Method to assess component contribution to toxicity of complex mixtures: Assessment of puberty acquisition in rats exposed to disinfection byproducts

A method based on regression modeling was developed to discern the contribution of component chemicals to the toxicity of highly complex, environmentally realistic mixtures of disinfection byproducts(DBPs). Chemical disinfection of drinking water forms DBP mixtures.Because of concerns about possible reproductive and developmental toxicity, a whole mixture(WM) of DBPs produced by chlorination of a water concentrate was administered as drinking water to Sprague–Dawley(S–D) rats in a multigenerational study. Age of puberty acquisition,i.e., preputial separation(PPS) and vaginal opening(VO), was examined in male and female offspring, respectively. When compared to controls, a slight, but statistically significant delay in puberty acquisition was observed in females but not in males. WM-induced differences in the age at puberty acquisition were compared to those reported in S–D rats administered either a defined mixture(DM) of nine regulated DBPs or individual DBPs. Regression models were developed using individual animal data on age at PPS or VO from the DM study. Puberty acquisition data reported in the WM and individual DBP studies were then compared with the DM models. The delay in puberty acquisition observed in the WM-treated female rats could not be distinguished from delays predicted by the DM regression model, suggesting that the nine regulated DBPs in the DM might account for much of the delay observed in the WM. This method is applicable to mixtures of other types of chemicals and other endpoints.     

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.     

Phytotoxicity of atrazine, isoproturon, and diuron to submersed macrophytes in outdoor mesocosms

The submersed macrophytes Elodea canadensis, Myriophyllum spicatum and Potamogeton lucens were constantly exposed over a five-week period to environmentally relevant concentrations of atrazine, isoproturon, diuron, and their mixture in outdoor mesocosms. Effects were evaluated investigating photosynthetic efficiency (PE) of the three macrophytes and growth of M. spicatum and E. canadensis. Adverse effects on PE were observed on days 2 and 5 after application. M. spicatum was found to be the more sensitive macrophyte. E. canadensis and P. lucens were less sensitive to atrazine, diuron and the mixture and insensitive to isoproturon. PE of M. spicatum was similarly affected by the single herbicides and the mixture demonstrating concentration addition. Growth of E. canadensis and M. spicatum was not reduced indicating that herbicide exposure did not impair plant development. Although PE measurements turned out to be a sensitive method to monitor PSII herbicides, plant growth remains the more relevant ecological endpoint in risk assessment.     

The effect of certain intrinsic and extrinsic variables on the acute toxicity of selected organophosphorus insecticides to the mummichog,Fundulus heteroclitus

Abstract

The acute toxicity of two organophosphorus (OP) insecticides, azinphosmethyl and acephate, was evaluated in the mummichog, Fundulus heteroclitus. In addition, the effects of certain intrinsic (sex) and extrinsic (salinity and multiple toxicant interraction) variables on the toxic response were also investigated. Azinphosmethyl was by far the most toxic of the two OP insecticides with a 96h LC50 ～ 100,000 x lower than that for acephate. Slight sex differences were observed in the sensitivity of mummichogs to each of the OP insecticides with male fish being marginally more sensitive. Significant effects of low salinity stress were observed only with acephate exposure and, in this case, low salinity appeared to be slightly protective. In general, all of the insecticide mixtures (azinphosmethyl/endosulfan, azinphosmethyl/fenvalerate and acephate/fenvalerate) tested exhibited simple additive toxicity.     

Predicting the aquatic risk of realistic pesticide mixtures to species assemblages in Portuguese river basins

Although pesticide regulatory tools are mainly based on individual substances, aquatic ecosystems are usually exposed to multiple pesticides from their use on the variety of crops within the catchment of a river. This study estimated the impact of measured pesticide mixtures in surface waters from 2002 and 2008 within three important Portuguese river basins (‘Mondego’, ‘Sado’ and ‘Tejo’) on primary producers, arthropods and fish by toxic pressure calculation. Species sensitivity distributions (SSDs), in combination with mixture toxicity models, were applied. Considering the differences in the responses of the taxonomic groups as well as in the pesticide exposures that these organisms experience, variable acute multi-substance potentially affected fractions (msPAFs) were obtained. The median msPAF for primary producers and arthropods in surface waters of all river basins exceeded 5%, the cut-off value used in the prospective SSD approach for deriving individual environmental quality standards. A ranking procedure identified various photosystem II inhibiting herbicides, with oxadiazon having the relatively largest toxic effects on primary producers, while the organophosphorus insecticides, chlorfenvinphos and chlorpyrifos, and the organochloride endosulfan had the largest effects on arthropods and fish, respectively. These results ensure compliance with European legislation with regard to ecological risk assessment and management of pesticides in surface waters.     

