A fluorescence-based bioassay for aquatic macrophytes and its suitability for effect analysis of non-photosystem II inhibitors

Background, Goals and Scope During the last years the miniaturization of toxicity test systems for rapid and parallel measurements of large quantities of samples has often been discussed. For unicellular algae as well as for aquatic macrophytes, fluorescence-based miniaturized test systems have been introduced to analyze photosystem II (PSII) inhibitors. Nevertheless, high-throughput screening should also guarantee the effect detection of a broad range of toxicants in order to ensure routinely applicable, high-throughput measuring device experiments which can cover a broad range of toxicants and modes of action others than PSII inhibition. Thus, the aim of this study was to establish a fast and reproducible measuring system for non-PSII inhibitors for aquatic macrophyte species to overcome major limitations for use. Methods A newly developed imaging pulse-amplitude-modulated chlorophyll fluorometer (I-PAM) was applied as an effect detector in short-term bioassays with the aquatic macrophyte species Lemna minor. This multiwell-plate based measuring device enabled the incubation and measurement of up to 24 samples in parallel. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, polycyclic aromatic hydrocarbons (PAHs) and pharmaceuticals and personal care products (PPCPs), which are often detected in the aquatic environment. The I-PAM was used (i) to establish and validate the sensitivity of the test system to the three non-PSII inhibitors, (ii) to compare the test systems with standardized and established biotests for aquatic macrophytes, and (iii) to define necessary time scales in aquatic macrophyte testing. For validation of the fluorescence-based assay, the standard growth test with L. minor (ISO/DIS 20079) was performed in parallel for each chemical. Results The results revealed that fluorescence-based measurements with the I-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples. The I-PAM enabled the recording of concentration-effect-curves with L. minor samples on a 24-well plate with single measurements. Fluorescence-based concentration-effect-curves could be detected for all three chemicals after only 1 h of incubation. After 4–5 h incubation time, the maximum inhibition of fluorescence showed an 80–100% effect for the chemicals tested. The EC50 after 24 h incubation were estimated to be 0.06 mg/L, 0.84 mg/L and 1.69 mg/L for paraquatdichloride, alizarine and triclosan, respectively. Discussion The results obtained with the I-PAM after 24 h for the herbicide paraquat-dichloride and the polycyclic aromatic hydrocarbon alizarine were in good accordance with median effective concentrations (EC50s) obtained by the standardized growth test for L. minor after 7 d incubation (0.09 mg/L and 0.79 mg/L for paraquat-dichloride and alizarine, respectively). Those results were in accordance with literature findings for the two chemicals. In contrast, fluorescence-based EC50 of the antimicrobial agent triclosan proved to be two orders of magnitude greater when compared to the standard growth test with 7 d incubation time (0.026 mg/L) as well as with literature findings. Conclusion Typically, aquatic macrophyte testing is very time consuming and relies on laborious experimental set-ups. The I-PAM measuring device enabled fast effect screening for the three chemicals tested. While established test systems for aquatic macrophytes need incubation times of ≥ 7 d, the I-PAM can detect inhibitory effects much earlier (24 h), even if inhibition of chemicals is not specifically associated with PSII. Thus, the fluorescence-based bioassay with the I-PAM offers a promising approach for the miniaturization and high-throughput testing of chemicals with aquatic macrophytes. For the chemical triclosan, however, the short-term effect prediction with the I-PAM has been shown to be less sensitive than with long-term bioassays, which might be due to physicochemical substance properties such as lipophilicity. Recommendations and Perspectives The results of this study show that the I-PAM represents a promising tool for decreasing the incubation times of aquatic macrophyte toxicity testing to about 24 h as a supplement to existing test batteries. The applicability of this I-PAM bioassay on emergent and submerged aquatic macrophyte species should be investigated in further studies. Regarding considerations that physicochemical properties of the tested substances might play an important role in microplate bioassays, the I-PAM bioassay should either be accompanied by evaluating physicochemical properties modeled from structural information prior to an experimental investigation, or by intensified chemical analyses to identify and determine nominal concentrations of the toxicants tested. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, PAHs and PPCPs which are often detected in the aquatic environment. Nevertheless, in order to ensure a routinely applicable measuring device, experiments with a broader range of toxicants and samples of surface and/or waste waters are necessary. ESS-Submission Editor: Dr. Markus Hecker (MHecker@Entrix.com)     

