Environmental occurrence, analysis, and toxicology of toxaphene compounds.

Toxaphene production, in quantities similar to those of polychlorinated biphenyls, has resulted in high toxaphene levels in fish from the Great Lakes and in Arctic marine mammals (up to 10 and 16 microg g-1 lipid). Because of the large variabiliity in total toxaphene data, few reliable conclusions can be drawn about trends or geographic differences in toxaphene concentrations. New developments in mass spectrometric detection using either negative chemical ionization or electron impact modes as well as in multidimensional gas chromatography recently have led researchers to suggest congener-specific approaches. Recently, several nomenclature systems have been developed for toxaphene compounds. Although all systems have specific advantages and limitations, it is suggested that an international body such as the International Union of Pure and Applied Chemistry make an attempt to obtain uniformity in the literature. Toxicologic information on individual chlorobornanes is scarce, but some reports have recently appeared. Neurotoxic effects of toxaphene exposure such as those on behavior and learning have been reported. Technical toxaphene and some individual congeners were found to be weakly estrogenic in in vitro test systems; no evidence for endocrine effects in vivo has been reported. In vitro studies show technical toxaphene and toxaphene congeners to be mutagenic. However, in vivo studies have not shown genotoxicity; therefore, a nongenotoxic mechanism is proposed. Nevertheless, toxaphene is believed to present a potential carcinogenic risk to humans. Until now, only Germany has established a legal tolerance level for toxaphene--0.1 mg kg-1 wet weight for fish.

Toxaphene, a complex mixture of polychlorinated camphenes, was first introduced in 1945 by Hercules Co. (Wilmington, DE) as Hercules 3965. Until the mid 1980s, it was mass produced and widely used as an insecticide, particularly in the cotton-growing industry. It was also used as a piscicide to control rough fish (undesired stock) in various water systems (1). The lipophilic, persistent, and volatile natures of toxaphene have contributed to its global dispersion throughout freshwater and marine environments. Traces of toxaphene have even been found in remote areas such as the Arctic (2) where the pesticide was never used. In addition to bioaccumulating in biota inhabiting these regions, toxaphene also has been detected in humans (3)(4)(5)(6)(7)(8)(9)(10). Toxaphene was banned by the U.S. Environmental Protection Agency (U.S. EPA) in 1982, an example that was followed by many countries. However, in the early 1990s toxaphene detected in marine fish in Europe caused concern about the relationship of human health and fish consumption. Therefore, increased attention has been focused on toxaphene, both in the analytic and toxicologic fields. Research in this field has received further impetus with the synthesis of individual compounds of toxaphene and their increasing commercial availability (11,12). Using individual standards, we can gain more insight into the transport, fate, and toxicological effects of toxaphene in the environment. Although identification of individual congeners provides more detailed information, it also leads to more complicated analyses. Another problem lies in the nomenclature of individual compounds. Proposals have been published recently for codes simpler than the systematic nomenclature now in use. These proposals will be discussed in this review.
In 1997 a European research project titled "Investigation into the Monitoring, Analysis and Toxicity of Toxaphene" (MATT) was initiated. As part of the project, an update of available knowledge on the developments in toxaphene analysis, new environmental data, and toxicology was prepared. To avoid duplication of the extensive review on toxaphene published by Saleh in 1991 (1), this review concentrates on developments since 1990.

Physical and Chemical Properties
Toxaphene (CAS No. 8001-35-2) was one of the main products produced by the Hercules Co. in the United States (1). The process of producing toxaphene consists of extracting crude ax-pinene from pine Manuscript received at EHP30 July 1998; accepted 20 October 1998. produces camphene, bornylene, and uo-terpineol. The camphene is then subsequently chlorinated under ultraviolet (UV) light to produce toxaphene. The average chlorine content is 67 to 69% (13). Structures of the main components of toxaphene are shown in Figure 1.
Toxaphene is a yellow, waxy solid and has a mild terpene odor, with softening occurring at a temperature range of 343 to 363K. Although readily soluble in most organic solvents, it is more soluble in aromatic than aliphatic hydrocarbons. Its average elemental composition is C1oH1oC18 (1). 1Toxaphene comprises at least 180 to 190 comnponents, most with the formula Cl(H18-nCln or C10H16-,Cln, where n is 6 to 10 (14). Buser et al. (15) report that polychlorobornanes (C10H18 nCL, n= [5][6][7][8][9][10][11][12] are formed as the main components in a Wagner-Meerwin-type rearrangement reaction. The peak area percentage of all components identified, measured using the electron capture detector (ECD), amounts to 50% of the total toxaphene area (1).
The commercial product is relatively stable but may be degraded by losing HCI or Cl2 with prolonged exposure to sunlight, alkali, or temperatures above 393K (16). Saleh (1) found that technical toxaphene does not undergo a serious change wvhen exposed to normal sunlight. Saleh and Casida (17) and Parlar et al. ( 18) reported that irradiation at wavelengths below 290 nm results in reductive dechlorination and dehydrochlorination; radiation above 290 nm does not appear to affect toxaphene composition. When adsorbed on silica, however, technical toxaphene is completely mineralized to C02 and HCl at 230 nm (19).
A specific gravity of 1.6 kg liter-1 has been reported for technical toxaphene (20). Vapor pressure and the log octanolwater partition coefficient (Ko,,) value have been estinmated to be comparable to that of hexachlorobenzene (HCB), 1.73 x 10-3 Pa at 298K (21), and a log KO,,, of 5.5 (22).
Howard (23) and Sullivan and Armstrong (24) recorded KOU, values of 4.82 to 6.4, respectively. A log Kol0, value of 6.44 was recorded by Hooper et al. (25). This is somewhat lower than that of technical polychlorinated biphenyl (PCB) mixtures but higher than those ofp,p'-DDT and its metabolites, suggesting that the bioconcentration of toxaphene is high. These data are diffictult to compare because of the variety of mixtures used. Bioconcentration factors (BCFs) of 2 x 106 have been observed by Kucklick et al. (26) for toxaphene in Arctic cod. This value is higher than that predicted from the log KOW' On the basis of their vapor pressure calculations, Wania and Mackay (27) suggested that toxaphene changes its chemical characteristic from gas phase to largely aerosol absorbed within the range of global environmental temperatures. At 298K, less than 10% is adsorbed to aerosols; at 253K, almost 90% is adsorbed. This implies that with a change in temperature, most toxaphene in the air condenses onto particles present in the atmosphere and thus becomes subject to wet and dry deposition. Toxaphene is transferred more rapidly from the atmosphere to soil and water at low temperatures.
Water solubility values of toxaphene have been reported with an equally broad spectrum and range from 0.4 mg liter-1 at 298K (28) to 0.55 to 3.3 mg kg-1 at 293 to 298K (24).
The most important factor determining the flux between the air-water interface is the Henry's law constant (H). Murphy et al. (29) measured H for a technical mixture of toxaphene congeners as 0.62 Pa m3 mole-1 at 293K. Using fugacity-based equations (22,30), the direction and magnitude of the flux can be calculated according to McConnell et al. (31), who assumed that the temperature slope determined by Tateya et al. (32) for PCBs is also valid for toxaphene. Using the H measurement, a toxaphene-specific intercept can be determined and from that a temperaturecorrected H can be obtained. This value allows the direction of the flux to be calculated. Such calculations suggest that up to 2 kg of material would be deposited in Lake Baikal, Russia, per month by gas exchange; the process is further enhanced by the low water temperatures of the lake (32). More accurate congener-specific H values are required to improve these estimates. This flux direction of air to water has also been recorded by Bidleman et al. (33). Hoff et al. (34) report that additional inputs via precipitation and particle deposition are likely to be 10 to 20 times less than those from gas absorption.
Over 180 companies are reported to have produced toxaphene since 1947 with various product names (1) ( Table 1).
In 1989 there were 168 registered uses of toxaphene in the United States (40) and more than 277 worldwide to control  65,534 167 major insect pests encountered in the production of agricultural commodities and crops. Its use in livestock dips as a miticide and in lakes as a piscicide to control rough fish populations has been widely reported (1). The interpolated total global use between 1950 to 1993 was 1330 x 106 kg and from 1970 to 1993, 670 x 106 kg (41). This estimation was based on data from the literature and on contacts with international agencies and researchers; data quality varies and shows large spatial and temporal gaps.
The United States (42), the Central American states, and the former Soviet states have recorded the highest usage of toxaphene. This may be because more detailed information on usage was received from these countries, whereas in other countries information often is not recorded or is kept confidential (41). El-Sebae et al. (28) report that toxaphene continues to be used in African countries, especially Ethiopia, Sudan, Tanzania, and Uganda where field runoff eventually flows into the Nile and ultimately into the Mediterranean Sea. These runoffs could be a source of future contamination. Information is lacking for other African countries.
In 1970 toxaphene was used in a formulation called polydophen, which was composed of 20% DDT and 40% toxaphene in a diesel fuel oil solvent. This was recommended as a substitute for DDT in Central Asia (31). Bidleman et al. (43) and Voldner and Schroeder (44) suggested that toxaphene application likely continues in the Soviet states, Mexico, Romania, Hungary, Poland, and the Indian subcontinent as well as many African nations, Nicaragua, and Mexico.
The most recent data available from the Food and Agricultural Organization of the United Nations (FAO) on toxaphene usage [as reported by Swackhamer et al. (45)] indicates that Korea and Mexico were the only countries using toxaphene into the 1980s; Mexico reported using 600 tonnes in 1985.
Although toxaphene is currently banned in many countries, Argentina and Mexico allow restricted use. Toxaphene was used only in small quantities in Sweden and has been banned since 1956 (46).
The Soviet government restricted the use of toxaphene in 1971. It is thought to still be in use as an insecticide for sugar beets, peas, potatoes, mustard, rape seed, and perennial herbs in the following formulation: 50% active ingredient, 30% oil, 15% amalgamate at 1.6 to 3.0 kg ha-1 during sprout stage (47). Voldner and Li (41) report that 1 x 108 kg of toxaphene has been used since 1970 in the former Soviet Union.
In 1956 toxaphene was recommended for nationwide use in Egypt as an insecticide to protect against cotton leafworm, pink bollworm, and spiny bollworm in cotton fields. Field efficacy was the only consideration for the use. It was applied as a formulated emulsifiable concentrate of toxaphene (60% chlorinated camphene) and used in four successive sprays during the cotton season. This method of administering the chemical caused maximum contamination of soil and can result in up to 20% being released into air, 20 to 50% into soil, and 20 to 50% into water systems. This can ultimately lead to air and groundwater pollution and to soil contamination. A concentration of 10 ppm has been reported in Egyptian soil, biota, and water (1). Although insecticide application doubled between 1956 and 1961, major crop losses were experienced as efficacy decreased and insect resistance increased as a result of removal of the insect's natural enemies. Egypt alone used 54x 103 kg toxaphene between 1956 and 1961, an estimated 25% of the non-U.S. toxaphene use (28). The resistance level of the cotton leafworm was 26-fold that of the laboratory controls. This led to Egypt banning toxaphene in 1961, not because of its environmental impact but because of the poor efficacy factor.
It was previously thought that chlorohydrocarbons were produced in the wood pulp industry from residual monoterpenes during the chlorobleaching process.
However, no evidence has been found of compounds identical to the main congeners in commercial toxaphene. This indicated Environmental Health Perspectives * Vol 107, Supplement 1 * February 1999 that toxaphene in fish did not come from chlorobleaching of pulp (48). However, chlorinated camphenes are present in pulp mill recipients (bleached Kraft pulp mill discharged organic matter in lake sediment) (49). Chlorine bleaching of wood pulp produces chlorinated compounds similar in composition to toxaphene but with lower chlorine content (50).

