Selected endocrine disrupting compounds (Vinclozolin,Flutamide, Ketoconazole and Dicofol): Effects on survival,occurrence of males,growth, molting and reproduction of <Emphasis Type="Italic">Daphnia magna</Emphasis>

BACKGROUND, AIM AND SCOPE: Pollution-induced endocrine disruption in vertebrates and invertebrates is a worldwide environmental problem, but relatively little is known about effects of endocrine disrupting compounds (EDCs) in planktonic crustaceans (including Daphnia magna). Aims of the present study were to investigate acute 48 h toxicity and sub-chronic (4-6 days) and chronic (21 days) effects of selected EDCs in D. magna. We have investigated both traditional endpoints as well as other parameters such as sex determination, maturation, molting or embryogenesis in order to evaluate the sensitivity and possible use of these endpoints in ecological risk assessment. MATERIALS AND METHODS: We have studied effects of four model EDCs (vinclozolin, flutamide, ketoconazole and dicofol) on D. magna using (i) an acute 48 h immobilization assay, (ii) a sub-chronic, 4-6 day assay evaluating development and the sex ratio of neonates, and (iii) a chronic, 21 day assay studying number of neonates, sex of neonates, molting frequency, day of maturation and the growth of maternal organisms. RESULTS: Acute EC50 values in the 48 h immobilization test were as follows (mg/L): dicofol 0.2, ketoconazole 1.5, flutamide 2.7, vinclozolin >3. Short-term, 4-6 day assays with sublethal concentrations showed that the sex ratio in Daphnia was modulated by vinclozolin (decreased number of neonate males at 1 mg/L) and dicofol (increase in males at 0.1 mg/L). Flutamide (up to 1 mg/L) had no effect on the sex of neonates, but inhibited embryonic development at certain stages during chronic assay, resulting in abortions. Ketoconazole had no significant effects on the studied processes up to 1 mg/L. DISCUSSION: Sex ratio modulations by some chemicals (vinclozolin and dicofol) corresponded to the known action of these compounds in vertebrates (i.e. anti-androgenicity and anti-oestrogenicity, respectively). Our study revealed that some chemicals known to affect steroid-regulated processes in vertebrates can also affect sublethal endpoints (e.g. embryonic sex determination and/or reproduction) in invertebrates such as D. magna. CONCLUSIONS: A series of model vertebrate endocrine disrupters affected various sub-chronic and chronic parameters in D. magna including several endpoints that have not been previously studied in detail (such as sex determination in neonates, embryogenesis, molting and maturation). Evaluations of traditional reproduction parameters (obtained from the 21 day chronic assay). as well as the results from a rapid, 4-6 day, sub-chronic assay provide complementary information on non-lethal effects of suspected organic endocrine disrupters. RECOMMENDATIONS AND PERSPECTIVES: It seems that there are analogies between vertebrates and invertebrates in toxicity mechanisms and in vivo effects of endocrine disruptors. However, general physiological status of organisms may also indirectly affect endpoints that are traditionally considered 'hormone regulated' (especially at higher effective concentrations as observed in this study) and these factors should be carefully considered. Further research of D. magna physiology and comparative studies with various EDCs will help to understand mechanisms of action as well as ecological risks of EDCs in the environment.     

Specific issues in health risk assessment of endocrine disrupting chemicals and international activities.

Specific issues in health risk assessment of EDCs and correlated international activities thereof are discussed. Risk characterization is a synthesis of all information including hazard assessment, dose-response relationship, and exposure information to identify clearly the strengths and weaknesses of the database, the criteria applied to evaluation and validation of all aspects of methodology, and the conclusions reached from the review of scientific information. In the case of EDCs, new models need to be developed taking into account possible new kinds of information, e.g., effects of EDCs on gene activation in response to hormonal challenge or effects on receptor expression. Such models should also account for homeostatic adaptive responses and consider the possibility of having windows of exposure for given effects. Work to compile and harmonize the definitions and terms appropriate to endocrine disruption will be conducted within the joint IPCS/OECD project on harmonization of risk/hazard assessment terminology reviews. The ICPS is the process of preparing a "State of the Science" report, and is implementing a global inventory of ongoing research on ECDs.     