Development and validation of a new fluorescence-based bioassay for aquatic macrophyte species

Bioassays with unicellular algae are frequently used as ecotoxicological test systems to evaluate the toxicity of contaminated environmental samples or chemicals. In contrast, aquatic macrophyte test systems are still rarely used as they are laborious to handle because species exhibit distinct ecological requirements. The aim of this study was to establish a fast and reproducible measuring system for aquatic macrophyte species to overcome those limitations for use. Thus, a newly developed pulse-amplitude modulated chlorophyll fluorometer (Imaging-PAM) was applied as an effect detection in short-term bioassays with aquatic macrophyte species. This multiwell-plate-based measuring device enables the incubation and measurement of up to 24 samples in parallel. The Imaging-PAM was used (i) to establish and validate the sensitivity of the test systems to three Photosystem II (PSII) inhibitors (atrazine, prometryn, isoproturon), (ii) to compare the test systems with established biotests for macrophytes and (iii) to define necessary time scales in aquatic macrophyte testing. The results showed that fluorescence-based measurements with the Imaging-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples and of toxicants effects of the PSII inhibitors tested on aquatic macrophytes. Measurements revealed a good correlation between obtained median effective concentrations (EC50s) for the new and the established biotest systems. Hence, the Imaging-PAM measuring device is a promising tool to allow fast chemical effect screening for high amounts of samples with little time and material and thus offers scope for high-throughput biotesting using aquatic macrophyte species.     

Monitoring heavy metal pollution by aquatic plants

Introduction

The copper bioaccumulation by the floating Lemna minor and by the completely submerged Ranunculus tricophyllus as a function of exposure time and copper concentration was studied, with the aim of proposing these species as environmental biosensors of the water pollution.

Results

The results show that both these aquatic angiosperms are good indicators of copper pollution because the copper uptake is the only function of metal concentration (water pollution).

Conclusion

Uptake behavior is reported as a function of the time and concentration, based on the results of a 3-year study. Kinetic evaluations are proposed.     

The impact of pesticides toward parrotfeather when applied at the predicted environmental concentration

Pesticides pose a serious risk for aquatic macrophytes in the environment. They are also detrimental to the rooted macrophytes used in bioassays for assessment. Currently, no data is available for impact of pesticides toward parrotfeather when present at the predicted environmental concentration. The calculated expected environmental concentration was applied to the plants and the effect was compared. Eight of the 18 pesticides showed significantly different impact. All of the other tested pesticides induced a significant change in pigment content of parrotfeather. The RQ values for risk quotient had a value higher than 0.5, so need regulatory action for environment. This study may be the first to evaluate the predicted environmental concentrations reported by pesticide registration in Europe. Additional studies are required to test all pesticides within one group since the compounds tested may depict a wide toxicity level. Furthermore, the tests should include more than one macrophyte, e.g. one rooted and one non-rooted species, in order to provide a better understanding on pesticide toxicity.     

An overall risk probability-based method for quantification of synergistic and antagonistic effects in health risk assessment for mixtures: theoretical concepts

Purpose

In the assessment of health risks of environmental pollutants, the method of dose addition and the method of independent action are used to assess mixture effects when no synergistic and/or antagonistic effects are present. Currently, no method exists to quantify synergistic and/or antagonistic effects for mixtures. The purpose of this paper is to develop the theoretical concepts of an overall risk probability (ORP)-based method to quantify the synergistic and antagonistic effects in health risk assessment for mixtures.

Method

The ORP for health effects of environmental chemicals was determined from the cumulative probabilities of exposure and effects. This method was used to calculate the ORP for independent mixtures and for mixtures with synergistic and antagonistic effects.

Results

For the independent mixtures, a mixture ORP can be calculated from the product of the ORPs of individual components. For systems of interacting mixtures, a synergistic coefficient and an antagonistic coefficient were defined respectively to quantify the ORPs of each individual component in the mixture. The component ORPs with synergistic and/or antagonistic effects were then used to calculate the total ORP for the mixture.

