Model-based experimental design for assessing effects of mixtures of chemicals

We exposed flour beetles (Tribolium castaneum) to a mixture of four poly aromatic hydrocarbons (PAHs). The experimental setup was chosen such that the emphasis was on assessing partial effects. We interpreted the effects of the mixture by a process-based model, with a threshold concentration for effects on survival. The behavior of the threshold concentration was one of the key features of this research. We showed that the threshold concentration is shared by toxicants with the same mode of action, which gives a mechanistic explanation for the observation that toxic effects in mixtures may occur in concentration ranges where the individual components do not show effects. Our approach gives reliable predictions of partial effects on survival and allows for a reduction of experimental effort in assessing effects of mixtures, extrapolations to other mixtures, other points in time, or in a wider perspective to other organisms.     

A comparison of mixture toxicity assessment: examining the chronic toxicity of atrazine, permethrin and chlorothalonil in mixtures to Ceriodaphnia cf. dubia

Pesticides predominantly occur in aquatic ecosystems as mixtures of varying complexity, yet relatively few studies have examined the toxicity of pesticide mixtures. Atrazine, chlorothalonil and permethrin are widely used pesticides that have different modes of action. This study examined the chronic toxicities (7-d reproductive impairment) of these pesticides in binary and ternary mixtures to the freshwater cladoceran Ceriodaphnia cf. dubia. The toxicity of the mixtures was compared to that predicted by the independent action (IA) model for mixtures, as this is the most appropriate model for chemicals with different modes of action. Following this they were compared to the toxicity predicted by the concentration addition (CA) model for mixtures. According to the IA model, the toxicity of the chlorothalonil plus atrazine mixture conformed to antagonism, while that of chlorothalonil and permethrin conformed to synergism. The toxicity of the atrazine and permethrin mixture as well as the ternary mixture conformed to IA implying there was either no interaction between the components of these mixtures and/or in the case of the ternary mixture the interactions cancelled each other out to result in IA. The synergistic and antagonistic mixtures deviated from IA by factors greater than 3 and less than 2.5, respectively. When the toxicity of the mixtures was compared to the predictions of the CA model, the binary mixture of chlorothalonil plus atrazine, permethrin plus atrazine and the ternary mixture all conformed to antagonism, while the binary mixture of chlorothalonil plus permethrin conformed to CA. Using the CA model provided estimates of mixture toxicity that did not markedly underestimate the measured toxicity, unlike the IA model, and therefore the CA model is the most suitable to use in ecological risk assessments of these pesticides.     

Peroxidase-mediated removal of endocrine disrupting compound mixtures from water

Several classes of oxidative enzymes have shown promise for efficient removal of endocrine disrupting compounds (EDCs) that are resistant to conventional wastewater treatments. Although the kinetics of reactions between individual EDCs and selected oxidative enzymes are well documented in the literature, there has been little investigation of reactions with EDC mixtures. This makes it impossible to predict how enzyme-mediated treatment systems will perform since wastewater effluents generally contain multiple EDCs. This paper reports pseudo-first order rate constants for a model oxidative enzyme, horseradish peroxidase (HRP), during single-substrate (k₁) and mixed-substrate (k_1-MIX) reactions. Measured values are compared with literature values of three Michaelis-Menten parameters: half-saturation constant (K_M), enzyme turnover number (k_CAT), and the ratio k_CAT/K_M. Published reports had suggested that each of these could be correlated with HRP reactivity towards EDCs in mixtures, and empirical results from this study show that K_M can be used to predict the sequence of EDC removal reactions within a particular mixture. We also observed that k_1-MIX values were generally greater than k₁ values and that compounds exhibiting greatest estrogenic toxicities reacted most rapidly in a given mixture. Finally, because K_M may be tedious to measure for every EDC of interest, we have constructed a quantitative structure-activity relationship (QSAR) model to predict these values. This model predicts K_M quite accurately (R² = 89%) based on two molecular characteristics: molecular volume and hydration energy. Its accuracy makes this QSAR a useful tool for predicting which EDCs will be removed most efficiently during enzyme treatment of EDC mixtures.     