Implications of research on endocrine disruption for the environmental risk assessment, regulation and monitoring of chemicals in the European Union

We assess the implications which research on endocrine disrupting chemicals (EDCs) has for the regulation of synthetic substances and for the protection of the environment, particularly under the forthcoming European Union (EU) REACH legislation. EDCs present regulatory problems inter alia because they can act additively at concentrations which are individually harmless, and they may have non-classical dose (concentration)-response relationships at low exposure levels. Furthermore, current in vivo testing routines were not specifically designed to assess the endocrine disrupting properties of chemicals, whilst in silico and in vitro methods have only limited applicability and availability for this purpose. We need to ensure that the assessment approaches specified in the draft REACH legislation and Technical Guidance are able to evaluate EDCs efficiently. However, it must also be recognised that environmental monitoring procedures in Europe will need to be improved to detect EDCs that have evaded identification, and where appropriate, control, under REACH.     

Evaluation of the Daphnia magna reproduction test for detecting endocrine disruptors

The Daphnia 21 d reproduction test is considered as a comprehensive and decisive test in the OECD Conceptual Framework for testing and assessment of endocrine disrupting chemicals (EDCs). However, how to interpret results of the Daphnia 21 d reproduction test for identification, risk assessment and testing strategy of EDCs remains an unsolved issue. This study analysed a total number of 135 published studies encompassing 86 known EDCs and non-EDCs with different modes of action. Our results show that the majority of effects on apical endpoints (survival, molting, growth, time to reproductive maturity, brood size, the number of broods, and the total number of offspring) do not seem to be EDC-specific. In contrast, the endpoint sex ratio is likely specific to juvenile hormones and their mimics. Variability is quantified for three most reported endpoints survival, the total number of offspring and sex ratio. Quantification of the endpoint sensitivity shows that the sensitivity of the sex ratio is lower than that of the total number of offspring. The Daphnia 21 d reproduction test gives insufficient information to conclude if a substance is an EDC or not. EDCs that are potent in assays in vitro may not be potent in the Daphnia 21 d reproduction test. We conclude that the Daphnia 21 d reproduction test is important for deriving No Observed Effect Concentrations for risk assessment but may produce false negatives in identification of EDCs when used on its own. A targeted testing strategy for selection of species, tests, and endpoints is suggested for identifying EDCs.     

Biological assessment of bisphenol A degradation in water following direct photolysis and UV advanced oxidation

Endocrine disrupting compounds (EDCs) are exogenous environmental chemicals that can interfere with normal hormone function and present a potential threat to both environmental and human health. The fate, distribution and degradation of EDCs is a subject of considerable investigation. To date, several studies have demonstrated that conventional water treatment processes are ineffective for removal of most EDCs and in some instances produce multiple unknown transformation products. In this study we have investigated the use of direct photolysis with low-pressure (LP) Hg UV lamps and UV+hydrogen peroxide (H(2)O(2)) advanced oxidation process (AOP) for the degradation of a prototypic endocrine disrupter, bisphenol A (BPA), in laboratory water. Removal rates of BPA and formation of degradation products were determined by high performance liquid chromatography (HPLC) analysis. Changes in estrogenic activity were evaluated using both in vitro yeast estrogen screen (YES) and in vivo vitellogenin (VTG) assays with Japanese medaka fish (Oryzias latipes). Our results demonstrate that UV alone did not effectively degrade BPA. However, UV in combination with H(2)O(2) significantly removed BPA parent compound and aqueous estrogenic activity in vitro and in vivo. Removal rates of in vivo estrogenic activity were significantly lower than those observed in vitro, demonstrating differential sensitivities of these bioassays and that certain UV/AOP metabolites may retain estrogenic activity. Furthermore, the UV/H(2)O(2) AOP was effective for reducing larval lethality in treated BPA solutions, suggesting BPA degradation occurred and that the degradation process did not result in the production of acutely toxic intermediates.     

