Electrophiles and acute toxicity to fish.

Effect concentrations in fish LC50 tests with directly acting electrophiles are lower than those of unreactive chemicals that act by narcosis. LC50 values of more hydrophobic reactive chemicals tend to approach those of unreactive chemicals. Quantitative studies to correlate fish LC50 data to physical-chemical properties indicate that LC50 values of reactive chemicals depend on hydrophobicity as well as chemical reactivity. In this paper, several examples will be given of chemical structures that are known as direct electrophiles. This classification might be useful to identify chemicals that are more effective at lower concentrations than unreactive compounds. Chemicals that require bioactivation are not included because almost no information is available on the influence of bioactivation on acute toxic effects in aquatic organisms.


Introduction
The toxic effects of electrophiles are based upon their reaction with nucleophilic sites in biological macromolecules, but these cannot be defined in terms of a single mechanism ofaction. The major effect following an acute exposure to a relatively high dose of an electrophile might be membrane irritancy. More chronic exposure to lower levels might induce cytotoxic effects related to the disturbance of various types of processes within and outside the cell. Many electrophiles have been implicated as genotoxic agents that may act as carcinogens. Several compounds are direct electrophiles, but for many chemicals, electrophiles are formed in vivo by metabolic activation (1). It comes as no surprise that much attention is directed to possible mutagenic and carcinogenic effects of electrophiles.
Most information on carcinogenicity, toxicity, and bioactivation processes has been derived from mammalan studies or from cellular in vitro systems isolated from mammals; much less is known about such processes in fish. It is questionable whether bioactivation is always important in acute toxicity tests with the aquatic species. LC50 concentrations of directly acting electrophiles are generally lower than those of unreactive organic chemicals. In this paper examples will be given of electrophilic chemical structures/moieties that are known to act as direct electrophiles. This classification might be useful in identifying chemicals that are very likely effective at lower concentrations than unreactive compounds. The structural requirements related to this particular mode of action are rather well defined. Chemicals that act by narcosis include: saturated aliphatic alcohols, saturated ketones, and chlorinated aliphatic (saturated) and aromatic hydrocarbons.
Many pollutants cause lethality at much lower concentrations than predicted by Eqs. (1) or (2) because they act through a specific mode of action or because they may interact directly or indirectly (after bioactivation) with nucleophiles. This paper summarizes those chemical substructures that possess directly reactive properties. The survey is restricted to directly reactive chemicals because little is known of the influence of bioactivation on acute toxic effects. This classification of reactive structures might be useful in identifying chemicals that are very likely to be more lethal than unreactive compounds at lower concentrations in acute toxicity experiments.

Chemically Reactive Substructures
Electrophiles can react with several types of nucleophiles. Amino (-NH2), hydroxy (-OH), and sulfhydryl (-SH) groups are the most important of these from a biological point of view because they are found in many biological macromolecules, such as in proteins and in organic bases in DNA. Electrophiles may react with a nucleophilic ligand by different mechanisms of reaction and some of these mechanisms are summarized in Table  1. These mechanisms include nucleophilic displacement reactions (scheme a), addition at a carbon-oxygen bond (scheme b) and addition at a carbon-carbon double bond (scheme c).
Information on directly reactive structures can be drawn from several sources. Many examples of reactive chemicals are given in monographs or review papers on mutagenic and carcinogenic effects of chemicals (10). Infornation on reactive chemicals is also given in literature on organic chemistry (11,12), enzyme inhibitors (13), alkylation agents (14), and sulfhydryl agents (15). The -SH group is only one example of a nucleophilic ligand, but it may be a good representative of nucleophiles in general. Organic chemicals that can react with -SH groups are also likely to be reactive towards other nucleophilic ligands such as -OH and -NH2.
The following survey of reactive electrophilic substructures is arranged first according to the atom or chemical group that can bind a nucleophile: acylation reaction, reaction with cyanate, reaction with carbonyl compounds, alkylation and arylation reactions, reaction with metal ions and organometallic compounds, and other miscellaneous reactions with sulfhydryl groups.
Within each ofthese classes, a division into subclasses can be made according to the specific substructures representing the actual reactive site. The notation of chemical structures shown is hydrogen suppressed unless hydrogen atoms constitute an essential part of the reactive moiety. In addition, the following abbreviations

Acylation Reactions
In an acylation reaction the end product is an acylated nucleophile such as in a reaction between a sulfhydryl group and acetylchloride in Eq. (3) (15).
Examples of chemicals that may react with nucleophiles by acylation (10,15) are given below: ketenes: acid halides: carboxylic acid anhydrides:

Reaction with Carbonyl Compounds
Chemicals with a carbonyl group such as an aldehyde react with R-SH (11) as follows: Carbonyl groups in aldehydes and lactones are especially reactive. These are much more reactive than, e.g., a C==O group in ketones. Alternatively a reaction with amino groups can lead to Schiff base fonnation. 0 aldehydes: -C-H Table 1. Three different mechanisms of reactions of chemicals with nucleophiles.

