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By measuring a battery of cytological and biochemical biomarkers in adult female perch (Perca fluviatilis), the city of Stockholm (Sweden) was investigated as a point source of anthropogenic aquatic pollution. The investigation included both an upstream gradient, 46 km westwards through Lake Mälaren, and a downstream gradient, 84 km eastwards through the Stockholm archipelago. Indeed, there was a graded response for most of the biomarkers and for the muscle concentrations of ΣPBDE, four organotin compounds and PFOS in the perch. The results indicated severe pollution in central Stockholm, with poor health of the perch, characterised by increased frequency of micronucleated erythrocytes, altered liver apoptosis, increased liver catalase activity, decreased brain aromatase activity, and decreased liver lysosomal membrane stability. Some biomarker responses were lowest in the middle archipelago and increased again eastwards, indicating a second, partly overlapping, gradient of toxic effects from the Baltic Sea.

Since the new millennium, a notion has developed in certain parts of society that environmental pollutants and their associated effects are under control. The primary objective of this investigation, performed in 2003, was to test whether this was actually the case in an industrialised region in the County of Västernorrland in northern Sweden with well-documented environmental pollution from past and present activities. This was performed by measuring a moderate battery of simple biomarkers in adult female perch at several stations. The point sources included sewage-treatment plants, pulp and paper mills, as well as other industries. The biomarkers included growth, somatic indices, gonad maturation status, gonad pigmentation, fin erosion, skin ulcers, and ethoxyresorufin-O-deethylase (EROD) activity in the liver. The results showed that the environmental pollutants and their associated effects were not under control. In fact, the health of the perch was impaired at all of the polluted stations. Many responses were unspecific with respect to underlying cause, whereas some effects on EROD activity and gonad maturation status were attributed to historical creosote pollution and current kraft pulp mill effluents, respectively. The data presented may also be used as reference values for future investigations of health effects in perch. © Springer Science+Business Media 2013. Language of Original Document: English

In a series of bio-effect-directed fractionation experiments, we investigated the potential toxicity of sediment extracts from a contaminated bay. A previous study investigated abnormalities and hepatic ethoxyresorufin O-deethylase (EROD) activities in rainbow trout (Oncorhynchus mykiss) larvae by exposing newly fertilized eggs to the total extract and to fractions separated by degree of aromaticity. A major part of the potential toxicity was isolated in a fraction containing polycyclic aromatic compounds (PACs). In this study, we prepared a synthetic PAC mixture with 17 com- monly analyzed polycyclic aromatic hydrocarbons (PAHs) in amounts equimolar to those found in the sediment PAC fraction. The 17 PAHs, which included 11 of the 16 United States Environmental Protection Agency (US EPA) priority PAHs, were unable to account for the toxicopathic effects observed and could explain less than 4% of the total EROD induction. The lack of a clear relationship between toxicopathic effects and EROD induction underlines the need for a battery of biomarkers for estimating environmental risk. These results reveal the limits of our knowledge regarding compounds responsible for potential toxicity in field situations. Resume : Dans une serie d'experiences de fractionnement dirige vers les bio-effets, nous avons etudie la toxicite po- tentielle d'extraits de sediments provenant d'une baie contaminee. Une etude anterieure avait examine les anormalites et les activites de l'ethoxyresorufine O-deethylase (activites EROD) hepatiques chez les larves de truites arc-en-ciel (Oncorhynchus mykiss) en exposant des oeufs fraichement fecondes a un extrait complet et a des fractions separees d'apres leur degre d'aromaticite. Une partie importante de la toxicite potentielle a pu etre isolee dans une fraction contenant des composes aromatiques polycycliques (PAC). Nous avons prepare dans notre etude un melange synthe- tique de PAC contenant 17 hydrocarbures aromatiques polycycliques (PAH), frequemment trouves dans les analyses, dans des quantites equimolaires a celles presentes dans la fraction PAC des sediments. Les 17 PAH, qui incluent 11 des 16 PAH sur la liste de priorite de l'EPA, ne peuvent expliquer les effets toxicopathiques observes et n'arrivent a elucider qu e4%d e l'induction totale d'activite EROD. L'absence de relation nette entre les effets toxicopathiques et l'induction d'EROD souligne la necessite d'avoir une batterie de biomarqueurs pour estimer les risques environnemen- taux. Nos resultats montrent les limites de nos connaissances sur les composes responsables de la toxicite potentielle dans les conditions de terrain.