Minimum ignition energy of medicinal powder – Florfenicol and Tilmicosin

This work aims to help improve the electrostatic safety design and explosion prevention of medical facilities. In this study, the minimum ignition energies (MIEs) of Florfenicol, Tilmicosin and mixtures of Florfenicol and Tilmicosin at ratios of 1:1, 1:2, 2:1 and 1:4 were measured in a Hartmann apparatus. The results demonstrated that the MIEs for Florfenicol, Tilmicosin and mixtures of Florfenicol and Tilmicosin at ratios of 1:1, 1:2, 2:1 and 1:4 are 200, 70, 180, 150, 200 and 110 mJ, respectively. Tilmicosin is more sensitive to static electricity, which is more dangerous than the other two powders examined in this paper. Furthermore, the MIEs of the mixtures are proportional to the Florfenicol content. For all powders, the MIE first decreased with the powder mass and later reached its minimum value. In addition, scanning electron microscopy (SEM), differential scanning calorimetry (DSC) were used to investigate the morphological specificity and thermal decomposition of the powders to elucidate the parameters governing the powder explosions further.     

Environmental impact assessment (EIA): Equilibrium fugacity calculations and worst case approach

A mathematical model UNITRISK which can be used for screening purposes and risk ranking was set up to calculate relative risk values of single chemicals and mixtures of chemicals towards single or mixtures of organisms via contamination of air, water, soil and food dose. The concentration values are calculated by applying the fugacity concept or a dose must be defined. The dose-response is assumed to be linear versus concentration and degradative processes are not considered which is the worst case. The assumption that equilibria between the environmental phases exist is simplifying the model and is also representing the worst case. A mean risk factor is calculated which is 1 if the endpoint values (LC₅₀, LD₅₀, ADI, etc.) are exceeded for the investigated organisms and man.     

Alcohol ethoxylate mixtures in marine sediment: Competition for adsorption sites affects the sorption behaviour of individual homologues

Mineral surfaces form the main sorption phase for alcohol ethoxylates (AEs) in marine sediment. Competition for adsorption sites is investigated for marine sediment and kaolinite clay using simple mixtures of AE homologues. For both sorbents, adsorption sites on mineral surfaces can be effectively blocked by an AE homologue with the strongest adsorption affinity. The strongly adsorbed AE, however, forms a second sorption phase to which weakly adsorbing AE will sorb, forming bilayers. An extended dual-mode model accounts for competition effects, while still based on sorption properties of individual compounds. Competition effects become apparent when total adsorbed concentrations reach ∼10% of the adsorption capacity. Deviations from individual sorption isotherms depend on affinity constants and dissolved homologue composition. Competition will not often occur in contaminated field sediments, with AEs concentrations usually far below the adsorption capacity, but will affect sorption studies, sediment toxicity tests or applications with nonionic surfactant mixtures.     

Predicting the aquatic risk of realistic pesticide mixtures to species assemblages in Portuguese river basins

Although pesticide regulatory tools are mainly based on individual substances, aquatic ecosystems are usually exposed to multiple pesticides from their use on the variety of crops within the catchment of a river. This study estimated the impact of measured pesticide mixtures in surface waters from 2002 and 2008 within three important Portuguese river basins(‘Mondego', ‘Sado' and ‘Tejo') on primary producers, arthropods and fish by toxic pressure calculation. Species sensitivity distributions(SSDs), in combination with mixture toxicity models, were applied. Considering the differences in the responses of the taxonomic groups as well as in the pesticide exposures that these organisms experience, variable acute multi-substance potentially affected fractions(ms PAFs) were obtained. The median msP AF for primary producers and arthropods in surface waters of all river basins exceeded 5%, the cut-off value used in the prospective SSD approach for deriving individual environmental quality standards. A ranking procedure identified various photosystem II inhibiting herbicides, with oxadiazon having the relatively largest toxic effects on primary producers, while the organophosphorus insecticides, chlorfenvinphos and chlorpyrifos, and the organochloride endosulfan had the largest effects on arthropods and fish, respectively. These results ensure compliance with European legislation with regard to ecological risk assessment and management of pesticides in surface waters.     

Hidden Markov models for circular and linear-circular time series

We introduce a new class of circular time series based on hidden Markov models. These are compared with existing models, their properties are outlined and issues relating to parameter estimation are discussed. The new models conveniently describe multi-modal circular time series as dependent mixtures of circular distributions. Two examples from biology and meteorology are used to illustrate the theory. Finally, we introduce a hidden Markov model for bivariate linear-circular time series and use it to describe larval movement of the fly Drosophila. Received: September 2003 / Revised: March 2004