Conclusions

An ORP-based method was developed to quantify synergistic and antagonistic effects in health risk assessment for mixtures. This represents a first method to generally quantify mixture effects of interacting toxicants.     

Bioaccumulation and trophic transfer of mercury in a food web from a large, shallow, hypereutrophic lake (Lake Taihu) in China

Purpose

Due to the fast development of industry and the overuse of agrichemicals in past decades, Lake Taihu, an important source of aquatic products for Eastern China, has simultaneously suffered mercury (Hg) contamination and eutrophication. The objectives of this study are to understand Hg transfer in the food web in this eutrophic, shallow lake and to evaluate the exposure risk of Hg through fish consumption.

Methods

Biota samples including macrophytes, sestons, benthic animals, and fish were collected from Lake Taihu in the fall of 2009. The total mercury (THg), methyl mercury (MeHg), ??¹³C and ??¹⁵N in the samples were measured.

Results and discussion

The signature for ??¹⁵N increased with the trophic levels. Along with a diet composed of fish, the significant relationship between the ??¹³C and ??¹⁵N indicated that a pelagic foraging habitat is the dominant pathway for energy transfer in Lake Taihu. The concentrations of THg and MeHg in the organisms varied dramatically by ??3 orders of magnitude from primary producers (macrophytes and sestons) to piscivorous fish. The highest concentrations of both THg (100 ng g^?1) and MeHg (66 ng g^?1), however, were lower than the guideline of 200 ng g^?1 of MeHg for vulnerable populations that is recommended by the World Health Organization (WHO). The daily intake of THg and MeHg of 92 and 56 ng day^?1 kg^?1 body weight, respectively, was generally lower than the tolerable intake of 230 ng day^?1 kg^?1 body weight for children recommended by the Joint FAO/WHO Expert Committee on Food Additives. Significant relationships between the ??¹⁵N and the logarithm of THg and MeHg showed an obvious biomagnification of Hg along the food web. The logarithmic bioaccumulation factor of MeHg in the fish (up to 5.7) from Lake Taihu, however, was relatively low compared to that of other aquatic ecosystems.

Conclusion

Health risk of exposure to Hg by consumption of fish for local residents is relatively low in the Lake Taihu area. Dilution of Hg levels in the phytoplankton induced by eutrophication is a possible factor inhibiting accumulation of MeHg in fish in eutrophic Lake Taihu.     

Relating cadmium concentrations in three macrophyte-associated freshwater invertebrates to those in macrophytes,water and sediments

We found that Cd concentrations in three species of macrophyte-associated invertebrates (the gastropods Bithynia tentaculata and Physa gyrina, and the amphipod Gammarus fasciatus) collected at twenty sites along the St. Lawrence River were correlated with Cd concentrations in their main food source, i.e. macrophytes and associated periphyton. Cd in these invertebrates was not significantly correlated with Cd concentrations in the sediments (even when corrections for iron oxide or organic carbon content were applied) or to calculated free-cadmium concentrations in the water. Cd levels in juveniles of B. tentaculata were very tightly linked to Cd concentrations in macrophytes, which is consistent with the close relationship that these organisms have developed with macrophytes. Linear models predicting Cd levels in juveniles of B. tentaculata from Cd levels in different macrophyte species were strong (R2 = 0.69-0.90). Analysis of covariance on these models showed no statistical difference of slope or intercept for any of the macrophyte species, except Vallisneria americana. We suggest that the macrophyte-periphyton complex is a key link in the transfer of cadmium to some aquatic invertebrates in the littoral zone of the St. Lawrence River and that macrophytes and their associated epiphytes should also be used as biomonitors.     

A tiered ecological risk assessment of three chlorophenols in Chinese surface waters

Introduction

The ecological risks posed by three chlorophenols (CPs), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP) in Chinese surface waters were assessed.

Materials and methods

This was achieved by applying a tiered ecological risk assessment (ERA) approach ranging from deterministic methods to probabilistic options to measured concentrations of CPs in surface water of seven major watersheds and three drainage regions in China and the chronic toxicity data for indigenous Chinese species.

