Hydractinia echinata test system. II. SAR toxicity study of some anilide derivatives of Naphthol-AS type

Chemosphere 82 (2011) 1578–1582

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Hydractinia echinata test system. II. SAR toxicity study of some anilide derivatives of Naphthol-AS type Sergiu Adrian Chicu a,1, Simona Funar-Timofei a,⇑, Georgeta-Maria Simu b a b

Institute of Chemistry Timisßoara of the Romanian Academy, B-dul Mihai Viteazul 24, RO-300223 Timisßoara, Romania University of Medicine and Pharmacy Victor Babesß Timisßoara, Faculty of Pharmacy, Piata Eftimie Murgu 2-4, RO-300034 Timisßoara, Romania

a r t i c l e

i n f o

Article history: Received 10 August 2010 Received in revised form 17 November 2010 Accepted 21 November 2010 Available online 16 December 2010 Keywords: Hydractinia echinata Naphthol-AS Class isotoxicity Omega software Conformational analysis

a b s t r a c t In this paper, a toxicity study for a series of anilides of Naphthol-AS type is presented. The toxicity of the model compounds was determined by using the Hydractinia echinata (Hydrozoa) test system. Conformational analysis of Naphthol-AS derivatives was performed to elucidate the possible enzymatic hydrolysis mechanism of these compounds. This mechanism occurs with different rates and always leads to a stoichiometric mixture of reaction products, consisting in the substituted amine and the corresponding a-hydroxy-carboxylic acid. With one exception, the toxicities of the reaction products are subadditive. Quite similar measured toxicity values, log(1/MRC50), led to their average calculated values, and thus to the establishment of class isotoxicity. This method represents a practical alternative useful for the reduction of experimental tests on animals to the lowest possible level, in accordance to the ‘3Rs’ (reduction, refinement and replacement) concept. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction One of the major concerns of the scientific research is the development of new methods for the evaluation of the toxicity in the case of persistent organic effluents, such as aromatic amines and their related dyes. The toxicity test methods currently used especially in biomedical research involve in vitro and in vivo experiments on animals, like: rodents, fish, monkeys, etc. This practice often causes death and possible distress of the subjects and presumes special equipment, a great deal of experimental work, and considerable time and expense (Anliker, 1979; Borros et al., 1999; Pielesz et al., 2002). For this reason, the development of a method for the toxicity evaluation based on biological tests on colonies of Hydractinia echinata appears as an attractive and real practical alternative. It has great advantages: simplicity, accessibility, reproducibility, time and experimental work considerably reduced. The H. echinata test system (Chicu et al., 2008) is more sensitive in comparison to the Daphnia-immobilisation test, as well as to the duckweedgrowth-inhibition test (on freshwater aquatic plants of the genus Lemna (duckweed)), which cannot differentiate the toxicity of some p-nonylphenols isomers (Preuss and Ratte, 2007). Moreover the toxicity studies by the marine colonies of H. echinata have been

⇑ Corresponding author. Tel.: +40 256 491818; fax: +40 256 491824. E-mail addresses: [email protected] (S.A. Chicu), [email protected] (S. Funar-Timofei). 1 Present address: Siegstr. 4, 50859 Köln, Germany. Tel.: +49 02234 78530. 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.11.057

carried-out for alkanes, aliphatic amines, benzene and some benzene derivatives (Chicu and Berking, 1997; Chicu et al., 2000, 2008; Simu et al., 2004; Chicu and Putz, 2009; Chicu and Simu, 2009). Generally synthetic dyes are widely used as colorants in the textile industry, as well as in many other fields, such as medicine, pharmaceutical industry, cosmetics, and food (Liu and Sun, 2008). When used as dyes, these compounds should fulfil several physicochemical requirements, most important being the solubility, affinity for cellulose fibre (Funar-Timofei and Schüürmann, 2002), as well as a low biodegradability. The presence of some aromatic amines with high toxicity among the products of biological degradation led to the interdiction of some dyes in the European Union (Eskilsson et al., 2002), in Germany the number of such amines being up to 20 (Moir et al., 2001). Generally the anilides of the 2-hydroxy-3-naphthoic acid are known as stable compounds from physico-chemical point of view. Through the waste waters they can reach the environment where by storage and subsequent degradation they can have unpredictable effects, and by assimilation/ contact they can be biodegraded to aromatic amines. Some of these amines can have mutagenic effect (Benigni et al., 1998; Suzuki et al., 2001), as in case of azo dyes via skin microflora, by experiments in vitro with Staphylococus aureus (Platzek et al., 1999) or via the human body, by the action of some enzymes including microsomal and soluble enzymes (Levine, 1991). The anilide of the 2-hydroxy-3-naphthoic acid (compound 1 in Table 1), also known as Naphthol-AS, exhibits an exceptional affinity for cotton fibres. This is the reason for which this compound is extensively used as coupling component in the synthesis of some