Alkylation and Arylation of SH Groups
The replacement of a hydrogen atom in a molecule by an alkyl group is termed alkylation. Many different organic chemicals react with nucleophiles by alkylation. The carbon atom through which the attachment is made must be saturated Eq. (6). Therefore, the replacement of a hydrogen atom, e.g., in a sulfhydryl group, by a ethyl group is a simple example of an alkylation (14).
The general structures of sulfonic, sulfuric, and phosphoric esters are indicated below (14). Phosphoric acid esters are well-known insecticides that act specifically by inhibiting acetylcholinesterase (AChE). The enzyme AChE is inhibited by phospho-rylation of a hydroxy group in serine (16,17), but organophosphates can also react by alkylation. Whether organophosphates act as alkylating or as phosphorylating agents is pH dependent (14).
Halogenated Acids, Amides, Ethers, Sulfides, and Amines. Halogen atoms are more easily substituted by other nucleophiles in the presence of activating substituents such as carboxy, amide, ether, sulfide, and amino groups. Halogenated acetates are acetamides (15), halogenated ethers, ethyl sulfides, and ethyl amines (14) are especially reactive to nucleophiles. The reactive character of propionates is lower because the activating influence weakens as the distance between the halogen atom and the activating group increases. Halogenated ethyl sulphides and amines are also known as sulfur and nitrogen mustards.  (7) The tendency of halogens in alkyl halides and aryl halides to be substituted by another nucleophile depends strongly on the presence of other substructures. Activation of the C-Hal bond is based on inductive effects (electron-withdrawing or donation) and mesomeric or resonance effects (electron redistribution). More details on the effects of these factors on chemical reactivity are given in general text books on organic chemistry (11,12).
ALKYL HALIDES AND ARYL HALIDES WITH ONLY C, H, AND HAL. Saturated alkyl halides are generally not very reactive towards nucleophiles and LC50 values of such chemicals are well predicted by QSAR equations for unreactive chemicals (4,5). In general, the reactivity of saturated alkyl halides increases as follows: halogen atom: I > Br > Cl > F and C C alkyl chain: C-C-Hal > C-C-Hal > C C-C-Hal > -C-Hal Methyl bromide is much more reactive than methyl chloride, and isopropyl bromide is a much more directly reactive agent than methyl bromide. The difficulty is in deciding which combination is directly reactive with nucleophilic groups in biological macromolecules. Unsaturated alkyl halides have a much higher tendency to react with nucleophiles than saturated alkyl halides. The position of the halogen atom in an unsaturated alkyl halide, however, strongly affects its reactive character. Halides, in which the halogen is directly attached to one ofthe unsaturated carbon atoms such as in vinyl chloride (C=C-Hal) are unreactive, while allyl halides (C=C-C-Hal) are very reactive. Also, benzyl halides (C6H5-CH2-Hal) are much more reactive than halogenated benzenes.
Therefore, the presence of the following substructures strongly increase the reactivity of alkyl halides or aryl halides: allylic group: -C==C-C-Hal benzylic group: C(ar)-C-Hal ALKYL HALIDES AND ARYL HALIDES WITH OTHER SUBSTITUENTS. In the section "Halogenated Acids, Amides, Ethers, Sulfides, and Amines," several examples were shown of possible activating influences of certain substituents on the reactivity of the aliphatic C-Hal bonds. Halogens attached directly to an aromatic carbon atom are usually unreactive. Nitro groups, however, especially in the 2 and 4 position, strongly enhance the tendency for halogens to be substituted.