Results and discussion

The results show that the risks of three chlorophenols are ranked PCP>2,4-DCP??2,4,6-TCP. PCP posed little ecological risk while 2,4-DCP and 2,4,6-TCP posed negligible or de minimis risk in Chinese surface water. However, the risks varied with different river basins, for example, PCP posed some ecological risk in the Yangtze, Huaihe, and Pearl Rivers. The magnitude of 2,4-DCP and 2,4,6-TCP pollution in North China was more serious than that in South China.

Conclusion

The probabilistic risk assessment approach, which can provide more information for risk managers and decision makers, was favored over the screening-level single-value estimate method. However, the results from all tiers of the ERA methods in the framework were consistent with each other.     

Arsenic accumulation and speciation in the submerged macrophyte Ceratophyllum demersum L.

Introduction

Ceratophyllum demersum L. is a widespread submerged macrophyte in aquatic environments.

Methods

Simulation experiments were conducted in the laboratory to investigate arsenic (As) accumulation, speciation, and efflux of C. demersum exposed to arsenate and arsenite solutions.

Results

Plant shoots showed a significant accumulation of As with a maximum of 862 and 963???g?As?g^?1 dry weight after 4?days of exposure to 10???M arsenate and arsenite, respectively. Regardless of whether arsenate or arsenite was supplied to the plants, arsenite was the predominant species in plant shoots. Furthermore, a dramatically higher influx rate of arsenate compared with arsenite was observed in C. demersum exposed to As solutions without the addition of phosphate (P). Arsenate uptake was considerably inhibited by P in this study, suggesting that arsenate is taken up by C. demersum via the phosphate transporters. However, arsenite uptake was unaffected by P and markedly reduced in the presence of glycerol and antimonite (Sb), indicating arsenite shares the aquaporin transport pathway. In addition, C. demersum rapidly reduces arsenate to arsenite in the shoot of the plant and extrudes most of them (>60?%) to the external solutions. The efflux of arsenite was much higher than that of arsenate; the former is supposed to be both active and passive processes, and the latter through passive leakage.

Conclusion

C. demersum is a strong As accumulator and an interesting model plant to study As uptake and metabolism due to the lack of a root-to-shoot translocation barrier.     

Natural Organic Matter Differently Modulates Growth of Two Closely Related Coccal Green Algal Species (6 pp)

Background

and Aim. Humic substances (HS) comprise the majority of dead and living organic carbon, including organisms. In the environment, they are considered to be chemically inert or at least refractory. Recent papers, however, show that HS (including natural organic matter – NOM, isolated by reverse osmosis) are natural chemicals which interact with aquatic organisms. They are taken up and cause a variety of stress defense reactions which are well known from man-made chemicals. These reactions include chaperon activation, induction and modulation of biotransformation enzymes, or induction of antioxidant defense enzymes. One specific reaction with freshwater plants is the reduction of photosynthetic oxygen release. In this contribution, we compare the susceptibilities (cell yield) of two closely related coccal green algae, Monoraphidium convolutum and M. minutum, towards various NOM isolates.

Methods

Cultures of M. convolutum and M. minutum were obtained from the algal collection of the Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, and from the Culture Collection of Algae, Göttingen, and maintained in a common medium. The cultures were non-axenic. The algae were exposed to 5 mg L-1 DOC of each humic material, an environmentally realistic concentration. Cell numbers were counted microscopically in Neugebauer cuvettes in 5 replicates on days 1, 4, 7, 10, 14, and 21.

Results and Discussion

Almost all NOM isolates modulated the growth of the algae. Only the NOM of a Norwegian raised peat bog lake did not reveal any significant effect with M. convolutum. In general, the results with two algal species are by no means uniform. For instance, Suwannee River NOM causes a decrease in cell density with M. minutum, but temporarily stimulates the growth of M. convolutum. The opposite applies to Aurevann NOM: Growth increase in M. minutum, but a bi-phasic response in M. convolutum. Different responses of both Monoraphidium species must be attributed to intrinsic factors of the algae rather than only to chemical features of the exposed materials, because the exposures were identical with both algal species. The reduction in growth yields can be explained as a herbicide-like mode of action that affects the photosystem II most prevalently. The growth promoting effect remains somewhat obscure. It may be due to (1) an increase in bioavailability of some trace nutrients in the presence of HS, (2) the release of some growth promoting substances by microbial or photochemical processing of the humic materials, and (3) a hormetic effect upon the exposure of HS. Hormesis means stimulation of organisms or metabolic activities when exposed to noxes in low concentrations. However, it is still open to discussion why the growth promotion only applies to one or the other, but not simultaneously to both Monoraphidium species.