Sea-run Baltic salmon ( Salmo salar ) populations are suffering from the M74 syndrome, a reproduction disorder affecting both broodfish and their progeny. The syndrome is usually manifested during the middle part of the yolk-sac fry stage and has been shown to be associated with a thiamine (vitamin B 1 ) deficiency. Development of the disease is reversible by thiamine treatments of broodfish or progeny. This study aimed at investigating the ability of the thiamine antagonist pyrithiamine, administered by microinjections 3 days after hatch, to cause M74-like signs i.e. typical clinical symptoms, high mortality rates and histopathological changes. Furthermore, the effects of pyrithiamine on hepatic activities of the thiamine-dependent enzyme transketolase (TK), the glucose-6-phosphate dehydrogenase (G6PDH) and the cytochrome P4501A (CYP1A) were evaluated. Six family groups with differing thiamine status were sampled on three occasions during the yolk-sac fry stage. All pyrithiamine exposed groups, with the exception of the one with the highest thiamine concentration, showed M74-like symptoms and suffered from high mortality. Enzyme activities were not different in pyrithiamine groups as compared with controls. However, the TK-activities were strongly associated with the thiamine concentrations. The G6PDH-activity demonstrated small variations with the highest activities in the M74-groups. The [TK]/[G6PDH]-ratios were considerably lower in the M74-groups than in the healthy controls, indicating an imbalance between the oxidative and the non-oxidative part of the pentose–phosphate shunt due to a deficit in thiamine. The pyrithiamine-injections induced several M74-like symptoms including incoordination, lethargy, whitened liver and yolk-sac precipitates. They also caused high mortality rates, in addition to lowered glycogen levels and increased prevalence of necrotic brain cells. Moreover, the study demonstrates that the TK, G6PDH and CYP1A-activities are associated with the thiamine content.

Dietary uptake and biological effects of decabromodiphenyl ether in the rainbow trout (Oncorhynchus mykiss)

Viviparous blenny ( Zoarces viviparous ) were exposed to chrysene and held in natural brackish water as well as in two different synthetic brackish waters, all with a salinity of 6.8%. After 10 weeks of exposure a number of different biomarkers was analysed. The result indicated increased ethoxyresorufin- O -deethylase activity and the formation of two different DNA adduct spots in the liver tissue, as a result of the chrysene exposure. Very similar results of the chrysene exposure were obtained in blenny held in the two synthetic brackish waters. No influence of the synthetically made waters compared to the natural brackish water could be observed on the biomarkers investigated in the study.

Newly fertilised eggs of sea trout (Salmo trutta) were injected with the thiamine antagonist oxythiamine or with oxythiamine in combination with 3,3′,4,4′-tetrachlorobiphenyl (PCB #77). Rainbow trout (Oncorhynchus mykiss) eggs were injected with PCB #77. A decrease in liver transketolase activity was used as a biomarker to indicate a physiological deficiency in thiamine. Exposure to oxythiamine caused deformities in the newly hatched larvae, as well as high mortality during the larval stages. Exposure to oxythiamine in combination with PCB #77 caused a synergistic higher frequency of disorders. Analysis of liver transketolase activity in the larvae injected with PCB #77 indicated that PCB #77 did not decrease the half-life of thiamine in the offspring. The data obtained indicate that oxythiamine increased the sensitivity to PCB #77 in the offspring, however through mechanisms other than those causing thiamine deficiency in feral fish.

Two polybrominated diphenyl ethers, 2, 4, 2′, 4′-tetrabromodiphenyl ether and 2, 4, 5, 2′, 4′-pentabromodiphenyl ether were added to the food given to two different groups of rainbow trout. The rainbow trout were fed for a period of 22 days. On days 6 and 22, a number of physiological and biochemical variables were investigated including condition factor, liver somatic index, spleen somatic index, hematocrit, differential count of leucocytes, haemoglobin and glucose in whole blood as well as the liver enzyme activities, glutathione reductase, catalase and ethoxyresorufin-O-deethylase. The results show that both these substances were relatively non-acutely toxic and several variables were unaffected. However, the blood variables hematocrit and blood glucose showed a slight influence from 2, 4, 5, 2′, 4′-pentabromodiphenyl ether after six days of exposure. Also the glutathione reductase activity in the liver decreased after 22 days of exposure. The 2, 4, 2′, 4′-tetrabromodiphenyl ether showed no influence on the investigated variables, with one striking exception. This structure caused a decrease of about 75% in ethoxyresorufin-O-deethylase activity in the liver after six days, and this effect was also observed after 22 days.

