Cholinesterase characterization and effects of the environmental contaminants chlorpyrifos and carbofuran on two species of marine crabs,Carcinus maenas and Pachygrapsus marmoratus

Among the most frequent targets for toxic effects of modern pesticides, namely organophosphates and carbamates, one may find cholinesterases (ChEs). ChEs exist in a wide variety of animals and have been used actively to discriminate among the environmental effects of different pollutant groups, including the aforementioned pesticides. This study had three purposes, namely (i) identifying the ChE forms present in tissues (eyes and walking legs muscle) of two crab species, Carcinus maenas and Pachygrapsus marmoratus; to (ii) determine the in vitro toxicological effects, and (iii) compare the sensitivity of such enzymatic forms towards commonly used anti-ChE pesticides, namely the organophosphate chlorpyrifos and the carbamate carbofuran. Our results showed that there was not a clear preference for any of the tested substrates in any of the tissues from both species. Furthermore, the ChE activity was almost completely suppressed following incubation with eserine and with the specific inhibitor BW284C51 in all tissues from both species. In vitro exposure to chlorpyrifos promoted a significant decrease in ChE activity in both species. Furthermore, the ChE activity was completely suppressed following incubation with carbofuran and chlorpyrifos. These results suggest that the major ChE forms present in tissues of both crab species show intermediate structural properties and activity patterns, halfway between classic acetylcholinesterase and pseudocholinesterases. However, the sensitivity of the found forms towards ChE inhibitors was established, and the responsiveness of such forms towards common anti-ChE chemicals was established. Both tested species seem to be promising test organisms to be used in marine and coastal scenarios of putative contaminations by anti-ChE chemicals, considering the here reported patterns of response.

     

Evaluation of the procedures for the measurement of water solubility and n-octanol/water partition coefficient of chemicals results of a ring test in Japan

A ring test by 19 labolatories in Japan was carried out in 1984 for the evaluation of the experimental manuals for the measurements of water solubility and partition coefficient (n-octanol/water). The experimental data obtained by the procedures in the manuals were collected by Environmental Agency, Japan. In the measurement of water solubility, this manual applies a modified batch method (the glass beads technique), whereas the OECD Test Guidelines suggests the use of the column elution method for chemicals having water solubility lower than 10 mg/L. Anthracene and flouranthene were used as test chemicals for the measurement of water solubility, and γ -hexachlorocyclohexane (γ -HCH) and 1,2,3-trichlorobenzene were used for partition coefficient. Good agreements were observed at a practical basis between the OECD methods and our methods for the results of mesurements of water solubility and partition coefficient. We concluded that the present ring test showed an advantage of the use of the glass beads method which requires only simple apparatus in water solubility measurement, and the HPLC method is very useful to measure partition coefficient because of the simplicity of procedure and the wide range of measurement, especially at higher values of partition coefficient.     

Field derived accumulation and release kinetics of DDTs in plants

Vegetation plays an important role in influencing the air/surface exchange of semivolatile organic compounds (SOCs). In order to predict the capability of different plant species to capture chemicals from the air, plant-air partition coefficients and kinetic accumulation parameters must be defined. In this study, potted plants of three different species were transferred to the vicinity of a source point for DDT, namely a contaminated area around a former production plant in Italy. Leaves were constantly sampled in order to follow the uptake from air over time. Later, the potted plants were transported to a location characterized by background diffuse air concentrations for the release phase. Coupling the experimental results with a two-compartment accumulation model it was possible to derive the kinetics parameters and the plant-air partition coefficient K(PA) for p,p'-DDT. The logK(PA) (on a mass/volume basis) ranged between 1.7 and 2.2 for the different species. The uncertainties related to the different phenomena involved in a field uptake/release experiment are discussed.     

An algebraic/graphical tool to compare ecosystems with respect to their pollution V: cluster analysis and Hasse diagrams

In case of large data matrices comparative evaluations of objects/regions with the technique of Hasse diagrams may be troublesome due to a messy system of lines in the graphical representation. Here fuzzy clustering leads to useful simplifications because regions with slightly different pollution pattern are grouped together. However, fuzzy clustering implies to introduce a threshold value for the membership of an object to a cluster and to select the best number of clusters. Therefore many arbitrarities evolve. Within the systematic study presented here we found that some objects are very stable against variations of the threshold value and the number of cluster whereas other objects behaves different. According to their behaviour we investigated a classification of the objects. Formal Concept Analysis shows that in some cases specific pollution pattern imply the membership to one of these classes. For example objects which are characterized by high Pb-, Zn-concentration and moderate S-concentration imply a high stability against variants of the clustering process. Further implications are described in the paper.     

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.     