Toxaphene Use in the Great Likes ofNorth America
Much of the research on toxaphene has been conducted in the Great Lakes of North America, with conflicting data on the sources of pollution. These range from the use of toxaphene as a piscicide to the contribution of the wood pulp industry in addition to atmospheric sources. Swackhamer et al. (53) report that approximately 1% or less of U.S. toxaphene use was in the Great Lakes basin (54). The rate of use in the basin was approximately 1 x 106 kg year`between 1970 and 1977 and peaked around 1977. Thus, the presence of toxaphene in the Great Lakes has been largely attributed to long-range atmospheric transport from the southern United States or from Central America followed by wet and dry deposition to the lakes (55,56).
Historical investigation of records on Lake Michigan revealed that 224 x 103 kg of toxaphene was used in the Green Bay watershed between 1950 and 1980, with most used as a pesticide on cropland but small amounts on livestock and in lakes as a piscicide. It has been noted that even if there were only a 1% runoff into Lake Michigan, this would represent a large fraction of the estimated inventory of toxaphene in the lake, i.e.,11 x 103 kg (57).
Inputs from the atmosphere to water surfaces such as the Great Lakes include dry fallout of particulate-associated contaminants, washout of gas phase and particulate phase contaminants by precipitation events, and gas transfer across the air-water interface (58). Oehme et al. (10) reported that the continual process of transport, deposition, revolatilization, and new transport along a decreasing temperature system results in accumulation of toxaphene in sediments because vapor pressure becomes so low that it restricts atmospheric transport.
In the 1960s several lakes in Wisconsin were treated with toxaphene to kill rough fish. Kidd et al. (9) reported that concentrations of toxaphene in fish in Laberge, Canada, were entirely due to atmospheric input followed by long food chain bioaccumulation giving rise to hazardous concentrations in fish. Kidd et al. (9) provide further information on possible sources and report that some contamination of Lake Ontario was due to surreptitious dumping.
Howdeshell and Hites (59) claim that the Niagara River is the main riverine source of sediment and water to Lake Ontario and therefore likely to be a source of some toxaphene in the lake; atmospheric deposition is also important.
Scheel (60) reports that toxaphenelike contaminants found in Michigan sport lakes may not be completely due to the presence of toxaphene compounds. It was suggested that they may be due to a mixture of chlorinated bicyclic monoterpenes, including the chlorinated pinenes, occurring as unwanted byproducts from chlorination of naturally occurring plant-derived product materials. Results suggest that not all chlorinated bicyclic monoterpenes found in fish tissue are the result of chlorinated camphenes or camphanes from toxaphene but may be from other sources such as the natural product family of bicyclic monoterpenes including pinene and borneol (61).

Nomenclature
For many years it was assumed that the pesticide toxaphene consisted primarily of chlorinated bornanes in addition to small amounts of chlorinated bornenes and even smaller amounts of chlorinated bornadienes (62). The existence of bornenes and bornadienes was based on data obtained with gas chromatography (GC) with negative chemical ionization mass spectrometry (NCI/MS) studies. Mass spectra with fragments 2 or 4 amu below the [M-Cl]ions of bornanes were interpreted as bornenes and bornadienes. However, new insights into synthetic pathways of technical toxaphene indicate the formation of camphenes and dihydrocamphenes (63). Therefore, the observed mass spectra probably should be attributed mainly to chlorinated camphenes and dihydrocamphenes. According to Saleh (62), the technical mixture also consists of small amounts of other chlorinated hydrocarbons and nonchlorinated hydrocarbons.
As can be seen from  (CHBs) (73), where several of these names only contain one of the groups present in the technical mixture. The variety of trade and common names used for toxaphene, in addition to the trivial names of various compound classes referred to in Table 1, complicate any nomendature system. Names no longer supported by International Union of Pure and Applied Chemistry (IUPAC), such as norbornanes and camphanes for bornanes and iso-camphanes for dihydrocamphenes, compound the naming problem.

Systematic Names
It is a complex task to formulate systematic names for all groups of compounds mentioned above ( Table 1) that conform to IUPAC rules. The structure of these compounds is given in Figure 1. The generally accepted systematic nomenclature for bornanes, according to IUPAC rules, is based on the following rules and agreements ( Figure IA) (74,75): * Numbering of the carbon atoms, as shown in Figure IA (as presented by IUPAC). * Substituents on the six-membered ring that point downward are in the endo position and substituents that point upward are in the exo position (the bridging carbon, C7, is above the ring). * The carbon atom above the C2-C3 bond is C9, the carbon above the C5-C6 bond is C8. * The lowest possible numbering should be applied. The carbon neighboring Cl is decisive for the direction of numbering. If both carbons next to C1 bear the same number of chlorine atoms, substitution of the next carbon in the ring is decisive in determining the direction of numbering. If these are also equivalent, the first carbon with an endo chlorine determines the direction of numbering.
* Enantiomers receive the same systematic nomenclature. For bornenes (Figure 1 B) and bornadienes ( Figure 1C) the following agreements and additonal rules should be applied: * If one double bond is present, the carbon atoms at this bond are numbered C2 and C3. * If two double bonds are present, the numbering of the six-membered ring should result in the lowest possible numbers, as with the bornanes. * As with the bornanes, the C9 carbon should be positioned over the C2-C3 double bond.
Hainzl (63) proposed systematic names based on a fixed numbering of the camphene skeleton ( Figure 1D,E). This approach is quite straightforward, resembles the bornane nomenclature, and is more user friendly. However, IUPAC does not yet support assigning these fixed numbers and, in addition, there still are no IUPAC rules for designating C8 and C9 orientations in camphene (76).

Nomencature Systems
Because chlorinated bornanes are the most abundant compounds in technical toxaphene, most attention has been devoted to them, both with regard to analytic method development and monitoring, and nomenclature. In the past systematic nomenclature of the bornane skeleton has been nonuniform because several authors have cited the IUPAC nomenclature incorrectly, particularly the C8 and C9 positions ( Figure 1) (74). Difficulties in formulating the correct systematic names for chlorinated bornanes were solved when IUPAC assigned definitive numbering for the carbon skeleton.
Because of the extensive systematic names for chlorinated bornanes (e.g., 2endo,3-exo,5-endo,6-exo, 8,9,9, 10, 10nonachlorobornane), isolated congeners were often designated by simpler names such as T12, Toxicant A, Toxicant Ac, Toxicant B, TOX8, and TOX9; however, a clear nomenclature system was lacking. Several authors proposed and used more systematic nomenclatures in attempts to remedy this situation. Table 3 gives an overview of these nomenclatures, which will be discussed below.
The nomenclature used by Burhenne et al. (11) and Hainzl et al. (77) is based on GC retention on a certain stationary phase. Consisting simply of a 2-digit code representing a peak in the technical mixture, the nomenclature can be applied to chlorinated bornanes, camphenes, and dihydrocamphenes.
Wester et al. (37) proposed a nomenclature system that is a mixture of the systems previously mentioned, with advantages that the structural information can be directly deduced and that the nomenclature is applicable to chlorinated bornanes as well as to chlorinated bornenes and bornadienes. The proposed system yields a code consisting of two parts. The digits in the first part of the code reflect the degree of chlorination of carbons C2 to C6, presented according to the rules listed in Table 4. C4, i.e., the third digit, can only have a code of 0 or 1. The digits in the second part indicate the number of chlorine atoms of C8 to CIO. The letter "B"precedes the 8-digit number in the case of bornanes. For example, the code for 2-endo,3-exo,5-endo,6-exo, 8,8,9, 10,10-nonachlorobornane is B[12012]- (212). The code for the conformation of  To differentiate both enantiomers "r" is proposed for clockwise numbering of the six-membered ring ( Figure 2A; B[30012]-(11 1)r ) and "s" for counterclockwise numbering ( Figure 2B; B[30012]-(1 1 1)s ), provided the bridging carbon atom, C7, is above the ring. The advantage of this notation is that it is related to the generally accepted notation for chirality (R/S), and enantiomers receive the same code. For racemates, the r/s notation can be left out. Another advantage of this system of nomenclature is the simplicity of establishing whether a congener has an enantiomer.
Wester et al. (37) extended their nomenclature to indude bornenes and bornadienes, which is easily done because of structural similarity. Only one chlorine atom can be attached to a carbon atom participating in a double bond. Only a "0" or "1" can be assigned to such a carbon. For example, 2,5-endo,6-exo, 8,9,9, Polychlorinated camphene and dihydrocamphene structures could not be represented by codes based on the system previously described because of the large differences among structures. However, Wester et al. (80) developed a coding system analog to their system for bornanes, bornenes, and bornadienes (37). The 122 numbering of the carbon atoms in the skeleton is the same as that proposed by Hainzl (83); however, the "a" and "b" indications of the substituents at CI0 (63) have been replaced by "'E" (trans) and "Z" (cis), respectively ( Figure 2A). As seen in Figure 1, this method bears a strong resemblance to the clockwise numbering of the bornane carbon skeleton (37). Figure 1D illustrates that it is not necessary to consider carbons C5 and C6 because they cannot be chlorinated. The first part of the code concerns the substituents at carbons Cl to C4, C7, and CG0. Carbons C1 and C4 can only have one chlorine substituent and these invariably will be in the endo position. Substitution at carbons C2 and C3 can be denoted according to the rules of Table 2.
For carbon C7, the positions of the substituents must be defined. A "0" is assigned for no substitution, "1" for substitution in the "a" position, "2" for substitution in the "b" position, and "3" for two substituents. For ClO the known "E"(trans) nomenclature corresponds with code 1 and "Z" (cis) with code 2. Carbons C8 and C9 are dealt with in the second part of the proposed code, which merely reflects the number of chlorine substituents at C8 and C9. Finally, the code is preceded with a "C" for camphenes (74), with enantiomers being distinguished by an "r" or "s"according to Wester (37). The general code then becomes C[code C1, code C2, code C3, code C4, code C7, code C10]-(code C8, code C9)r/s. The same logic used previously can be applied to the dihydrocamphenes ( Figure  1E). There is no need to consider carbon C5, as chlorination cannot occur. For the first part of the code, the same rules are applied for carbons Cl to C4 and C7 as for the chlorinated camphenes. Carbon C6 can only have one substituent, which can be in the endo or exo position, and the rules in Table 2 were applied. However, if C6 is not chlorinated, the position of its hydrogen atom is unclear, in which case a subscript selected according to the rules of Table 2 is used to denote the endo or exo position of the hydrogen atom. The second part of the code deals with C8 to Cl0, with the code reflecting the number of chlorine substituents. Finally, the code is preceded by DC (dihydrocamphenes) and enantiomers are distinguished by adding an "r" or "s" according to Wester (37). The general code then becomes DC[code Cl, code C2, code C3, code C4, code C6H6, code C7]-(code C8, code C9, code C1O)r/s.

