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The conjugated hormones in this case had to be brought into a form which guarantees the chemical stability of the hormones and permits processing of the hormones into a tablet. In the experiments, it was found that by spraying the hormone concentrate the conjugated hormones could be applied homogeneously to the auxiliaries by the fluidized bed technique on carriers such as microcrystalline cellulose or mixtures of microcrystalline cellulose with lactose.

A provided from a collecting campaign urine concentrate was sprayed on to microcrystalline cellulose or on to a mixture of microcrystalline cellulose and lactose and the hormones thereby applied to the carrier or the excipient mixture. This process was carried out in a fluidized bed.

The particle size and porosity of the active substance granule was regulated by the supply and exhaust air temperature and the spray rate. The spraying rate was selected accordingly in order to maintain the aforementioned ranges. For producing dry extracts of natural mixtures of conjugated estrogens a fluidized bed apparatus Strea 1 was used in these experiments, dry extract per batch can be produced with about 1 kg. The aqueous, a natural mixture of conjugated estrogens containing solution extract was added to the top-spray method in the fluidised bed apparatus. Manufacture of a dry extract with a content of 45 mg conjugated estrogens per g dry extract for originals g of the carrier.

Alle 3 Versuche verliefen unproblematisch. All three trials were problematic. The spraying times were in Experiment 1 at 83 minutes in experiment 2 was 46 minutes and in test 3 at 35 minutes. This experiment is a repeat of Experiment 1, it should be by the Checks whether a finer dry extract can be produced by reducing the spraying rate. The dry extract in sieve analyzes proved to be compared with the one obtained in experiment 1 dry extract as a fine see summary of the results of the tests. Detailed results on the hormone balance in the experiments 1 to 4 are summarized in Tables I to IV.

In principle, it has been found that with an initial charge of g to g Avicel PH as the carrier a continuous and rapid application of the extract is possible Tests 1 to 3. For the employed from to g varied extract volumes were the spraying times for these tests 35 to 83 minutes. Hieraus ergaben sich Auftragsmengen von 0,55 g bis 0,64 g Feststoff aus dem Extrakt pro g Avicel Mittelwert: 0,59 g. From this coating amounts of 0.

Um in einem weiteren Versuch Versuch 5 den vorgegebenen Soll-Gehalt von 45 mg konjugierte Oestrogene pro g Trockenextrakt einzuhalten bzw. In order in a further test Test 5 to comply with the preset desired content of 45 mg conjugated estrogens per g dry extract or to determine limits for maximum quantities of active substance coatable, the template Avicel PH was reduced to Aufgetragen werden sollte g Extrakt.

Hierbei traten bis ca. Here sprayed extract came to about g, no problems, as up as in the previous experiments 1 to 4 again was present at that amount, an application rate of 0. Bei ca. At about g extract sprayed a coating amount of 0. Until this application amount, the extract was spray largely unproblematic. Thereafter, the spraying has been greatly reduced, since at this amount of solids from the extract exceed the amount of excipient and showed the product from that point tackiness.

The pure spraying time was more than 5 hours. Zusammenfassend kann daher gesagt werden, dass bis zu einem Auftrag von 0,6 g Feststoff aus dem Extrakt pro g Avicel der gesamte Extrakt verarbeitet sein sollte. In summary it can be said, therefore, that should be processed up to a coverage of 0.

At about 0. Thereafter, a reduction of the spray application and a corresponding adjustment of the remaining parameters is required. Versuch 4 stellt eine Wiederholung von Versuch 1 dar. Test 4 is a repetition of Test 1. Here, lower spraying rate and thereby higher exhaust air temperature and a lower exhaust air humidity was prepared, a finer trituration by changing the parameters. In this test was from an application quantity of 0. The sprayed amount of extract at this time was about 40 wt. Ab ca. Tabelle I table I. The preparation of a dry extract in the fluidized-bed apparatus, even with support materials of different grain size distribution, is not problematic.

Index of /

So the drying process does not affect the stablety of hormones. As the tests show it is possible to process large hormone-containing extract amounts of 2 to 4 kg within a short time, that is to apply to carriers and to dry it. Here it was found that on presentation of eg Avicel as carrier a job to about 0. It further spray tests were carried out to apply an aqueous solution extract containing a natural equine conjugated estrogens mixtures in different concentrations, at a microcrystalline celluloses selected from the group pharmaceutical carrier.

The properties of the aqueous solution extracts used A to F drug solutions and the process parameters used in each case are shown in Table V. Other properties of the obtained in the form of a solid, free-flowing dry extract pharmaceutical preformulations are given in Table VI. The obtained in the form of a solid, free-flowing dry extract pharmaceutical preformulations were subjected to a sieve analysis.

Sieve analysis was performed using a Retsch sieve shaker under the following conditions: 5 min. Sieving, pulse time 3 s, vibration height 1. Were used with a mesh size of microns sieves. The results of the sieve analysis are given in Table VII. Extraktisg conc.. Supply air temperature. Properties obtained according to Example 2 of pharmaceutical preformulations which contain a natural mixture of conjugated equine estrogens as solid, free-flowing dry extract fraction. Versuch Nr. Test No. Sieve analyzes obtained according to Example 2 of pharmaceutical preformulations in the form of solid, free-flowing dry extracts.

Um die galenische Weiterverarbeitbarkeit der im Beispiel 1 durch Wirbelschichttechnologie hergestellten Trockenextrakte bzw. In order to test the galenic further processability of the dry extracts or preformulations produced in Example 1 by fluidized-bed technology, the dry extracts or preformulations were mixed with further tabletting auxiliaries and compressed to form matrix tablets. It was found that the mixtures could be homogeneously distributed in a matrix tablet and compressed. The composition of the carrier material used for the conjugated estrogens as carrier, for example the mixture of microcrystalline cellulose with lactose, the particle size and the porosity of the active substance granules and the particle size distribution in this case affect the quality of the compressibility and the release profile of the hormones out of the matrix are released.

A pharmaceutical preformulation for tabletting, characterized a by an active substance content of a mixture of natural, conjugated, equine estrogens from the urine of pregnant mares, wherein the active substance content i is defined per amount of a pharmaceutical support material, and. The preformulation according to claim 1, characterized in that the active substance content a is calculated as the dry matter DM of an extract containing the mixture of natural, conjugated, equine estrogens from the urine of pregnant mares total hormone content, including the free estrogens and other solids , based on the amount of the pharmaceutical support material in the preformulation, and is in the range of 0.

