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Cytotoxicity of 109 chemicals to goldfish GFS cells and relationships with 1-octanol/water partition coefficients
Abstract
The midpoint cytotoxicity (NR50 values) of 109 chemicals including alcohols, aromatics, phenols and pesticides to goldfish scale GFS cells were compared with the fish acute toxicities and with the 1 - octanol/water partition coefficient (Pow). The NR50 values were significantly correlated to the available acute toxicity data for fathead minnow (n=31, r=0.958, s=0.480, F=325) and guppy (n=29, r=0.957, s=0.482, F=294).The Pow gave a good correlation in simple linear regression analysis for all compounds excluding 8 compounds that the NR50 values could not be obtained for their low solubility in culture medium.For 29 narcotic type of compounds, the regression analysis revealed a high correlation between the NR50 values and Pow, and appeared to give a baseline cytotoxicity.And also, the regression analyses between NR50 and Pow for some types of compounds were discussed.These results suggest that NR assay is useful for prediction of acute toxicity to fish and studies of quantitative structure-activity relationships (QSARs).
... To assess the toxic potential of the compounds investigated their hydrophobicity was determined. This is usually expressed in terms of the logP value, which gives the partitioning of a compound over an octanol/water two-phase standard system Saito et al., 1993). The logP values were calculated according to the hydrophobic fragment constants given by Rekker and de Kort (1979). ...
... A promising result is that the toxicity of this group of organic compounds can also be estimated by calculating the logP values of the chemicals. A quantitative structure-toxicity relationship (QSTR) between the hydrophobicity of organic solvents and their toxic potential has often been described for different organic compounds and with different test systems (Beltrame et al., 1984;Heipieper et al., 1995;Liu et al., 1982;Oikawa et al., 1985;Saito et al., 1993). However, these methods require repeat testing for every new group of organic solvents. ...
We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 69–73, 2001
... Fish cell lines sensitivity has been investigated comparing in vitro EC 50 values (obtained from different basal cytotoxicity tests) with in vivo acute lethality LC 50 values. In most of the studies (Bols et al., 1985;Babich et al., 1990;Castaño et al. 1994Castaño et al. , 2003Lange et al., 1995;Saito et al., 1991Saito et al., , 1993Saito et al., , 1994Segner, 2004;Schirmer, 2006) the correlation coefficients (r) are usually higher than 0,80 (positive linear correlation). However, in absolute terms, the in vitro EC 50 values are, on average, one or two orders of magnitude higher than corresponding fish acute toxicity values. ...
In the frame of European Regulation REACH, all chemical substances with a volume of production higher than 1 t per year, must be tested for their ecotoxicity. In addition to tests with algae and crustaceans, a large number of toxicity tests with fish has to be conducted. Due to the good correlation found between the in vitro data and the in vivo fish data (Castaño and Gómez-Lechón, 2005), the use of established fish cell lines can represent an alternative tool to acute fish bioassay for toxicity screening of chemicals.
... [Cetin et al., 2006]. c [Saito et al., 1993]. d [Hansch et al., 1995]. ...
[1] Consumption of pesticides in China has increased rapidly in recent years, however occurrence and fate of current-use pesticides (CUPs) in China coastal waters are poorly understood. Globally banned pesticides, so-call historic-use pesticides (HUPs), are still commonly observed in the environment. In this work, air and surface seawater samples taken from the Bohai and Yellow Seas in May, 2012 were analysed for CUPs including trifluralin, quintozene, chlorothalonil, dicofol, chlorpyrifos and dacthal, as well as HUPs (hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs) and endosulfan). CUP profile in both air and seawater samples generally reflected their consumption patterns in China. HUPs in the air and seawater samples were in comparable levels as those of CUPs with high detectable amount. α-Endosulfan, dicofol and chlorothalonil showed strong net deposition likely resulting from their intensive use in recent years, while CUPs with low consumption amount (quintozene and dacthal) were close to equilibrium at most samplings sites. Another CUP with high usage amout (i.e. chlorpyrifos) underwent volatilization possibly due to its longer half-life in seawater than that in air. α-HCH and γ-HCH were close to equilibrium in the Bohai Sea, but mainly underwent net deposition in the Yellow Sea. The net deposition of α-HCH could attributed to polluted air pulses from the East China identified by air mass back trajectories. β-HCH showed net volatilization in the Bohai Sea, which was driven by its relative enrichment in seawater. HCB either slightly favored net volatilization or was close to equilibrium in the Bohai and Yellow Seas.
... It is a breakdown product as well as a chemical intermediate in the production of organophosphorus pesticides (methyl parathion, parathion, fenitrothion and EPN) and analgesic paracetomol (N-acetyl-p-aminophenol) [1,[3][4][5][6][7][8][9][10][11][12]. Because it is carcinogenic [13], mutagenic [14][15][16][17] and cyto-and embryotoxic to mammals [18][19][20], there are urgent needs for innovative an-alytical tools/devices to facilitate its detection in case of an accidental release during these industrial activities. ...
A novel microbial biosensor/electrode for highly selective, sensitive and rapid quantitative determination of p-nitrophenol (PNP) was developed using PNP-degrading organisms immobilized in a membrane on a dissolved oxygen electrode. Moraxella sp. specifically oxidizes PNP while consuming oxygen. A change in oxygen concentration was determined by a Clark oxygen electrode and correlated to the PNP concentration. The sensor signal and response time were optimized with respect to the buffer pH, temperature, time of cell growth and weight of cells immobilized. The best sensitivity and response time were obtained using a sensor constructed with 0.3mg of cells and operating in pH 7.5, 20mM phosphate buffer. Using these conditions, the biosensor was used to measure as low as 14ppb (0.1μM) of PNP extremely selectively without interference from structurally similar compounds, such as phenol, nitrophenols and chlorophenols. The biosensor had very good storage and multiple use stability when stored in the operating buffer at 4°C.
... 4-Nitrophenol is rapidly biodegraded with a half-life between 1 and 10 days in soil and 1-8 days in water environment [10]. It is well known that 4-nitrophenol has carcinogenic [26], mutagenic [27][28][29][30], cyto-and embryotoxic effects on mammals [28,[31][32][33] (i.e. mammalian lethal dose LD 50 is 247 mg kg −1 oral, and 920 mg kg −1 through the skin [10]), and reliable analytical methods for rapid determination of 4-nitrophenol are therefore a great need. ...
A selective competitive flow-immunoassay for monitoring of 4-nitrophenol, a demonstrated carcinogenic and mutagenic compound, is presented. The immunochemical detection principle is based on the competition between a fixed amount of labelled 4-nitrophenol derivative (tracer) and the analyte for a limited amount of antibody binding sites. The separation of free and bound tracer fractions is performed in a restricted-access (RA) column with subsequent fluorescence detection of the labelled immune complex. Optimisation of parameters, e.g. incubation time, flow rate, column size, type of antibody and tracer and their concentrations, was performed. The cross-reactivity for about 30 phenols, nitro-derivatives and other compounds was estimated. No major interferences except for 2,4-dinitrophenol and mercury(II) ions were observed. The optimised assay allowed a detection limit of 0.5 μg l−1 4-nitrophenol (3.5 nM) with a sensitivity of 5.7 mg−1 l, a sample throughput of 30 injections h−1 and a dynamic range between 5 and 1000 μg l−1 4-nitrophenol. The assay was further tested on different sample matrices such as phosphate buffer, tap-, and rainwater without the need for any pre-treatment step.
... Fisher et al. (1993) found a significant correlation between log K OW and acute sediment toxicity of 12 insecticides (i.e., organophosphorous and carbamate insecticides) to the midge Chironomus riparius. Similarly, Saito et al. (1993) found a significant correlation between the log K OW of 101 chemicals (i.e., alcohols, aromatics, phenols and pesticides) and the acute midpoint cytotoxicity to goldfish scale cells. Thus, pesticides with increasing K OW values will be expected to be more acutely toxic to exposed aquatic organisms. ...