Fuzzy system models combined with nonlinear regression for daily ground-level ozone predictions

This study focuses on applying a Takagi–Sugeno fuzzy system and a nonlinear regression (NLR) model for ozone predictions in six Kentucky metropolitan areas. The fuzzy “c-means” clustering technique coupled with an optimal output predefuzzification approach (least square method) was used to train the Takagi–Sugeno fuzzy system. The fuzzy system was tuned by specifying the number of rules and the fuzziness factor. The NLR models were based in part on a previously reported, trajectory-based hybrid NLR model that has been used for years for forecasting ground-level ozone in Louisville, KY. The NLR models were each composed of an interactive nonlinear term and several linear terms. Using a common meteorological parameter set as input variables, the NLR models and the Takagi–Sugeno fuzzy systems model exhibited equivalent forecasting performance on test data from 2004. For all 2004 ozone season forecasts for the six metropolitan areas, the mean absolute error was 8.1 ppb for the NLR model and 8.0 ppb for the Takagi–Sugeno fuzzy model. When a nonlinear term (which was part of the NLR model) was included in the fuzzy model, the combined NLR–fuzzy model had slightly better performance than the original NLR model. For all 2004 metropolitan area forecasts, the mean absolute error of the NLR–fuzzy model forecasts was 7.7 ppb. These small differences may be statistically significant, but for practical purposes the performance of the fuzzy models was equivalent to that of the NLR models.     

Correlations between experimental and predicted equilibrium distribution coefficient of chlorobenzene and chlorophenol compounds in soil-water systems using partial solubility parameters

Many pesticides are degraded to become chlorinated aromatic compounds in soils. Equilibrium distribution of chlorobenzene and chlorophenol compounds in soil-water systems of Yangmingshan loam, Pingcheng silty clay loam and Annei silty loam was studied with the integral distribution equilibrium equation involving the partial solubility parameters of the chemicals. If the adsorption of chemicals on soils is partitioning in soil organic matter surrounding the soil mineral particles, the absorption constant (Kd) of a chemical in soil-water system could be stated as the distribution coefficient (or partition constant, Koc) of the chemical in the two adjunct immiscible phases--water and soil organic matter. The distribution coefficient (Koc) of chemicals calculated from the integral distribution equilibrium equation agrees well with the experimental adsorption coefficient (Kd, or experimental Koc) of chemicals determined in this study, for all the three different types of soils in water according to multiple-regression analysis. Reference data of Karger or Tijssen are employed to estimate the Koc for both polar and non-polar chemicals. The integral distribution equilibrium equation can exactly describe the distribution behavior of nonionic compound of chlorobenzenes and chlorophenols in soil-water systems.     

Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates

National and international chemical management programs are assessing thousands of chemicals for their persistence, bioaccumulative and environmental toxic properties; however, data for evaluating the bioaccumulation potential for fish are limited. Computer based models that account for the uptake and elimination processes that contribute to bioaccumulation may help to meet the need for reliable estimates. One critical elimination process of chemicals is metabolic transformation. It has been suggested that in vitro metabolic transformation tests using fish liver hepatocytes or S9 fractions can provide rapid and cost-effective measurements of fish metabolic potential, which could be used to refine bioconcentration factor (BCF) computer model estimates. Therefore, recent activity has focused on developing in vitro methods to measure metabolic transformation in cellular and subcellular fish liver fractions. A method to extrapolate in vitro test data to the whole body metabolic transformation rates is presented that could be used to refine BCF computer model estimates. This extrapolation approach is based on concepts used to determine the fate and distribution of drugs within the human body which have successfully supported the development of new pharmaceuticals for years. In addition, this approach has already been applied in physiologically-based toxicokinetic models for fish. The validity of the in vitro to in vivo extrapolation is illustrated using the rate of loss of parent chemical measured in two independent in vitro test systems: (1) subcellular enzymatic test using the trout liver S9 fraction, and (2) primary hepatocytes isolated from the common carp. The test chemicals evaluated have high quality in vivo BCF values and a range of logK(ow) from 3.5 to 6.7. The results show very good agreement between the measured BCF and estimated BCF values when the extrapolated whole body metabolism rates are included, thus suggesting that in vitro biotransformation data could effectively be used to reduce in vivo BCF testing and refine BCF model estimates. However, additional fish physiological data for parameterization and validation for a wider range of chemicals are needed.     