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Table 1 Hydractinia echinata test system: experimental toxicities (log(1/MRC50)), calculated average values (C) and their difference (DM) of some Naphthol-AS derivatives, of type: ArIC(@O)N(X)-ArII. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

C.I.a 37 505 37 520 37 521

37 511 37 525

CAS

ArI

ArII

X

log(1/MRC50)

C

DM

92 726 92 773 135 615

3-OH-2-naphthoic acid 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Naphthol 2-Anthracene 2-Hydroxydibenzofuran 2-OH-carbazole

– Phenyl 2-Me-phenyl 4-Me-phenyl 2-Et-phenyl 2-Cl-phenyl 3-Cl-phenyl 4-Cl-2-Me-phenyl 3-Br-phenyl 3-(triFMe)-phenyl 2-Nitro-phenyl 3-Nitro-phenyl 4-Nitro-phenyl 3-Me-phenyl 4-MeO-phenyl 2,4-diMeO-phenyl 2,5-diMeO-phenyl 5-Cl-2,4-diMeO-phenyl 4-COOH-phenyl Phenyl 1-Naphthyl 2-Naphthyl 2-Me-phenyl 2,5-diMeO-phenyl 4-Cl-phenyl

– H H H H H H H H H H H H H H H H H H CH3 H H H H H

3.29 3.35c 5.70c 3.64 5.44

3.55 5.54 3.55 5.54

0.20 0.16 0.09 0.10

92 762

37 515 37 516

135 659

37 535

92 795

37 545 37 550

92 739 92 728

37 560 37 565 37 585 37 605 37 600

132 683 135 648 1 830 779

b

3.38 3.47 3.65 3.85

3.55 3.55 3.55 3.55

0.17 0.08 0.10 0.30

b

5.33 3.85 4.29 3.35 5.47 3.42 5.32 3.28c 3.88 5.94c 4.81 3.52 3.49 7.05

5.54 3.55 3.55 3.55 5.54 3.55 5.54 3.55 3.55 5.54 5.54 3.55 3.55 4.55

0.21 0.30 0.74 0.20 0.07 0.13 0.22 0.23 0.33 0.40 0.73 0.03 0.06 2.52

a

C.I. – colour index number. The toxic effect is not noticed in experimental conditions. Examples of average measured values, characterizing the reproducibility of the experimental measurements (in parentheses): for No. 2: 3.35 (3.58; 3.11), for No. 3: 5.70 (6.00; 5.39), for No. 19: 3.28 (3.23; 3.32), for No. 21: 5.94 (6.15; 5.72). b

c

azo compounds which can be obtained directly on the fibre, at low temperatures. These dyes are essentially insoluble and are included in the class of vat dyes or azotols. They exhibit nice and bright shades and are characterized by good fastness to light and washing, as compared, for instance to those of anthraquinonic dyes. Elkins et al. (2000) mentioned compound features necessary for binding to a peptidase: one carbonylic oxygen atom, a hydrophobic center, and an aminic nitrogen atom (hydrogen acceptor area). A low energetic conformation and an adequate solubility are, also, required (Milne et al., 1998). From structural point of view, the Naphthol-AS molecule exhibits most of these requirements. Due to the presence of strong polarisable carbonylic group, the hydrolysis reaction of the chromophoric ACOANHA group occurs enzymatically by a probable mechanism similar to that of the hydrolysis of the peptidic bond (Alberts et al., 1983; Wilmouth et al., 2001). Besides, the rate of formation of intermediate biodegradation products will depend on the rate and intensity of the substrate–enzyme interaction. The aims of the present work are the comparative determination of the toxicities of some Naphthol-AS derivatives using the H. echinata biological test system and the proposition of a possible hydrolysis reaction mechanism of these compounds, as well as the eventuality of the apparition of the additivity phenomenon which could characterize the reaction products mixture. Conformational analysis was performed by molecular mechanics calculations to obtain minimum energy structures possibly involved in the hydrolysis mechanism.