Dissolved organic matter (DOM) represents an important carbon phase in coastal environments and influences the partitioning of organic contaminants. In this study, we evaluated the role of salt-marsh sediment-derived DOM vs. DOM-free seawater on the acute and chronic toxicity of three pesticides (chlorothalonil—CHTH, chlorpyrifos—CHPY and fipronil—FIP) to the meiobenthic copepod Amphiascus tenuiremis. Acute toxicity was evaluated via standard 96-h median lethal concentration (LC50), while chronic toxicity was evaluated for 16 days using a 96-well microplate life-cycle bioassay. DOM significantly reduced (p<0.05) acute toxicity of CHTH and CHPY to male and female copepods relative to copepods exposed in DOM-free seawater. In contrast, DOM elevated the acute toxicity of FIP to male copepods. In chronic exposures with/without DOM, CHTH and CHPY did not significantly affect copepodite-to-adult development. In these treatments, plus controls, the majority (95%) of copepodites developed into adults by 8 days. Individuals exposed to FIP in the presence of DOM showed a slower development rate than FIP individuals in DOM-free seawater. Overall, FIP exposed copepodites developed into adult copepods 4 days later than controls. CHTH, CHPY and FIP significantly reduced reproductive success by 33%, 31% and 89%, respectively. DOM, however, mitigated 30% (p=0.006) and 20% (p=0.05) of the reproductive failure attributed to CHTH and CHPY. FIP-induced reproductive failure was high (∼80%) in all exposures regardless of DOM presence/absence. An exponential growth model predicted that exposure to CHTH, CHPY and FIP in the absence of DOM significantly reduced (≥38%) projected naupliar production relative to DOM-free controls. In the presence of DOM, naupliar production under CHTH and CHPY exposures was not significantly different (p>0.05) from control projections. These results indicate that DOM generally reduced the acute and chronic toxicity of CHTH and CHPY to A. tenuiremis, while certain compound-specific pesticide (FIP): organic associations may enhance acute toxicity of FIP, particularly to male copepods.
... Researchers from a number of disciplines have created specialized datasets of pesticide effects on the environmental parameters and biota most relevant to their fields (e.g. Atkins et al., 1981;Royce et al., 1984;Mayer and Ellersieck, 1986;Meyers and Schiller, 1986;Lal and Lal, 1988;Theiling and Croft, 1988;Shiu et al., 1990;Herner, 1992;Nellessen and Fletcher, 1992;Wauchope et al., 1992;Saito et al., 1993;Smith, 1993;Madhavi et al., 1994;Koppert Biological Systems, unpublished data;Matteoni and Elliott, unpublished data). These datasets are compiled from the work of single research groups or from the published results of ecotoxicological tests and experiments. ...
Systems which assess the comparative environmental impact of pesticides may enable farmers and policy makers to choose effective practices for pest control with the least detrimental impact. This paper reviews a number of approaches that have been proposed by research, planning and regulatory agencies for assessing pesticide impacts. Major issues and challenges in developing assessment models include how to incorporate and synthesize a broad range of environmental parameters in creating an integrated assessment; how to evaluate, integrate and present economic and environmental information within one assessment framework; and how to strike a balance between the advantages of the ease of use of simpler systems with the information-richness of more complex systems that may be prohibitively difficult to use. We conclude that universal answers to these questions are not possible because of different specific objectives, methods and decision-making contexts of particular assessment systems.
... a 2008: http://www.cdpr.approximatelygov/docs/pur/purmain.htm b Barceló et al. (1994), Hazardous Substances Data Bank 2008, Saito et al. (1993) ...
Two hundred sediment samples were collected and their toxicity evaluated to aquatic species in a previous study in the agriculturally dominated Central Valley of California, United States. Pyrethroid insecticides were the main contributors to the observed toxicity. However, mortality in approximately one third of the toxic samples could not be explained solely by the presence of pyrethroids in the matrices. Hundreds of pesticides are currently used in the Central Valley of California, but only a few dozen are analyzed in standard environmental monitoring. A significant amount of unexplained sediment toxicity may be due to pesticides that are in widespread use that but have not been routinely monitored in the environment, and even if some of them were, the concentrations harmful to aquatic organisms are unknown. In this study, toxicity thresholds for nine sediment-associated pesticides including abamectin, diazinon, dicofol, fenpropathrin, indoxacarb, methyl parathion, oxyfluorfen, propargite, and pyraclostrobin were established for two aquatic species, the midge Chironomus dilutus and the amphipod Hyalella azteca. For midges, the median lethal concentration (LC₅₀) of the pesticides ranged from 0.18 to 964 μg/g organic carbon (OC), with abamectin being the most toxic and propargite being the least toxic pesticide. A sublethal growth endpoint using average individual ash-free dry mass was also measured for the midges. The no-observable effect concentration values for growth ranged from 0.10 to 633 μg/g OC for the nine pesticides. For the amphipods, fenpropathrin was the most toxic, with an LC₅₀ of 1-2 μg/g OC. Abamectin, diazinon, and methyl parathion were all moderately toxic (LC₅₀s 2.8-26 μg/g OC). Dicofol, indoxacarb, oxyfluorfen, propargite, and pyraclostrobin were all relatively nontoxic, with LC₅₀s greater than the highest concentrations tested. The toxicity information collected in the present study will be helpful in decreasing the frequency of unexplained sediment toxicity in agricultural waterways.
The discharge of huge amount of chemicals from industries into the environment has led to toxicity towards different living species. Therefore, risk assessment of these chemicals is essential. In order to comply with the ethical issues, in this present work, we have developed quantitative structure-activity relationship (QSAR) models for cytotoxicity against GFS (goldfish scale) tissue (Crassius auratus) and enzymatic activity against PLHC-1 cell line (topminnow hepatoma cell line) (Poeciliopsis lucida). The final models were developed by means of PLS (Partial Least Squares) regression method applying only ETA (extended topochemical atom) descriptors. The results obtained from various validation parameters (obtained from the both datasets) suggested that the developed models are statistically robust and predictive. From the insights obtained from the models developed from the Neutral Red dye (NR) dataset, it can be concluded that presence of bulky atoms, unsaturation, branching and hetero atoms (most importantly N, Cl) enhance the cytotoxicity towards the Goldfish scale tissue. On the other hand, in case of the Ethoxyresorufin-O-deethylase (EROD) dataset, presence of higher electronegative atoms (O, Cl), polycyclic aromatic hydrocarbons (PAHs) with more number of rings and absence of polar groups and hydrogen bond acceptors enhance enzymatic activity of the PLHC-1 cell line.
Liverpool John Moores University and FRAME recently conducted a research project sponsored by Defra on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for using alternative methods (both in vitro and in silico) for environmental (aquatic) toxicity testing. The manuscript reviews tests based on fish cells and cell lines, fish embryos, lower organisms, and the many expert systems and QSARs for aquatic toxicity testing. Ways in which reduction and refinement measures can be used are also discussed, including the Upper Threshold Concentration — Step Down (UTC) approach, which has recently been retrospectively validated by ECVAM and subsequently endorsed by the ECVAM Scientific Advisory Committee (ESAC). It is hoped that the application of this approach could reduce the number of fish used in acute toxicity studies by around 65–70%. Decision-tree style integrated testing strategies are also proposed for acute aquatic toxicity and chronic toxicity (including bioaccumulation), followed by a number of recommendations for the future facilitation of aquatic toxicity testing with respect to environmental risk assessment.
Quantitative structure‐toxicity relationship (QSTR) models were built for two in vitro endpoints: cytotoxicity and enzymatic activity of diverse chemicals to goldfish (Crassius auratus) scale tissue (GFS) and topminnow (Poeciliopsis lucida) hepatoma cell line (PLHC‐1), respectively. The data sets were based on experimental cytotoxicity measured with uptake of 3‐amino‐7‐dimethylamino‐2‐methylphenazine hydrochloride dye (Neutral Red assay) representing lysosomal damage and enzymatic activity measured with Ethoxyresorufin‐O‐deethylase (EROD) induction potency. The descriptors were calculated with DRAGON 6 and SPARTAN 10 software packages. Descriptor selection was made by ‘All Subset’ and Genetic Algorithm‐based features implemented in QSARINS software. The proposed QSTR models validated both internally and externally. Additionally, the QSTR models generated for cytotoxicity and EROD induction potency were used to predict the relevant endpoint values for external set chemicals with structural coverage of 95.0 % and 92.1 %, respectively. A strong correlation of experimental in vivo fish lethality data with predicted in vitro cytotoxicity and EROD induction potency values for external set chemicals was found. It was concluded that the proposed QSTR models might be useful to provide an initial screening and prioritization for these diverse chemicals. Also, regarding the strong correlations between predicted in vitro and experimental in vivo data, the use of QSTR predictions as an alternative to the acute fish toxicity assessment can be claimed.