Whole effluent toxicity assessment at a wastewater treatment plant upgraded with a full-scale post-ozonation using aquatic key species

Ozonation as final wastewater (WW) polishing step, following conventional activated sludge treatment is increasingly implemented in sewage treatment for contaminant degradation to prevent surface water pollution. While the oxidative degradation of chemicals has been extensively investigated, the in vivo toxicological characteristics of ozonated whole effluents are rarely a matter of research.In the present study, whole effluents were toxicologically evaluated with an in vivo test battery before and after full-scale ozonation and subsequent sand filtration on site at a treatment plant. One aquatic plant (duckweed, Lemna minor) and five invertebrate species of different systematic groups (Lumbriculus variegatus, Chironomus riparius, Potamopyrgus antipodarum, Daphnia magna) were exposed to the effluents in a flow-through-designed test system with a test duration of 7-28 d.None of the considered toxicity endpoints correlated with the pollutant elimination. A tendency towards an increased toxicity after ozonation was apparent in three of the test systems showing [statistically] significant adverse effects in the L. variegatus toxicity test (decrease in reproduction and biomass). After sand filtration, adverse effects were reduced to a similar level like after conventional treatment. Solely the Daphnia reproduction test revealed beneficial effects after ozonation in combination with sand filtration.Results of the test battery indicate the formation of adverse oxidation products during WW ozonation. L. variegatus appeared to be the most sensitive of the five test species. Sand filtration effectively removes or detoxifies toxic oxidation products, as toxic effects were subsequently reduced to the level after conventional treatment.     

Influence of hydroxypropylcyclodextrins on the toxicity of mixtures

We studied the influence of hydroxypropylcyclodextrins (HPCDs) on the toxicity of some mixtures. Using the Photobacterium phosphoreum toxicity test, the joint toxicological effect for Mixture I (containing p-nitrobenzaldehyde and 1-nitronaphthalene) and Mixture II (containing p-nitrobenzaldehyde and malononitrile) were determined in water and in aqueous solutions of HPCDs. The results indicate that, although the toxicological joint effect for Mixture I (simple addition) differs from that of Mixture II (synergism), alpha- and beta-HPCD can significantly reduce the toxicity of the test compounds, whereas gamma-HPCD has only a slight effect. Explanations for these observations are given that invoke the molecular structure of the individual chemicals as well as the structures of HPCDs. This provides information to assist the application of HPCDs in remediation of environmental pollution.     

A flow-through passive dosing system for continuously supplying aqueous solutions of hydrophobic chemicals to bioconcentration and aquatic toxicity tests

A continuous supply of water with defined stable concentrations of hydrophobic chemicals is a requirement in a range of laboratory tests such as the OECD 305 protocol for determining the bioconcentration factor in fish. Satisfying this requirement continues to be a challenge, particularly for hydrophobic chemicals. Here we present a novel solution based on equilibrium passive dosing. It employs a commercially available unit consisting of ∼16 000 polydimethylsiloxane (PDMS) tubes connected to two manifolds. The chemicals are loaded into the unit by repeatedly perfusing it with a methanol solution of the substances that is progressively diluted with water. Thereafter the unit is perfused with water and the chemicals partition from the unit into the water. The system was tested with nine chemicals with log K_OW ranging from 4.1 to 6.3. The aqueous concentrations generated were shown to be largely independent of the water flow rate, and the unit to unit reproducibility was within a factor of ∼2. In continuous flow experiments the aqueous concentrations of most of the study chemicals remained constant over 8 d. A model was assembled that allows prediction of the operating characteristics of the system from the log K_OW or PDMS/water partition coefficient of the chemical. The system is a simple, safe, predictable and flexible tool that generates stable aqueous concentrations of hydrophobic chemicals.     

Distribution of PAHs and PCBs to dissolved organic matter: high distribution coefficients with consequences for environmental fate modeling

Dissolved organic carbon/water distribution coefficients (K(DOC)) were measured for a selection of PCBs with octanol/water partition coefficients (K(OW)) ranging from 10(5.6) to 10(7.5). A solid phase dosing and sampling technique was applied to determine K(DOC) to Aldrich humic acid. This technique is in particular suitable for determining the distribution of very hydrophobic chemicals to complex matrices like humic acids. The K(DOC) values were calculated from the experimental data using a linear model. Determined K(DOC)'s were evaluated in relation to octanol/water partition coefficients of the test compounds, and compared to literature data. Measured K(DOC) values were somewhat higher than literature data, which can probably be attributed to the overestimation of freely dissolved aqueous concentration as a result of incomplete phase separation in other studies, and to the unique character of Aldrich humic acid as a "sorbent" or co-solute or to the fact that Aldrich humic acid is not a typical DOC, and other (adsorption) processes can occur. This study reports DOC distribution coefficients that belong to the highest ones ever measured. In addition, the DOC distribution was discussed in relation to current risk assessment modeling.     