Analytical Methods
Mostly toxaphene levels are determined that may lead to a large overor underestimatation of the true concentration, as the peak pattern of the sample under study does not resemble that of the standard.
Peak patterns may be considerably altered in the environment (84,85), but there also are large differences among standards for the commercially available technical toxaphene. Using various technical standards, Carlin and Hoffman (86) found variations between 19 and 131% compared to their laboratory standard. Furthermore, detector response generally is not equal for all congeners. The most relevant question may be: What does a total concentration imply when the composition is unknown?
Because of this the trend at present is toward using congener-specific approaches, which is possible after the first isolation and synthesis of individual compounds (87,88). Currently, about 30 individual congeners are commercially available. For comparison of monitoring results, it is important that authors report the full analytical procedure used, as different methods can yield large variations in results, which could lead to incorrect conclusions.

Exration
Little attention has been paid to the efficiency of extraction procedures. However, it is thought that extraction procedures suitable for related compounds such as PCBs, DDT, and chlordanes could also be used for toxaphene compounds because of lipophilic and structural similarities (73). Pre-Sparaton and Clean-Up Several stationary phases have been used in the sample preparation for residue analysis. Aluminium oxide (89) and gel permeation chromatography (GPC) (90) or a combination of the two (91-93) can be used to remove lipids from the sample. Florisil (48,90,94) and silica gel (7,89,95) (97). When silica fractionation is used, individual congeners should be used to establish the volume range of the toxaphene fractions and to evaluate recoveries; low recoveries for certain congeners may occur when only a technical mixture is used for optimization (69,98). de Boer et al. (69) used columns of 2.5 g SiO2'2% H20 (w/w); most of the toxaphene compounds, including the most relevant congeners, were eluted in a second fraction of 12 ml diethyl ether/iso-octane (20:80, v/v) after a first fraction of 13 ml iso-octane that contained mostly PCBs. Only 1 to 2% PCBs were present in the toxaphene fraction, which did not seriously interfere with the toxaphene quantification using ECD. The entire clean-up procedure resulted in recoveries of 80 (202) was divided over the two fractions (about 40% in the first fraction and 60% in the second fraction), with an overall recovery of 85 to 95%. In a collaborative study to determine four bornane congeners in fish oil, gel permeation was used followed by adsorption chromatography on silica gel (99). The silica gel clean-up was performed using 1.0 g silica deactivated with 1.5% water. The toxaphene compounds were collected with the PCBs and some organochlorine pesticides in the first fraction and eluted with 8 ml hexane/toluene (65:35, v/v). Although the results of this study were obtained using GC-ECD, the recoveries were 77 to 100%, and the relative standard deviations of reproducibility were 18 ± 4, 24 ± (92). They eluted the silica column before the hexane/toluene fraction with 8 ml hexane in which the PCBs and p,p'-DDE were recovered. Some chlordane/nonachlor and p,p '-DDT and B[12012]- (202) were also found in that fraction. Krock et al. (96) improved on this method by using 8.0 g activated silica. The sample was eluted with 48 ml hexane to remove PCBs. This was followed by elution with 50 ml hexane/ toluene (65:35, v/v) in which the toxaphene compounds were recovered. Injection Alder et al. (100) reported that injector temperature should not exceed 513K because severe decomposition of compounds may take place. Bartha et al. (101) recommend an injector temperature below 523K. Care should be taken with active sites in the liner and the injector. It is recommended that the optimal temperature be verified by a series of simple tests, as there is much variation in injector geometry. Alawi et al. (102) showed that response factors obtained using splitless injection are lower than those obtained using on-column injection. Bartha et al. (101) reported that using pressure pulse injection (PPI) at 498K resulted in response factors 4 times that of those obtained with splitless injection. This was especially significant for compounds with a low vapor pressure and long retention times [e.g., B[30030]-(122)] (101). With this technique, the time the compounds spend in the injector is short, so there is less chance of degradation.  (99).

Gas Chromatographic Separation
Krock et al. obtained a relatively good separation using a very nonpolar Sil-2 stationary phase (comparable to squalene) (96), the same elution order as on the more polar DB-5 columns was found (103). The CP-Sil 2 phase was successfully used to a temperature as high as 563K, although the supplier advised a maximum temperature of 473K. No alteration of retention times was observed after analysis of several hundred toxaphene compounds on this phase (101 (112), it was found that compounds with alternating endo-exo substitution elute earlier than compounds with two chlorines at C2 and C5 (103).
Nikiforov et al. (106) split the bornane skeleton into two parts, the six-membered ring, "Ring," and the three methyl groups, "Metil." By comparing available retention indices (RI) to those from a DB-5-type phase with the substitution of these two parts of the molecule, several correlations were found and the following conclusions were drawn:  1 12) and (21 1) < (1 12). The use of heart-cut multidimensional gas chromatography (MDGC) (108) offers a possibility to overcome coelution problems due to the large amount of congeners. By transferring heart-cuts from a separation performed on a DB-5-type phase to a 15% dimethylsilicone, 85% polyethylene glycol (DX-4; J&W Scientific) phase, in addition to a polyethylene glycol terephthalic acid ester (FFAP; Hewlett Packard) and a 10% cyanopropyl, 90% biscyanopropyl polysiloxane (Rtx-2330; Restek Corp., Bellefonte, PA) phase for further separation (a multidimensional set-up) (69), a large number of peaks were observed in the secondary chromatograms, which indicated that the resolution offered by a single column is insufficient and can easily contribute to false-positive results, especially when nonselective ECD is used for quantification. There were no large differences between the column combinations; the DB-5-Rtx-2330 Environmental Health Perspectives * Vol 107, Supplement 1 * February 1999  (69) did not observe degradation effects on this phase; this was also true after reevaluation of data and further experiments with this stationary phase ( Figure 3). This may be partly because a shorter column was used (15 m instead of 30 m), which limits exposure time of the components to a high temperature, 493K. It is extremely time consuming to analyze several compounds in a complex sample using a multidimensional set-up, even when a system is available that has several parallel traps for storage of heart-cuts (108). If the speed of the secondary separation is high enough to separate a cut from the first dimension while the next cut is being collected, it will then be possible to record a connecting set of secondary chromatograms. The complete two-dimensional chromatogram can be constructed from the secondary chromatograms, similar to that in thin-layer chromatography. A method with this capability is called comprehensive (110). A comprehensive separation uses the whole two-dimensional separation space to generate resolution provided that the individual separations are based on different interactions (i.e., are not correlated). For a method to be comprehensive, it is necessary that the first dimension be sampled at least every peak width by the second separation dimension. The first dimension can then be constructed from the secondary chromatograms (111,112 (202) with GC-NCI/MS in the SIM mode l N/1 include the formation of (M-OCI)-fragments of PCBs; false-positive signals may be caused in part by chlordanes and the appearance of higher chlorinated bornane congeners (67,73,115). Krock  impact (El) mode and will lead to false positive results. Structural information is, of course, much more limited in the NCI mode. The ECD is an attractive alternative detector, however, and as ECD is less selective than MS detection, an even more efficient separation will be necessary. The profiles obtained with flame ionization detection are similar to those obtained with full-scan El/MS and have a low response dependency on the chlorine substitution pattern; however, only the latter technique has the selectivity and sensitivity necessary for residual analysis (92). NCI/MS has a completely different peak profile that is probably caused by the higher variation in response factors for individual congeners (11).
When using the ECD, removal of interfering compounds is a prerequisite.
PCBs, for example, are present at high concentrations in most environmental samples, which may also contain toxaphene compounds. In addition, PCBs have rinated bornane congeners was obtained using NCI/MS (102). It was tentatively found that a 2,2,5,5 substitution of chlorobornane congeners ([30030]) had a negative effect on the NCI/MS response (81,116). Buser (202) in penguin and harbor seal samples. Most toxaphene congeners produce fragments with m/z = 125 under El conditions; this ion, together with ions at m/z= 159,195, and 231, is considered to be characteristic of toxaphene congeners (62). In contrast with quadrupole or double focusing MS/MS in which tandem mass spectrometry is accomplished through space, Saturn 4D MS/MS uses the time dimension to accomplish MS/MS. The isolation of precursor ions and further dissociation takes place in the same chamber (m/z locking) but at a different time. This reduces loss of precursor ions and hence provides better sensitivity. The major ion in the daughter spectrum of m/z= 159 is a fragment at m/z= 125. However, PCBs and some organochlorine compounds also  produce this fragment in the MS/MS mode. Therefore, it would appear that the ion at m/z = 89 (dechlorinated monochlorotropylium ion), which orginates from the m/z= 125 ion, would be more useful for quantification of toxaphene congeners (117). However, coelution of compounds that produce this ion cannot be observed. Furthermore, the response factors with this method vary considerably for individual cogeners [B[12012]- (202)  using GC-NCI/MS. Alder and Vieth then reanalyzed the sample using NCI/MS and obtained a value of 5210 pg kg-1, which is close to the value reported by Fowler et al. (118). They concluded that this large difference between the concentrations found is caused by the large difference in response factors between congeners with NCI, which gives a positive bias to the results when they are compared with those using ECD, which has a smaller difference between response factors. Rantio et al. (48) also showed that NCI/MS gave generally higher results than ECD. However, the results demonstrated a linear relationship between the two detection techniques, which made it possible to compare the result obtained. A higher response for NCI/MS was also reported by Wideqvist et al. (46), especially when the degree of chlorination was higher. In contrast, Xu et al. (119) found that GC-ECD gave results identical to those for GC-NCI/MS for quantification of individual chlorobornanes in fish samples. A possible explanation for these, which at first are contradictory observations, could be different standards used in combination with the detector, which could influence the result to a large extent, as shown by Carlin and Hoffman (86). For example, it is possible to obtain the same results for GC-ECD and GC-NCI/MS with one standard, but largely differing results with another. Another explanation can be differences between the MS configuration used in the studies.
ECD determination of total toxaphene is subject to insufficient selectivity, whereas NCI/MS is subject to variable response factors. Using indicator compounds as a basis for calculation of total concentration was suggested as a way to obtain precise and comparable data (92). However, this approach can only be used successfully when the indicator compounds do not coelute with other compounds. Coelution of suggested indicator compounds was shown by heart-cut multidimensional gas chromatography (69). Depending on the sample type, up to 10 peaks were found when the analyte peak was further separated on a second, different column.