Conclusion

and Perspectives. Exposure of the closely related coccal green algal species to humic material changes their growth characteristics. Since the reactions are not consistent within the two species and the various humic materials, it seems that the less sensitive species is favored by HS exposure. The environmental relevance, however, is subject to future studies.     

Aquatic ecotoxicity assessment of a new natural formicide

Background, aim and scope

Agrochemicals could reach aquatic ecosystems and damage ecosystem functionality. Natural formicide could be an alternative to use in comparison with the more toxic formicides available on the market. Thus, the objective of this study was to assess the ecotoxicity of the new natural formicide Macex? with a battery of classical aquatic ecotoxicity tests.

Material and methods

Bacteria (Aliivibrio fischeri), algae (Pseudokirchneriella subcapitata), hydra (Hydra attenuata), daphnids (Daphnia magna), and fish (Danio rerio) tests were performed in accordance with international standardized methodologies.

Results

In the range of formicide concentrations tested (0.03 to 2.0?g?L^?1) EC₅₀ values varied from 0.49 to >2.0?g?L^?1, with P. subcapitata being the most sensitive species and H. attenuata and D. rerio the most tolerant species to this product in aqueous solutions.

Conclusions

This new formicide preparation can be classed as a product of low toxicity compared to the aquatic ecotoxicity of the most common commercialized formicides.     

Hydroponic uptake of atrazine and lambda-cyhalothrin in Juncus effusus and Ludwigia peploides

Phytoremediation encompasses an array of plant-associated processes known to mitigate contaminants from soil, sediment, and water. Modification of pesticides associated with agricultural runoff includes processes directly associated with aquatic macrophytes in addition to changes in soil geochemistry and associated rhizospheric degradation. Remediation attributes of two vegetative species common to agricultural drainages in the Mississippi Delta, USA, were assessed using atrazine and lambda-cyhalothrin. Concentrations used in 8-d hydroponic exposures were calculated using recommended field applications and a 5% runoff model from a 0.65-cm rainfall event on a 2.02-ha field. While greater atrazine uptake was measured in Juncus effusus, greater lambda-cyhalothrin uptake occurred in Ludwigia peploides. Maximum pesticide uptake was reached within 48h for each exposure and subsequent translocation of pesticides to upper plant biomass occurred in macrophytes exposed to atrazine. Sequestration of 98.2% of lambda-cyhalothrin in roots of L. peploides was measured after 8d. Translocation of lambda-cyhalothrin in J. effusus resulted in 25.4% of pesticide uptake partitioned to upper plant biomass. These individual macrophyte remediation studies measured species- and pesticide-specific uptake rates, indicating that seasonality of pesticide applications and macrophyte emergence might interact strongly to enhance mitigation capabilities in edge-of-field conveyance structures.     

Effect of chlorsulfuron on growth of submerged aquatic macrophyte Potamogeton pectinatus (sago pondweed)

Research has been conducted on the effect of chlorsulfuron on non-target plants but little information is available on its effects on aquatic macrophytes. Potamogeton pectinatus (sago pondweed) is an ecologically important submerged aquatic macrophyte found in freshwater bodies. Many species of wildlife use this plant as a food source. The objective of this study was to measure the phytotoxic effects of chlorsulfuron on sago pondweed. P. pectinatus plants were exposed to chlorsulfuron at 0, 0.25, 0.50, 1.0, or 2.0 ppb, in an environmental growth chamber. Plants exposed to 0.25 ppb chlorsulfuron showed a reduction in length (76%), number of leaves (50%), and number of stems (50%), compared to control plants. A reduction (47%) was also observed in the length of stems produced by plants treated with > or = 0.50 ppb chlorsulfuron. Significant reductions in wet and dry weights, and increased mortality were observed on treatments with > or = 1.0 ppb chlorsulfuron.     