Organo Metallic Compounds
Metal ions of Cu, Ag, Au, Zn, Cd, Hg, Sn, Pb, As, and Sb show a high affinity for sulfhydryl groups and according to Torchinsky (15) can react as follows: R-SH + M+ --R-S-M + H+ (9) Also, organic compounds derived from these elements may react with SH groups. The number of organic groups, attached to the central metal atom, will depend on the valence state of the metal. Examples of wellknown organo metallics include organo mercury, organo lead, and organo tin compounds.
(R{C)n M+ Related structures, but those not derived from metal ions, are alkylating ammonium and sulfonium compounds (14). The reactivity of such compounds ammonium compounds: --C-N--C sulfonium compounds: -C-S--C depends on the basicity of the heteroatom and on the nature of the alkyl group. In a unimolecular process the more substituted alkyl groups will tend to be displaced, while in a bimolecular mechanism a nucleophile will attack at a less substituted group (14).

Other Miscellaneous Reactions with Sulfhydryl Groups
The oxidation of sulfhydryl groups in thiols can produce disulfides or sulfonic acids. Mild oxidizing agents will produce disulfides, while strong oxidizing agents result in the formation of sulfonic acids Eq.

Reactive Intermediates and Acute Toxicity
The mutagenic or carcinogen activity of many chemicals is based on reactive intermediates forned by metabolic activation. Examples of chemicals that may be metabolized to reactive intermediates are summarized in Table 2. Although the role of reactive intermediates in carcinogenicity is quite evident, the influence ofbioac- tivation on acute toxic effects is unclear. Aromatic amines, for example, can form reactive intermediates (18), but Veith and Broderius (19) have shown that effects of several aromatic amines in LC50 tests with fish are very similar to those produced by narcotics. Also the mutagenic effect of chlorinated alkanes and alkenes is based on reactive intermediates such as epoxides (20,21) and conjugates with glutathione (22). LC50 values of several chlorinated alkanes and alkenes to fish, however, are well-predicted by QSAR equations derived for chemicals that act by narcosis (Table 3). Other examples, however, suggest that bioactivation may also be important in acute toxicity tests. LC50 values of nitroaromatics, for example, correlate very well with their tendency to be reduced (23). The low LC50 values of dinitroaromatics, in particular, may be related to their high tendency for reduction (Table 3). Also, the high toxicity of unsaturated alcohols, as indicated in Table 3, is considered to be related to activation to a, ,B-unsaturated aldehydes and ketones (24,25). In general, however, little is known of the possible role of bioactivation in acute toxicity tests with fish. The information, available from mammalian studies, cannot be simply translated to LC50 tests with aquatic species.

Some Quantitative Correlations for Fish LC50 of Reactive Chemicals
It is well known that reactive chemicals are lethal at lower concentrations than unreactive compounds with equal K., values. The LC50 data for several classes of reactive electrophilic chemicals have been analyzed by QSAR and the derived equations are presented in Table  4. LC50 data of a series ofreactive alkyl halides correlate much better with rate constants (k) of a reaction with 4-nitrobenzyl pyridine (4-NBP) than with Ko, (26).
Reactivity towards 4-NBP has also been applied in correlations between mutagenicity and alkylating potency  showed a high correlation with K0w and the introduction of a reactivity descriptor did not improve the correlation (31).The observation that Kow alone is a good descriptor might indicate, as suggested by Deneer, that "possibly the rate of uptake of the compounds is the rate limiting process in the case of the compounds studied" (31). An example of a QSAR study for chemicals that probably are activated to reactive intermediates is given by Lipnick et al. (24). They observed that the toxicities of allylic and propargylic alcohols are much lower than those calculated from a QSAR equation for narcosistype chemicals. It was proposed that the allylic and propargylic alcohols are activated to the corresponding aldehydes and ketones that can react with nucleophiles by addition at the conjugated carbon-carbon double or triple bond.
Thus, it has been demonstrated that, in general, the LC50 values of electrophilic chemicals such as alkyl halides, epoxides, aldehydes, and unsaturated alcohols are lower than the LC50 values of corresponding unreactive chemicals. Further, it is obvious that to obtain significant correlations for these reactive chemicals, it is necessary to include descriptors related to their electrophilic reactivity. An interesting aspect of the QSAR equations for aldehydes and epoxides is the fact that observed LC50 values tend to approach LC50 values of chemicals that act by narcosis as Kow increases (29,31). Similar effects are also observed with esters (32), epoxides (30), and unsaturated alcohols (24,25). It seems as if the effects of more hydrophobic reactive chemicals are associated with narcosis. This phenomenon might be related to differences in distribution, with more hydrophobic chemicals partioning into lipid phases such as membranes.