It is of ongoing interest to evaluate real-time instruments for monitoring water contaminants for source water control and water reuse system performance applications. Portable fluorescence sensors have the potential to provide instantaneous measurements of fluorescent organic compounds present in chemical and biological pollutants. Comparisons between one-dimensional fluorescence data from portable sensors and data obtained from higher sensitivity benchtop fluorometers, capable of three dimensional spectral acquisition, are needed to evaluate the performance of portable instruments. We tracked fluorescence intensities in titration experiments with diverse pollutants, including two volatile organic compounds (diesel, gasoline), three pharmaceuticals (ibuprofen, caffeine, lopinavir), and a pesticide (isoxathion), added to creek water, tertiary treated wastewater, and water from different treatment stages at a water reuse facility. Our results indicated that optical filters with wavelength bands in the tryptophan-like and tyrosine-like peak regions were able to track the peaks of most organic chemicals studied. Fluorescence intensities measured with both portable and benchtop instruments were highly correlated for all samples and all contaminants (R² > 0.90). In the final product water of the water reuse facility, both portable and benchtop fluorometers displayed high sensitivity, being able to discern contaminants from the background at concentrations as low as 0.2 ppb. However, in creek water, post-ozone water, and tertiary effluent, pollutant detection limits were much higher for both portable and benchtop fluorometers. Therefore, in waters with substantial background fluorescence, fluorescence-based sensors may be best suited for tracking higher concentrations of organic contaminants, such as those found in chemical spills in source waters.
Uso di colture cellulari per la valutazione ecotossicologica delle sostanze chimiche ai fini del Regolamento REACH: manuale per il mantenimento della linea stabilizzata di pesce RTG-2 (Rainbow Trout Gonad)
I l Regolamento comunitario REACH (Registration, Evaluation and Authorisation of Chemicals) n.1907/2006, riguardante la valutazione, l'autorizzazione e la restrizione delle sostanze chimiche, oltre a voler in primo luogo assicurare un elevato livello di protezione della salute umana e dell'ambiente, ha come obiettivo la promozione e lo sviluppo di metodi alternativi ai saggi in vivo, per la valutazione dei pericoli che le sostanze chimiche comportano. In vari capitoli del Regolamento si specifica che le informazioni da raccogliere in merito alla tossicità ed ecotossicità delle sostanze chimiche dovranno essere acquisite ricorrendo, ove possibile, a mezzi diversi dai test su animali vertebrati (art. 13), perché questi REACH: applicazione di metodi alternativi di saggi in vitro Lo sviluppo di metodi alternativi in vitro costituisce uno degli obiettivi del regolamento REACH sulle sostanze chimiche. Le colture di cellule sono un sistema di facile impiego per valutazioni preliminari di composti tossici. A tale scopo, è presentato il progetto ISPRA (già APAT) riguardante l'uso di linee cellulari di pesce per valutazioni ecotossicologiche devono essere considerati come ultima risorsa (art. 25), adottando inoltre, disposizioni per limitare ripetizioni inutili dei test, attraverso, ad esempio, la condivisione dei dati ottenuti mediante prove sperimentali (art. 30). Per metodi alternativi si intende, sia idonei metodi in vitro, che modelli di relazioni qualitative o quantitative struttura-attività o dati relativi a sostanze strutturalmente affini (art. 13 e Alleg. XI). In primo luogo, idonei significa metodi in vitro vali-dati (tabella 1), come ad esempio il test di irritazio-ne cutanea che utilizza epidermide ricostituita con lo strato corneo funzionale o il test di mutagenicità con Salmonella typhimurium (test di Ames). In ambito ecotossicologico, l'unico metodo valida-to è l'Upper Threshold Concentration (UTC) che comporta una strategia per ridurre l'uso dei pesci nella valutazione della tossicità acquatica acuta. I criteri per la validazione dei metodi in vitro sono indicati nella Guidance Document n° 34 dell'OECD (2005) e una volta validati essi possono sostituire completamente o parzialmente quelli eseguiti in vivo sugli animali. In secondo luogo, sono considerati idonei i test in fase di elaborazione sufficientemente avanzata secondo i criteri riconosciuti a livello internazionale, come ad esempio quelli adottati dal Centro Europeo per la Convalida dei Metodi Alternativi (ECVAM) per l'immissione di una prova nel processo di pre-validazione (ECHA, 2008). Molte ragioni di tipo tecnico, scientifico, economico ed etico supportano la necessità di sviluppo dei metodi con colture di cellule in vitro in ambito
In order to quantitatively examine the lipophilicity of the widely used organophosphorus pesticides (OPs) chlorfenvinphos (CFVP), chlorpyrifos-methyl (CPFM), diazinon (DZN), fenitrothion (FNT), fenthion (FT), isofenphos (IFP), profenofos (PFF) and pyraclofos (PCF), their partition coefficient (Kp) values between phosphatidylcholine (PC) small unilamellar vesicles (SUVs) and water (liposome-water system) were determined by second-derivative spectrophotometry. The second-derivative spectra of these OPs in the presence of PC SUV showed a bathochromic shift according to the increase in PC concentration and distinct derivative isosbestic points, demonstrating the complete elimination of the residual background signal effects that were observed in the absorption spectra. The Kp values were calculated from the second-derivative intensity change induced by addition of PC SUV and obtained with a good precision of R.S.D. below 10%. The Kp values were in the order of CPFM>FT>PFF>PCF>IFP>CFVP>FNT⩾DZN and did not show a linear correlation relationship with the reported partition coefficients obtained using an n-octanol-water system (R(2)=0.530). Also, the results quantitatively clarified the effect of chemical-group substitution in OPs on their lipophilicity. Since the partition coefficient for the liposome-water system is more effective for modeling the quantitative structure-activity relationship than that for the n-octanol-water system, the obtained results are toxicologically important for estimating the accumulation of these OPs in human cell membranes.
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This study evaluated in vitro cytotoxicity of 5 pesticides, including 2 herbicides, 2 germicides, and an insecticide, as an alternative to the fish acute toxicity test. The in vitro cytotoxicity was tested using a neutral red uptake (NRU) assay with epithelioma papulosum cyprini (EPC) cells that originated from the epidermal tissue of Cyprinus carpio (common carp). An in vivo fish acute toxicity test was conducted according to OECD Test Guideline No. 203 using Aphyocypris chinensis (Chinese bleak), Oryzias latipes (Japanese medaka), and C. carpio. The results showed that the sensitivity of the cell viability assay for the pesticides was similar to the fish acute test in ranking order despite having approximately 10 times less absolute sensitivity. The r^2 correlation values were calculated as 0.38 (p
The uncertainties on whether dicofol can be identified as a persistent organic pollutant (POP) in terms of its long-range transport (LRT) potential and global distribution, are always a controversial topic during international regulation deliberations. The lack of monitoring data in remote background regions necessitates a model-based evaluation approach for assessing the global distribution of dicofol. However, few model simulations are available at present, as there is no inventory available for global historical usage of dicofol that has sufficiently high spatial and temporal resolution. To describe the current status of global emission, we first developed an inventory of global dicofol usage for the period of 2000–2012 at 1° × 1° latitude/longitude resolution. We then assessed the LRT potential of dicofol by calculating its Arctic Contamination Potential using the Globo-POP model. In addition, we simulated the global mass distribution and the fate of dicofol in the environment using the BETR-Global model at 15° × 15° latitude/longitude resolution. Our estimated inventory established that over the period of 13 years, a total of 28.2 kilo tonnes (kt) of dicofol was applied and released into the environment. East and Southeast Asia, the Mediterranean Coast, and Northern and Central America were identified as hotspots of usage and release. Dicofol exhibited a higher Arctic Contamination Potential than several confirmed Arctic contaminants, and a larger current volume of consumption than most existing POPs. The results of our BETR-Global simulation suggest that (i) dicofol can indeed be transported northward, most likely driven by both atmospheric and oceanic advections from source regions at midlatitudes, and (ii) dicofol will be enriched in remote background regions. Continuous use of dicofol in source regions will result in exposure both locally and in remote regions, and the examination of the potential for adverse effects is therefore of paramount importance. Proactive restrictions at the international level may be warranted.
A vast number of chemicals require environmental safety assessments for market authorisation. To ensure acceptable water quality, effluents and natural waters are monitored for their potential harmful effects. Tests for market authorisation and environmental monitoring usually involve the use of large numbers of organisms and, for ethical, cost and logistic reasons, there is a drive to develop alternative methods that can predict toxicity to fish without the need to expose any animals. There is therefore a great interest in the potential to use cultured fish cells in chemical toxicity testing. This review summarises the advances made in the area and focuses in particular on a system of cultured fish gill cells grown into an epithelium that permits direct treatment with water samples.