The pharmaceutical preformulation according to claim 2, characterized in that the residual moisture in the preformulation is a maximum of 3. The pharmaceutical preformulation according to claim 1, characterized in that the active substance content standardized to the main hormone components contains the conjugated hormones in the following proportions: The pharmaceutical preformulation according to claim 1, characterized in that the pharmaceutical support material is microcrystalline cellulose, or a mixture of microcrystalline celluloses.

The pharmaceutical preformulation according to claim 1, characterized in that the pharmaceutical support material is a mixture of microcrystalline cellulose with lactose. The pharmaceutical preformulation according to claim 7, characterized in that the weight ratio of microcrystalline cellulose to lactose is in the range of to , preferably in the range of about 7.

The pharmaceutical preformulation according to one of the previous claims, characterized in that the mean bulk volume of the preformulation is in the range of 1. The preformulation according to one of the previous claims, characterized in that the mean bulk weight bulk density of the preformulation is in the range of 0. The pharmaceutical preformulation according to one of the previous claims, characterized in that the preformulation has a particle size distribution characterized by sieve analysis as a function of the sieve mesh size of about 0.

The method according to claim 14, characterized in that the aqueous solution, containing the active substance which is used contains, in addition to water, one or more water-miscible organic solutions, preferably lower alcohols, as an additional solvent.

The method according to claim 15, characterized in that the aqueous solution, containing the active substance which is used, is a substantially pure aqueous solution of the mixture of natural, conjugated, equine oestrogens. The method according to claims 14 to 16, characterized in that the aqueous solution having an active substance content, which is used a is calculated as a dry matter of the mixture of natural, equine, conjugated oestrogens total hormone content, including the free oestrogens and other solids , and is in the range of about 3.

Type grade. PH PH Dichte: Density:. Vorlage: Template:. Ablufttemperatur: Exhaust air temperature:. Oestrogene estrogens. Freie Estrogene : Free estrogens:. Trockenextrakt dry extract. Estron estrone. Equilin equilin. Equilenin equilenin. Gesamthormongehaft Gesamthormongehaft. Summe Haupthormone 3 Total main hormones 3. Freie Estrogene Free estrogens. Gesamthomlongehalt Gesamthomlongehalt. Gesamthormongehalt Total hormone content. VA -1 VA VA-2 VA VA-3 VA VB-1 VB VB-2 VB Biodegradation studies of estrogens with activated sludge form different sources showed a higher removal rate in sludge from a municipal STPs compared to the removal rate in sludge from an industrial STPs.

This confirms the importance of an adapted microbial population in the biological removal of estrogens. No significant degradation of 4- 14 C -estradiol was observed in an anoxic test system with the same type of sludge. The mineralisation rate of ethinylestradiol is low compared to estradiol. Investigations of samples taken from either the influent or the effluent from primary sedimentation tanks and final effluent from STPs in Canada, Germany, Italy, the Netherlands, Brazil and Japan show that the concentrations of estrogens are within the same ranges 85 86 87 88 The removal of estradiol and estriol are generally more extensive than the removal of estrone and ethinylestradiol.

The removal efficiency of the four estrogens in the study of the Italian STPs, i. It is not possible to make any conclusion concerning the amount removed by biodegradation. It is obvious from the results obtained in batch tests that removal of the estrogens from the water phase in STPs may occur by degradations as well as sorption to sludge particles. The degradation of APnEO is initiated by sequential cleaving of ethoxylated units. Under aerobic conditions the resulting products are alkylphenols, mono- and diethoxylates and the more hydrophilic carboxylates.

Carboxylation may occur in both the alkyl and ethoxy side chains of the molecules. AP seems to be degradable under aerobic conditions. The transformation of APnEO under anaerobic conditions oxygen free conditions as under anaerobic digestion of sludge results in production of mono- and diethoxylates and finally APs. APs are not further degraded under anaerobic conditions This results, generally, in extremely high concentration of APs in anaerobically digested sludge, which may be reduced by introducing a post-aeration step The mono- and diethoxylates as well as APs are hydrophobic compounds, which also are removed from the water streams within STPs by sorption to sludge particles.

The primary sedimentation did not significantly alter the distribution between the different metabolites alkylphenol, alkylphenolethoxylates and carboxylated alkylphenols APnEC. However, APnEC was the most abundant group of the metabolites in the effluent after the activated sludge treatment. Investigations of eleven STPs in Switzerland showed that approx.

Therefore, the potential pool of AP in the effluent from STPs may be considerably higher than known today. Biodegradation studies of bisphenol A have shown that the compound should be easily degraded under aerobic condition in the activated sludge tank of STPs. However, degradation under anaerobic or anoxic conditions is much more unlikely to occur. It can also be expected that an amount of the bisphenol A received by STPs will be removed from the water phase by sorption.

The fate of the compounds in STPs is not only correlated with the intrinsic properties of the compounds, i. However, treatment conditions of the STPs studied are often not completely described. Hydraulic retention time HRT , sludge retention time SRT , temperature, denitrification, nitrification, and phosphate elimination will all have an important bearing on the plants efficiency Comparing data within single studies, in which the sampling techniques and analytical methods are identical, was thought to give the best basis for evaluation of the importance of different treatment processes.

The present assessment of the effects of the type of STPs was therefore mainly based on studies with investigations of STPs with different processes. Generally, the effluent concentrations of estrogens and alkylphenol compounds AP-c seem to decrease by upgrading the STPs to nutrient removal and by using other tertiary treatment process as e.

Analysis of influent samples from this STP showed correspondingly high concentrations. It can be concluded that the use of low technology plants, especially plants of very low technology in the so-called "open land" can be expected to result in relatively high concentrations of estrogens and xenoestrogens in the mixing zone of a recipient compared to plants of higher technology. The plant is upgraded to nutrient removal.

It has not been possible to explain this high concentration on the basis of the available data However, long retention in the biological treatment step should increase the time of degradation. Furthermore, a long SRT of e. However, an investigation of influent and effluent samples from three Dutch STPs based on the activated sludge system showed that the highest removal efficiencies of estrone and estradiol were obtained in the two plants with the highest HRTs 18 and 26 h and SRTs 11 and 20 d Apparently, there is a tendency to higher concentrations of especially estrogens in effluents from the U.

However, the data in the literature do not allow any final conclusions regarding potential differences between countries. Influence of advanced sewage treatment processes on removal efficiencies. Conventional sewage treatment is not an effective barrier to trace contaminants like estrogens and xenoestrogens.