Endocrine disrupting chemicals accumulate in earthworms exposed to sewage effluent

Endocrine disrupting chemicals (EDCs) can alter endocrine function in exposed animals. Such critical effects, combined with the ubiquity of EDCs in sewage effluent and potentially in tapwater, have led to concerns that they could be major physiological disruptors for wildlife and more controversially for humans. Although sewage effluent is known to be a rich source of EDCs, there is as yet no evidence for EDC uptake by invertebrates that live within the sewage treatment system. Here, we describe the use of an extraction method and GC-MS for the first time to determine levels of EDCs (e.g., dibutylphthalate, dioctylphthalate, bisphenol-A and 17beta-estradiol) in tissue samples from earthworms (Eisenia fetida) living in sewage percolating filter beds and garden soil. To the best of our knowledge, this is the first such use of these techniques to determine EDCs in tissue samples in any organism. We found significantly higher concentrations of these chemicals in the animals from sewage percolating filter beds. Our data suggest that earthworms can be used as bioindicators for EDCs in these substrates and that the animals accumulate these compounds to levels well above those reported for waste water. The potential transfer into the terrestrial food chain and effects on wildlife are discussed.     

Complex mixtures that may contain mutagenic and/or genotoxic components: a need to assess in vivo target-site effect(s) associated with in vitro-positive(s)

A battery of short-term in vitro assays and/or in vivo protocols to evaluate single-agent mutagenicity and/or genotoxicity is available. However, a protocol to assess the effect(s) of complex mixtures in vivo following a positive test finding in vitro remains difficult. Complex interactions may occur in vivo because component pharmacokinetics increases the unpredictability of pharmacodynamic outcomes. The question arises as to whether in vitro mutagenic component(s) of a complex mixture, probably unidentified, reach target organ(s) in vivo at a sufficient concentration. To address the issue of an in vitro positive, standard in vivo chromosome damage assays to test both mixtures and fractions could be conducted but, to assess site-of-contact effects, the alkaline single cell-gel electrophoresis ("comet") assay or DNA reactivity (e.g., (32)P-postlabelling of DNA adducts) might be employed. A newer approach may be the derivation of a "biochemical-cell fingerprint" of potential target sites using infrared microspectroscopy. There is interest in platforms such as gene expression, proteomics, epigenomics or metabolomics as biomarkers of signature genotoxic or non-genotoxic mechanisms. One still needs to address whether a mutagenic and/or genotoxic component reaches a target organ. An approach to track levels of target-organ exposure may be to radio-label components with a short-lived positron-emitting radionuclide. The parent compound retains its physicochemical properties whilst allowing non-invasive in vivo tissue-specific imaging. However, determining target-organ concentration(s) and effect(s) in vivo remains a difficult challenge.     

Occurrence and treatment trials of endocrine disrupting chemicals (EDCs) in wastewaters

This study demonstrates that both synthetic and natural endocrine disrupting chemicals (EDCs) (e.g., bisphenol A, estrone and 17beta-estradiol) were found in the crude wastewaters from two wastewater treatment works (WwTWs). Conventional biological processes can lower EDCs concentrations to several tens to hundreds ngl(-1). Since natural EDCs (e.g., estrone and 17beta-estradiol) have biological activity and adverse impact on the environment at extremely low concentrations (several tens of ngl(-1)), further treatment after conventional biological processes is required. Preliminary trials with ferrate(VI) and electrochemical oxidation process demonstrated that both processes can effectively reduce EDCs to very low levels, ranging between 10 and 100ngl(-1), but the former is more effective than the latter to reduce COD concentration in wastewater for given studying conditions.     

The current practice of health risk assessment: potential impact on standards for toxic air contaminants