Acute and chronic toxicity of benzotriazoles to aquatic organisms

Purpose

Resulting from their intensive use as corrosion inhibitors in aircraft deicing and anti-icing fluids (ADAF) and for silver protection in dishwasher detergents benzotriazoles (BTs) are widespread in European surface waters. The current study aimed on an ecotoxicological characterization of 1H-benzotriazole (1H-BT) and 5-methyl-1H-benzotriazole (5MBT).

Methods

Acute and chronic OECD guideline tests were conducted with primary producers (Desmodesmus subspicatus, Lemna minor) and two daphnia species (Daphnia magna, Daphnia galeata) to characterize the hazard of these chemicals. Additionally, the estrogenic activity of both BTs was analyzed in vitro using a recombinant yeast estrogen screen (YES).

Results

Both BTs revealed significant effects in acute and chronic experiments, but exhibited no estrogenic activity in the YES. The algal growth test displayed an inhibited cell number increase with effect concentration (EC) values of EC₁₀ 1.18 and 2.86?mg?l^-1 for 1H-BT and 5MBT, respectively. In the Lemna test, EC₁₀ values were 3.94?mg?l^-1 (1H-BT) and 2.11?mg?l^-1 (5MBT). D. magna was also affected with EC₅₀ (48?h) values of 107?mg?l^-1 for 1H-BT and 51.6?mg?l^-1 for 5MBT. D. galeata was more sensitive with an EC₅₀ (48?h) of 14.7?mg 1H-BT l^-1 and 8.13?mg 5MBT l^-1. In the 21-day reproduction tests with D. magna, the EC₁₀ for 5MBT was 5.93?mg?l^-1 while 1H-BT showed no adverse effects. D. galeata turned out to be more sensitive in the chronic study with EC₁₀ values of 0.97?mg?l^-1 for 1H-BT and 0.40?mg?l^-1 for 5 MBT.

Conclusion

Because BTs are regularly found in the aquatic environment at lower ??g l^-1 concentrations reflecting their persistence and poor elimination during wastewater treatment processes, a preliminary risk assessment was conducted. There is little indication that BTs pose a risk for aquatic ecosystems at current exposure levels during most of the year. However, it cannot be excluded that in winter with a higher usage of ADAFs environmental concentrations may well exceed the level that is considered safe for aquatic organisms.     

Toxic masking and synergistic modulation of the estrogenic activity of chemical mixtures in a yeast estrogen screen (YES)

Background, aim and scope

Estrogenic and non-estrogenic chemicals typically co-occur in the environment. Interference by non-estrogenic chemicals may confound the assessment of the actual estrogenic activity of complex environmental samples. The aim of the present study was to investigate whether, in which way and how seriously the estrogenic activity of single estrogens and the observed and predicted joint action of estrogenic mixtures is influenced by toxic masking and synergistic modulation caused by non-estrogenic chemical confounders.

Materials and methods

The yeast estrogen screen (YES) was adapted so that toxicity and estrogenicity could be quantified simultaneously in one experimental run. Mercury, two organic solvents (dimethyl sulfoxide (DMSO) and 2,4-dinitroaniline), a surfactant (LAS-12) and the antibiotic cycloheximide were selected as toxic but non-estrogenic test chemicals. The confounding impact of selected concentrations of these toxicants on the estrogenic activity of the hormone 17ß-estradiol was determined by co-incubation experiments. In a second step, the impact of toxic masking and synergistic modulation on the predictability of the joint action of 17ß-estradiol, estrone and estriol mixtures by concentration addition was analysed.

Results

Each of the non-estrogenic chemicals reduced the apparent estrogenicity of both single estrogens and their mixtures if applied at high, toxic concentrations. Besides this common pattern, a highly substance- and concentration-dependent impact of the non-estrogenic toxicants was observable. The activity of 17ß-estradiol was still reduced in the presence of only low or non-toxic concentrations of 2,4-dinitroaniline and cycloheximide, which was not the case for mercury and DMSO. A clear synergistic modulation, i.e. an enhanced estrogenic activity, was induced by the presence of slightly toxic concentrations of LAS-12. The joint estrogenic activity of the mixture of estrogens was affected by toxic masking and synergistic modulation in direct proportion to the single estrogens, which allowed for an adequate adaptation of concentration addition and thus unaffected predictability of the joint estrogenicity in the presence of non-estrogenic confounders.