Enantiomers
Usually enantiomer ratios (ERs) are used to express the ratio in which the enantiomers are present. The peak area/height of the (+)-enantiomer is divided by that of the (-)-enantiomer (120)(121)(122)(123). When the conformation of the enantiomers eluting from a chromatographic system is not known, as with enantiomers present in toxaphene, the ER is often expressed as the peak area/height of the first eluting enantiomer divided by that of the second (124). Using the quotient of the two enantiomers gives an undefined result when the second enantiomer is not detected. de Geus et al. (125) observed this and therefore divided the second enantiomer by the first. Of course, this approach only shifts the problem. It would be better to divide by the detection limit (which does not equal zero) when a compound is not found, but this can lead to very high or low numbers. In addition, because of the reciprocal-like scale, ERs larger than unity appear to deviate more than ERs smaller than unity (e.g., 6.7 and 5.0 vs 0.15 and 0.20). To avoid these disadvantages, the (+)-enantiomer or the first eluting enantiomer can be expressed as a proportion of the sum of the two (126 (124). Comparison of the EFs of different congeners in combination with their molecular structures can help us gain insight into the metabolism of these compounds. When determining EFs of chlorinated bornanes in biota, the possibility cannot be excluded that the values found are not merely due to metabolism in the species studied because a change during previous disposition in the food chain is also possible. Feeding studies in which the species of interest is exposed to (racemic) mixtures of known composition would eliminate this problem. As an alternative, in vitro assays can be used in which microsomes are incubated with the compounds of interest. The microsomes contain the cytochrome P450-dependent monooxygenase enzyme systems involved in enantioselective and nonenantioselective biotransformation. Boon et al. (127,128) successfully used such an approach to study the a-chiral biotransformation of toxaphene congeners by microsomes from harbor seal, whitebeaked dolphin, sperm whale, and laysan albatross.
Separation should be enantioselective as well as isomer specific to determine EFs. Unfortunately, this doubles the number of peaks to be separated (125). A tert-butyldimethylsilylated P-cyclodextrin phase, introduced by Blum and Aichholz (129), has been shown to give a good enantiomer separation of toxaphene compounds (15,104,124,125,130). However, enantiomer separation of bornane congeners is still a rather empirical task and the selection of a convenient stationary phase is determined primarily by trial and error Environmental Health Perspectives * Vol 07, Supplement * February 999 (131). It has been shown that columns based on heptakis(2,3,6-0-tert-butyldimethylsilyl)-,-cyclodextrins are especially suitable for the separation of polychlorinated bornane enantiomers (15,124,132,133). Unfortunately, this stationary phase is not very well defined and batchto-batch differences have been observed (134). Vetter et al. (135) compares several enantioselective phases for the separation of toxaphene compounds.
The obtainable enantiomer resolution depends on the column oven temperature profile. It was found that this phase can be used up to a temperature of 535K in a programmed run. However, at lower temperatures the obtained resolution is much higher (130). Baycan-Keller and Oehme (104) showed that a temperature ramp of 1K resulted in much better separations compared to one of 10K. This was also found by de Geus et al. (125). Unfortunately, slow temperature programs lead to very long run times, which can be a problem when compounds with low concentrations must be detected.
Most attention has been devoted to measuring the EFs of B[12012]-(202) and B[12012]-(212) ( Table 6) (15,137). However, studies by Vetter et al. (130) and de Geus et al. (125) show that other compounds can be much more interesting 'Enantiomer fraction is the abundance of the first eluting enantiomer relative to the abundance of the sum of both enantiomers (126). hData from Kallenborn et al. (124). cData from Alder et al. (136). didentical extract determined with GC-ECD.
because they show more enantioselective activity (Table 7). Parlar et al. (139) report that all parent compounds in toxaphene occur as racemates. Buser and Muller (140) showed that some compounds are present in the technical formulation (Melipax) in nonracemic compositions. However, interferences from other compounds cannot be exduded, even in the MS/MS method they used. Vetter et al. (130) isolated the compound B[21020]-(022) from Melipax. The mass spectrum of this compound showed no significant impurities; however, the first eluting enantiomer was significantly more abundant than the second. The EF was 55.8 ± 0.6%. Furthermore, the authors showed that the EF of this compound was 50.0% in a cod liver extract from the Baltic. If a synthesized standard with a racemic composition were used, it might be concluded that no enantioselective process took place. This demonstrates the importance of carefully choosing the standard. On the other hand, deviation of this compound from the racemic value should also appear in other technical formulations because Melipax accounts for only 5% of the global toxaphene production (141)  Parlar et al. (139) document EFs of several chlorinated bornanes in cod liver oil, herring, halibut, caviar, and redfish samples obtained from Karlson and Oehme (107), Kallenborn et al. (124), and Alder et al. (136). The EFs show little variation-50.2 ± 1.2%; therefore, Parlar et al. conclude that no significant degradation of toxaphene enantiomers takes place in Table 7. Enantiomer fractions.a Percentage reported in the literature. Weddell seal (female, subadult) 52 55 77 Weddell seal (male, adult) 55 57 72 Weddell seal (male, adult) 52 57 74 Leopard seal (unkown, adult) 57 (136) show that the EF value of these compounds in warm-blooded species (human milk and cynomologus monkey adipose) deviate from unity. This is in accordance with the observed EF of 57.3% for B[12012]- (212) in Antarctic penguins by Buser and Muller (140). This could indicate a more efficient metabolism present in these species compared to that in other species (fish).