Synonyms and antonyms of Sorption in the German dictionary of synonyms

There has, therefore, been an increasing focus on the use of more advanced treatment processes with the objective to remove the contaminants. No significant differences were observed using sand filtration or microfiltration on the same type of effluent For some reverse osmosis membranes, retention was similar to nanofiltration, but others showed a very high and stable retention of the compounds. Microfiltration also showed initial almost complete retention followed by a drop as expected. The obtained results are currently being confirmed on larger scale systems The preliminary results of a pilot-scale process show a positive treatment effect regarding endocrine disrupters.

The operating costs of the production of ozone were estimated to be between 0. It can be concluded that more knowledge concerning the fate of both estrogens and xenoestrogens within STPs are needed. There should be high quality studies of influent, effluent, sludge and internal streams of STPs using standardized sampling procedures and validated analytical methods. The studies shall be linked to concurrent comprehensive monitoring of overall STP performance e.

STPs with different treatment processes should be studied with the purpose of understanding the influence of different process designs and operation criteria. The monitoring of full-scale plants should be accompanied by studies in pilot- and laboratory-scale. Possible non-sewage effluent related sources of estrogens to the aquatic environment. Besides sewage effluent other sources of estrogens to the aquatic environment might have to be considered. Two potential sources are the use of manure from life stock and sludge from sewage treatment plants as fertilisers on fields.

Publications — Institut für Mikrosystemtechnik - IMTEK

Insufficient knowledge exist, however, on the extent of runoff of estrogens with drain water and it is not possible yet to assess whether these are considerable sources of estrogenic activity to the surface waters. I Europa er feminisering af hanfisk udsat for spildevand set i England 1 , Sverige 3 , Norge 4 , Tyskland 5 , Holland 6 , Frankrig 7 ; 13 , Spanien 8 ; 14 og Danmark 2.

Desuden har man gjort observationerne af feminisering blandt vildtlevende fiskebestande hos flere arter incl. Kontrollerede eksponeringer af fisk for spildevand i f. Feminisering er blevet fundet i forskellige udviklingsgrader blandt fisk fra milde til alvorlige forstyrrelser af det hanlige forplantningssystem. De vigtigste alkylphenoler mht. Nye resultater har dog indikeret nedsat befrugtningsevne blandt intersex-skallehanner i England 57 ; Efter denne periode udskiller kvinder i gennemsnit ca.

Alkylphenoler anvendes hovedsageligt i produktionen af alkylphenolethoxylater APnEO , tris nonylphenyl phosphite og alkylphenol-formaldehydkondensationsresiner Alkylphenolethoxylater nedbrydes relativt let til alkylphenoler og er derfor vigtige kilder til alkylphenoler I Danmark vil nonylphenolethoxylater og nonylphenol kunne udledes fra formulering og anvendelse i bl. Hovedparten af bisphenol A anvendes som kemisk byggeblok i produktionen af polycarbonat og epoxyresiner.

Det er ikke muligt at vurdere, hvor stor en andel af denne fjernelse, der skyldes biologisk nedbrydning. Nedbrydningen af APnEO initieres ved sekventiel fraspaltning af ethoxylatenhederne. Under aerobe forhold er de resulterende produkter alkylphenol, mono- og diethoxylater samt de mere hydrofile carboxylater. Det er derimod mere usandsynligt, at der sker en nedbrydning af bisphenol A under anaerobe og anoxiske forhold. Driftsomkostningerne for ozonproduktion blev estimeret til mellem 0,90 and 1,60 EURO pr.

Der er p. The intersex condition was characterised by a presence of both early and developed stages of egg cells oocytes , and the presence of the female duct, the ovarian cavity, in the testes of presumptive male fish. The ovarian cavity is the structure within the ovary to which mature eggs are released before being passed on to the oviduct.

In some cases the male sperm duct was further missing. The study showed a correlation between the degree of intersex and the concentration of sewage effluent in the river indicating that compounds in the sewage effluent were responsible for inducing the intersex condition.

Synthesis of vitellogenin by male or immature fish is an internationally used marker for estrogenic exposure. Vitellogenin is normally produced by female fish during their reproductive cycle. It is induced in the liver by the endogenous estradiol production of the female, excreted to the blood stream and incorporated into the developing oocytes in which it is degraded to yolk proteins. These act as nutrition for the developing larvae Males and immature fish normally contain no or very little vitellogenin in plasma possibly due to very low circulating levels of estradiol. If male fish are exposed to xeno- estrogens this will, however, induce the synthesis of vitellogenin which in severe cases can reach levels as high as or higher than found in sexually mature females.

A second species of cyprinid fish, the gudgeon Gobio gocio has also been demonstrated to show intersex in English rivers The frequency was not as high as demonstrated among roach from the same rivers. This is despite the fact that the gudgeon is a bottom living fish which could be expected to be exposed to compounds both in the water phase and in the sediment. However, in line with the high frequency of disruption in roach, the most severe cases of intersex among gudgeon were found among fish from this site and the male fish also had elevated levels of vitellogenin.

In general, however, this species seems to be less sensitive compared to roach towards endocrine disruption. The observations of feminised feral fish from English rivers have led to studies of roach populations in both Denmark and France and of populations of other fish species in other European countries and USA. Among the roach populations from two clean lakes with no known effluent discharge intersex occurred in 4.

A tendency to a higher degree or extent of feminisation was found in intersex individuals from the three sewage effluent affected streams, but only significantly at one site, compared to the extent in intersex fish from the two control sites. In general, a milder degree of feminisation was found in the examined Danish roach populations compared to that observed in English roach.

Occurrence of the female ovarian cavity was not observed and only primary growth stages of oocytes were seen from few to a maximum of per testes section.

Dr. John Lee (Teil6): Die ganze Wahrheit über Oestrogene, Progesteron, Testosteron...,

In accordance with the Danish study, a study of roach from 4 rivers in the north eastern part of France concluded a lower incidence of intersex compared to English roach. In France estrogenic effects have also been observed in a preliminary study of another fish species the chub Leuciscus cephalus from one river 7. This study is, however, a study based on relatively few fish from each site. Brown trout Salmo trutta is another fresh water species which has been studied in Denmark with regard to endocrine disruption 2.