Different behaviours in the solubilization of polycyclic aromatic hydrocarbons in water induced by mixed surfactant solutions

Water solubility of polycyclic aromatic hydrocarbons (PAHs), viz, naphthalene and phenanthrene, in micellar solutions at 25 °C was investigated, using two series of different binary mixtures of anionic and nonionic surfactants. Tween 80 and Brij-35 were used as nonionic surfactants whereas fatty acids or amphiphilic cyclodextrins (Mod-β-CD) synthesized in our laboratory were used as anionic ones. Solubilization capacity has been quantified in terms of the molar solubilization ratio and the micelle-water partition coefficient, using UV-visible spectrophotometry. Anionic surfactants exhibited less solubilization capacity than nonionics. The mixtures between Tween 80 and Mod-β-CD did not show synergism to increase the solubilization of PAHs. On the other hand, the mixtures formed by Tween 80 and fatty acids at all mole fractions studied produced higher enhancements of the solubility of naphthalene than the individual surfactants. The critical micellar concentration of the mixtures of Tween 80/sodium laurate was determined by surface tension measurements and spectrofluorimetry using pyrene as probe. The system is characterized by a negative interaction parameter (β) indicating attractive interactions between both surfactants in the range of the compositions studied.     

Mechanisms of purple moor-grass (Molinia caerulea) encroachment in dry heathland ecosystems with chronic nitrogen inputs

We analysed growth strategies (biomass allocation, nutrient sequestration and allocation) of heather (Calluna vulgaris) and purple moor-grass (Molinia caerulea) seedlings in monocultures and mixtures in relation to N, P, and N + P fertilisation in a greenhouse experiment in order to simulate a heath’s pioneer phase under high airborne nitrogen (N) loads. N fertilisation increased the total biomass of both species in monocultures. In mixtures, M. caerulea sequestered about 65% of the N applied, while C. vulgaris suffered from N shortage (halving of the total biomass). Thus, in mixtures only M. caerulea will benefit from airborne N loads, and competition will become increasingly asymmetric with increasing N availability. Our results demonstrate that the heath’s pioneer phase is the crucial tipping point at which the competitive vigour of M. caerulea (high belowground allocation, efficient use of belowground resources, shortened reproductive cycles) induces a shift to dominance of grasses under increased N availability.     

Influence of earthworm mucus and amino acids on tomato seedling growth and cadmium accumulation

The effects on the growth of tomato seedlings and cadmium accumulation of earthworm mucus and a solution of amino acids matching those in earthworm mucus was studied through a hydroponic experiment. The experiment included four treatments: 5 mg Cd L⁻¹ (CC), 5 mg Cd L⁻¹ + 100 mL L⁻¹ earthworm mucus (CE), 5 mg Cd L⁻¹ + 100 mL L⁻¹ amino acids solution (CA) and the control (CK). Results showed that, compared with CC treatment, either earthworm mucus or amino acids significantly increased tomato seedling growth and Cd accumulation but the increase was much higher in the CE treatment compared with the CA treatment. This may be due to earthworm mucus and amino acids significantly increasing the chlorophyll content, antioxidative enzyme activities, and essential microelement uptake and transport in the tomato seedlings. The much greater increase in the effect of earthworm mucus compared with amino acid treatments may be due to IAA-like substances in earthworm mucus.     

UraniumVI sorption behavior on silicate mineral mixtures

UraniumVI sorption experiments involving quartz and clinoptilolite, important mineral phases at the proposed US nuclear waste repository at Yucca Mountain, NV, were conducted to evaluate the ability of surface complexation models to predict UVI sorption onto mineral mixtures based on parameters derived from single-mineral experiments. The experiments were conducted at an initial UVI aqueous concentration of approximately 2.0 x 10(-7) mol.l-1 (0.1 mol.l-1 NaNO3 matrix) and over the pH range approximately 2.5 to approximately 9.5. The UVI solutions were reacted with either quartz or clinoptilolite only, or with mixtures of the two minerals. The experiments were carried out under atmospheric pCO2(g) conditions (in loosely capped containers) or under limited pCO2(g) (in capped containers or in a glove box). Data from sorption experiments on quartz at atmospheric pCO2 conditions were used to derive UVI binding constants for a diffuse-layer surface complexation model (DLM). The DLM was then used with surface area as a scaling factor to predict sorption of UVI onto clinoptilolite and clinoptilolite/quartz mixtures under both atmospheric and low pCO2 conditions. The calculations reproduced many aspects of the pH-dependent sorption behavior. If this approach can be demonstrated for natural mineral assemblages, it may be useful as a relatively simple method for improving radionuclide transport models in performance-assessment calculations.     