Intedabowry Study
A German collaborative study (142) with contaminated milk fat undertaken in the mid-1980s demonstrated the analytic difficulties and uncertainties in analysis of technical (total) toxaphene by packed column GC-ECD. Andrews found that in many laboratories only about 15 to 30% of toxaphene components were eluted from silica or Florisil columns with a nonpolar solvent. This was thought to be the main source of the large variation between laboratories (98). In a German intercalibration experiment, recoveries of 77 to 100% with a relative standard deviation of reproducibility of 23 (9.2-50.5%) were found for four indicator compounds in a fatty matrix. On the basis of these results, the method was recommended for routine analysis in food inspection in Germany (99). In a recent QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) laboratory performance study with four toxaphene congeners in standard solutions, most of the 15 participants reported satisfactory results (143). Indicator Compounds Ideally, toxicity should play a major role in the selection of indicator compounds. Unfortunately, little is currently known about acute and chronic toxicity of individual congeners to mammals. Occurrence determines, in combination with toxicity, whether a compound is important. Stereochemistry may play an important role since the biologic disposition of enantiomers varies (125) (Table 7). Boon et al. (128) showed that B[12012]-(202) and B[12012]-(212) did not yield a positive response in a mutatox test, whereas technical toxaphene and B[30012]-(1 1 1) did yield a positive response. The latter compound is only detected in low concentrations in wildlife samples (69,99,117).
Next to these parameters, analytic convenience is important. The compounds should be detectable without the interference of other compounds when common extraction, clean-up, and separation/detection procedures are used. The compounds should also be commercially available (92).
In practice, the availability of standards and analytic convenience dictate the  122), for the same purpose. Because this compound was found to degrade easily in the detector and is only present in minor amounts in technical formulations, it is not useful as an indicator compound (92). Measuring the individual indicator compounds on a single GC column presents a problem in that several compounds may be present in one peak, as demonstrated by heart-cut multidimensional GC (69).
Instead of measuring the toxaphene compounds in all fish for consumption, samples from important species and fishing areas can be selected to answer the question of human intake of these compounds, as was done in a large study by Alder et al. (100). As an alternative, Alder  Most of the available information about toxaphene concentrations in biota is referred to in terms of total toxaphene. However, since the number and pattern of congeners in environmental samples is substantially different from those in the technical mixture (as a result of environmental and metabolic modification) (88,113), values for total toxaphene should be considered only indicative. Table 8 gives an overview of the total toxaphene levels in biota samples as reported in the literature. Toxaphene concentrations plateaued after a period of steady increase through the 1970s, but its incidence continued to increase; residues were present at 88% of the stations sampled from 1980 to 1981 (162). From 1978 to 1979, toxaphene concentrations were highest in lake trout (Salvelinus namaycush) samples from Lakes Michigan and Superior, with typical concentrations of 5 to 10 pg g-' lipid (163).
From 1980 to 1981, concentrations were generally lower, 2 to 5 pg g-1 lipid. Concentrations of toxaphene declined in trout and smelt from the Great Lakes between 1982 and 1992 except for fish from Lake Superior (159). Little toxaphene has been used in the Great Lakes basin alone. The main input is thought to be through atmospheric transport from the southern United States or Central America, followed by wet and dry deposition (53,115). Atmospheric transport was probably also responsible for residues detected in fish from lakes in Alaska. Several other reports conclude that toxaphene is carried in the atmosphere from the site of application and its accumulation is widespread in freshwater and marine fish (90).
Toxaphene was also the major organochlorine residue in Canadian Arctic marine invertebrates and fish. Arctic cod (Boreogadus saida) in three eastern Arctic locations had concentrations of toxaphene 5to 10-fold higher than those for DDT or PCB (146). Musial and Uthe (84) found that levels of CHBs in Arctic cod liver were about 2-fold lower than those in Atlantic cod (Gadus morhua). Bidleman et al. (2) reported levels of toxaphene to be equivalent to those of PCBs in zooplankton and in amphipodes collected from an ice island in the Arctic Ocean. Other organochlorines had lower concentrations. Toxaphene was found to be a major contaminant in Atlantic cod liver and herring (Clupea harengus) muscle from eastern Canadian waters, with levels similar (lipid weight basis) to those for PCB but generally higher than those for DDT (84). Toxaphene was not detected in deep-sea (Canadian waters) scallop (Placopecten magellianicus) (84).
High levels of toxaphene were reported for white-beaked dolphins (Lagenorhynchus albirostris) and pilot whales (Globicephala malaene) collected from 1980 to 1982 from the coast of Newfoundland (152). This was explained by the increased use of toxaphene during the 1970s. Toxaphene levels were higher than those for other organochlorines such as PCBs and DDT Most of the peaks in the toxaphene standard were not present in dolphin blubber, an indication of the dolphin's considerable metabolism and/or selective accumulation of some isomers and/or metabolites. Two peaks accounted for about 50% of the toxaphene peaks (probably GC-EI/MS). Toxaphene also was the major organochlorine contaminant detected in blubber of Arctic belugas (Delphinapterus leucas) (150). Little geographic variation in the concentration of toxaphene was observed in five different areas (East Hudson Bay, Cumberland Sound, West Hudson Bay, Beaufort Sea, and Jones Sound). Geographic comparisons of toxaphene levels for belugas are difficult because belugas migrate over relatively long distances and spend most of the year at the ice edge rather than at the locations where they were sampled. Belugas collected from the north coast ofAlaska had higher toxaphene concentrations in blubber (151) than PCBs, DDTs, and chlordanes in the same samples. Males had higher concentrations of toxaphene than females and the oldest male had a higher concentration than the youngest male. Transplacental transfer to the fetus and through lactation to the nursing pups are the most probable causes of the lower toxaphene levels in females compared to males. Stern et al. (165) identified the two major recalcitrant toxaphene congeners in aquatic biota from beluga blubber as B[12012]-(202) and B[12012]- (212). Their sum constituted 28 to 34% of total toxaphene in arctic char, 53% in burbot, and 81 to 89% in beluga whale blubber from the Canadian Arctic.
Toxaphenes were the dominating organochlorines in narwals (Monodon monoseros) collected from 1982 to 1983 from northern Baffin Island in the Canadian Arctic (94) and was composed of two major components, an octachloroborane and a nonachlorobornane. The pattern of organochlorines in tissue suggests that narwals are exposed proportionally to more volatile compounds and may be less able to metabolize some of these compounds than odontocetes living closer to sources of these contaminants.
Toxaphene was measured in landlocked Arctic char and ringed seal (Phoca hispida) from Greenland (145). Char from the east coast of Greenland had toxaphene levels that were significantly higher than those in char from areas of the west coast. However, overall levels of toxaphene in muscle were low. Seals displayed no significant geographic variation in toxaphene levels, presumably because of their relatively high biotransformation capacity for toxaphene (128).
Zell and Ballschmiter (4) analyzed fish from different regions to characterize organohalogens in pristine aquatic environments. They found toxaphene in spawn of Arctic char (S. alpinus) from a lake in the Tyrolean Alps, pike (Esox lucius) from northwest Ireland, sturgeon (Acispenser stellatus) from the Caspian Sea, salmon (Salmo salar) from Ireland and Alaska, and in the livers of Antarctic cod (Dissostichus eleginoides) from South Georgia. They indicated that the global pollution by toxaphene could be as widespread or more so than compounds like those in the DDT and PCB groups. The pattern of toxaphene spread was modified to a variable extent compared to that of technical mixtures. Samples from the North Atlantic Ocean and the Caspian Sea contained levels about 10-fold higher than those from samples of feeding primarily on the endemic whitefish or omul (Coregonus autumnalis) and planktivorous sculpin (Comephorus dybowskii). Toxaphene in biota ranged from 1.1 to 2.3 pg g-' lipid in sculpin and seal, respectively, indicating little biomagnification of toxaphene from fish to seal. Toxaphene patterns in seals were degraded to a greater extent than those in fish but retained several prominent congeners. These results are in agreement with degradation studies by Boon et al. (128).
Levels of toxaphene and other organochlorine pesticides have been analyzed in tilapia (Sarotherodon mossambicus) and guapote (Cichlasoma managuense) collected in 1991 from Lake Xolotlan in Nicaragua (157). The carnivorous tilapia contained concentrations of toxaphene 4 to 5 times that in the omnivorous guapote. Located on the shore of the lake was a factory producing toxaphene, which may have contributed to some of the high levels of toxaphene.
Jansson et al. (149) reported total toxaphene residues in Arctic Char (S. fontinalis) from lake Vattern in southern Sweden, and in grey seal (Halichoerus gryphus) and herring (C. harengus) from the Baltic Sea. Fish from the different areas gave similar chromatograms, indicating widespread input of toxaphene to the whole region through the atmosphere. This finding was supported by Paasivirta and Rantio (166), who compared toxaphene levels in salmon from the Arctic and the Baltic and found no significant difference. Similarly, levels of toxaphene in cod liver did not differ. Toxaphene has not been used as a pesticide in Scandinavia. Andersson and Wartanian (72) analyzed toxaphene in blubber samples from various seal species collected from the Baltic and the west coast of Sweden. Toxaphene levels in Baltic seals were higher than those in animals from the west coast of Sweden. Comparison of the data for adult and juvenile seals revealed, in addition, to agerelated variation in contamination, i.e., toxaphene levels in adult Baltic ringed seals were significantly higher than those in adult grey seals from the same region and 5 to 10 times than those in juvenile ringed seals from the same region. Andersson et al. (148) reported no geographic differences in concentrations of toxaphene from animals in the Arctic region with those in corresponding species in the Baltic.
Several reports on levels of toxaphene in fish and fish products from Europe show the ubiquitous presence of toxaphene in all 132 types of fish (89,167,168). High residues of toxaphene in fish and fish products from Europe were reported by Muller et al. (167), who showed that toxaphene concentrations in herring and mackerel (Scomber scombrus) from the North Sea and the relatively remote waters west and northwest and of Ireland and the Shetland Islands exceeded the German tolerance level, which was 0.1 mg kg-l on a lipid basis or 0.01 mg kg-l wet weight (ww) at that time. van der Valk and Wester (89) conducted a study in fish from northern Europe. Highest toxaphene concentrations were found in herring oil from the Baltic (7 pg g-' lipid).
Toxaphene levels in cod liver showed an upward trend from the southern to the northern North Sea, increasing from 0.4 to 1 jig g-' lipid. This finding was somewhat unexpected, as the northern North Sea usually is considered less polluted than the southern North Sea. de Boer and Wester (7) report that toxaphene has almost never been used in Western Europe. Accumulation of toxaphene in northeastern Atlantic waters may be attributable to aerial transport from the American continent.
The authors also reported that Baltic her-  (Table 9).
Icelandic cod liver contained the highest concentrations (Table 9). Toxaphene in the livers of hake from west of Ireland and herring muscle and dolphin blubber from the North Sea were all studied by de Boer et (Table 9).  (Table 9). Highest residue concentrations were found in marine fish with moderate-to-high fat content such as halibut, herring, redfish, and mackerel. The sum of the indicator compounds in sardines and in fish with lean muscle tissue levels (Alaska pollock, saithe, hake, and cod) were low. Farmed salmon from Chile showed lower levels of the three compounds than salmon from the northern hemisphere. Eel    both fresh water and marine biota all over the world. Also, at remote areas long distances from toxaphene sources, the levels in biota can be quite high. These findings illustrate the importance of long-range transport, perhaps through the atmosphere, in the global spreading of this group of contaminants.

Toxicology
Since the late 1940s, reports have been published addressing the toxicity of the chlorinated camphenes to fish, bi mammals (172)(173)(174)(175)(176). In ac toxaphene was found to elicit m and carcinogenic properties in ma test systems, thereby posing a t humans (25,177).