In one stream with low sewage effluent load, Voel Brook, an increased level of vitellogenin was found among males. Whether the different testes structure compared to most control fish also was a consequence of hormonal exposure or is due to other factors such as a different age structure among fish from Voel or exposure to other chemicals is not known. Vacuolation in sertoli cells in the brown trout can be the result of the reabsorption of sperm cells which have not been spawned in the previous spawning cycle or absorption of degenerated sperm cell stages from the present reproductive cycle ; In Spain, male carp Cyprinus carpio has been examined for feminising effects in two rivers, Anoia and Cardener, with high and low to moderate presence of estrogens, respectively 8 ; In accordance with the higher estrogenic load in the Anoia river, male carp from this river showed a higher plasmatic vitellogenin concentration than males from Cardener River.

Intersex fish with simultaneous presence of testicular and ovarian tissue were further observed among fish from River Anoia.

Degradation of Estrogens in Sewage Treatment Processes

In this case the presence of high vitellogenin levels in the male fish was not consequently indicative of gonadal effects since intersex fish both were found to have vitellogenin levels higher than females and levels not significantly different from control males. A large national survey in the Netherlands of occurrence and effect of estrogens and xenoestrogens in the aquatic environment has examined the reproductive health of approximately bream Abramis brama 6. High levels of vitellogenin were detected in male bream from several freshwater sites including the rivers Rhine and Meuse and in three major river sedimentation areas.

The highest vitellogenin concentrations were found among males from a small stream, Drommel, from which the largest percentage of intersex was also recorded. Initial results from other smaller Dutch waters, which have not been published yet, have indicated that in one other stream, equally high levels of vitellogenin and intersex in male bream as in the Drommel were found. Generally, estrogenic effects appeared at half the selected locations 6. The bream population in the river Elbe in Germany has also been examined for endocrine disruption at nine different sites from Hamburg to the Czech border 5.

Weak estrogenic effects were seen along the entire length of the river since higher levels of vitellogenin in male bream were found at all sites compared to levels in males from a control lake with no domestic or industrial effluent input. The strongest estrogenic influence, detected as the highest vitellogenin induction times the level in controls , was found at locations characterised by high levels of effluent discharge to the river discharges from STPs with , and 1,, population equivalents P.

Suppression of reproductive function was also assessed by altered steroid hormone status lowered 17 b -estradiol in females and lowered ketotestosterone in males and lowered gonadosomatic index GSI 6 in both sexes. In general it was concluded that disruption of the endocrine system of the bream population was observed but only the higher levels of vitellogenin were ascribed to estrogenic exposure whereas other effects might have happened through multiple mechanisms also involving other groups of chemicals such as polyaromatic hydrocarbons PAHs and polychlorinated biphenyls PCBs also known to exist in higher levels in the Elbe.

Levels of estrogens were not measured 5. Other studies of carp Cyprinus carpio and walleye Stizostedion vitreum have demonstrated higher vitellogenin levels in males collected near discharges from STPs In both species lower levels of serum testosterone were also detected in male fish. In walleye a delay in spermatogenesis relative to fish from control sites was observed and there were indications that viable sperm was not being produced in males from the STP site.

These fish were therefore apparently completely sex reversed males Due to their low endogenous levels of estrogens, male fish are considered to be the most sensitive sex towards reproductive disrupting effects caused by estrogenic compounds in sewage effluent. There does, however, exist some observations indicating that endocrine disruption of female feral fish has also taken place.

New results on English roach from two of the most affected rivers in which males had developed intersex have also demonstrated a higher incidence of oocyte atresia or degeneration in the sewage effluent-exposed females compared to control females. Sex hormone levels were also disturbed since the estradiol concentration was only half that measured in control fish illustrating that the female reproductive system was also affected Another report on female reproductive effects come from a study in Sweden reported in , which described endocrine disruption in fish from a lake which was receiving leakage water from a nearby refuse dump The situation is therefore not entirely comparable to the exposure cases with STP, but included since strong indications of endocrine disruption were seen.

Effects were observed in both males and females but with the most pronounced effects in females. Among the sexually mature females a lower aromatase activity in the brain and altered steroid levels in plasma were detected. Testosterone levels were lower in fish from the leakage water affected lake and a concomitant tendency to a lower level of plasma estradiol was observed. The authors suggested that the reduced level of estradiol might be a result of the lower brain aromatase 9 activity and that the reduced estradiol production could have lowered the production of vitellogenin and therefore inhibited the development of oocytes in the females.

Changed steroid levels have also been reported in female walleye from a sewage effluent affected site in the USA A number of caging experiments where fish have been placed in streams in close proximity to the STP outlets and experiments where fish have been exposed to various controlled dilutions of sewage effluent have verified the estrogenic and feminising capacity of sewage effluent from several STPs on male fish. Dose-response relationships among effluent dilution and feminising effects have been demonstrated 4 ; 24 ; ; ; along with consequences of exposure time A series of cage experiments in England where male rainbow trout Oncorhynchus mykiss were placed in cages for three weeks in vicinity of discharges from STPs were the first to demonstrate the estrogenic nature of UK sewage effluent being discharged to English rivers through observations of up to a 10,fold induction of vitellogenin in male fish ; The survey of six rivers demonstrated induction of vitellogenin in caged fish in five of these rivers.

In one river the estrogenicity was shown to persist as far as 5 km from the discharge point but was in the other rivers in a three week trial only observed in immediate vicinity of the STPs Fractionation of the sewage effluent and chemical analysis of the water have subsequently shown that the compounds responsible for the observed feminisation in most cases primarily were natural and synthetic estrogens, 17 b -estradiol, oestrone and ethinylestradiol In the case of one river, however, outlets of alkylphenolic chemicals with industrial sewage from wool scouring and other textile industries 34 were assessed to contribute to the majority of the estrogenic activity.

Estrogens from humans are excreted in a hormonally inactive form as steroid glucoronides and sulphates primarily via urine. The steroid conjugation takes place in the liver and is a normal metabolic pathway used to facilitate the excretion of compounds from the body In humans estrogens are primarily excreted as estrogen glucuronide conjugates. Women may excrete around 2. Smaller quantities are also excreted with faeces in For more information regarding women as a source of estrogens see section The microbial activity in the sewage treatment works and possibly also in the sewage before it reaches the treatment works results, however, in a deconjugation of the steroid thereby restoring the estrogenicity of the steroid components of the sewage effluent Cage experiments using juvenile rainbow trout have also demonstrated the estrogenic nature of Swedish sewage effluent from a relatively small STP which mainly receives domestic sewage 3.