3

2

5

3

2

3

3

2. Materials and methods Fig. 1. Structure of Naphthol-AS derivatives.

The test substances, Naphthol-AS derivatives (Fig. 1 and Supplementary material), were synthesized at the Institute of Chemistry of Timisoara of the Romanian Academy, Romania. They

were used such as or as solutions of known concentrations in methanol or synthetic seawater.

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The test conditions and method were identical to those described in previous work (Chicu et al., 2000). The larvae of H. echinata were exposed for 3 h to seawater containing CsCl and simultaneously one of the test substances. The percentage of animals that underwent metamorphosis (development into polyps) was determined the next day. The concentration of the test substance (expressed in M), was varied in such a way that we were able to determine the concentration at which the frequency of induction was reduced by 50% with respect to a control. This concentration was termed MRC50 (Metamorphosis Reducing Concentration) and it was expressed as the reciprocal value of its logarithm (log 1/MRC50). Their average (C) concentrations (Table 1) were calculated. The accuracy of experimental (log 1/MRC50) data was checked by their standard deviations, which were determined by the Microsoft Office Excel 2003 program. For each substance triplicate experiments were performed for each concentration assessment and each experiment was repeated at least twice. 3. Theory/calculation 3.1. Conformational analysis The molecular structures of 24 arylamides of Naphthol-AS type (presented in Table 1) were modeled by the conformational search ability of the Omega v.2.3.2 (OpenEye Scientific Software, Santa Fe, NM 87507) program (only structures having toxic effect on H. echinata test system were considered). Omega employs a rule-based algorithm (Tresadern et al., 2009) in combination with variants of the Merck force field 94 (Halgren, 1999). SMILES notation was used as program input. The following parameters were used for the conformer generation with Omega v.2.3.2: a maximum of 200 conformers per compound, an energy cutoff of 10 kcal mol1 relative to global minimum identified from the search. The force field used was the 94s variant of the Merck Molecular force field (MMFF) with coulomb interactions and the attractive part of the van der Waals interactions. To avoid redundant conformers, any conformer having a RMSD fit outside the range between 0.1 and 0.5 Å to another conformer was removed. 4. Results and discussion The energetically most stable structures are those in which an intramolecular hydrogen bond is formed between the hydroxilic hydrogen and amidic oxygen atoms, except compound 25 (Fig. 2), where the hydrogen bond is formed between the hydroxilic oxygen and amidic hydrogen atoms and compound 20, which does not form any hydrogen bond. The amidic bond lies out of the naphthalene plane. The torsion angle CArI(OH)CArI(CONH)C(@O)Nam (where CArI(OH) and CArI (CONH) are the two naphthalene carbon atoms attached to the hydroxilic, respectively amidic groups; C(@O) – the amidic carbon atom and Nam – the amidic nitrogen atom, in Table 1) are close to