Toxicokindtcs and Biotansfori
The use of toxaphene as a pisci discontinued after the discovo toxaphene was persistent in the environment and its presence pi successful restocking of treated lakes with desirable fish (178,179). However, experi-Reference mental information is scarce on the depuration of toxaphene in fish and their (165) residue kinetics. Delorme et al. (180) stud-(100) ied the elimination rate of toxaphene and two of the more persistent congeners, B[12012]-(202) and B[12012]-(212), in lake trout and white suckers in a natural ecosystem following intraperitoneal injection of technical toxaphene (7 pg g-' for (169) white suckers; 3.5 and 7 pg g-1 for lake (119) trout). The estimated half-lives for total toxaphene were 524 days for white suckers and 232 (high dose) and 322 (low dose) days for lake trout. Half-lives for the two (100) congeners in trout were 294 and 376 days (high dose) and 316 and 367 days (low dose), respectively. In white suckers, only  (169) was injected intravenously into normo- (100) and hypolipidemic mice. In normolipidemic mice, most of the radioactivity initially was found in the liver and adrenals either in the absence or presence of LDL or (102) HDL. Four hours after application, the (100) radioactivity was redistributed into the adipose tissue. Notably, lower amounts of radioactivity were found 20 min after mice were injected with toxaphene in combination with HDL than in mice injected with 14C-toxaphene-LDL, suggesting a more efficient metabolism and disposal of toxaphene when HDL was used as a carrier. Mohammed and co-workers initially found irds, and less 14C-radiolabeled toxaphene in the liver Idition, and adrenals and more in the kidney and utagenic heart of hypolipidemic mice (181). 14Cmmalian Toxaphene was redistributed mainly to the hreat to liver and only in small amounts to adipose tissue 4 hr after injection. According to the authors, these results indicate that changes onatlOl in the lipid pattern may influence tissue cide was distribution of toxaphene. Mohammed ery that et al. (181) also studied the distribution of aquatic 14C-radiolabeled toxaphene among liporevented protein fractions in vitro and in vivo using Environmental Health Perspectives * Vol 107, Supplement 1 * February 1999 human and rat plasma. In rat 37 to 52% of radioactivity was recovered in the HDL fraction, whereas 18 to 52% was associated with the albumin-rich bottom fraction (BF) both in vivo and in vitro. In contrast to distribution in the rat, the in vitro distribution of 14C-toxaphene among human lipoprotein fractions is relatively homogeneous. In the BF, 26% of radioactivity was found, whereas in the HDL, LDL, and very low-density lipoproteins (VLDL) fractions, 27, 29, and 18% of radioactivity were recovered, respectively. Reductive dechlorination or dehydrochlorination and, in some cases, oxidation, have been shown to be the major mechanisms by which toxaphene is metabolized in microorganisms as well as in insects, birds, aquatic organisms, and mammals (1,182). Degradation of toxaphene in the soil proceeds rather slowly under aerobic conditions, whereas under anaerobic conditions toxaphene is more easily degraded (139).  (111), one of the two end-metabolites of the six bornanes tested. In addition, Fingerling et al. (36) showed that none of the components tested were degraded in autoclaved soil, indicating that degradation is mediated primarily by microorganisms.
In contrast to the identification of dechlorination products formed from toxaphene components as well as technical toxaphene under anaerobic conditions in soil, reports on the isolation and characterization of oxygen-containing products is scarce. Fingerling and Parlar (183)  According to Saleh (1), hepatic microsomal mixed-function oxidases are most important in toxaphene metabolism in mammals, followed by glutathione S-transferases. Chandra and Durairaj (184) showed that in addition to inducing cytochrome P450 and aniline hydroxylase activity in the liver, toxaphene also induces activity of these enzymes in the kidney. Therefore, the authors speculate that toxaphene alone may be metabolized in the liver as well as in the kidney.
In an attempt to evaluate the role of phase I biotransformation in the bioaccumulation process of toxaphene, Boon (122). Neither toxaphene nor the four congeners were metabolized in vitro using hepatic microsomes of the sperm whale. Interestingly, the authors' results showed that the in vitro capacity of microsomes derived from the different species to metabolize technical toxaphene, reflects the decreasing number of peaks in the toxaphene residues of wildlife extracts. Aquatic Toxicity Toxaphene is highly toxic to aquatic organisms. It was found that in general saltwater fish are more sensitive to toxaphene then freshwater fish (mean acute toxicity values of 0.07 pg liter-i and 1.6 pg liter-1, respectively) (1). Keller (185) studied the acute toxicity of several pesticides, including toxaphene in freshwater mussels (Anodonta imbecilis), and compared their sensitivities to those in common test organisms such as Daphnia magna, Cerio dubia, and fathead minnow (Pimephales promelas). The 96-hr LC50 for A. imbecilis exposed to toxaphene was 0.74 mg liter-. Compared to the other organisms tested, A. imbecilis is less sensitive to toxaphene. The acute toxicity levels for toxaphene in most aquatic organisms range from 1 to 40 pg liter-1 (1).
Interestingly, addition of sediment to the test chambers drastically reduced the toxicity of toxaphene to A. imbecilis. Thus, susceptibility of A. imbecilis to toxaphene toxicity appeared to vary depending on whether concentrations were sedimentor aqueous-bound.
Application of toxaphene to lakes as a piscicide has caused direct as well as indirect damage to the ecosystem. Direct damage includes disappearance of target as well as nontarget organisms inhabiting toxapheneexposed waters. Indirect damage occurred when application of toxaphene resulted in some cases in replacement of native organisms by a new population of organisms, which modified the structure of the ecosystem. Miskimmin and Schindler (186) examined the response to toxaphene application and stocking with a nonnative fish species on total chironomids, Chaoborus spp., planktonic Cladocera in a mesotrophic lake (Peanut Lake, north basin), and a eutrophic lake (Chatwin Lake) in central Alberta, Canada. The response in these lakes was compared to that in a lake that had not been treated (Peanut Lake, south basin). The authors studied some invertebrates prior to application of toxaphene during 1961 to 1962 and examined recovery of the community in the following 30 years by analyzing sediment cores from the lakes. They found that as a result of toxaphene application (0.0184 ppm) to Chatwin Lake, planktonic Cladocreans decreased in abundance and dominance changed from smallto large-body types. No short-term effects were detected by examining sediment cores from the toxaphene-treated Peanut Lake (0.0075 ppm). In the absence of native fish and during trout stocking, large invertebrates became dominant in both treated lakes. Residual toxicity and/or predation by stocked fish in both lakes probably resulted in low population levels of Chaoborus spp. throughout the 1960s. Long-term changes in invertebrates in both lakes probably were a result of the manipulation of fish communities rather than effects of residual toxicity.   (195) conducted between 1950 and 1980. As reviewed by Saleh (1), the acute LD50 of toxaphene to laboratory mammals ranged from 5 to 1075 mg kg-', depending on the species studied and the route of exposure. In addition, female rats appeared to be somewhat more sensitive to toxaphene exposure than male rats. Among the most prominent symptoms observed in laboratory animals acutely intoxicated by toxaphene are generalized epilepticlike convulsions starting with excessive saliva production followed by vomiting and muscle spasms. In time, the frequency of convulsions increased. Finally, animals became exhausted and died from respiratory failure (173). Pathologic changes upon toxaphene exposure may include degeneration of the brain, spinal cord, and pulmonary edema (1). Combination Toxicity. Because toxaphene was widely used as a pesticide, in addition to other pesticides, the toxicity of toxaphene alone as well as in combination with other widely used pesticides was evaluated in ICR mice after 14 days of oral administration or 90 days in drinking water (195,196). Overall, decreases in body weight as well as increases in liver to body weight ratios were observed in mice exposed to toxaphene and toxaphene-containing mixtures. Visually, no pathologic changes were observed in tissues from treated animals. However, proliferation along with dilatation and fragmentation of the endoplasmatic reticulum and scattering of ribosomes in the liver were pronounced. Cotreatment of mice with toxaphene and parathion resulted in higher levels of inhibition of serum cholinesterase (serum ChE) activity than did treatment of mice with toxaphene alone for up to 3 days after initial exposure. In contrast, an increase of serum ChE activity was observed in mice cotreated with toxaphene and 2,4dichlorophenoxyacetic acid (2,4-D) compared to mice treated with toxaphene alone. Phenobarbital-induced sleeping time was reduced in mice exposed to toxaphene and toxaphene-containing mixtures, whereas no reduction was observed in mice exposed to either parathion or 2,4-D This was probably because exposure to toxaphene-containing mixtures induces the hepatic mixed-function oxygenase (MFO) system. It cannot be determined from these studies whether the combination of toxicity of toxaphene and other pesticides is synergistic or antagonistic in nature or the result of effects manifested by their components individually.