The STP had chemical and biological treatment steps but no anaerobic denitrification step and processed sewage effluent from a smaller number of people Still, a very high induction of vitellogenin was found in male fish after an exposure period of two weeks in the stream. Both estradiol, estrone, ethinylestradiol, nonylphenol and bisphenol A could be detected in the bile of the fish.

Chemical analyses of the stream water showed high levels of all three estrogens but with ethinylestradiol at a 45 times higher concentration 4. Ethinylestradiol was therefore considered to be responsible for most of the estrogenicity of the examined effluent. Very high levels of EE2 detected in a final STP effluent in Texas were also considered the likely cause of elevated vitellogenin levels, reduced GSI and reduced secondary sexual characteristics in fathead minnows Pimephales promelas kept for three weeks in cages in a treatment wetland close to the outlet of effluent No information were given regarding the treatment steps of the STP from which the effluent was coming.

On the contrary, no effects on the same parameters in a similar study of fathead minnow were observed when caged fish were exposed in situ at riverine sites to representative effluents from central Michigan sewage treatment plants All plants were using at least secondary treatment technologies and at some sites tertiary treatment such as sand or charcoal filters were also applied.

Chemical analysis of possible estrogenic compounds in the sewage effluent or surface water was not performed. Different sensitivities of different fish species have to be taken into consideration when using cage studies to assess or confirm the estrogenic capacity of sewage effluent.

In the dutch study mentioned above where a high degree of feminisation was observed in wild bream from the small stream, Drommel, cage experiments in the same stream with either carp or rainbow trout showed high vitellogenin induction in rainbow trout but not in carp when male fish were placed in cages for 21 days 6. The results from the cage experiments with rainbow trout did, however, confirm that the feminisation of feral bream could be ascribed to sewage effluent exposure. The Danish studies which have examined the estrogenicity of sewage by caged fish have in general found no or a relatively weak induction of vitellogenin in the sewage exposed fish.

In the last mentioned study no induction was, however, not seen in two other species, rainbow trout and eel Anguilla anguilla. This is despite the observation of relatively high frequencies of intersex among roach from Kristrup Landkanal. The explanation for this discrepancy might be differences in exposure time for the two species. Further, for the Atlantic croaker Micropogonias undulatus , it has been demonstrated that the estrogen receptor in the testis is saturated at four times lower concentrations of estrogens and a number of estrogenic compounds compared to the estrogen receptor in the liver, in which the vitellogenin synthesis is induced.

Alterations in the gonads such as intersex or sex reversal might therefore be more sensitive indicators of an estrogen effect than vitellogenin. In line with the last study just mentioned, a number of controlled studies with graded concentration of sewage effluent have further demonstrated a dose-response relationship between vitellogenin induction or intersex and the percentage of sewage effluent verifying again the estrogenicity of sewage effluent from several European countries and adding proof to the sewage effluent as the source of compounds causing the feminisations of wild fish.

Sewage effluent from a municipal sewage plant which receives domestic sewage from Oslo was estrogenic to rainbow trout at concentration of 2.


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In the same study effluent from an oil refinery treatment works was estrogenic to rainbow trout. Concerns about endocrine disruption by effluent from pulp mills have mainly been in the direction of masculinising effects, which has been seen in a number of cases ; Both estrogenicity and androgenicity of the effluent were observed in the Swedish study since exposure to the pulp mill effluent besides inducing vitellogenin also resulted in a higher proportion of males.

This indicates the presence of both estrogenic and androgenic compounds in the water. In roach, it has further been shown that vitellogenin synthesis induced by sewage effluent besides being dose dependent also depends on the exposure period The percentage of the sewage effluent which was able to induce vitellogenin in mature adult roach fell from In the sewage effluent used in this study the levels of estrogens were 1. Sewage from the same STP could in another study induce a dose-response dependent development of intersex in roach when exposing juvenile fish from 50 to days after hatch.

The fish developed the female duct called the ovarian cavity also detected in wild roach 1 at a threshold concentration of This feature seemed to be irreversible since depuration of the effluent exposed fish in clean water for days did not result in any alteration in the duct formation. No induction of oocytes was induced in the testes of any male fish in the present study and therefore the causality of the oocytes in the testes in wild roach still needs complete elucidation. Marine organisms have been expected to be less affected by endocrine disruption from estrogenic discharges to the marine environment compared to freshwater fauna due to the usually high degree of dilution of sewage effluent in estuaries and open waters A number of surveys have, however, demonstrated feminisation of some marine species, predominantly in flounder Platichthys flesus The flounder has been chosen as a suitable marine test monitoring species because it lives in close contact with the sediment in which it buries itself and it lives most of the time in the estuaries migrating only to sea for breeding One disadvantage with this species is, however, that controlled laboratory studies using water exposure have demonstrated the flounder to be less sensitive towards estrogens compared to for instance the rainbow trout Still, elevated levels of vitellogenin has been detected in flounder from several industrialised estuaries in England especially in Tyne, Tees and Mersey compared to reference sites in a number of studies conducted over a period of 6 years ; 21 ; The routes by which flounder is exposed to environmental estrogens are not fully understood.

Exposure through the food chain might contribute to the observed effects in flounder since experiments where flounders were fed mussels held in the Tees estuary for 3 months showed a fold increase in plasma vitellogenin Exposure of male flounder to water and sediment in cages at two sites of the Tees and Tyne for three weeks did not induce vitellogenin production Occasionally high vitellogenin levels have also been detected among males collected offshore.

The reason for this is unclear but might rather be due to migration of males from the more polluted estuarine areas than caused by local pollution offshore Similar observations of single very high levels of vitellogenin among male flounder from several near cost areas in Denmark have been made How these results should be interpreted is also very uncertain and a need for more knowledge on seasonal and age related vitellogenin levels in this species is needed.

Intersex has also been detected among male flounder in the English surveys. Other gonad changes which have been reported from flounder are abnormal testes morphology such as disorganised lobules 10 , vacuolation, thickening of connective tissue and lack of germ cells pointing toward an inhibited spermatogenesis A higher proportion of degenerating oocytes were seen among females from Tyne in the same study. In a Dutch study of offshore, estuarine and inland locations including flounders, levels of vitellogenin were found to be low among males at most investigated sites 6 except at two sites in the North Sea Canal, an industrialised port zone which receives effluent from sewage treatment works.

Here moderately elevated levels of vitellogenin were found among the examined males in a fall but not a spring sampling. None of the captured males had oocytes in their testes. Based on these results it was therefore concluded that estrogenic effects in the marine waters of the Netherlands were few compared to the situation reported from the English and Welch Coast. Both a higher level of vitellogenin and intersex were found in male flounder from the Tokyo Bay which receives large amounts of industrial and domestic sewage effluent. As mentioned, studies of feminising effects on marine fish species have mainly concentrated on the flounder but a few results on other species have been reported.