±149–151°, except compounds 20 and 25, for which this torsion angle value is of 60°, respectively 30°. In all compounds the atoms of the amidic group are in the same plane with the phenylic moiety of the ArII (Fig. 1) molecular fragment. The molecules of the studied series of Naphthol-AS derivatives contain a central amidic (peptidic) group substituted at the carbonyl carbon atom by ortho-hydroxy-naphthalene, anthracene or heterocyclic (of carbazol or dibenzofuran type) moieties (ArI), and at the amino nitrogen atom by phenyl radicals (ArII) (Fig. 1). The hydrolysis reaction products (except those obtained from compound 20) of Naphthol-AS compounds are primary amines and the corresponding a-hydroxy-carboxylic acid derivative, in stoichiometric mixtures. In experimental conditions, at pH = 8.2, the functional groups will be protonated/ionizated, or neutral, depending on the pKa. The resulted amines will be ionized at pKa > 8.2 and the concentration of the neutral form of these compounds increases with decreasing pKa values. In the case of the acid groups, the phenomenon is inverted. According to literature the non-protonated species are more toxic than the protonated one (Sinks et al., 1998), both species having however a contribution to the general toxicity (Zhao et al., 1998; Cronin et al., 2000). The measured toxicity values log(1/MRC50) (Table 1) are compared to their calculated average values (C), considering that toxicity is constant over a range of ±0.5 logarithmic units (log u.), as compared to the calculated toxicity value. The difference between the experimental and the calculated average toxicities (DM) describes the way by which the toxicity effectiveness is close to each other and their dependences to some structural configurations (e.g. ortho, meta, para in the ArII moiety, see Fig. 1). According to Table 2, even if 12 Naphthol-AS derivatives exhibit greater experimental effective toxicity values, (F(x + y))e, as compared to the individual values of the hydrolysis products, (F(x)i – for the resulted acid and F(y)i – for the resulted amine), one could not expect the additivity of toxicity values (F(x + y)e P F(x + y)t), where F(x + y)t correspond to the theoretical values, as suggested by Backhaus et al. (1999). Only the derivative 25 exhibits a toxicity effectiveness which can be considered as a superadditive (or synergistic) effect, for which F(x + y)e P F(x + y)t, although this fact has not been entirely cleared up until now. These results have contradictory conclusions. Thus, according to literature data, the additivity of estrogenic compounds appear even at individual concentrations which do not cause detectable toxic effects (Payne et al., 2000) and some results concerning the synergistic effects in the case of xenoestrogen mixtures (Fernandez-Alba et al., 2002) or mixtures of methyl-tert-butyl ether and pesticides (Hernando et al., 2003), have been confirmed in a proportion of 30–60% or withdrawn, due to their low reproducibility (Witorsch, 2002). Also, the studies made on the human beings indicate that in case of mixtures which contain hydrophilic and lipophilic species, the lipophilic ones facilitate a high absorption of the hydrophilic ones, with unexpected effects for the singular compound (Zeliger, 2003). As result, the study of mixture toxicities is

Fig. 2. Minimum energy structure of compound 9 (left) and 25 (right). Hydrogen bonds are expressed by dashed line.

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Table 2 Individual (F(x)i and F(y)i), experimental (F(x + y)e), and theoretical (F(x + y)t) toxicity values of the enzymatic hydrolysis reaction products of a series of Naphthol-AS derivatives.a Compound name N-AS N-AS-D N-AS-RT N-AS N-AS-MCA N-AS-RT N-AS-RL N-AS-BS N-AS-AH N-AS N-AS-BO N-AS-SW N-AS-LB

ArI (x) 3-OH-2-naphthoic acid

Carbazole

F(x)i 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.29 3.64a

ArII (y)

F(y)i

Aniline 2-Me-aniline 4-Me-aniline 2-Et-aniline 3-Chloraniline 4-Cl-2-Me-aniline 4-MeO-aniline 3-Nitroaniline 4-Nitroaniline 4-Carboxyaniline 1-Naphthylamine 2-Naphthylamine 4-Cl-aniline

b

3.00 3.00b 2.84 3.50b 4.27 2.94b 3.32 2.77 2.85 3.34 3.79 3.57 2.94

F(x + y)e

F(x + y)t

SUAD

3.35 5.70 3.64 5.44 3.38 3.47 3.35 5.33 3.85 3.32 5.93 4.81 7.05

6.55 6.79 6.13 6.79 7.56 6.23 6.61 5.66 6.14 6.63 7.08 6.86 6.58

o o o o o o o o o o o o

SINE

o

a

F(x)i and F(y)i: individual log(1/MRC50) toxicity values; SUAD-subadditive toxicity values (for which F(x + y)e P F(x + y)t); e = experimental; t = theoretical; SINE-synergic toxicity values (for which F(x + y)e P F(x + y)t). b Calculated values, e.g.: aniline = 2-Me-aniline = 4-Me-aniline, 4-Cl-2-Me-aniline = 4-Cl-aniline (same class isotoxicity).