Mammalian Toxicity
Neurotoxicity. Neurotoxic effects of toxaphene exposure such as effects on behavior and learning have been reported to occur (1). The mechanisms underlying neurotoxicity, however, are little understood. In guinea pig, Chandra and Durairaj (194) observed histological changes in the guinea pig brain, e.g., hypoxic (disorganization) and anoxic (enlargement) changes in the neurones, upon exposure to toxaphene. Depletion of cytoplasmic organelles in the oligodendritic cells of the cerebrum was observed in guinea pigs exposed to 2 mg kg-1 toxaphene, whereas disfigurement of myelin in the brain occurred when they were exposed to the high 5 mg kg-' day-' dose. In a subsequent study, Chandra and Durairaj (197) investigated the impact of acute and subacute toxicity of toxaphene on the lipid profile in brain, liver, and kidney of guinea pig. An increase in neutral lipids and cholesterol and a reduction of phospholipids was observed in the brain. The individual phosphoglycerides phosphatidylinositol, sphingomyelin, and phosphatidic acid increased in both the acute and subacutely intoxicated guinea pig brain. On the basis of their studies, Chandra and Durairaj (197) postulated that the observed effects of toxaphene on lipid contents in brain, liver, and kidney led to membrane damage. In addition, alterations in phospholipids and cholestrol content were thought to be an adaptive mechanism to cope with the stress due to toxaphene intoxication. Furthermore, they argued that the increase of sphingomyelin in the brain might be related to neurotoxic symptoms, as an increase in sphingomyelin inhibits the permeability of the membrane to small molecules and ions.
Chandra and Durairaj (184) also observed reduced ATPase and acetylcholinesterase (AChE) activities in the brain on acute and subacute exposure of guinea pigs to similar concentrations of toxaphene. Addressing the mode of action of the neurotoxic effects of toxaphene, Chandra and Durairaj discussed that inhibition of AChE can result in neural and neuromuscular disorders. In addition, respiratory failure, which leads to hypoxic and anoxic changes, would eventually result in decreased phosphorylation and ATP production, as evidenced by inhibition of ATPases. Toxaphene in vitro inhibits brain and kidney ATPases in mammals, as well as in fish, and insects (1). In contrast to the observed effects on brain AChE activity in the guinea pig, little effects on brain ChE activity were observed in mice treated with toxaphene and toxaphene-containing mixtures (195). The exposure of mice to toxaphene or a toxaphene-containing mixture did not result in pathologic changes in brain and liver at the light microscopic level. Table  11 gives an overview of neurologic, reproductive, and endocrine effects caused by exposure to toxaphene.
Nephrotoxicity. The effects of toxaphene exposure on the kidney of mammals were observed in a number of studies. In the 1992 study by Chandra and Durairaj (194), a single administration of 300 mg toxaphene kg`bw to guinea pigs resulted in no observable changes in the ultrastructure of the kidney 72 hr after exposure. In a subacute exposure study, 2 or 6 mg toxaphene kg-l day-l administered for 60 days led to vacuolization in cells of the collecting system and glomerulus, degeneration of corticol tubular cells, vacuolization, and an increase in the number of mitochondria of tubular epithelial. From this study, the authors evaluated the toxaphene-induced nephrotic changes as an adaptive mechanism in the guinea pig to cope with a disturbance in membrane-associated glycoproteins and glycolipid metabolism in liver and kidney. In a study on the impact of acute and subacute effects of toxaphene on the lipid profile in kidney, Chandra and Durairaj (197) observed an increase in phosphatidylcholine, phosphatidylinositol, and phosphatidic acid levels accompanied by a decrease in cardiolipin and sphingomyelin contents. However, no alterations in other phosphoglyceride contents were found. Both acute and subacute exposure of the guinea pig to toxaphene resulted in reduced ATPase and AChE activities in the kidney (184). This study also indicated that toxaphene may be metabolized in the kidney in addition to the liver, as an enhanced cytochrome P450 content and induced aniline hydroxylase activity were found in the kidney when exposed to toxaphene.
Hepatotoxicity. Several studies have shown that toxaphene or toxaphenecontaining mixtures induce a number of hepatic biotransformation enzymes. Toxaphene and combinations of toxaphene with parathion (5 mg kg-') and/or 2,4-D (50 mg kg-l) induced hepatic enzymes such as cytochrome P450, benzo[a]pyrene hydroxylase, and aliesterase in mice after 7 days of oral exposure. Furthermore, the in vitro biotransformation of parathion and paraoxon was effectively enhanced using hepatic 9000 g supernatant from mice exposed to toxaphene (202). Toxaphene and toxaphene-containing mixtures also decrease the phenobarbital-induced sleeping time in mice, suggesting an effect of toxaphene on CYP2B-type metabolizing enzymes (195). These studies show that the toxaphene-induced increase of appropriate biotransformation enzymes, including cytochrome P450, potentially stimulates the metabolism of a number of other xenobiotics and consequently may even reduce their toxicity.
A single dose of 300 mg toxaphene kg-' bw in guinea pig did not result in histopathologic or ultrastructural changes of the liver, whereas administration of 2 or 5 mg kg-l day-l for 60 days led to a relative increase in liver weight, chronic venous congestion, mononuclear infiltration, and fatty changes in hepatocytes (194). The effect of subacute toxicity of 2 and 5 mg toxaphene kg`day-l on the hepatic lipid profile was a decrease of phospholipids without significant alterations in glycolipid, neutral lipids, and cholestrol levels (197). Notably, in this study the acute dose of 300 mg kg`bw resulted in piloerection, sedation, crouching, clonic-tonic convulsions, and death within 72 hr. The changes observed in the lipid profile were thought to be an adaptive mechanism to cope with stress associated with toxaphene intoxication. In a similar experiment, toxaphene also reduced hepatic ATPase and AChE activities and interfered with collagen and calcium metabolism (184). Reproductive Effects. Few data are available on the effects of toxaphene on reproduction in mammals and fish. Few or no effects were found in mammals to indicate interference of toxaphene with reproduction (203)(204)(205). Recently, the effects of toxaphene on reproduction were studied in sexually mature female zebrafish after being fed toxaphene-contaminated food (0.02, 0.23, and 2.2 pg g-l fish day-l) for 2 weeks (206). In the highest dose group, all fish died within 24 hr; 9 of 14 fish died in the group exposed to 0.23 pg g-' fish day' between days 8 and 12. Other toxic effects observed in the parent fish were skin discoloration, subcutaneous hemorrhages, and curved backbones in the vertical plane. With regard to reproductive success, a nonsignificant decrease in mean total number of eggs spawned was observed. No differences in reproductive success were observed, as assessed by percentage of viable eggs 24 hr after fertilization, percentage of embryo mortality, and percentage of eggs hatching 72 hr after fertilization. In contrast, toxaphene produced a dose-related decrease in the percentage of oviposition for female zebrafish. Hence, it was concluded that dietary exposure of zebrafish to toxaphene affects their reproductive process. Endocrine Toxicity. A recent concern about many environmental pollutants is that they might have endocrinelike properties. Environmental xenobiotics that mimic steroidal hormones have been implicated in the increasingly high incidence of breast cancer and other gender-specific disorders (207)(208)(209). To determine whether environmental chemicals act as exogenous hormones in the American alligator, Possible estrogenic or antiestrogenic potencies of toxaphene either alone or in combination with other pesticides were studied in a number of in vitro systems by other authors. The effect of toxaphene on the aromatase enzyme complex, which converts androgenic to estrogenic enzymes, was studied by Drenth et al. (211) in the human choriocarcinoma cell line JEG-3. Aromatase activity did not decrease as a result of toxaphene exposure. The expression of estrogen-regulated mRNA-stabilizing factor (E-RmRNASF) in toxaphene-treated leghorn rooster liver was studied by determining the stability of apolipoprotein II (apoIl) mRNA in vitro. It was shown that toxaphene prevented estrogen stimulation of E-RmRNASF expression, acting as an antiestrogen (212). Toxaphene also inhibits the binding of progesterone, dexamethasone, and testosterone to their respective receptors (IC20 values of 68.4, 4.2, and 3.5 pM, respectively) isolated from eggshell gland mucosa of the domestic owl (213).
In contrast to the antiestrogenic potencies, weakly estrogenic potencies of toxaphene were observed in a number of other in vitro test systems. In the human E-screen test, 10 pm of toxaphene was shown to be weakly estrogenic (0.0001, as potent as estradiol). Interestingly, a morethan-additive estrogenic response was observed in the human E-screen test after administration of a mixture of 10 estrogenic chemicals including toxaphene (214,215). Bonefeld-J0rgenson et al. (216) conducted transient gene expression studies using a chimeric reporter construct containing one estrogen-responsive element (ERE) to expression of the chloramphenicol acetyltransferase (CAT) gene in human breast cancer cells. They found that technical toxaphene (10 pM), as well as the toxaphene congener B[12012]-(212) (10 pM), acted as an antiestrogen that blocked the action of estrogens by inhibiting the ER:ERE-activated gene transcription.
In a study by Ramamoorthy et al. (217), minimal estrogenic potencies of toxaphene and no synergistic effects of combinations of toxaphene and other pesticides were observed. Induction of CAT activity was not observed in MCF-7 human breast cancer cells transiently transfected with plasmids containing estrogen-responsive 5'-promotor regions from either rat creatine kinase B or human cathepsin D genes after treatment with a combination of toxaphene (1 0-8 _ 10-5 M) or cotreated with toxaphene and dieldrin (equimolar concentrations, 10-5 M). Furthermore, no estrogenic response was found in the uterus of a 21-day-old female B6C3F, mouse after oral exposure to toxaphene (2.5-275 ,mole kg-' bw) or to toxaphene in combination with equimolar concentrations of dieldrin. In contrast to the results obtained with the systems previously mentioned, Ramamoorthy et al. (217) observed a slight estrogenic effect in an estrogen-responsive reporter system in yeast-expressing mouse estrogen receptor 2.5 hr after treatment with toxaphene (2.5 10-5 M) or mixtures of toxaphene with endosulfan, dieldrin, or chlordane. The latter treatments were not synergistic. In contrast, no estrogenic effect was observed in yeast-expressing human estrogen receptor treated with toxaphene alone or in combination with other pesticides.