Comparative acute freshwater hazard assessment and preliminary PNEC development for eight fluorinated acids

Short-term 48, 72 and 96-h aquatic toxicity tests were conducted to evaluate the acute toxicity of eight fluorinated acids to the cladoceran, Daphnia magna, the green alga, Pseudokirchneriella subcapitata, and the rainbow trout, Oncorhynchus mykiss or the fathead minnow, Pimephales promelas. The eight fluorinated acids studied were tridecafluorohexyl ethanoic acid (6:2 FTCA), heptadecafluorooctyl ethanoic acid (8:2 FTCA), 2H-dodecafluoro-2-octenoic acid (6:2 FTUCA), 2H-hexadecafluoro-2-decenoic acid (8:2 FTUCA), 2H,2H,3H,3H-undecafluoro octanoic acid (5:3 acid), 2H,2H,3H,3H-pentadecafluoro decanoic acid (7:3 acid), n-perfluoropentanoic acid (PFPeA) and n-perfluorodecanoic acid (PFDA). The results of the acute toxicity tests conducted during this study suggest that the polyfluorinated acids, 8:2 FTCA, 8:2 FTUCA, 6:2 FTCA, 6:2 FTUCA, 7:3 acid and 5:3 acid, and the perfluorinated acids PFPeA and PFDA, are generally of low to medium concern based on evaluation of their acute freshwater toxicity (EC/LC50s typically between 1 and >100 mg L⁻¹) using the USEPA TSCA aquatic toxicity evaluation paradigm. For the polyfluorinated acids, aquatic toxicity generally decreased as the number of fluorinated carbons decreased and as the overall carbon chain length decreased from 12 to 8. Acute aquatic toxicity of the 5 and 10 carbon perfluorocarboxylic acids (EC/LC50s between 10.6 and >100 mg L⁻¹) was greater or similar to that of the 6-9 carbon perfluorocarboxylic acids (EC/LC50s > 96.5 mg L⁻¹). This study also provides the first report of the acute aquatic toxicity of the 5:3 acid (EC/LC50s of 22.5 to >103 mg L⁻¹) which demonstrated less aquatic toxicity than the 7:3 acid (EC/LC50s of 0.4-32 mg L⁻¹). The cladoceran, D. magna and the green alga, P. subcapitata had generally similar EC50 values for a given substance while fish were typically equally or less sensitive with the exception that PFPeA was most toxic to fish. Predicted no-effect concentrations (PNECs) were estimated using approaches consistent with REACH guidance and when compared with available environmental concentrations, these PNECs suggest that the fluorinated acids tested pose little risk for aquatic organisms.     

Spectroscopic changes on fulvic acids from a kraft pulp mill effluent caused by sun irradiation

Large volumes of wastewater with a high organic load are generated by the pulp and paper industry that negatively affect the quality of receiving waters. The main waste products in the pulp mill effluents are lignin derived macromolecular compounds, which are similar to natural humic substances and very resistant to wastewater treatments. Fulvic acids (FA) represent the higher percentage of these humic substances and it was observed that solar irradiation modify their properties. Several analytic tools, namely, UV–Visible, molecular fluorescence and FTIR spectroscopies, were used to assess the effect of solar exposition on fulvic acids from a kraft pulp mill effluent. It may be concluded that sun irradiation may alter to a high extent the physicochemical properties of macromolecular organic matter, namely fulvic acids, released by kraft pulp mill effluents. After solar exposition, the aromaticity decreases, the aliphatic structures become more oxygenated, and the fulvic acids from the pulp mill effluent remaining in solution are more similar to aquatic fulvic acids from non polluted sites.     

Spectroscopic changes on fulvic acids from a kraft pulp mill effluent caused by sun irradiation

Large volumes of wastewater with a high organic load are generated by the pulp and paper industry that negatively affect the quality of receiving waters. The main waste products in the pulp mill effluents are lignin derived macromolecular compounds, which are similar to natural humic substances and very resistant to wastewater treatments. Fulvic acids (FA) represent the higher percentage of these humic substances and it was observed that solar irradiation modify their properties. Several analytic tools, namely, UV–Visible, molecular fluorescence and FTIR spectroscopies, were used to assess the effect of solar exposition on fulvic acids from a kraft pulp mill effluent. It may be concluded that sun irradiation may alter to a high extent the physicochemical properties of macromolecular organic matter, namely fulvic acids, released by kraft pulp mill effluents. After solar exposition, the aromaticity decreases, the aliphatic structures become more oxygenated, and the fulvic acids from the pulp mill effluent remaining in solution are more similar to aquatic fulvic acids from non polluted sites.     