Since the Bhopal incident, the public has placed pressure on regulatory agencies to set community exposure limits for the dozens of chemicals that may be released by manufacturing facilities. More or less objective limits can be established for the vast majority of these chemicals through the use of risk assessment. However, each step of the risk assessment process (i.e., hazard identification, dose-response assessment, exposure assessment, and risk characterization) contains a number of pitfalls that scientists need to avoid to ensure that valid limits are established. For example, in the hazard identification step there has been little discrimination among animal carcinogens with respect to mechanism of action or the epidemiology experience. In the dose-response portion, rarely is the range of "plausible" estimated risks presented. Physiologically based pharmacokinetic (PB-PK) models should be used to understand the difference between the tissue doses and the administered dose, as well as the difference in target tissue concentrations of the toxicant between rodents and humans. Biologically-based models like the Moolgavkar-Knudson-Venzon (MKV) should be developed and used, when appropriate. The exposure assessment step can be significantly improved by using more sensitive and specific sampling and analytical methods, more accurate exposure parameters, and computer models that can account for complex environmental factors. Whenever possible, model predictions of exposure and uptake should be validated by biological monitoring of exposed persons (urine, blood, adipose) or by field measurements of plants, soil, fish, air, or water. In each portion of an assessment, the weight of evidence approach should be used to identify the most defensible value. In the risk characterization, the best estimate of the potential risk as well as the highest plausible risk should be presented. Future assessments would be much improved if quantitative uncertainty analyses were conducted. Procedures are currently available for making future assessments. By correcting some of these shortcomings in how health risk assessments have been conducted, scientists and risk managers should be better able to identify scientifically appropriate ambient air standards and emission limits.     

Rapid resolution liquid chromatography-tandem mass spectrometry method for the determination of endocrine disrupting chemicals (EDCs), pharmaceuticals and personal care products (PPCPs) in wastewater irrigated soils

A multiresidue analytical method was developed for the determination of 9 endocrine disrupting chemicals (EDCs) and 19 pharmaceuticals and personal care products (PPCPs) including acidic and neutral pharmaceuticals in water and soil samples using rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS). Solid phase extraction (SPE), and ultrasonic extraction combined with silica gel purification were applied as pretreatment methods for water and soil samples, respectively. The extracts of the EDCs and PPCPs in water and soil samples were then analyzed by RRLC-MS/MS in electrospray ionization (ESI) mode in three independent runs. The chromatographic mobile phases consisted of Milli-Q water and acetonitrile for EDCs and neutral pharmaceuticals, and Milli-Q water containing 0.01 % acetic acid (v/v) and acetonitrile: methanol (1:1, v/v) for acidic pharmaceuticals at a flow rate of 0.3 mL/min. Most of the target compounds exhibited signal suppression due to matrix effects. Measures taken to reduce matrix effects included use of isotope-labeled internal standards, and application of matrix-match calibration curves in the RRLC-MS/MS analyses. The limits of quantitation ranged between 0.15 and 14.08 ng/L for water samples and between 0.06 and 10.64 ng/g for solid samples. The recoveries for the target analytes ranged from 62 to 208 % in water samples and 43 to 177 % in solid samples, with majority of the target compounds having recoveries ranging between 70–120 %. Precision, expressed as the relative standard deviation (RSD), was obtained less than 7.6 and 20.5 % for repeatability and reproducibility, respectively. The established method was successfully applied to the water and soil samples from four irrigated plots in Guangzhou. Six compounds namely bisphenol-A, 4-nonylphenol, triclosan, triclocarban, salicylic acid and clofibric acid were detected in the soils.     

中国地表饮用水水源地有机类内分泌干扰物污染现况分析

有机类内分泌干扰物(EDCs)是一类对人类和生物内分泌系统产生干扰,并可造成其紊乱的特殊外来物质,对人类和生物健康有极大的危害.以与人类生活密切相关的地表饮用水水源地中的有机类EDCs为研究重点,对中国目前地表饮用水水源地EDCs污染状况、污染物种类及其来源及可能的污染途径进行了评述.目前,中国各地区地表饮用水水源地均有有机类EDCs检出.其中以有机氯农药六六六及其异构体、DDT及其代谢产物和多氯联苯检出率最高,且某些地区检测浓度相当高,主要来源是农药使用和污水排放.国内外对地表饮用水水源地EDCs突发污染事件的研究均较少,应引起高度重视.     

Environmental and biological monitoring of endocrine disrupting chemicals.

Trends toward an increase of adverse health effects on reproductive organs have been reviewed. An urgent need has been recognised to establish validated in vivo and in vitro screening assays to test for hormonal activities of chemicals. Relevant existing OECD guidelines have been reviewed and enhancements of some of these have been identified, mainly to test for estrogenic and androgenic activity of chemicals. The problems connected to monitoring activities are outlined, particularly for ambient and biological monitoring. Indeed, the problem of assessing human exposure to endocrine disrupting chemicals through environmental chemical analysis tends to a very high level of complexity. This has been illustrated through the example of one single subclass of endocrine disrupting compounds (EDCs), the organohalogen compounds. Valid biological markers are also needed to be effectively used in epidemiological studies and risk assessment. A multidisciplinary approach and the collaboration among experts in the field of clinical biochemistry, toxicology, and epidemiology is required.     