CarcinogeWncty
In the past, much effort has been expended on studying the carcinogenic properties of toxaphene. Table 12 gives an overview of carcinogenic and mutagenic data of toxaphene presented in the literature. Toxaphene was found to be highly carcinogenic in rat and mice and induced malignant liver tumors, reticulum cell sarcomas, sarcomas in the uterus, neoplasms in the reproductive system and/or mammary gland, and neoplasms in the pituitary, adrenal, and thyroid glands (1,177). The National Cancer Institute conducted a study in which neoplasms were found in the thyroid gland of the rat (218). To investigate whether the increased incidence of thyroid tumors observed in the rat in the National Cancer Institute bioassay of toxaphene had a nongenotoxic etiology, Waritz et al. (219) studied the thyroid function and thyroid tumors in male Crl:Cd BR (Sprague-Dawley-derived) rats orally exposed to 75 mg toxaphene kg-1 day-1 for 28 days (100 mg toxaphene kg-' day-' was administered for the first  24 hr Brain PKC activity (mouse) 200 pM Induction of mouse brain PKC activity. (222) Abbreviations: GJIC, gap junctional intercellular communication; HBEC, human breast epithelial cells.
Environmental Health Perspectives * Vol 107, Supplement 1 * February 1999 4 days). Rats were sacrificed at days 0, 7, 14, and 28 of exposure. A significant timedependent increase in serum thyroid-stimulating hormone levels was found, whereas there were no changes in serum levels of T3, T4, rT3, and corrected rT3. They observed a time-dependent increase in thyroid follicular cell hypertrophy and intrafollicular hyperplasia and a decrease in thyroid follicular cell colloid stores, both characteristic of a hyperactive thyroid. Considering that toxaphene has the characteristics of a phenobarbital-type inducer of the cytochrome P450 enzyme system, the authors concluded that the increase in thyroid follicular neoplasia in toxaphene-treated rats probably was caused by a nongenotoxic mechanism such as that believed to be responsible for thyroid tumor increases in rats chronically treated with phenobarbital. Because this type of mechanism for thyroid neoplasia is not known to occur in humans, the authors also conclude that it becomes increasingly unlikely that toxaphene presents a hazard as a thyroid carcinogen for humans.
In an attempt to further elucidate the mechanism of toxaphene-induced hepatocarcinogenicity, Hedli et al. (223) investigated two potential mechanisms: peroxisomal proliferation, which has been invoked as a nongenotoxic mechanism of hepatocarcinogenicity, and DNA adduct formation. After oral treatment of CD/1 mice for 7 days with toxaphene (0-100 mg kg-1 day-1), no increases in immunodetectable levels of CYP4A1 were detected, suggesting that peroxisomal proliferation is not involved in the toxicity of toxaphene. Furthermore, no evidence was found for DNA adduct formation in the liver of toxaphene-treated mice. On the basis of this study, the authors suggest that the hepatocarcinogenic properties of toxaphene may be exerted through a nongenotoxic or promotional mechanism rather than through a genetic mechanism.
Although in vivo no evidence for a genetic mechanism for toxaphene-induced tumor formation was found, in vitro studies showed that toxaphene is genotoxic in mammalian cell systems and mutagenic in the Ames Salmonella test without requiring metabolic activation by liver homogenates (1). More recently, Steinberg et al.  1 1 1), for mutagenic activity in Salmonella typhimurium strains TA98 and TA100 using a validated microsuspension procedure instead of the usual plateincorporated procedure. Toxaphene was mutagenic only in the TA100 strain at concentrations of 2,500, 5,000, and 10,000 pg m1-1. In contrast, toxaphene was also mutagenic to strain TA98 at a concentration of 10,000 pg plate-1 when using the plateincorporated assay. Using the microsuspension method, none of the four tested toxaphene congeners showed mutagenic activity in strain TA100 at any of the concentrations tested (maximum concentration: 10,000 pg ml-1). A dose-dependent (10-10,000 kg plate-') increase in His revertants was also observed in strains TA97, TA98, TA100, TA102, and TA104 by Schrader et al. (225) (122), was also demonstrated by Boon et al. (128) using the Mutatox assay. Addition of rat S9 fraction or microsomes of harbor seal and albatross decreased the genotoxic potential of the tested congeners and toxaphene. More in vitro evidence for genotoxicity was found by Sobti et al. (226) showing toxaphene-induced sisterchromatid exchange (SCE) in cultured lymphoblasts. In contrast, Schrader et al. (225) could not demonstrate convincing evidence of a toxaphene-induced (1-10 pg ml-l) dose-dependent SCE induction at the HGPRT gene locus in V79 cells.
Knowing that cell-cycle delay may interfere with the expression of genotoxicity, Steinel et al. (220) studied the effect of cell-cycle delay on the induction of SCE by toxaphene in Chinese hamster lung (Don) cells. They found that toxaphene exhibited a doseand time-dependent decrease in cell-cycle progression. At similar concentrations of toxaphene, higher numbers of SCEs were observed and dose and treatment time relationships were demonstrated. Hence, SCE induced by toxaphene was not masked by mitotic delay and longer toxaphene treatment times were not necessary in Don Chinese hamster cells. Nevertheless, the authors support recommendations for prolonged incubation times in SCE assays affected by mitotic delay.
To study a promotional mechanism rather than a genetic mechanism for toxaphene-induced tumor formation, Kang et al. (221) studied the inhibition of gap junctional intercellular communication (GJIC) by toxaphene. Noncytotoxic concentrations of toxaphene (0-10 pg ml-1) inhibited GJIC in normal human breast epithelial cells reversibly in a dose-dependent manner after 90 min of exposure. In an attempt to determine how toxaphene inhibited GJIC, Kang and co-workers (221) examined Cx43 protein in cells treated with toxaphene. A reduction in the number of gap junctional plaques and induction of hypophosphorylation of Cx43 in normal human breast epithelial cells were observed at toxaphene concentrations that affected GJIC. In addition, these studies also showed that toxaphene inhibits GJIC through a nonestrogen receptor mechanism, as the cells used in these studies do not express the estrogen receptor. An alternative working hypothesis suggesting a central role for protein kinase C (PKC) has been proposed for skin tumor promotion (227). Moser and Smart (222) examined the potency of some hepatocarcinogenic organochlorine pesticides to stimulate PKC in vitro in mouse brain, hepatic, and epidermal homogenates.
Two hundred pM toxaphene increased brain PKC 469-fold. The induction was phospholipid and calcium dependent. It is premature to conclude from this result, however, that stimulation of PKC activity is involved in toxaphene-induced hepatic tumor promotion.
Toxaphene, a Human Risk Factor As mentioned previously, toxaphene is carcinogenic in rats and mice and also has been proven to be mutagenic (1,178). Such findings have led to the assumption that toxaphene poses a risk as a human carcinogen. Human exposure to toxaphene occurs mainly through the consumption of contaminated fish or by occupational exposure.
Data are scarce on the risk to humans from toxaphene exposure (1). Brown et al. (228) and Cantor et al. (229) evaluated the association between elevated risk of leukemia and non-Hodgkin's lymphoma (NHL) among farmers and exposure to pesticides and other agricultural chemicals and concluded that there is an elevated risk of NHL among farmers. Risk increased in cases in which farmers personally handled, mixed, or applied pesticides, did not use protective clothing, and when more specific active mixures of pesticide exposure were used. Chemicals most strongly associated with risk of NHL were carbaryl, chlordane, DDT, diazinon, dichlorvos, lindane, malathion, nicotine, and toxaphene.
Although studies like these contribute to our knowledge about the toxicity of toxaphene for humans, difficulties arise in the interpretation of human risk. In an International Agency for Research on Cancer evaluation of the carcinogenic risk of toxaphene to humans, toxaphene was regarded a carcinogenic risk to humans on the basis of evidence that toxaphene is carcinogenic in rats and mice and, despite the lack of adequate data, humans (230). To date most studies on carcinogenicity of toxaphene have been conducted using technical toxaphene mixtures. Human exposure, however, is mainly through consumption of toxaphene-contaminated fish. Composition of toxaphene mixtures is changed from original technical mixtures through weathering conditions and internal metabolism. Human exposure, therefore, is to a mixture other than technical toxaphene. The toxic and carcinogenic properties of fishborne residues of toxaphene are unknown. Under the auspices of the European Union (EU)-funded project MATT, our laboratories are involved in a semichronic exposure study in a joint effort to produce and isolate fish (cod)-based toxaphene residues that are chemically characterized and toxicologically evaluated, particularly for genotoxicity (in vitro) and tumor promotion capacity.

Legislation of Toxaphene in Food
In 1976 a European directive regulating residues of toxaphene in fruits and vegetables (0.4 mg kg-') was issued (231) that was integrated into the national food laws of all member states in the EU. At that time toxaphene was still used as a pesticide. In 1982 the European maximum residue limit (MRL) for fruits and vegetables was extended to some food of animal origin such as meat and meat products, milk and milk products, and animal edible fat in the German MRL ordinance (232). During that period no reports or data about toxaphene residues were published. On the basis of growing toxicologic concerns when toxaphene was internationally classified as a compound possibly carcinogenic to humans (1), the European MRL for fruits and vegetables was further reduced in 1993 to 0.1 mg kg-1 ww (233), equal to the limit of determination of common residue analysis methods. Thus, residues of toxaphene should not be found in these foods. In 1994 during implementation of this regulation into the German MRL Ordinance, this strict MRL was extended to all food of animal origin (234) including fish and fish products. For fatty fish (lipid content > 10%) the MRL was set at 0.1 mg kg-1 lipid weight, for lean fish with a lipid content > 10%, the MRL was set as at 0.01 mg kg-' ww. In general the previously mentioned regulations were based on total toxaphene levels. At the beginning of the 1990s a sensitive residue analysis method by GC-ECD and GC-NCI/MS using three individual chlorinated bornane congeners, B[12012]- (202), B[12012]- (212), and B[30030]- (122), was developed in Germany (11,92). The method was applied in routine analyses of many German laboratories and validated by an interlaboratory exercise (99,235). First reports indicated that relatively high concentrations of these toxaphene congeners were in some fish from the North Atlantic, an area from which much of Germany's fish stock is derived (100). It was obvious that some edible fish would exceed this low MRL. Therefore, the new regulation for fish, fish products, and mussels was suspended until the end of 1996 (234,236) to give legislators time to determine the level at which the MRL should be established to take into account the questions of, on the one hand, an acceptable level of consumer protection and, on the other hand, the necessary supply of fish and fish products. In the interim, data about the contamination of all edible fish by the three indicator congeners were collected and evaluated in order to calculate the average toxaphene intake through consumption of fish (0.22 jg person-1 day'1) (237). At present there is no acceptable daily intake (ADI) value for toxaphene for use in conducting a risk assessment study. Therefore, the average toxaphene intake was compared with the lowest no-observed adverse effect level (NOAEL) considering a sufficient high safety factor (-25,000-50,000). In 1997 a new concept was incorporated into the German MRL for toxaphene in food of animal origin. The MRL for fish and fish products was set at 0.1 mg kg4l ww on the basis of the sum of the three indicator congeners (238); the MRL for all other food of animal origin was set at 0.1 mg kg-l on the basis of total toxaphene. The German government plans to adjust the MRL in the future on the basis of the toxaphene indicator congener concept. The German ordinance is the first national MRL for fish on the basis of toxaphene congeners.
The United States and Canada are the only countries to have established tolerance levels for toxaphene in food consumed by humans. The U.S. tolerance level was set at 5 mg kg-1 ww; however, this was withdrawn in the early 1990s. Instead of using a tolerance level, Canada uses an ADI value of 0.2 pg kg-1 bw. The calculated daily intake values from the results of Alder et al. (100) stay below this Canadian acceptable daily intake.