples. Field observations of fish exposed to these amounts showed a prominent skewing of sex ratios. The data suggest the hypothesis that there are three patterns of testosterone transport in the environment. (1) Testosterone associated with estrone, ethinylestradiol and estriol which is characteristic of sewage effluent; (2) Testosterone associated with estrone and estradiol which is characteristic of runoff from cattle pasture and manure fertilized fields; and (3) Testosterone alone, characteristic of leaching from soil and baseflow. Other sources of steroidal hormones beside domestic animals are wildlife such as deer and goat species as well as the larger birds such as storks. Although the contribution to the overall hormonal load is probably not significant, measurement of fecal steroid using the same techniques as for water, can give information on the degree of stress, behavior patterns and reproductive capacity of the herd or flock. Similarly legumes, which respond to both the presence of estrogen and fungal infection with a rise in phytoestrogens, can be measured using the same techniques. We therefore suggest that hormone measurements can not only define sources of pollution but give a general picture of what is happening in a ecosystem. Unlike many of the organic compounds which are studied, a great deal is known about the origin of hormones and their properties. Since the tests are in general simple, rapid and inexpensive and the expertise to do them widely available (any medical laboratory), the water manager easily utilize this as a tool for managing a watershed.

220

References Casey, F.X.M., Hakk, H., Simunek, J., Larsen, G.L., 2004, Fate and transport of testosterone in agricultural soils, Environmental

Science and Technology 38:790-798. Christiansen, L.B., Winther-Nielsen, M, Helwig C., 2002, Feminisation of fish. The effect of estrogenic compounds and their fate in

sewage treatment plants and nature, Environmental Project No. 729, Danish Environmental Protection Agency. Available at: http://www.mst.dk/udgiv/publications/2002/87-7972-305-5/html/default_eng.htm

Colucci, M.S., Bork H., Topp, E., 2001, Persistence of estrogenic hormones in agricultural soils: I. 17β-estradiol and estrone J.

Environ. Qual. 30:2070-2076. Colucci, M.S., Topp, E., 2001, Persistence of estrogenic hormones in agricultural soils: II. 17α-ethinylestradiol. J. Environ. Qual.

30:2077-2080. Durham E.J., Lambright C., Wilson V., Butterworth B.C., Kuehl D.W., Orlando E.F., Guillette, LJ, Gray LE, Ankley G.T., 2002,

Evaluation of androstenedione as an androgenic component of river water downstream of a pulp and paper mill effluent. Environ. Toxicol. Chem. 21:1973-1976.

Drewes J.E., Shore L.S., Concerns about pharmaceuticals in water reuse, groundwater recharge, and animal waste, in Daughton

C.G., Jones-Lepp T., eds, Pharmaceuticals and Personal Care Products in the Environment: Scientific and Regulatory Issues, Symposium Series 791, American Chemical Society, Washington, D.C., 2001, pp. 206-228.

Finlay-Moore, O., Hartel, P.G., Cabrera, M.L., 2000, 17 beta-estradiol and testosterone in soil and runoff from grasslands amended

with broiler litter. J Environ. Qual. 29:1604-1611. Jürgens, M.D., Holthaus, K.I.E., Johnson, A.C., Smith, J.J.L., Hetheridge, M., Williams, R.J., 2002, The potential for estradiol and

ethinylestradiol degradation in English rivers. Environ. Toxicol. Chem. 21:480-488. Kotrschal, K., Dittami J., Hirschenhauser, K., Mostl, E., Peczely, P., 2000,. Effects of physiological and social challenges in different

seasons on fecal testosterone and corticosterone in male Domestic Geese (Anser domesticus). Acta Ethologica 2:115–122. Kotrschal, K., Dittami J., Hirschenhauser K., Mostl E., Peczely P., 2002, Corticosterone metabolites can be measured moninvasively

in excreta of european stonechats (Saxicola torquata rubicola). The Auk 119:1167–1173. Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E. M., Zaugg, S., Barber, L.B., Buxton, H.T., 2002, Pharmaceuticals, hormones,

and other organic wastewater contaminants in U.S. streams,1999-2000: A national reconnaissance, Environmental Science and Technology 36:1202-1211.