Two species of sand gobies Pomatoschistus minutus and P. MIPS is a condition in which the male urogenital papilla a secondary sexual organ used for depositing sperm has developed villi at the papilla tips which resembles the villi on the female papilla used for oviposition. This is a condition which has been demonstrated to be inducible by controlled exposure to estradiol The development of sperm cells takes place along the entire length of the tubule periphery.

At maturity, spermatozoa are released to the tubule lumen and transported to the sperm duct The observations of feminisation of fish exposed to sewage effluent, indicating that the effluent contains estrogenic compounds, have led to attempts of quantifying the estrogenic activity of the effluent. Fractionation techniques followed by in vitro assays have been used to quantify the estrogenic activity of the sewage effluent. The fractions responsible for the estrogenic activities in the in vitro assays have in some cases further been subjected to chemical analyses leading to identification of the active chemicals 25 - a procedure called toxicology identification evaluation TIE.

The following chapter will summarise the results made on estrogenicity of sewage effluent with numerous in vitro assays and compare these with the actual concentrations measured in the sewage effluent where these have been made. Additional results on estrogen concentrations from chemical analysis of sewage effluent and surface water will be presented in chapter 5.

The additive behaviour of the estrogenic activity of single substances in a mixture has been demonstrated and is the basis for quantitatively assessing the total contents of estrogenic substances in an environmental sample by the use of in vitro assays. A number of cell based in vitro assays have been applied which uses cells transfected with an estrogen receptor controlled reporter gene 30 ; 30 ; Among these is the YES assay using yeast cells transfected with the human estrogen receptor ER and the reporter gene b -galactosidase Other assays use transfected human breast cancer cells transfected i.

The human breast cancer cells, of which different strains have been used, already contain the human estrogen receptor. The E-screen, in which proliferation of human breast cancer cells MCF-7 as a response to estrogen is measured, has also been used to determine the estrogenicity of sewage effluent and surface water ; One drawback which has to be considered when using these types of assays is, however, that antiestrogens which might also be present in the water samples can bind to the estrogen receptor and counteract the estrogenic response.

An underestimation of the actual estrogenic potential of the water source might therefore take place. This has been demonstrated earlier This might also lead to an underestimation of the actual feminising potential of a water source. In another study EEQs of between 0. This study indicated rather constant inputs of estrogenic substances via STP effluent to the rivers. This is not in agreement with an English study which demonstrated very variable estrogen levels in sewage effluent analysed over a period of 8 months The municipal STP processed sewage from , inhabitants and was reported to have a capacity of , population equivalents P.

Chemical analysis of the effluent water detected 4- tert -octylphenol 4- t OP , 4-nonylphenol NP and bisphenol A in the effluent at concentrations from 0. This indicated that most of the estrogenicity of the effluent was due to the presence of natural and synthetic estrogens Estrogenicity suspected to impact fish has, however, been demonstrated for another large German river, the Rhine, despite a times lower YES estrogenicity of the river water compared to the effluent. Chemical analysis detected estradiol at relevant concentrations of 3.

Natural and synthetic estrogens have also been considered responsible for the observed estrogenicity of sewage effluent in other studies. Sewage effluent from seven STPs which discharged into English Rivers were demonstrated to contain three fractions with estrogenic activity when these were tested with the YES assay Chemical analysis isolated 17 b -estradiol, estrone and ethinylestradiol from these fractions.

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These levels have led to the conclusion that most effects on freshwater feral fish observed in English rivers are a result of natural estrogens excreted from women. The effluent tested in this study contained little or no agricultural input and all natural estrogen input was believed to be of human origin.

The feminising effects which have been found in English estuaries in feral flounder also seem to be related to the presence of the natural hormone estradiol in the effluent The estrogen equivalent levels were also found to be higher in Dutch fresh water compartments and biota compared to coastal and marine environments The estrogenic activity of 2. This lake has been described as one of the most polluted water bodies of China and both municipal and industrial sewage from a city of 4 mio inhabitants are discharged into the bay.

Estradiol at concentrations from 1. In effluents from three municipal wastewater treatment plants in Michigan, four point source locations and five locations in Lake Mead, EEQs of 1. Estradiol and ethinylestradiol accounted for 88 to In a Korean river total estrogenic activity was 0. In a Japanese river EEQs of 3. In a South African study of 25 selected water and sediment samples using the YES assay, estrogenic potencies ranged from below detection limit 0. The last results which will be mentioned from studies assessing the in vitro estrogenicity are on sewage and surface water in the Netherlands and Belgium.

The Dutch national survey recently reported 0. A study of the estrogenic activity of Flemish rivers and effluent surprisingly found the highest estrogenic potency in the surface water samples compared to the effluent As described above the use of in vitro assays has demonstrated estrogenicity of sewage in numerous countries and in a large number of these studies the major part of the in vitro estrogenicity was assigned to the presence of natural and synthetic estrogens in the samples.

Therefore the following sections describing environmental concentrations in relation to known dose-response relationships regarding endocrine disrupting effects will concentrate on the natural estrogens 17 b -estradiol, estrone and estriol, the synthetic estrogen ethinylestradiol and among xenoestrogens the more potent alkylphenols and bisphenol A.

The present section will describe the occurrence of estrogens and xenoestrogens in the sewage effluent and what is known about their occurrence and fate when they reach the aquatic environment. Some information on the actual concentrations in both final effluent and surface water has already been given in chapter 4 in relation to the in vitro estrogenicity of the water samples.

Sorption: Only sorption of the free estrogens was measured, as the conjugated estrogens are more hydrophilic and thus are not believed to sorb to sludge to a great extent. Stability in sterile water: Stability of all five estrogens was measured in sterile water as a control experiment possible sorption to glass walls or other experimental artefacts and to establish a baseline for stability of estrogens. If estrogens degrade abiotically within a certain time, they are not likely to exist longer in any environment than this experiment shows. Stability in activated sludge suspensions: Stability of five estrogens in activated sludge suspensions were tested under aerobic conditions.

However, to reduce the number of samples that had to be measured, experiments with addition of E1 were omitted. It is well known from other studies that in activated sludge E2 is oxidized very fast to E1. Therefore, the degradation of E1 could be followed in the experiment where E2 was added. Stability of the free estrogens with activated sludge suspensions were tested under anaerobic conditions.