Removal of estrone, 17α-ethinylestradiol,and 17ß-estradiol in algae and duckweed-based wastewater treatment systems

Background, aim, and scope  

Many pollutants have received significant attention due to their potential estrogenic effect and are classified as endocrine disrupting compounds (EDCs). Because of possible ecological effects and increased attention for water reuse schemes, it is important to increase our understanding of the EDC removal capacities of various wastewater treatment systems. However, there has so far been little research on the fate and behavior of EDCs in stabilization pond systems for wastewater treatment, which represent an important class of wastewater treatment systems in developing countries because of their cost-effectiveness. The aim of this work is to study the fate and behavior of EDCs in algae and duckweed ponds. Because the synthetic hormone 17α-ethinylestradiol (EE2) and the natural hormones estrone (E1), as well as 17β-estradiol (E2), have been detected in effluents of sewage treatment plants and been suggested as the major compounds responsible for endocrine disruption in domestic sewage; E1, E2, and EE2 were therefore chosen as target chemicals in this current work.     

Research to develop a predicting system of mammalian subacute toxicity (3) construction of a predictive toxicokinetics model

A new predictive toxicokinetics model was developed to estimate subacute toxicity (target organs, severity, etc.) of non-congeneric industrial chemicals, where the chemical structures and physico-chemical properties are only available. Thus, a physiological pharmacokinetics model, which consists of blood, liver, kidney (these were experimentally found as major toxicological targets), muscle and fat compartments , was established to simulate the chemical concentrations in organs/tissues with pharmacokinetic parameters by means of Runge-Kutta-Gill algorithm. The pliarmacokinetic parameters, i.e. absorption rate, absorption ratio, hepatic extraction ratio of metabolism and renal clearance were calculated by using separately established Quantitative Structure-Pharmacokinetics Relationship equations. The developed predictive model was then applied to simulations of 43 non-congeneric industrial chemicals. The chemical concentrations in organs/tissues after single oral administration were simulated, and their maximum concentrations (Cmax's) and area tinder the concentration-time curves (AUC's) were calculated.Fast Inverse Laplace Transform was newly applied for the purpose of simulation of 28-day repeated dose toxicity.Simulated concentrations of 28 days repeated dose were, however, found to be the same as those of simple repetitions of a single administration per day because of the short half-lives of non-congeneric industrial chemicals.A comparison of subacute toxicity data with Cmax's and AUC's in a single dose scenario suggested that the organs/tissues with relatively high concentrations of tested chemical substances were the most sensitive targets within a chemical.Chemical concentrations in liver, for instance, were correlated with the severity of hepatotoxicity among the chemicals. It was also suggested that to improve and widen the present approach, data of metabolite and reactivity of non-congeneric industrial chemicals to organs/tissues, receptors, etc. should be incorporated into the model.     

Evaluation of the fish short term reproduction assay for detecting endocrine disrupters

In a fish testing strategy, positive results of the fish short term reproduction assay (FSTR), often trigger a definitive test like the fish sexual development test (FSDT) or the fish full life cycle test (FFLC), entailing ethical and economic problems. This study analysed 137 studies encompassing 35 chemicals with different modes of actions (MOAs). Variability is quantified for MOA endpoints vitellogenin (VTG) and secondary sex characteristics (SSCs) as well as for apical endpoints. Two MOA endpoints could indicate estrogenic, anti-estrogenic, androgenic, anti-androgenic and steroidogenesis activities. Great variability, however, has been observed for chemicals with anti-androgenic and steroidogenesis activities, suggesting that TG229/230 may not be sensitive enough to detect these types of chemicals and may produce false negatives. Changes in apical endpoints like fecundity are not limited to endocrine disrupting chemicals (EDCs). Non-EDCs could induce the similar effects on these apical endpoints. If elucidating MOA is needed, targeted in vitro MOA tests are suggested. Positive in vitro MOA results trigger a definitive test, which could be used for confirmation of the MOA in vivo and for deriving a no observed effect concentration (NOEC). Based on positive MOA results of TG229, a definitive test such as the FSDT or the FFLC is still needed, because the current TG229 has limitation on the derivation of a NOEC. An extended TG229 with more power to detect reproduction effects, as recently proposed in the OECD test guideline program, would improve the possibility to derive a NOEC and increase its usefulness in risk assessment.     