important and needs intense cooperation among toxicologists, model developers and pharmacologists (Groten et al., 2001). In the case of experiments carried-out in vivo with H. echinata, the hydrolysis mechanism of anilides of Naphthol-AS type, occurs under the influence of some specific enzymes. The probable reaction mechanism takes place similarly to the catalytic one which is known in the case of Chymotrypsin (Komiyama and Bender, 1979; Wilmouth et al., 2001; Liu et al., 2006; Otte et al., 2009), characterized by the Ser195–His57–Asp102 catalytic triad, when used as proteolytic enzyme, acting in the digestive systems of mammals and other organisms. Thus, the hydrolysis occurs: (1) by the formation of the enzyme–substrate complex and the appearance of an oxyanionic intermediate of the carbonylic carbon atom, with tetrahedric structure and (2) by the water molecule intervention and appearance of reaction products. Thus, in accordance to the literature data the following hydrolysis mechanism is proposed: in the first step, the active center of the anilide is generated by shifting of the electrons to the oxygen atom (except compound 25) of the carbonyl group. The positively charged carbonylic carbon atom will turn from the planar sp2 hybridized form to the sp3 tetrahedric one, at which the SerAO fragment will make a nucleophilic attack, with the formation of an intermediate complex with tetrahedric structure, with rotation of carbon–nitrogen bond into a position favorable for the nitrogen protonation. In the second step a water molecule is activated and splitted up by the action of the specific protease. The hydroxyl group replaces the serinic fragment at the carbonylic carbon atom (at the experimental pH value of 8.2 the hydroxy group exhibits a stronger nucleophilic character, as compared to the polar water molecule) and forms the hydroxy carboxylic acid. The electrons of the carbon–nitrogen bond move to the nitrogen atom, with formation of the amine and the proton lead to the formation of Ser195. One could take into account that this mechanism, as well as the effectiveness of the enzymatic catalysis, also depend on the presence of hydrogen bonds formed at the enzymatic center (Gerlt et al., 1997), which are characterized by stability, small distances, and an easier hydrogen transfer, respectively (Perrin and Nielson, 1997) and on the orientation of the p-orbitals of the anilidic nitrogen atom (Petkov et al., 1978). The experimental results suggest as important for the anilide toxicity the position of ArII substituents. Thus, substituents from positions 30 (e.g. in compounds 7, 9, 10, 14, except compound 12) and 40 (e.g. in compounds 4, 13, 15, 19) determine lower toxicity values (having the C value of 3.55). Compounds substituted in position 20 (e.g. in compounds 3, 5), or 20 and 40 (in compounds 16 and 18, except compounds 8 and 17), had higher toxicities values (with

C value of 5.54). Compounds 21 and 22, which have a bulkier ArII moiety behave likely to those having substituents in positions 20 , respectively 20 and 40 . It is more difficult to discuss compounds 23 and 24 from this point of view, which have specific ArI fragment. Thus, in the case of the ArII moiety, the substituents located in the 20 position will generate an ‘‘acceleration’’ of the hydrolysis reaction rate, probably due to the proximity stereo-electronic influences which are specific to the oxyanionic tetrahedric intermediate. In the case of the substituents located in the 30 and/or 40 positions, these influences are lower. Compounds 20 and 25 do not form any intramolecular hydrogen bond, having an average toxicity values of 3.55 and of 4.55, respectively. Generally, according to the results presented in Table 2, the experimental measured toxicity, F(x + y)e, is more or less higher as compared to the individual toxicities of the anionic component resulted from 3-OH-2-carboxylic acid, F(x)i, and the neutral or ionized species resulted from aniline or substituted anilines in positions 20 30 or 40 , F(y)i. The additivity phenomenon (F(x + y)e P F(x)i + F(y)i) could be noticed only in the case of N-AS-LB derivative. Being a singular case, it suggests that in fact, the additivity phenomenon appears subsidiary in case of faster reaction kinetics. The Naphthol-AS exhibiting alike log(1/MRC50) values lead to average calculated values, quite similar to those of some derivatives characterized by antimalaric properties (Kim et al., 1979) or of the amino-salts of some carboxylic acids (Chicu et al., 2008), when one could consider a class isotoxicity. The calculated average values have a dynamic character, and they could undergo some changes, depending on the appearance of new experimental data. H. echinata test system proves itself as being fast and with good reproducibility, even in the case of the toxicity determination of Naphthol-AS derivatives similarly to phenol derivatives (Chicu and Simu, 2009), in accordance to the ‘3Rs’ (reduction, refinement and replacement) concept (Balls et al., 1995). 5. Conclusion The development of toxicity evaluation of dye precursors, as well as of dyes, involving biological tests on colonies of H. echinata constitutes an important contribution to the integration of new materials, for the improvement of the security and the quality of life, by preventing the noxious effects of the persistant organic effluents on the human health and environment. H. echinata (hydrozoa) test system was used to evaluate the toxicity of anilides of Naphthol-AS type. Conformational analysis was