Again, the experiment with addition of E2 was used to measure degradation of E1. It was not possible within the constraints set by time and resources to measure degradation of conjugated estrogens under anaerobic conditions. It is likely that aerobic or anaerobic conditions will not affect the degradation of conjugated estrogens.

Upon arrival at the laboratory about three or one hours later, respectively, the sludge was immediately washed three times using tap water US EPA standard, see Section 4. The sludge was then freeze dried. Further, the temperature was measured in the tank of the STP from which the sludge was taken. The data from the characterisation of the collected sludge are shown in table Table Characterisation parameters for the activated sludge used in all experiments. On arrival at the laboratory about three hours later, the sludge was immediately aerated until the total solids concentration was determined and the batch experiment reactors could be started.

The batch reactors were started about 12 hours after the sludge was taken from the activated sludge tank of the STP. The data from the characterisation of the collected sludge are shown in Table 4—1. In the experiment, the sorption equilibrium between sludge and water was allowed to adjust. A range of estrogen concentrations were tested. The water used was similar to the artificial sewage described by Nyholm et al. An experiment was carried out to determine the minimal time for establishing equilibrium between the water phase and solid phase activated sludge.

Based on this experiment the equilibration time for the experiment was set to 3 hours. This experiment was not performed on sludge from Lundtofte STP. Instead, the sorption experiment was allowed to equilibrate for 6 h in experiments with sludge from Lundtofte SPT. This standard suggests that a range of equilibrium concentrations are produced by adding different amounts of sludge to bottles with a fixed concentration of the compound. The reduction in aqueous concentration of the measured compound provided by the sorption process is used to establish the different equilibrium concentrations needed to experimentally determine a sorption isotherm.

This experimental design was attempted with sludge from Lundtofte STP varying in concentrations from 0. However, it was found that aqueous equilibrium concentrations varied relatively little less than two decades. Given the normal analytical random error and additional variation caused by inhomogeneity of the sludge, the isotherms were poorly described. It was therefore decided to use a modified experimental design still based on varying the ratios of compound concentration to sludge concentration as prescribed in the standard.

We used different concentrations of steroid estrogens covering four decades spaced equally on a logarithmic scale and equilibrated with a fixed sludge concentration of 1. Since the analytical method was not able to quantitate the estrogens precisely over 4 decades of concentrations, different sample volumes were extracted ranging from 1 to ml. The volumes and starting concentrations used for the experiments are shown in table The modified experimental design reduced the random variation compared to the scale of the isotherms and provided a more precise determination of isotherms.

Table Sample volumes and initial concentrations used for the different concentration replicates in the experiments for determining the sorption isotherms of E1, E2 and EE2. C O is the nominal starting concentration of each steroid estrogen. One replicate was made for each steroid estrogen at each concentration level. Sample volumes varied from 1 ml to ml to allow exact quantification of a wide range of concentrations. Samples were filtered before extraction by solid phase extraction SPE and analysed as described in Section 4.

One experiment was made for each chemical except EE2 for which the experiment was made in quadruplicate in order to determine the variability between reactors rather than studying this substance in particular detail. In the experiment with E1, E2 was also measured and likewise E1 was measured in the experiment with E2 since E1 and E2 can be transformed to each other by simple oxidation or reduction reactions. Since an analytical method was not available for analysing estrogen conjugates E1 was measured in experiments with EGlu and ESul.

The main degradation reaction for these two conjugates is expected to be hydrolysis which forms E1. Experiments were performed in 1 l glass reactors as described by Nyholm et al. Samples were taken after 5 min, 1 h, 12 h, 24 h, 48 h, 72 h and 96 h. The two compounds expected to be most persistent with activated sludge, E1 and E1-Sul, were also sampled after h. Figure Schematic drawing of the glass reactor used for all degradation experiments. Samples of ml were extracted with SPE and analysed as described in Section 4.

No filtering or clean-up was used. The degradation of E2 and EE2 were investigated in diluted activated sludge with oxygen and nitrate as oxidation source. EGlu and ESul were investigated in diluted activated sludge only with oxygen. One experiment was made for each chemical except EE2 in aerobic reactors for which the experiment was made in triplicate to determine variability between the reactors.

In the experiment with E2, E1 was also measured since E2 can be transformed to E1 by a simple reduction reaction. Experiments were performed in 1 l glass reactors with 0. The activated sludge was added 12 h prior to initiating the experiment started to give the sludge bacteria time to adapt to the oxidation sources O 2 or NO 3 - in the reactors. Samples were taken after 3 min, 6 min, 20 min, 40 min, 90 min, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h. Further, six blank samples and six control samples were taken. During the experiment nitrate was measured and replenished at least twice daily.

The particles in the sample was loaded on top of the cartridge rather than removed by filtration before the extraction as was done in the sorption experiment. This made it possible to elute the particle bound steroid estrogens together with the steroid estrogens on the SPE column. This was done in order to extract all estrogens present in the sample rather than only the estrogens dissolved in the water phase. The chemical environment in the batch reactor was characterised by measuring O 2 and pH with selective electrodes and nitrate with a colorimetric test kit see appendix 1 for details on instrumentation several times a day during the experiments.

The stability of the biodegradation activity in the sludge slurry over the experimental period was further estimated by measuring the cumulative biological oxygen demand BOD over 24 h in samples taken from reactors with both types of RedOx regimes every day of the experiment. The cartridge was then freeze dried. The estrogens were eluted from the cartridge with 5 ml acetone. Samples from the abiotic stability experiment were treated exactly as samples for the sorption experiment except that no solids had to be removed.

Samples were spiked with ng of deuterated standards d4-E1, d5-E2 and d5-EE2 as extraction standards. The sample containing both water and sludge were then loaded onto a solid phase extraction cartridge. The particles in the sample was loaded on top of the cartridge rather than removed by filtration before extraction according to a method described by Ternes et al.

Then all the water had been drawn through the cartridge the sludge was left on top of the column. The cartridge was then dried by freeze drying. The estrogens were eluted from the cartridge with 2 x 2. The reduced extract was purified by passing in through a custom made 1 g silica gel column with 5 ml acetone:hexane mixture Andersen et al. The clean-up removed most of the coloured matrix from the extract. In the method evaluation of the analytical procedure for this sample type it was found that the use of silica gel clean-up was essential for being able to detect the estrogens in this matrix rich sample type.