Lee, L.S., Strock, T.J., Sarmah, A.K., Rao, P.S.C., 2003, Sorption and dissipation of testosterone, estrogens, and their primary

transformation products in soils and sediment, Environmental Science and Technology 37:4098-4105. Lintelmann, L., Katayama, A., Kurihara, N., Shore, L., Wenzel, A., 2003, Endocrine disruptors in the environment (IUPAC

Technical Report). Pure and Applied Chemistry 75:631-681. Peterson, E.W., Davis, R.K., Orndorff, H.A., 2000, 17 beta-estradiol as an indicator of animal waste contamination in mantled karst

aquifers. J. Environ. Qual.; 29:826-834. Segner, H., Caroll, K., Fenske, M., Janssen, C.R., Maack, G., Pascoe, D., Shäfers, C., Vandenbergh, G.F., Watts M., Wenzel A., 2003,

Identification of endocrine-disrupting effects in aquatic vertebrates and invertebrates: report from the European IDEA project, Ecotoxicological and Environmental Safety 54 (302-314.

Shore, L., Kapulnik, Y., Ben-Dov, B., Fridman, Y., Wininger, S., Shemesh, M., 1992, Effects of estrone and 17ß-estradiol on

vegetative growth of Medicago sativa. Physiol. Plant. 84: 217-222 Shore,L.S., Gurevich M., Shemesh M., 1993, Estrogen as an environmental pollutant. Bull. Environmental Contamination and

Toxicology 51: 361-366. Shore, L.S., Correll D., Chakroborty P.K., 1995a, Fertilization of fields with chicken manure is a source of estrogens in small streams,

in Steele K., ed., Animal Waste and the Land-Water Interface, Lewis Publishers, Boca Raton, Florida, pp. 49-56. Shore, L.S., Kapulnik, Y., Gurevich, M., Wininger, S., Badamy, H., Shemesh, M., 1995b, Induction of phytoestrogens production in

Medicago sativa leaves by irrigation with sewage water. Environ. Exper. Bot. 35:363-369. Shore, L.S., Hall D.W., Shemesh, M., 1997, Estrogen and testosterone in ground water in the Chesapeake Bay Watershed.

Dahlia Greidinger Inter. Symp. on Fertilization and the Environment, pp. 250-255, Technion, Haifa, Israel. Shore, L.S., Shemesh, M., 2003, Naturally Produced Steroid Hormones and their Release into the Environment. Pure and

Applied Chemistry 75:1859–1871.

221

Shore,L.S., Reichman, O., Shemesh, M., Wenzel, A., Litaor, M.I., 2004, Washout of accumulated testosterone in a watershed, Science of the Total Environment, in press.

Wenzel, A, Kuechler, Th., Mueller, J., 1998, Konzentrationen oestrogen wirksamer Substanzen in Umweltmedien. Report. Project

sponsored by the German Environmental Protection Agency; Project No 216 02 011/11 (In German). Williams, R.J., Johnson, A.C., Smith, J.J.L., Kanda, R., 2003, Steroidal estrogen profiles along river stretches arising form sewage

treatment works discharges. Environ. Sci. Tech. 37: 1744-1750. Biographical Sketch Laurence Shore, Ph.D. Laurence Shore is a Senior Scientist at the Kimron Veterinary Institute, Bet Dagon, Israel. His original papers on the source and transport of estrogen and testosterone in the environment have become core papers in this field. He has served on international committees dealing with endocrine disruptors. In addition to his environment studies, Dr. Shore has published many papers in animal reproduction, in particular the effects of phytoestrogen ingestion on fertility in domestic animals. Dr. Shore is a native of Philadelphia and has been resident in Israel for some 30 years. Mailing address: Dept. of Hormone Research, Kimron Veterinary Institute, Bet Dagan, POB 12 , Israel Email address: shorel@int.gov.il Telephone: 972-3-9681775 Fax: 972-3-9681721