Four automatic compounds were evaluated in laboratory studies to investigate their accumulation and toxicant-induced changes in the rate of heat dissipation in the freshwater invertebrates Chironomus riparius and Lumbriculus variegatus. The sublethal energetic response detected by direct calorimetry was related to tissue chemical concentration by the threshold model and an attempt was made to apply the critical body residue (CBR) concept. Below the compound-specific tissue threshold concentration or CBR, no correlations were found between the dose and the metabolic rate, and the slopes of the regression were close to zero. Above the threshold, depending on the chemical, metabolic rate either increased or decreased. An increase in heat output produced by 2,4-dinitrophenol (2,4-DNP), pentachlorophenol (PCP), and 2,4,5-trichlorophenol (2,4,5-TCP) was closely correlated with the dose. The order of toxicity for these phenols was 2,4-DNP = PCP > 2,4,5-TCP, which reflects the interaction of compounds` lipophilicities and acidities and their combined influence on bioaccumulation and effects on the energy-transducing membrane by uncoupling oxidative phosphorylation. A decrease in the heat output caused by 1,2,4-trichlorobenzene (1,2,4-TCB) was more variable relative to dose. Also, 1,2,4-TCB required a much higher molar tissue threshold concentration ({approximately}2.0 {micro}mol/g wet weight) than required by phenols to generate the response. Both the metabolic response and the chemical threshold value were those expected to result from narcosis. Results suggest that calorimetric measures can identify not only the integrated physiologic response but also have some resolution of the mechanism of toxic effects.
Zusammenfassung Es wurde die toxische Kombinationswirkung von Xenobiotika unterschiedlicher chemischer Substanzklassen auf menschliche Fibroblasten
untersucht. Subtoxische 4-Chloranilin-Konzentrationen verstärkten die Toxizität von 2,4-Dichlorphenoxyessigsäure (2,4-D, Dimethylammonium-Salz)
additiv. Hingegen erhöhte ein Gemisch aus subtoxischen Konzentrationen von 4-Chloranilin, Dicofol und 4-Chlorphenol die Toxizität
von 2,4-D synergistisch. Die drei Komponenten des Gemisches wurden hierbei in Konzentrationen eingesetzt, die jeweils 1/3
ihrer NOEC-Werte (No Observed Effect Concentration) entsprachen. Die Kombinationseffekte von 4-Chloranilin, Dicofol und 4-Chlorphenol
in subtoxischen Einzelkonzentrationen (ohne 2,4-D) waren ebenfalls synergistisch. Die starken Lipophilitätsunterschiede der
kombinierten Substanzen werden als mögliche Ursache der synergistischen Effekte diskutiert.
A review is presented of the physical-chemical properties of the chlorophenols and alkyl phenols, including acid dissociation constant, aqueous solubility, vapour pressure, octanol-water partition coefficient, Henry's law constant or air-water partition coefficient, bioconcentration factor and sorption partition coefficient. Graphical, rather than numerical correlations are presented of these properties, using additive LeBas molar volume as a molecular descriptor. Fairly reliable quantitative structure-property relationships are shown to exist which can be used for predictive purposes. The fates of selected chemicals in an evaluative environment are also described, including the estimated effect of pH on environmental partitioning. In general, the phenols tend to be primarily associated with soils and water and have persistencies controlled by degradation rates in these media. They are not subject to appreciable evaporation because of their low air-water partition coefficients. As pH increases, the tendency of dissociation increases, especially for phenols of relatively low pK, such as pentachlorophenol. The net effect is to enhance transport from air to soil and water, to reduce partitioning from water to sediment and to cause a slight overall reduction in persistence. There is little effect on fate in soil. The overall effect of pH change is quite complex and requires careful scrutiny of multimedia model results. It is recommended that the dependence of organic carbon-water, lipid-water, and octanol-water partitioning on pH and the presence of cations other than H+ be more thoroughly investigated and the degradation rates of these compounds in soil and water be determined, especially at high concentrations.
A four‐step solid‐phase extraction (SPE) method is presented for toxicity‐directed fractionation of industrial wastewater. This fractionation procedure serves as a key step for identifying unknown organic toxicants in complex samples. Toxicity was determined as luminescence inhibition of Vibrio fischeri using microtiter plates. This method was compared to standard tests in glass cuvettes using both 37 standard chemicals and 24 wastewater fractions with EC 50 values covering five orders of magnitude. Results of both methods correlated well.
22 tannery wastewater samples were sequentially extracted using C 18 e and polystyrene‐divinylbenzene phases in combination with pH‐changes. Final solid‐phase filtrates showed low inhibition, so toxicity of inorganics could be neglected. Using 1/ EC 50 values, the SPE eluates showed clearly different toxicity patterns. Even in eluates of the fourth extraction step, high toxic effects could be observed. In several cases, luminescence inhibition was increasing at the anaerobic treatment step compared to the corresponding untreated samples. After aerobic treatment, toxicity of most wastewater fractions was greatly diminished.
HPLC/DAD analyses of the wastewater fractions showed a fair separation concerning compound polarity. However, the samples were still too complex to identify single compounds responsible for the detected toxicity. Therefore, a further clean‐up step accompanied with toxicity testing is needed.
AUTOLOGP(TM) (Version 4.0) forWindows(TM) 3.1 or Windows(TM) 95 is a log P calculator using a neural network model designed from 7200 molecules described by means of the autocorrelation method. The characteristics of the software are underlined and its simulation performances are presented through different examples. Comparisons with other models are also performed.
Partition coefficients for phenol; o-, m-, and p-cresols; 3,5-xylenol; and α-naphthol in an ionic liquid/water two-phase system were measured at 298.2 K. 1-Butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]) was used as the ionic liquid. Further, the infinite dilution partition coefficients were also determined by extrapolation. The infinite dilution partition coefficients were correlated by regular solution theory. Interaction parameters between phenolic compounds and [bmim][PF6] pairs were determined by plotting the infinite dilution partition coefficients against the solubility parameters of the phenolic compounds. The correlated results are in good agreement with the experimental data.
Quantum mechanical molecular descriptors and physical properties have been used to estimate log Kow values for a range of substituted phenols using multilinear regression. The quantum mechanical molecular descriptors were derived from a commercially available software package. Two models have been proposed. The first model involves molecular mass and EHOMO as independent variables whereas the second model combines the following molecular descriptors: molecular mass, melting point, charge on oxygen atom, EHOMO, ELUMO, molecular volume, total surface area, refractivity, and polarizability. The equations were derived using a set of 14 reference phenolic compounds with their log kow values being the average of several experimental values reported in the literature. The predictive power of the derived model equations was compared to a set of 18 test compounds, whose log Kow values have been estimated by an isocratic liquid chromatography (LC) procedure, log Kow values of the test compounds estimated on the basis of the two equations showed good correlation with LC estimated log Kow values.
Indole and its derivatives form a class of toxic recalcitrant environmental pollulants. Sporotrichum thermophile was grown in a persolvent fermentation system containing a large amount of indole. The medium contained up to 20% by volume soybean oil and up to 2gL−1 indole. Most of the indole was partitioned in the organic solvent layer. When the organism was grown in the medium containing indole at 1gL−1, indole was totally consumed after 6 days. Under a fed–batch fermentation process where daily batches of indole (1gL−1) supplemented the microbial culture for 4 days, the biodegradation level was 3.0gL−1. These values make this process promising and worthy of further investigation for the microbial degradation of other toxic compounds.
1-Octanol–water partition coefficients (Kow) of s-triazines and some of their degradation products were determined using two reversed-phase high-performance liquid chromatography (HPLC) methods. In the first method, an octadecyl column was coated with 1-octanol and eluted with 1-octanol–saturated water. Using this method, a maximum logKow of 2.86 could be determined. For higher values a recently developed gradient HPLC method was used. The determined 1-octanol–water partition coefficients are helpful for studies and mathematical models dealing with the fate of s-triazine herbicides and their degradation products in the environment.
The OECD test guideline 203 for determination of fish acute toxicity requires substantial numbers of fish and uses death as an apical endpoint. One potential alternative are fish cell lines; however, several studies indicated that these appear up to several orders of magnitude less sensitive than fish. We developed a fish gill cell line-based (RTgill-W1) assay, using several measures to improve sensitivity. The optimized assay was applied to determine the toxicity of 35 organic chemicals, having a wide range of toxicity to fish, mode of action and physico-chemical properties. We found a very good agreement between in vivo and in vitro effective concentrations. For up to 73% of the tested compounds, the difference between the two approaches was less than 5-fold, covering baseline toxicants but as well compounds with presumed specific modes of action, including reactivity, inhibition of acetylcholine esterase or uncoupling of oxidative phosphorylation. Accounting for measured chemical concentrations eliminated two outliers, the hydrophobic 4-decylaniline and the volatile 2,3-dimethyl-1,3-butadiene, with an outlier being operationally defined as a substance showing a more than 10-fold difference between in vivo/in vitro effect concentrations. Few outliers remained. The most striking were allyl alcohol (2700-fold), which likely needs to be metabolically activated, and permethrin (190-fold) and lindane (63-fold), compounds acting, respectively, on sodium and chloride channels in the brain of fish. We discuss further developments of this assay and suggest its use beyond predicting acute toxicity to fish, e.g. as part of adverse outcome pathways to replace, reduce or refine chronic fish tests.