Quantitative structure-activity relationships for the inhibition toxicity to root elongation of Cucumis sativus of selected phenols and interspecies correlation with Tetrahymena pyriformis

The comparative toxicities of selected phenols to higher plants Cucumis sativus were measured and the negative logarithm molar concentration of the root elongation median inhibition (IRC50) were derived. Quantitative structure-activity relationships (QSARs) were developed to explore the toxicity influencing factors and for predictive purpose. The toxicity data, fell into two classes: polar narcosis and bio-reactive. For polar narcotic phenols, a highly significant two-parameter QSAR based on 1-octanol/water partition coefficient (logKow) and energy of the lowest unoccupied orbital (E(lumo)) was derived (IRC50 = 0.77 log Kow - 0.39E(lumo) + 2.36 n = 22 r2 = 0.89). The five bio-reactive chemicals proved to show elevated toxicity due to their typical substructure involved diverse reactive mechanisms. In an effort to model all chemicals, a robust multiple-variable QSAR combining logKow, E(lumo) and Qmax, the most negative net atomic charge, was developed (IRC50 = 0.65 logKow - 0.72E(lumo) + 0.23Qmax + 2.81 n = 27 r2 = 0.94), indicating that hydrophobicity, electrophilicity and hydrogen bond interaction contribute mainly to the phytotoxicity. The toxicological data was compared with Tetrahymena pyriformis 2-d population growth inhibition toxicity (IGC50) and excellent interspecies correlations were observed both for the polar narcotics and for five reactive chemicals (for polar narcotics: IRC50 = 0.95IGC50 + 1.07 n = 16 r2 = 0.89; for bio-reactive chemicals: IRC50 = 0.98IGC50 + 2.19 n = 5 r2 = 0.97; and for all: IRC50 = 0.93IGC50 + 1.63 n = 21 r2 = 0.87). This suggested that T pyriformis toxicity could serve as a surrogate of C. sativus toxicity for phenols and interspecies correlation also could be established for reactive chemicals.     

Application of the criteria for classification of existing chemicals as dangerous for the environment

The criteria for classification and labelling of substances as “dangerous for the environment” agreed upon within the European Union (EU) were applied to two sets of existing chemicals. One set (sample A) consisted of 41 randomly selected compounds listed in the European Inventory of Existing Chemical Substances (EINECS). The other set (sample B) comprised 115 substances listed in Annex I of Directive 67/548/EEC which were classified by the EU Working Group on Classification and Labelling of Existing Chemicals. The aquatic toxicity (fish mortality,Daphnia immobilisation, algal growth inhibition), ready biodegradability and n-octanol/water partition coefficient were measured for sample A by one and the same laboratory. For sample B, the available ecotoxicological data originated from many different sources and therefore was rather heterogeneous. In both samples, algal toxicity was the most sensitive effect parameter for most substances. Furthermore, it was found that, classification based on a single aquatic test result differs in many cases from classification based on a complete data set, although a correlation exists between the biological end-points of the aquatic toxicity test systems.     

In vitro biotransformation of surfactants in fish. Part I: linear alkylbenzene sulfonate (C12-LAS) and alcohol ethoxylate (C13EO8)

Developing regulatory activities (e.g., REACh, [DGEE. 2003. Directorates General Enterprise and Environment. The new EU chemicals legislation REACH. DG Enterprise, Brussels, Belgium. (http://www.europa.eu.int/comm/enterprise/reach/index_en.htm)]) will require bioaccumulation to be assessed for thousands of chemicals. Further, there is increasing pressure to reduce, refine or replace animal tests. Given this scenario, there is an urgent need to evaluate the feasibility of in vitro systems to supply data useful for bioaccumulation estimation. Subcellular and cellular hepatic systems were tested to determine the biotransformation of two surfactants: C12-2-LAS (2-phenyl dodecane p-sulfonate) and an alcohol ethoxylate C13EO8 (Octaethylene glycol monotridecyl ether). The subcellular systems tested were liver homogenates and microsomes from the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Cellular systems consisted of primary hepatocytes from the common carp (Cyprinus carpio) and PLHC-1 cells, hepatocarcinoma cells from the desert topminnow (Poeciliopsis lucida). All in vitro systems were exposed to radiolabeled test compounds and assayed for biotransformation using liquid scintillation and thin layer chromatographic methods. First-order kinetics were used to estimate rates of biotransformation. Bioconcentration of test materials in fish were predicted using an in vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model commonly used to predict bioaccumulation in fish. Subcellular biotransformation rates for each of the surfactants were greatest with microsomes. Cellular loss rates exceeded subcellular rates, leading to lower predicted BCF values. Predicted BCFs corresponded closely to measured values in several fish species, verifying the utility of in vitro systems in refining Kow-only-based BCFs via the inclusion of biotransformation rates.