Even after the clean-up some parts of the matrix were still left and made the ionisation efficiency for the MS detector unstable. This problem could be satisfactory solved by using deuterated standards of each of the analytes. As the deuterated standards were chemically exactly like the analyte they represent the variation in ionisation efficiency was exactly matched by the same variation in the ionisation of the standard. For each sample series a range of concentrations of steroid estrogens and extraction standards spiked to samples of drinking water was used to create a standard curve.

These spiked samples were extracted and cleaned with the same procedure as the real samples. Thus all systematic losses in the extraction and clean-up procedure for the samples were repeated on the standards and, hence, the effects will cancel each other. Further, each sample series included blank samples and spike recovery samples. The extraction standards E2-acetate AE2 and deuterated standards were used in an in-method statistical control system as described by Ternes et al.

For each steroid estrogen a characteristic mother ion formed from the molecule in the ionisation head of the MS-MS is selected by the mass detector. This ion is fragmented in the MS-MS and two characteristic daughter ions formed in the fragmentation process are selected for identification and quantification see Figure and Table Only when both ions were present in the correct ratio between the two, the signal could be used for quantification. A high certainty for quantification of the correct analyte is inherent to the chosen quantification method.

Each analyte had to elute at the correct retention time in the LC system. Then the ionisation should produce an ion with a correct mass, and fragmentation of this ion should produce two new ions with correct masses. Each concentration profile obtained in the abiotic and activated sludge stability experiment was fitted to first order reaction expressions as shown in Box 1 Figure This was done by integrating the expression with appropriate limits and fit the resulting expression to the observed concentration profiles.

Since E1 and E2 are degraded very fast in this experimental setup, it is difficult to measure the degradation rate precisely. Therefore, the net loss of the steroid estrogen structure was also determined by fitting the first order degradation expression to the sum of the concentrations of E1 and E2.

This should be interpreted as the effective rate constant for loss of the estrogenic structure. Figure Box 1: Fitting formulas for first order reaction rate constants and half-lives. The results of these experiments are shown in Figure It is therefore concluded that the sorption reaction had reached equilibrium after two hours. The three steroid estrogens are similar in size and lipophilicity, therefore the sorption kinetic should be similar for the three. It is assumed that the apparent lower water concentrations of E2 at six hours reaction time is an artefact of the random error on the concentration determination since no explanation based on the physical-chemical properties can be given.

Based on this experiment the equilibrium time used for the experiments to determine the sorption isotherms was set to three hours. Isotherms from the sorption experiments with the three steroids are shown in Figures and The Freundlich sorption isotherm defines the equilibrium between concentrations of a chemical in water and solid Schwarzenbach et al. Equation 1: The Freundlich equation for describing sorption isotherms Schwarzenbach et al. The parameters for the Freundlich equation were calculated by non-linear curve fitting as shown in Table This suggests that when these constants are used for calculating conditional K d -values and estimating the sorbed fractions of steroid estrogens tables and , the errors on the calculated results should be in that range.

Estimation of the errors on parameters with non-linear fitting using a generalised method is a very weak statistical method, which tends to give estimates of variation much higher than what is intuitively expected from the variation in the data. This is due to the non-specificity of the method as it designed to apply to all types of non-linear expressions.

K d -values can be calculated for representative water concentrations based on the Freundlich isotherms as:. Equation 2: Formula for generating conditional K d -values for steroid estrogens at different water concentrations from the Freundlich equation. By inserting relevant values for water concentrations of steroid estrogens into Equation 2 a table of K d -values can be generated as shown in Table It should be noted that the lower concentrations chosen for the table are extrapolations below the experimental concentrations range, which may hide systematic deviation, not covered in the experiment.

In the stability experiments with activated sludge the content of dry solids DS is 0. Equation 3 Equation for calculating the sorbed fraction of the total steroid estrogen concentrations at different densities of activated sludge MLSS. If the MLSS in the activated sludge tank and the excess sludge production mass of activated sludge removed from the STP per volume of treated sewage RESS is known the fraction of the total steroid estrogen load into the activated sludge tank that will be removed at equilibrium assuming no degradation occurs can be calculated as follows Equation 4.

This figure is derived from the daily production of excess sludge divided by the daily wastewater flow. If these values are used, the removal of steroid estrogens with activated sludge can be estimated as shown in table The sorbed fractions in the activated sludge tank have implications for the predictions of the degradation of the steroid estrogens in the activated sludge tank, which is discussed in Section 5. In the stability experiments with sludge the MLSS was 0. Presumably bacteria only degrade the dissolved steroid estrogens.

Further, if the desorption from the sludge takes place at a lower rate than that of the degradation in water, the desorption reaction becomes the rate limiting step for the overall degradation of the steroid estrogens. The predicted sorbed fractions also have consequences for the design of the analytical procedures for such experiments.

Thus, if one wishes to measure the steroid estrogens in a sample from an activated sludge tank, both water and sludge should be extracted and analysed. Contrary to this, if the sample was taken from the effluent, the error introduced by only extracting the water phase could maximally be 3. A practical implication of the above is that when the concentrations of effluent suspended solids are below the EU or national regulations, the sorbed fraction is of minor significance.

As shown in Figure no clear tendency for reduction of the three steroid estrogens were seen. Also no trace of E1 was seen in the experiment with E2. This could have indicated that auto-oxidation of E2 occurred. Similarly, no trace of E2 could be found in the experiment with E1.

This would have occurred if abiotic reduction of E2 occurred during the test. No clear sign of formation of E1 by hydrolysis of either EGlu or ESul was seen in the stability experiment. These values are shown in table Table First order reaction constants and half-lives for steroid estrogens in the abiotic experiments. Significant degradation of the five steroid estrogens was found except for ESul. E2 was degraded the fastest followed by E1. It appears that the only degradation process for E2 is oxidation to E1 since the sum of E1 and E2 was approximately constant in the first minutes of the degradation experiments with E2.

EE2 was the most stable of the estrogens. The degradation experiment with EE2 was made as triplicate independent experiments and therefore the variability between reactors could be estimated. The half lives in the three experiments varied between 10 h and 12 h. The stability of EGlu was not much higher than the stability of E1. Therefore only a small peak in concentrations of E1 was found in the experiment with EGlu.

In the experiment with ESul no significant amount of E1 was detected during the three days of incubation. It is therefore concluded that ESul is degraded slower than E1 in this experiment. The first order reaction rate constants obtained by fitting the concentration curves can be used to calculate half lives of each test compound using the formula in Box 1 Figure