The cytotoxicites of 13 anilines and 10 aldehydes to goldfish scale GFS cells were determined with the neutral red assay. The sequence of cytotoxicity was based on the concentration of chemicals that reduced uptake of neutral red by 50 % (NR50 values). Among a series of aniline derivatives, the parent aniline molecule was the least cytotoxic, with potency increasing with the progressive incorporation of chlorine atoms or with the alkyl chain length into the aromatic ring structure. Among a series of aldehyde derivatives, methanal (formaldehyde) and propanal, which have less than two carbon atoms of the alkyl chains, were more cytotoxic than predicted from the alkyl chain length. The in vitro cytotoxicity of these chemicals except for methanal was found to be significantly correlated to their in vivo acute toxicity to guppies. Moreover, NR50 values were significantly correlated with 1-octanol/water partition coefficients (Pow) excluding the lower aliphatic aldehydes.
In vitro cytotoxicity of chlorophenols to Cyprinus carpio brain (CCB) cells derived from carp and Oryzias latipes fin (OLF 136) cells derived from medaka was examined with the neutral red (3 amino 7 dimethylamino 2-methylphenazine hydrochloride) incorporation assay Results were compared with previous cytotoxicity data of chlorophenols to goldfish (Carassius auratus) scale (GFS) cells derived from the goldfish There were excellent correlations between the 24 h NR50 values of chlorophenols toward the three kinds of cells (r2 > 0 94)
The thermal resistance traits of three clonal lines of the silver crucian carp Carassius langsdorfii (SCC-1, -2 and -3) were investigated. Individual juvenile fish reared at 20°C were exposed to thermal stress at 36.5 ± 0.5°C and their death times during this exposure were measured. The death times of SCC-2 and SCC-3 fish were 66.6 ± 31.2 and 144.7 ± 49.8 min, respectively. In contrast, all SCC-1 fish survived under these conditions. Furthermore, the thermal sensitivity of primary culture cells from each clone was determined at 37, 40 or 43°C by the Trypan blue assay. Under all treatment conditions, the thermal sensitivity of the SCC-1 primary cultures was lower than those of the others. These results estimated the correlation between the in vivo and in vitro thermal resistances. Therefore, the use of primary culture cells to evaluate thermal resistance could be a useful method of selection breeding.
Four aromatic compounds were evaluated in laboratory studies to investigate their accumulation and toxicant-induced changes in the rate of heat dissipation in the freshwater invertebrates Chironomus riparius and Lumbriculus variegatus. The sublethal energetic response detected by direct calorimetry was related to tissue chemical concentration by the threshold model and an attempt was made to apply the critical body residue (CBR) concept. Below the compound-specific tissue threshold concentration or CBR, no correlations were found between the dose and the metabolic rate, and the slopes of the regression were close to zero. Above the threshold, depending on the chemical, metabolic rate either increased or decreased. An increase in heat output produced by 2,4-dinitrophenol (2,4-DNP), pentachlorophenol (PCP), and 2,4,5-trichlorophenol (2,4,5-TCP) was closely correlated with the dose. The order of toxicity for these phenols was 2,4-DNP = PCP > 2,4,5-TCP, which reflects the interaction of compounds' lipophilicities and acidities and their combined influence on bioaccumulation and effects on the energy-transducing membrane by uncoupling oxidative phosphorylation. A decrease in the heat output caused by 1,2,4-trichlorobenzene (1,2,4-TCB) was more variable relative to dose. Also, 1,2,4-TCB required a much higher molar tissue threshold concentration (∼2.0 μmol/g wet weight) than required by phenols to generate the response. Both the metabolic response and the chemical threshold value were those expected to result from narcosis. Results suggest that calorimetric measures can identify not only the integrated physiologic response but also have some resolution of the mechanism of toxic effects.
IntroductionMathematical Models for Environmental Fate and Exposure of ChemicalsAssessment Methods of the Toxic Effects of ChemicalsFish Cytotoxicity AssayHazard Assessment of Chemicals in JapanConclusion and Future ProspectsAcknowledgements
A suspension-cultured fish cell line was applied to develop a rapid and convenient cytotoxicity assay to evaluate toxicity of a large number of chemicals for fish. Suspension-cultured fish cells, CHSE-sp cells, and monolayer-cultured fish cells, CHSE-214 cells were used. Cytotoxicity of 11 synthesized chemicals chosen from various chemical classes was determined by the neutral red assay. A good correlation was obtained between cytotoxicity values of 11 chemicals to the CHSE-sp cells and to the CHSE-214 cells. Also, good correlations were observed between in vitro toxicity data obtained from the assay using two types of cultured fish cells and in vivo toxicity data obtained from a database on fathead minnow and rainbow trout. From these results, the method using suspension-cultured fish cells used in this study was considered applicable for a screening test method prior to in vivo testing.
Cytotoxicity testing of non-hydrosoluble chemicals poses a major problem because cells are always cultured in aqueous media. The test chemicals were, therefore, emulsified by ultrasonication, before treating the cells for 2h with different concentrations. The cytotoxicity of 19 lipophilic solvents, belonging to structurally unreleated chemical classes, was measured on cultured FHM (fathead minnow fish) cells by the neutral red uptake inhibition assay. The results are expressed as the NI50, which is the concentration of test compound required to induce a 50% reduction in neutral red uptake. A correlation coefficient r=0.86 was obtained when the NI50 values were compared with fish lethality data (LC50) obtained on golden orfe by Juhnke and Lüdemann (1978). Hence it appears that emulsification of lipophilic chemicals by ultrasonication allows to measure their cytotoxicity on cultured cells, and that this method can be considered as a valuable alternative tool for fish lethality testing.
Whole cells of Pseudomonas sp. were employed as the primary sensing element of a microbial biosensor suitable for online estimation of p-nitrophenol based on a Clark type oxygen electrode. In this process p-nitrophenol adapted Pseudomonas cells were immobilized on the polycarbonate membrane having pore size of 0.4 μm and placed on the tip of Clark type dissolved oxygen (DO) electrode to measure the change in oxygen concentration due to degradation of p-nitrophenol and consumption of oxygen by the Pseudomonas cells. The present study was carried out, keeping in view the possible use of the biosensor for field and online measurements of p-nitrophenol. It was tried to overcome the major limitations of the microbial biosensors viz. slow response, fixed temperature and requirement of a buffered solution of fixed strength and pH by developing a p-nitrophenol adapted Pseudomonas sp. microbial biosensor in a wide range of temperature and pH. The biosensor is suitable for quantification of p-nitrophenol in the concentration range of 10–50 μM with the varying pH (7.5–8.5) and temperature (25–40 °C) in short response time (7.0 min).
This investigation has been performed for the account of the Directorate-General for Environmental Protection, Directorate for Chemicals, Waste and Radiation, in the context of the project 'International and national environmental quality standards for substances in the Netherlands', RIVM-project no. 601782.
The growth of Acinetobacter calcoaceticus 69-V on the alcohols ethanol, n-propanol, n-pentanol, n-hexanol and on phenol caused an alteration in its fatty acid composition leading to a gradual increase in the degree of saturation of the C16 acids from 55.4% to 83.5%, which (apart from phenol-grown cells) correlated to an increase in the resistance of the electron-transport phosphorylation against the effect of 2,4-dinitrophenol. These changes are in principle paralleled by changes observed when the growth temperature was increased in the sequence 20, 30 and 40C with acetate as the carbon and energy source. However, in cells grown at 40C, as in the case of phenol-grown cells, resistance decreased. This effect could be caused by an increase in the fluidity of the target membrane since, by contrast, the increase in sensitivity induced by growth at 40C can be partially annulled by provoking a decrease in fluidity by performing the inhibition measurements at a lower temperature (20C). Both the degree of saturation of the fatty acids and the fluidity of the cytoplasmic membrane are features that should enable the resistance of Acinetobacter calcoaceticus 69-V to xenobiotics to be predicted.
Molecular modeling techniques were used to establish relationships between the octanol/water partition coefficient (K
ow) and molecular properties of 90 herbicides with several types of chemistry. The Kow values were obtained from the literature. Various molecular properties were calculated by quantum mechanical methods using molecular modeling software. The quantitative structure activity relationship (QSAR) analysis of all herbicides showed that K
ow was dependent on bulk (van der Waals volume, VDWv) and electronic (dipole moment, ; superdelocalizability of highest occupied molecular orbital, SHOMO; nucleophilic superdelocalizability, SN) properties, and the model explained 68% of the variation in K
ow. Herbicides were broadly divided into six families (ureas, acid amides, triazines, carbamates, diphenyl ethers, and dinitroanilines) based on structural similarities, and separate equations were established for each group. The QSAR models accounted for 74 to 98% of the variation in K
ow within these six groups. Applicability of these models was tested for some herbicides. The QSAR models produced estimates that correlate well with experimental values and appear to be specific to structurally similar compounds.