Estrogenic and dioxin-like potency in each step of a controlled landfill leachate treatment plant in Japan

The estrogenic activity (by E-screen bioassay), the concentrations of PCBs, PCDDs/PCDFs (and their resulting toxicity equivalents, TEQ) and several endocrine disrupting chemicals (EDCs: e.g., bisphenol A, nonylphenol, Butyl benzylpthalate (BBP), di-n-butylphthalate (DBP), 17alpha-ethynyl-estradiol or 4-octylphenol) have been analyzed from leachates of each step (before treatment, after biodegradation/sedimentation and after charcoal treatment) of a controlled landfill leachate treatment plant. The comparison of the effluent of the examined landfill leachate treatment plant with water from a nearby river in this study indicated no additional dioxin-like (e.g., TEQ: 0.027 compared to 1.01 pg TEQ/l; PCBs: 1.2 compared to 3.9 ng/l) or estrogenic impact (2.8 compared to 3.5 ng estradiol equivalents EE/l; analyzed by E-screen bioassay) from the leachate treatment plant into the surrounding water environment. The impact of dioxin-like compounds from uncleaned leachates into the final cleaned effluents could be sufficiently reduced by the leachate treatment plant for PCDDs (75%), PCDFs (62%), dioxin-like PCBs (97%), and the sum of TEQ (78%). The leachate treatment plant also achieved a reduction of the estrogenic activity as determined by E-screen (from 4.8 to 2.8 ng EE/l = 42%), by GC/MS for bisphenol A (>96% and nonylphenol (>98%) or by ELISA for estradiol (>80%). Additionally, for the validation of the E-screen, five known endocrine disrupting chemicals (EDCs: bisphenol A, BBP, DBP, 17 alpha-ethynyl-estradiol, 4-octylphenol) were analyzed. The EC50 values and estradiol equivalents factors (EEFs) for the five EDCs determined in this study were comparable to previously published data. The combined biological and chemical trace analysis data have provided valuable information on the relative contribution of natural, synthetic, and non-steroidal anthropogenic chemicals to the estrogenic and dioxin-like activity in leachates from a wastewater treatment plant, and water from a nearby river.     

Distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals in wild fish species from Dianchi Lake, China

The distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals (EDCs) were studied in various tissues of wild fish species from Dianchi Lake, China. In muscle tissue, 4-tert-octylphenol, 4-cumylphenol, 4-nonlyphenol and bisphenol A were detected in fish from each sampling site, with maximal concentrations of 4.6, 4.4, 18.9 and 83.5 ng/g dry weight (dw), respectively. Steroids (estrone, 17β-estradiol 17α-ethynylestradiol and estriol) were found at lower levels (<11.3 ng/g dw) and less frequently in muscle samples. The highest concentrations of steroids and phenols were found in liver, followed by those in gill and the lowest concentration was found in muscle. The field bioconcentration factors (BCFs) of phenols were calculated in fish species ranged from 18 to 97. Moreover, the measured tissue concentrations were utilized in order to estimate water concentration of steroids (4.4-18.0 ng/L). These results showed that steroidal and phenolic EDCs were likely ubiquitous contaminants in wild fish.     