In vivo endocrine effects of naphthenic acids in fish

Oil pollution from various sources, including exploration, production and transportation, is a growing global concern. The highest toxicity of hydrocarbon pollutants is associated with the water-soluble phase compounds, including naphthenic acids, a known component found in all hydrocarbon deposits. Recently, naphthenic acids (NAs) have shown estrogenic and anti-androgenic effects in vitro. For this reason we investigated the potential effects of two commercial mixtures of naphthenic acids on fish in vivo, using the three-spined stickleback (Gasterosteus aculeatus) as a model species.     

Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers

In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24 h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).     

Biodegradation of naphthenic acids by rhizosphere microorganisms

Naphthenic acids are components of most petroleums, including those found in the Athabasca Oil Sands of northeastern Alberta. Some naphthenic acids that are solubilized during bitumen extraction from oil sands are acutely toxic to a variety of organisms. Four-month enrichment cultures obtained from the rhizospheres of five plant species native to Alberta, and established with the addition of bitumen (0.5%) as the sole carbon source, revealed a high potential for aerobic degradation of a Merichem commercial preparation of naphthenic acids. Changes in the concentration and composition of the naphthenic acids mixtures during incubation were followed using high-performance liquid chromatography and gas chromatography-electron impact mass spectrometry. Concentrations did not significantly change in the sterile control, but they decreased by up to 90% after 10 days of incubation in the viable cultures. Lower molecular mass naphthenic acids were preferentially degraded, while the proportion of high molecular mass acids increased during incubation. By day 17, the most abundant ions were derived from cellular membranes, corresponding to an increase in microbial numbers in the cultures as naphthenic acids were metabolized. This study is the first to demonstrate the biodegradation potential of microorganisms from rhizosphere soils to biodegrade naphthenic acids.     

Influence of n-alkanes and petroleum on fatty acid composition of a hydrocarbonoclastic bacterium: Marinobacter hydrocarbonoclasticus strain 617

This study concerns the effects of various long-chain n-alkanes, n-alkane mixtures and Arabian Light crude oil on the fatty acid (FA) composition of a sedimentary marine bacteria (Marinobacter hydrocarbonoclasticus strain 617), growing under aerobic conditions. The cultures with n-alkanes, as compared with soluble carbon sources, led to greater amounts of saturated and methyl branched FA (mainly belonging to a delta10 series). We observed the appearance or increase of saturated and unsaturated FA with the same carbon chain length (CCL) as the n-alkane carbon source (maximum for n-alkane CCL corresponding to the 'range' of the de novo synthesized fatty acids). We also observed a strong control of the oddness/evenness of the CCL of the FA by the oddness/evenness of the n-alkane. A n-alkane utilization index, (saturated + branched)/monounsaturated fatty acids (SAFA + BFA/MUFA) enabled discriminating between soluble carbon sources and hydrocarbons.     

Comparative aryl hydrocarbon hydroxylase induction activities of commercial PCBs in Wistar rats and rat hepatoma H-4-II E cells in culture

The activities of several commercial PCB mixtures as inducers of microsomal aryl hydroarbon hydroxylase (AHH) and ethoxyresorufin 0-deethylase (EROD) in male Wistar rats and rat hepatoma cells were dependent on the chlorine content (and congener distribution) of the mixtures. The was a correlation between the $\text{in vivo}$ and $\text{in vitro}$ induction potencies of these mixtures; the most active mixtures, Aroclors 1242, 1248 and 1254 contained 42–54% by weight of Cl whereas formulations with higher or lower (Aroclors 1221 and 1268 respectively) chlorine content exhibited lower activities. This corresponds with studies reported for PCB isomers and congeners in which the most active compounds are tetra-hexachlorobiphenyls.     

Paper electrophoretic separations of some common pesticides : Determination of monocrotophos,rogor and malathion in soil samples

Abstract

Paper electrophoretic movements of a number of pesticides belonging to organophosphorus, organochlorine and pyethroid groups in various acid background electrolytes have been studied. The effect of pH and pKa of the acids on the movement of these pesticides have also been studied. It has been observed that the movement of most of the pesticides is enhanced with increase in the degree of ionisation of the acids (pKa) studied as background electrolytes. The movement also increases with increase in the pH of acids. On the basis of differential movement of pesticides towards cathode and anode, a number of separations have been achieved from binary mixtures. Monocrotophos, rogor and malathion have been determined quantitatively (28.6 ‐ 29.2 μg) in alcoholic extracts of soil samples.     

Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI FT-ICR MS): Characterization of Complex Environmental Mixtures

Structural and chemical characterization of compounds within complex environmental mixtures provides unique insights into the roles of these compounds in different environmental processes. Here, an emerging technique in environmental chemistry, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS), is described and its applications to the molecular characterization of humic acids and petroleum products are reviewed and examined. ESI is a low-fragmentation ionization technique that preferentially ionizes polar functional groups prior to mass spectrometric analysis. This technique allows the characterization of intact polar macromolecules that are inaccessible to standard chromatographic techniques. ICR MS is an ultrahigh resolution and mass accuracy MS technique based on the detection of ion cyclotron motion within a magnetic field. The combination of ESI FT-ICR MS with other structural techniques such as nuclear magnetic resonance (NMR) allows the unprecedented identification and characterization of polar macromolecules in environmental mixtures and will find numerous applications within environmental chemistry and forensics.     

Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI FT-ICR MS): Characterization of Complex Environmental Mixtures

Structural and chemical characterization of compounds within complex environmental mixtures provides unique insights into the roles of these compounds in different environmental processes. Here, an emerging technique in environmental chemistry, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS), is described and its applications to the molecular characterization of humic acids and petroleum products are reviewed and examined. ESI is a low-fragmentation ionization technique that preferentially ionizes polar functional groups prior to mass spectrometric analysis. This technique allows the characterization of intact polar macromolecules that are inaccessible to standard chromatographic techniques. ICR MS is an ultrahigh resolution and mass accuracy MS technique based on the detection of ion cyclotron motion within a magnetic field. The combination of ESI FT-ICR MS with other structural techniques such as nuclear magnetic resonance (NMR) allows the unprecedented identification and characterization of polar macromolecules in environmental mixtures and will find numerous applications within environmental chemistry and forensics.     

Influence of Sulfur Dioxide and Hydrogen Fluoride as a Mix or Reciprocal Exposure on Citrus Growth and Development

The influence of exposure to mixtures of SO₂ and HF on Koethen sweet orange and mixtures and alternate exposure to these gases on Satsuma mandarin were tested using a rotating fumigation greenhouse. Effects of HF-SO₂ mixtures on linear growth and leaf area of Koethen orange were additive, not synergistic. No necrosis was observed on Koethen oranges exposed to HF, SO₂, or a mixture of HF and SO₂. Effects of the mixture on chlorosis of Satsuma mandarin foliage was also not synergistic. No significant difference in linear growth of Satsuma mandarin was found among all treatments. Alternate exposure to SO₂ followed by HF produced no synergistic injury to Satsuma mandarin. Satsuma mandarin appeared more sensitive than Koethen orange to HF, SO₂, and mixtures of these two gases using degree of chlorosis and leaf abscission as the criteria of sensitivity. If iinear growth and leaf area were the principal criteria considered, Koethen orange would appear more sensitive.     

A statistical comparison of naphthenic acids characterized by gas chromatography-mass spectrometry

Naphthenic acids are complex mixtures of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, with the general chemical formula C_nH_2n+zO₂, where n is the carbon number and Z specifies a homologous family. These acids have a variety of commercial uses, including being used as wood preservatives. They are found in conventional and heavy oils, and in the oil sands of northeastern Alberta, Canada. Naphthenic acids are major contributors to the toxicity of tailings waters that result from the oil sands extraction process. Eight naphthenic acids preparations (four from commercial sources and four from the oil sands operations) were derivatized and analyzed by gas chromatography–mass spectrometry. The composition of each mixture was summarized as a three-dimensional plot of the abundance of specific ions (corresponding to naphthenic acids) versus carbon number (ranging from 5 to 33) and Z family (ranging from 0 to −12). The data in these plots were divided into three groups according to carbon number (group 1 contained carbon numbers 5–14, group 2 contained carbon numbers 15–21, and group 3 contained carbon numbers 22–33). A t-test, using arcsine-transformed data, was applied to compare corresponding groups in samples from various sources. Results of the statistical analyses showed differences between various commercial naphthenic acids preparations, and between naphthenic acids from different oil sands ores and tailings ponds. This statistical approach can be applied to data collected by other mass spectrometry methods.