The thermal tolerances of the ayu Plecoglossus altivelis and Japanese flounder Paralichthys olivaceus were evaluated by assaying the primary culture cells and the free cells from their caudal fins (at 43 °C for 2 h) in comparison with the whole body tolerances of juveniles (at 30 °C for the ayu and 32 °C for the Japanese flounder). Primary culture cells and free cells were assayed separately and similar results were obtained for each type of cells. The thermal tolerance values observed in the whole body were significantly correlated with the values obtained from both the primary culture cells and the free cells. These results suggest that there is a significant correlation between the in vivo and in vitro thermal tolerances in these fish. In the case of the ayu, significant differences were observed between two lines for the mean tolerance values evaluated by all three methods. This finding suggests that cell assays with primary culture cells or free cells from a caudal fin clip could be useful for evaluating the genetic variation and genetic mechanisms of thermal tolerance of fish.
The isomerization of cis to trans unsaturated fatty acid in Pseudomonas putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences, was tested as an indicator for toxicity of 10 organic compounds (aromatics and aliphatic alcohols). A direct correlation was observed between the hydrophobicity of the compounds (logP), concentration dependent growth inhibition, and the trans/cis ratio of unsaturated fatty acids. The application of this system is discussed in terms of an indicator for toxicity and environmental stress particularly during bioremediation processes.
Many processes in modern biotechnology, particularly biotransformations and environmental bioremediation, are hindered by the toxic effects of organic solvents on whole cells. These compounds dissolve in the cell membrane, disturbing its integrity and effecting specific permeabilization. The hydrophobicity of a compound, expressed as its log P value, is a good indicator of toxicity. Substances with a log P value in the range 1–5 are, in general, toxic to whole cells. However, in recent years, there have been several reports of bacteria exhibiting resistance to toxic solvents. The main adaptative reactions are alterations in the composition of the membrane, particularly changes in fatty-acid composition, phospholipid headgroups, and in the protein content. One of the key processes in the adaptation of some Pseudomonas strains, enabling them to tolerate organic solvents appears to be the isomerization of cis- into trans-unsaturated fatty acids. A greater understanding of these adaptations should eventually allow biotransformation reactions to be carried out in inhospitable two-phase systems incorporating an organic phase.
The determination of octanol-water partition coefficients (log K(ow)) is important for the prediction of the fate of organic pollutants in the environment. Traditionally, log K(ow) values are determined by shake-flask, estimated by, e.g., HPLC retention data, or calculated, e.g., from ClogP. In this paper, an alternative approach is reported that allows log K(ow) to be estimated from solid-phase microextraction (SPME) data. Previously reported attempts to correlate SPME data with log K(ow) are discussed. The results obtained in this work for six phenols, using an 85 μm polyacrylate-coated fiber, indicate that SPME is a viable method for estimating log K(ow) values <3.5.
The cytotoxicity of phenol and 14 chlorophenols (CPs) were examined in goldfish scale (GFS) cells, a fibroblastic cell line derived from goldfish scales. MTT reduction and intracellular LDH activities were examined as cytotoxicity indicators. Cell densities at inoculation were 1.0 x 10(5) cells/ml in the MTT assay and 0.5 x 10(5) or 0.1 x 10(5) cells/ml in the LDH assay. No change in the midpoint cytotoxicity concentration (IC(50)) was observed in either assay at the same order of cell density (0.5-1.0 x 10(5) cells/ml). However, at low chemical concentrations, the LDH assay was more sensitive than the MTT assay. In addition, for many CPs, the LDH assay at 0.1 x 10(5) cells/ml was more sensitive than the MTT and LDH assays at the higher cell density levels. The correlation coefficients (r) for all IC(50) determinations were greater than 0.97. Good correlations between these assays and the neutral red (NR) assay were also observed (r > 0.97). The 1-octanol/water partition coefficient (log P) gave good correlation coefficients (r > 0.90) with IC(50) values of both the MTT and LDH assays in simple linear regression analyses. The study demonstrates that both the MTT and LDH cytotoxicity assays with GFS cells, together with the NR assay, can be applied to screen the acute toxicities of chemicals.
The growth inhibitions of the yeast strains Pichia sp., Rhodotorula sp., Rhodotorula
rubra and Saccharomyces
cerevisiae (baker) by aniline (A), and the chloroanilines 4-C1-A; 2,4-Cl2-A; 2,6-Cl2-A; 3,5-Cl2-A; 2,3,4-Cl3-A; 2, 3, 5,6-Cl4-A; and 2,3,4,5,6-Cl5-A were determined. In all cases, the toxicities were found to increase in the order A < 4-C1-1 < 2,6-Cl2-A < 2,4-Cl2-A < 3,5 Cl2-A < 2,3, 4-Cl3-A. Linear structure-activity correlations were obtained for the toxicites as dependent on the octanol/water partition coefficients. The chloroaniline toxicities are also linearly correlated with those to Photobacterium phosphoreum and with the growth inhibitions by the corresponding chlorophenols to the same yeast species.
The 96-hr LC50 values of 21 substituted phenols for the guppy (Poecilia reticulata) were determined at pH levels 6-8 and related to the lipophilicity defined as log P from the 1-octanol/water system, and to the delta pKa value (pKa of phenol-pKa). Log P was the more important parameter and exhibited a good correlation with log(1/LC50) at pH levels. The contribution of delta pKa when introduced as a second parameter into the regression equation was dependent on the pH of water: at pH 6 it was positive but turned negative as the pH was raised to 8. If the LC50 values were corrected for ionization using an empirically formulated relation between toxicity and pH, the resulting regression equation could be used to predict the toxicity at any pH from 6 to 8. When corrected for ionization, log BCF (the bioconcentration factor) of 8 phenols was highly correlated with log P but not with delta pKa. The regression of log BCF on log P sufficed to explain the regression of toxicity on lipophilicity.
The influence of pH on the toxicity of ionizable chemicals to fish has been described with a formula, developed on basis of Tabata's ideas. The toxicity of 11 chlorophenols to guppies has been determined at 3 pH-values and calculated with this formula at another pH. QSARs can be calculated for the LC50's at each of these pH-values, with log Poct and pKa as variables.
LC50-experiments have been conducted using guppies subjected to 72 industrial pollutants. The correlation of the LC50 with several expressions of the hydrophobicity of these chemicals has been studied. Calculated log Poct-values appeared to satisfy more than HPLC retention indices, solubility data or molecular connectivity indices. One QSAR, with log Poct as the only variable, gave good estimations of the toxicity of most of the tested compounds with log Poct less than 6. No LC50 could be determined for solutions of compounds with log Poct greater than 6.
Subjects covered in the 21 papers of the workshop proceedings include QSAR of azaarenes, aromatic amines, chloroanilines, benzenes, biphenyls, phenols, and hydrocarbons, non-electrolyte herbicides, para-substituted phenols, oils, and other compounds with a variety of aquatic and terrestrial species. Included are papers on acute, subacute, chronic, and mutagenic effects of compounds, on comparative structure-toxicity relationships, pattern recognition, the environmental hazard profile analysis, QSAR of BOD, the role of QSAR in ecotoxicology, data evaluation and research needs for contaminant fate modelling ickes, and interrelations of physical-chemical with environmental partitioning and bio-concentration processes. Papers are abstracted separately. -from Publisher
Acute toxicities and hatching inhibitions of embryo by 14 chlorophenols from mono-Cl to penta-Cl and phenol to Japanese Medaka, Oryzias latipes, have been determined as 96 h-LC50 and 15 d-EC50, respectively and studied in terms of quantitative structure-activity relationship (QSAR). The 6 physico-chemical parameters of chlorophenols employed in this study were : n-octanol/water partition coefficient, log Pow ; dissociation constant, pKa ; Hammett constant, Σσ ; index of valence molecular connectivity, 1Xv ; perimeter of the efficient cross section of molecule, ΣD ; melting point, F. On the whole, acute toxicity and hatching inhibition of chlorophenols increase with a degree of chlorination and ortho-isomers appear as less toxic than meta-and para-isomers. QSAR study shows that log Pow, a parameter of hydrophobicity, gives the best correlation with the activity. With regard to each chlorophenol, 96 h-LC50 of Oryzias latipes agrees with that of another kind of fish, Lebistes reticulatus (guppy). High correlations were found among 96 h-LC50 and 15 d-EC50 values of Oryzias latipes, EC50 values of growth inhibition of green algae, Selenastrum capricornutum and EC50 values of immobilization of Daphnia magna, Daphnia carinata and Daphnia pulex.