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performed by the Omega software to derive energetically stable structures, further used to explain the possible enzymatic hydrolysis mechanism: the chromophoric group ACOANHA is transformed into an oxyanionic tetrahedric intermediate during the rate determinant stage, followed by the formation of a new covalent bond in the presence of water, by the protonation at the nitrogen atom and the nucleophilic attack exercised by the hydroxyl group located at the positively charged carbon atom. The reaction products, e.g. the aromatic amine and the hydroxyl-carbonilic acid, always appear in stoichiometric proportion, by the total displacement of the electrons to the nitrogen atom of the CAN bond. The experimental results show that generally, an acceleration of the reaction rate generated by the substituents located at position 20 of the ArII moiety, as compared to those located in positions 30 and 40 , respectively, lead to toxicity differences of up to 2 log u. It could be possible that the reaction rate also depends on the orientation of the p-orbitals of the amidic nitrogen atom. The additivity phenomenon was noticed only in case, of N-AS-LB derivative (Table 2), but it is not sure that this fact was caused by the individual toxicity of the carbazole moiety. The biological differences between H. echinata and superior organisms do not allow simple comparisons and extrapolations. However, the obtained results can be useful for the configuration of future in vivo experiments in order to reduce the number of test animals to the absolutely necessary level, in accordance to the ‘3Rs’ concept: reduction, refinement and replacement. The test system for the toxicity evaluation based on biological tests on colonies of H. echinata does not imply environmental problems, is reproducible, rapid, simple, and accessible and represents a practical alternative method for biomedical researches, as well as for the toxicity assays of chemical compounds. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chemosphere.2010.11.057. References Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J.D., 1983. Molecular Biology of the Cell. Garland Publishing, Inc., New York, London. Anliker, R., 1979. Ecological assessment of dyes with particular reference to ETAD’s activities. J. Soc. Dyers Colour. 9, 317–326. Backhaus, T., Scholze, M., Grimme, L.H., 1999. The single substance and mixture toxicity of quinolones to the bioluminescent bacterium Vibrio fischeri. Aquat. Toxicol. 49, 49–61. Balls, M., Goldberg, A.M., Fentem, J.H., Broadhead, C.L., Burch, R.L., Resting, M.F.W., Frazier, J.M., Hendriksen, C.F.M., Jennings, M., van der Kamp, M.D.O., 1995. The three Rs: the way forward. ATLA-Altern. Lab. Anim. 23, 838–866. Benigni, R., Passerini, L., Gallo, G., Sino, M., 1998. QSAR models for discriminating between mutagenic and nonmutagenic aromatic and heteroaromatic amines. Environ. Mol. Mutagen. 32, 75–83. Borros, S., Barbera, G., Biada, J., Agullo, N., 1999. The use of capillary electrophoresis to study the formation of carcinogenic aryl amines in azo dyes. Dyes Pigments 43, 189–196. Chicu, S.A., Berking, S., 1997. Interference with metamorphosis induction in the marine cnidaria Hydractinia echinata (hydrozoa): A structure–activity relationship analysis of lower alcohols, aliphatic and aromatic hydrocarbons, thiophenes, tributyl tin and crude oil. Chemosphere 34 (8), 1851–1866. Chicu, S.A., Putz, V.M., 2009. Köln–timisoara molecular activity combined models toward interspecies toxicity assessment. Int. J. Mol. Sci. 10 (10), 4474–4497. Chicu, S.A., Simu, G.M., 2009. Hydractinia echinata test system. I. Toxicity determination of some benzoic, biphenylic and naphthalenic phenols. Comparative SAR-QSAR study. Rev. Roum. Chim. 54 (8), 659–669. Chicu, S.A., Herrmann, K., Berking, S., 2000. An approach to calculate the toxicity of simple organic molecules on the basis of QSAR analysis in Hydractinia echinata (Hydrozoa, Cnidaria). Quant. Struct.-Act. Rel. 19, 227–236. Chicu, S.A., Grozav, M., Kurunczi, L., Crisan, M., 2008. SAR for amine salts of carboxylic acids to Hydractinia echinata test system. Rev. Chim.-Bucharest. 5, 582–587.

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