Discussion

The modified YES proved to be a reliable system for the simultaneous quantification of yeast toxicity and estrogen receptor activation. Experimental results substantiate the available evidence for toxic masking as a relevant phenomenon in estrogenicity assessment of complex environmental samples. Synergistic modulation of estrogenic activity by non-estrogenic confounders might be of lower importance. The concept of concentration addition is discussed as a valuable tool for estrogenicity assessment of complex mixtures, with deviations of the measured joint estrogenicity from predictions indicating the need for refined analyses.

Conclusions

Two major challenges are to be considered simultaneously for a reliable analysis of the estrogenic activity of complex mixtures: the identification of known and suspected estrogenic compounds in the sample as well as the substance- and effect-level-dependent confounding impact of non-estrogenic toxicants.

Recommendations and perspectives

The application of screening assays such as the YES to complex mixtures should be accompanied by measures that safeguard against false negative results which may be caused by non-estrogenic but toxic confounders. Simultaneous assessments of estrogenicity and toxicity are generally advisable.     

漂浮植物塘处理农村分散生活污水的应用

通过现场实验评价了漂浮植物塘配合化粪池处理农村分散生活污水的效果。实验结果表明,漂浮植物有效地抑制了污染水体中藻类的生长;在平均水力停留时间为36 d,COD、TN、TP平均污染负荷分别为3.1、0.86和0.056 g/(m2.d)的条件下,大薸塘对COD、TN、TP的平均去除率分别为68.5%、89.9%和85.2%,出水COD、TN和TP平均浓度分别为47、4.15和0.40 mg/L,达到GB18918-2002中的一级A标准。在水乡地区利用漂浮植物与农村宅河构造漂浮植物处理系统,是一种深度处理化粪池出水、控制农村生活污水对河道造成污染的有效措施。     

Contaminants of food

A scheme has been developed to rank 70 industrial organic chemicals in order of their priority for further study as potential contaminants of food. Numerical scales were developed for the following seven key criteria concerning environmental issues, food and toxicity:

-Production volume

-Pattern of usage

-Possible fate in the environment

-Likelihood of chemical entering the food chain

-Mechanism of entry into the food chain

-Persistence and accumulation in the food chain

-Toxicity.

Each chemical was assigned a score for the above criteria, which were combined to give an overall ranking for the chemicals. This scheme has been endorsed by the MAFF Steering Group on Chemical Aspects of Food Surveillance. It will be used in the assessment of relative priorities for further non-statutory surveillance for these contaminants in the UK food supply.     

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

Background, goals, and scope

In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17??-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.

Methods

A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and ??negative?? chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.

Results

With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.

Discussion and conclusions

With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).

Perspectives

Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.     

Perfluorooctane sulfonate increases β-oxidation of palmitic acid in chicken liver

Purpose

Perfluorooctane sulfonate (PFOS) belongs to a group of chemicals called perfluoroalkyl acids that have been extensively used in various applications such as stain and oil resistant treatments for fabrics, fire-fighting foams, and insecticides. These chemicals present an environmental and health risk being present in many samples both in wildlife and humans. In this study, we investigate the effect of PFOS on fatty acid ??-oxidation in developing chicken embryos.

Methods

Fertilized chicken eggs were exposed in ovo to PFOS at day?4 of incubation. On day?10, the eggs were dissected and livers were incubated in vitro with ³H-palmitic acid for 2?h. The media were collected, and after clean up, the amount of tritiated water was measured with liquid scintillation counting to determine the rate of palmitic acid ??-oxidation.

Results

PFOS was found to induce fatty acid ??-oxidation at doses starting from a lowest observed effect level (LOEL) of 0.1???g/g egg weight. Maximum induction of 77?% compared to control was seen at 0.3???g/g.

Conclusions

The administered doses in which effects are seen are around and even lower than the levels that can be found in wild populations of birds. General population human levels are a factor of two to three times lower than the LOEL value of this study. The environmental contamination of PFOS therefore presents a possibility of effects in wild populations of birds.