Narcosis is a reversible state of arrested activity of protoplasmic structures caused by a wide variety of organic chemicals. This nonspecific mode of toxic action was found predominant in acute toxicity studies of industrial chemicals and fish. This paper presents 96-h LC50 values for 65 industrial chemicals including alcohols, ketones, ethers, alkyl halides, and substituted benzenes. The common mode of action permitted the development of a structure–toxicity relationship as follows: log LC50 = −0.94 log P + 0.94 log (0.000068P + 1) −1.25 where P is the n-octanol/water partition coefficient. The data show that the toxicity of the chemicals to fish is directly comparable with the toxicity in mammals when expressed as chemical activity.
GF-Scale (GFS) cells, a fibroblastic cell line derived from the scale of goldfish, were used for the determination of the cytotoxicity of chlorophenols (CPs) and the quantitative structure-activity relationships (QSAR) studies. As the cytotoxicity end point, the amount of neutral red retained by viable cells after exposure to chemicals was quantified. The sequence of cytotoxicity based on the concentration of chemicals that reduced uptake of neutral red by 50% (NR50) was penta-Cl (PCP) > 2,4,5-Cl3 > 2,3,4-Cl3 > 2,3,4,6-Cl4 > 3,5-Cl2 > 3,4-Cl2 > 2,4-Cl2 > 2,5-Cl2 > 2,3-Cl2 > 2,4,6-Cl3 > 3-Cl > 4-Cl > 2,6-Cl > 2-Cl > phenol. The in vitro cytotoxicity of these chemicals was found to be significantly correlated to their in vivo acute toxicity to aquatic species, and NR50 values were correlated with six physicochemical parameters of chlorophenols. Log Pow (n-octanol/water partition coefficient) gave the best correlation in simple linear regression analysis, as is frequently stated in toxicity studies with aquatic animals. Multiparametric linear regression equations yielded improved correlation coefficients and predictive capabilities, including the log Pow and pKa. These results suggest that in vitro fish cytotoxicity assays using the GFS cell lines are useful for ecotoxicity screening of aquatic pollutants.
The effects of 57 phenols, anilines and aliphates on yeast growth rate as well as on structural and functional properties of the plasma membrane have been examined. The different systems correlated with each other and with data from higher eucaryotic organisms. Close relations between the activity of the chemicals in the different biological test systems and structural parameters, especially hydrophobicity, were observed.
In this paper a scheme is presented that makes it possible to classify a large number of organic pollutants into one of four classes, viz: [1] inert chemicals, [2] less inert chemicals, [3] reactive chemicals and [4] specifically acting chemicals. For chemicals that are thus classified as belonging to one of these four classes it is possible to calculate either an expected effect concentration (inert chemicals), such as the LC50, or an expected range of possible effect concentrations, from a compound's octanol/water partition coefficient (Log Kow). For chemicals that cannot be classified as belonging to one of these four classes no prediction can be made.This approach can be implemented to estimate aquatic effect concentrations, which can be used to derive preliminary environmental quality objectives, or for the prioritisation of chemicals for subsequent testing. Moreover, these estimates could be of great value in risk and hazard assessment. To our opinion, this approach represents the current state-of-the-art in estimation methods for aquatic toxicology. This paper is especially focused on identifying the limits of applicability of Quantitative Structure-Activity Relationships for predicting aquatic toxicity, as, according to Hart (1991) it “…is important not to exaggerate the accuracy of prediction for models…” [and] “…to make clear to the user the limitations of the relationship.”
This study was undertaken to test the ability of the molecular connectivity model to predict the level of acute toxicity of alcohols, ethers, aldehydes, ketones, nitriles, aliphatic and aromatic amines, halogenated hydrocarbons, substituted benzenes, and phenols for fish. We obtained good linear correlation between the valence zero-order molecular connectivity index (0χv) and 96-h LC50 data for a large group of commercial chemicals. Our results and their comparison with previously published models demonstrate that the molecular connectivity model is an accurate predictive tool for assessing the level of acute toxicity of a wide range of commercial chemicals. It can be confidently used to rank potentially hazardous chemicals and to create priority testing lists. In addition, it is shown that the molecular size of chemicals is directly proportional to the fish acute toxicity and that is the most important structural feature that accounts for 85% of the variation in the measured fish acute toxicity data. Finally, an attempt was made to critically evaluate and compare all published QSAR models for predicting acute toxicity for fish.
The cytotoxicities of 45 pesticides including 18 organophosphates and 7 carbamates to GF-Scale (GFS) cells, derived from the scale of goldfish (), were determined with the 24-hr neutral red assay. The assay revealed that 13 pesticides were classified into the highly cytotoxic group (NR50 ≦ 10 mg/L), while 17 pesticides were into the highly cytotoxic group (NR50 ≦ 10 mg/L), while 17 pesticides were moderate (10 < NR50 ≦ 100 mg/L) and 15 were relatively low (100 mg/L < NR50). Carbamates were less cytotoxic (200 mg/L < NR50) than organophosphates. And the 24-h NR50 values for the cytotoxicity to GFS cells were compared to published 48-h LC50 values for the acute lethal toxicity of the same pesticides to carp. A significant correlation was found between the NR50 and LC50 values (n=34, r=0.848, s=0.381, F=81.5) excluding 11 pesticides that neither NR50 nor LC50 values were not obtained for their low solubility. This result demonstrates that the in vitro cytotoxicity assays using GFS cells are useful for the prediction of the acute toxicity of pesticides to fish.
The logarithm of the retention time (log RT) of organic chemicals on a permanently bonded (C-18) reverse-phase high-pressure liquid chromatography system is shown to be linearly related to the logarithm of the n-octanol/water partition coefficient (log P). A rapid, inexpensive technique based on reverse-phase high-pressure liquid chromatography has been developed to estimate the n-octanol/water partition coefficient of organic chemicals. The system consists of a preparative Micro-Pak® C-10 reverse-phase column eluted with a 15% water/85% methanol solvent flowing at 2 ml min−1 at room temperature.A linear calibration of the logarithm of retention time with the logarithm of the partition coefficient (log P) is attained by using a mixture of benzene, bromobenzene, biphenyl, bibenzyl, p,p'DDE, and 2,4,5,2′,5′-pentachlorobiphenyl as reference standards of known log P. Using a calibration mixture, the log P of other organic chemicals were estimated with a mean accuracy of 22.8% of the log P values reported in the literature. The technique permits estimation of log P in a maximum of 25 min and does not require a knowledge of the structure of the chemical for the estimate.
Cultured fish cells can be used in a variety of cytotoxicity and genotoxicity assays for the preliminary testing of environmental chemical hazards that may be hazardous to the aquatic biota. Such assays can also be used to evaluate synergistic and antagonistic interactions between combinations of test agents and to establish structure-activity relationships for series of related chemicals. A range of fish cell lines are available for use in such assays and a variety of endpoints may be used. To detect toxicants that require bioactivation the chosen cell line must have significant P-450 activity, or a metabolizing component must be incorporated into the assay. Fish cells in culture respond to the same chemical mutagens and clastogens that are genotoxic to mammalian cells in culture. However, since fish cells in culture are eurythermic, they represent a unique system for studying temperature as a parameter in mediating the genotoxicity and the cytotoxicity of a test agent.
The neutral red (NR) cytotoxicity assay has been applied to the determination of structure-activity relationships (SARs). The in vitro cytotoxic potency of a series of alcohols with varying chain lengths, of chlorinated phenolics, of nitro- and methyl-containing phenolics and of chlorinated toluenes could be directly correlated with the lipophilicity of the molecules, expressed in terms of their log octanol/water partition coefficients (log P values). Dinitrotoluenes were outliers, in that their cytotoxicity was not a function of their lipophilicity. The sequence of cytotoxicity for a series of divalent cationic metals was a function of softness: the softer the metal (i.e. the lower its chemical softness parameter, sigma(p)), the greater its cytotoxicity. The SAR models developed with the NR assay were consistent with similar models developed in vivo. The NR assay has been further modified to include, when useful, total protein determinations.