Potential Impact on Standards for Toxic Air Contaminants

Since the Bhopal incident, the public has placed pressure on regulatory agencies to set community exposure limits for the dozens of chemicals that may be released by manufacturing facilities. More or less objective limits can be established for the vast majority of these chemicals through the use of risk assessment. However, each step of the risk assessment process (i.e., hazard identification, dose-response assessment, exposure assessment, and risk characterization) contains a number of pitfalls that scientists need to avoid to ensure that valid limits are established. For example, in the hazard identification step there has been little discrimination among animal carcinogens with respect to mechanism of action or the epidemiology experience. In the dose-response portion, rarely is the range of “plausible” estimated risks presented. Physiologically based pharmacokinetic (PB-PK) models should be used to understand the difference between the tissue doses and the administered dose, as well as the difference in target tissue concentrations of the toxicant between rodents and humans. Biologically-based models like the Moolgavkar-Knudson-Venzon (MKV) should be developed and used, when appropriate. The exposure assessment step can be significantly improved by using more sensitive and specific sampling and analytical methods, more accurate exposure parameters, and computer models that can account for complex environmental factors. Whenever possible, model predictions of exposure and uptake should be validated by biological monitoring of exposed persons (urine, blood, adipose) or by field measurements of plants, soil, fish, air, or water. In each portion of an assessment, the weight of evidence approach should be used to identify the most defensible value. In the risk characterization, the best estimate of the potential risk as well as the highest plausible risk should be presented. Future assessments would be much improved if quantitative uncertainty analyses were conducted. Procedures are currently available for making future assessments. By correcting some of these shortcomings in how health risk assessments have been conducted, scientists and risk managers should be better able to identify scientifically appropriate ambient air standards and emission limits.     

A new challenge—development of test systems for the infochemical effect

Background, aim, and scope

Many—if not all—organisms depend on so-called infochemicals, chemical substances in their surroundings which inform the receivers about their biotic and abiotic environment and which allow them to react adequately to these signals. Anthropogenic substances can interfere with this complex chemical communication system. This finding is called infochemical effect. So far, it is not known to what extent anthropogenic discharges act as infochemicals and influence life and reproduction of organisms in the environment because adequate testing methods to identify chemicals which show the infochemical effect and to quantify their effects have not been developed yet. The purpose of this article is to help and find suitable test designs.

Main features

Test systems used in basic research to elucidate the olfactory cascade and the communication of environmental organisms by infochemicals are plentiful. Some of them might be the basis for a quantified ecotoxicological analysis of the infochemical effect. In principle, test systems for the infochemical effect could be developed at each step of the chemosensory signal transduction and processing cascade.

Results

Experimental set-ups were compiled systematically under the aspect whether they might be usable for testing the infochemical effect of single chemicals in standardized quantifying laboratory experiments. For an appropriate ecotoxicological assessment of the infochemical effect, experimental studies of many disciplines, such as molecular biology, neurobiology, physiology, chemical ecology, and population dynamics, should be evaluated in detail before a decision can be made which test system, respectively which test battery, might be suited best. The test systems presented here are based on the knowledge of the genetic sequences for olfactory receptors, binding studies of odorants, signal transmission, and reactions of the receivers on the level of the organisms or the populations. The following basic approaches are conceivable to identify the role of an infochemical: binding studies to the odorant-binding protein or to the odorant receptor binding protein (e.g., by in situ hybridization and immunohistochemical studies), measurement of electrical signals of the receptor cells in the tissue (e.g., electroolfactograms, electroantennograms), registration of phenotypic changes (e.g., observation under the microscope), behavioral tests (e.g., in situ online biomonitoring, use of T-shaped olfactometers, tests of avoidance responses), measurement of population changes (e.g., cell density or turbidity measurements), and multispecies tests with observation of community structure and community function. The main focus of this study is on aquatic organisms.

Discussion

It is evident that the infochemical effect is a very complex sublethal endpoint, and it needs further studies with standardized quantitative methods to elucidate whether and to what extent the ecosystem is affected. The collection of approaches presented here is far from being complete but should serve as a point of depart for further experimental research.

Conclusions

This article is the first to compare various approaches for testing the infochemical effect. The development of a suitable test system will not be easy as there are a multitude of relevant chemicals, a multitude of relevant receptors, and a multitude of relevant reactions, and it must be expected that the effective concentrations are very low. The chemical communication is of utmost importance for the ecosystem and justifies great endeavors to find solutions to these technical problems.

Recommendations and perspectives

The infochemical effect is a new chapter in ecotoxicology. Will a new endpoint, the so-called infochemical effect, be required in addition to the actual standard test battery of Annex 5 to Commission Directive 92/69/EEC (EC 1992)? Finding the answer to this question is a big challenge that could be met by a comprehensive research project.