Quantitative structure-activity relationships (QSARs) were developed for nonreactive chemical toxicity to each of four groups of bacteria of importance in environmental engineering: aerobic heterotrophs, methanogens, Nitrosomonas, and Microtox. The QSARs were based on chemicals covering a range of structures and including important environmental pollutants (i.e., chlorinated and other substituted benzenes, phenols, and aliphatic hydrocarbons). QSARs were developed for each chemical class and for combinations of chemical classes. Three QSAR methods (groups of chemical describing parameters) were evaluated for their accuracy and ease of use: log P, linear solvation energy relationships (LSER), and molecular connectivity. Successful QSARs were found for each group of bacteria and by each method, with correlation coefficients (adjusted r2) between 0.79 and 0.95. LSER QSARs incorporated the widest range of chemicals with the greatest accuracy. Log P and molecular connectivity QSARs are easier to use because their parameters are readily available. Outliers from the QSARs likely due to reactive toxicity included acryls, low pKa compounds, and aldehydes. Nitro compounds and chlorinated aliphatic hydrocarbons and alcohols showed enhanced toxicity to the methanogens only. Chemicals with low IC50 concentrations (log IC50 mumol/liter less than 1.5) were often outliers for Nitrosomonas. QSARs were validated statistically and with literature data. A suggested method is provided for use of the QSARs.
The Na+/K+-ATPase of Chinese Hamster Ovary (CHO) cells, a plasma membrane bound protein was used as a test system to evaluate the toxicity of several phenol derivatives on membranes. Taking only 2 physico-chemical parameters into consideration, viz., the logarithm of the octanol/water partition coefficient as an indicator for the lipophilicity and the sigma-Hammett constant as a measure for the polarity of the phenol substitutes, it was possible to predict the toxicity with high significance. A multivariate regression analysis calculated a correlation coefficient of 0.99. The results confirm studies performed in our laboratory on cytotoxicity and on functional membrane proteins of fungal and mammalian cells [1,2], suggesting a common mechanism of toxicity by the action of hydrophobic xenobiotics on biomembranes. Taking into account the different sensitivities of the test systems, Quantitative Structure-Activity Relationship (QSAR) analyses could help to explain the basic toxicity of several classes of environmental chemicals.
The suitability of the fathead minnow (FHM) epithelial cell line for use as the target (indicator) system in in vitro cytotoxicity assays was evaluated using several endpoints. The organometal diethyltin dichloride served as the representative test agent. The concentration of diethyltin dichloride which resulted in a midpoint toxicity was 3.5 microM in a 3-day cell growth assay, 3.8 microM in the 24-hr neutral red assay, and 16.5 microM in a 4-hr cell detachment assay. The neutral red assay was used to compare the relative sensitivities of the FHM cells (exposed at 34 degrees C) with those of bluegill sunfish (BF-2) cells, a fibroblastic cell culture (exposed at 26 degrees C), in the presence of different classes of test agents frequently occurring as aquatic pollutants. For both fish species the sequence of potencies of the test agents was in the order of organometals greater than pesticides approximately equal to polychlorinated biphenyls greater than polynuclear aromatic hydrocarbons greater than phenolics. Overall, the FHM cells were more sensitive than were the BF-2 cells. However, there was a better correlation between the in vitro cytotoxicity data for the BF-2 cell culture and LC50 data for bluegill sunfish than between similar data for the FHM cell line and fathead minnows.
The in vitro cytotoxicities of various polycyclic aromatic hydrocarbons (PAHs) to bluegill sunfish BF-2 cells were determined with the neutral red assay, which was modified by the incorporation of an S-9 microsomal fraction. Whereas the PAHs per se were weakly cytotoxic, the presence of the S-9 fraction in the incubation mixture increased the cytotoxicity of many of the PAHs, presumably due to the formation of active metabolites. In addition, the cytotoxicity of the PAHs (whether in the absence or presence of the S-9 microsomal fraction) was potentiated if the 6 h exposure was at 37, rather than at 26 degrees C.
BF-2 cells, an established fibroblastic cell line derived from the caudal fin of bluegill sunfish (Lepomis macrochirus), were exposed to 18 metal salts. Cytotoxieity was assayed by the neutral red (NR) technique. Based on the concentration of metal that reduced the uptake of neutral red by 50% (NR50), the rank order of Cytotoxieity for the cationic metals was silver > mercury > cadmium > zinc > copper > cobalt > nickel > lead > tin > manganese > chromium (trivalent), and for the anionic metal complexes it was arsenite > dichromate > chromate > arsenate > selenite > permanganate > selenate. There was a strong correlation (r = 0.909) between the NR50 ranking for the divalent metal cations and their chemical softness parameters (σp). A good correlation (r=0.833) was also found between thein vitro NR50 Cytotoxieity values for the cationic metals and thein vivo water-borne LC50 values for bluegill. There was no correlation, however, between NR50 and LC50 data for the anionic metal complexes, primarily due to the unusual tolerance, as reported in the literature, of fish for hexavalent chromium salts.
The cytotoxicity of cadmium toward cultured bluegill fry (BF-2) cells was determined using several assay endpoints. The concentrations of cadmium causing a 50% decrease in colony formation, cell replication, uptake of neutral red, population growth (as determined by protein analysis), and uptake of [3H]uridine and 50% detachment of cells (as determined by protein analysis) were 0.03, 0.04, 0.08, 0.09, 0.12, and 0.21 mM cadmium, respectively. The neutral red assay was used to compare the relative sensitivities of bluegill BF-2 cells and RTG-2 cells, derived from the rainbow trout, toward four metals. The concentrations of cadmium, zinc, copper, and nickel causing a 50% reduction in the uptake of neutral red were 0.08, 0.19, 0.55, and 2.0 mM, respectively, with the BF-2 cells and 0.18, 0.64, 1.45, and greater than 10.0 mM, respectively, with the RTG-2 cells. The RTG-2 cells were less sensitive to the metals, in particular to nickel. The less stringent temperature requirements for growth, their greater sensitivity to pollutants, and their markedly shorter doubling time in vitro make the BF-2 cells the preferable cell line for ecotoxicity screening of aquatic pollutants.
A method is described which combines the use of a visual morphological cytotoxicity assay with a quantitative neutral red (NR) spectrophotometric test, for the assessment of the effect of toxic agents on 3T3 cells in culture. These sensitive and reproducible assays lend themselves to a screening procedure of potential toxicants which can help reduce the use of animals for toxicity testing.
Quantitative structure-activity relationships were calculated for the inhibition of bioluminescence of Photobacterium phosphoreum by 22 nonreactive organic chemicals. The inhibition was measured using the Microtox test and correlated with the partition coefficient between n-octanol and water (Poct), molar refractivity (MR), and molar volume (MW/d). At log Poct less than 1 and greater than 3, deviations from linearity were observed. Introduction of MR and MW/d improved the quality of the relationships. The influences of MR or MW/d may be related with an interaction of the tested chemicals to the enzyme system which produces the light emission. The sensitivity of the Microtox test to the 22 tested compounds is comparable to a 14-day acute mortality test with guppies for chemicals with log Poct less than 4. The inhibition of bioluminescence by a mixture of the tested compounds was slightly less than was expected in case of concentration addition. The Microtox test can give a good estimate of the total aspecific "minimum toxicity" of polluted waters. When rather lipophilic compounds or pollutants with more specific modes of action are present, this test will underestimate the toxicity to other aquatic life.
Quantitative structure-activity relationships (QSARs) were calculated for the acute lethal toxicities (14-day LC50) to the guppy (Poecilia reticulata) of anilines, with several hydrophobicity characteristics and the Hammett sigma constant as parameters. Calculated and experimental Poct values and II constants were used as hydrophobicity characteristics. Introduction of Hammett constants improved the quality of the QSARs. Together with the QSAR studies the toxicities of mixtures of the anilines were determined. The toxicities of three mixtures did not deviate from concentration addition.
The n-octanol/water partition coefficient (Pow) for organic chemicals which do not form the hydrogen bond was considered on the basis of London's dispersion force, because the dispersion force is the main component of van der Waals-type attractive forces between solute and solvent molecules. The dispersion force is a function of both the ionization potential and the molar refraction. As the variation of the ionization potential is small in comparison with that of the molar refraction, the latter is the most important factor to determine the value of Pow. Thus the relationship between Pow and the molar refraction can be established. The relationship was confirmed for some halogenated alkanes, alkenes and aromatics, and polycyclic aromatic hydrocarbons by analysis of observed values of Pow and calculated values of the molar refraction. Estimation of Pow from the molar refraction is advantageous for the preliminary hazard assessment of new chemicals, because direct measurement of Pow is laborious and time consuming for lack of information about analytical methods.
The acute median lethal concentrations of equitoxic mixtures of 8 and 24 toxicants with diverse modes of action to guppies were determined. To quantify the joint toxicity, the results are expressed by means of the Mixture Toxicity Index (MTI). The toxicity of the mixtures was near concentration addition. Concentrations of the chemicals of about 0.1 of their LC50's contributed to the toxicity of the mixtures.
Aquat.Toxicol.Hazard Assess. 8th Symp.
- Lipnick