Rainbow trout were exposed to an n -hexane extract of engine exhaust condensate by intra peritoneal (i.p.) injection or by feeding with contaminated home-made cod chips. Exposure levels were calculated and chosen to reflect those conditions possibly experienced by fish in their natural habitats. Disruption of biological functions has been observed at different levels of biological organization including cellular and subcellular processes (DNA-adduct levels and enzyme activity) and physiological functions (carbohydrate metabolism). Female and male fish may be affected to a varying degree by these types of pollutants.

To test the hypothesis that cod do not reproduce normally in the Baltic Sea, a comparative study using cod from relatively unpolluted areas in the Barents sea was performed. During the spawning period cod were caught by trawling, then stripped and fertilised directly on the boat. The fertilised egg batches were transported to the laboratory immediately, and held under optimal conditions for cod embryo and larval development. The data obtained indicates that reproductive success is dramatically lower in cod from the Baltic Sea compared with those from the Barents Sea. The disturbances might be correlated to the female individuals rather than the males.

A 2-cycle outboard engine was run in a tank to simulate boatload conditions. After appropriate dilution, exhaust water was led into another tank where fish were exposed. In addition, exposure from extract obtained by acetone/hexane extracted water containing exhaust was used in intraperitoneal injection of juvenile fish as well as in embryo injection experiments. The different areas of investigation were enzymatic disturbances, genotoxicological effects and reproduction disturbances. The investigation so far indicates that the biological effects of the exhaust from 2-cycle outboard engines is a serious threat to the environment. Toxicological effects have been measured in the liver, kidney and blood.

Abnormalities of a gill cover bone, the operculum, were found in perch Perca fluviatilis from an area affected by pulp mill effluents in the Gulf of Bothnia, Baltic Sea, Sweden. One of the abnormalities was expressed as a shortening of the distal part of the operculum, and was termed opercular deformity. This deformity was quantified by taking two measurements of the operculum. The second abnormality was manifested as craterous formations on the operculum. Both abnormalities were studied using a sample of opercula collected in the pulp mill effluent area between the years 1982 and 1989. The maximum prevalences of the abnormalities, 34% for the craterous formation and 20% for the opercular deformity, were found in 1983 and 1984, respectively. In a reference area the prevalences were 1% and 1.4%, respectively. After 1984, the prevalences decreased together with prevalences of other fish diseases and deformities in the same area. This coincided with a reduction in concentrations of effluent. The observed high prevalence of the opercular deformity and the craterous formations were attributed to the effects of the pulp mill effluent. In addition, an analysis of the biochemical composition opercula from perch caught in another pulp mill effluent area showed a dose-dependent alteration of several constituents, indicating a relationship with exposure to the effluent. This study shows the potential of opercular abnormalities to monitor effects of pulp mill effluents, and also, it is suggested, for retrospective studies on large samples of opercula used for age determination in ecological studies of fish.

In estuaries, hypoxic conditions and pollution are among the major factors responsible for the declines in habitat quality, yet little is known about their combined effects on estuarine organisms. In this study, to investigate single and combined effects of hypoxia and contaminated sediment, the Baltic amphipod Monoporeia affinis was exposed for 5–9 days to four different combinations of oxygen conditions (moderate hypoxia vs. normoxia) and contamination (polluted vs. unpolluted sediments) at environmentally realistic levels. To detect oxidative stress, a suite of biomarkers was used – antioxidant enzymes [superoxide dismutases (SOD), catalase (CAT), and glutathione S-transferases (GST)], acetylcholinesterase (AChE), lipid peroxidation status (TBARS concentration), protein carbonyl content (PCC), and DNA strand breakage (DNA-SB). To assay effects at the organism level, we used RNA:DNA ratio as a proxy for growth and metabolic rate and mortality. There were significant increases in CAT and SOD activities and TBARS levels in response to both moderate hypoxia and contaminated sediment, while GST increased and AChE decreased in response to the contamination only. Significant positive correlations were observed among the antioxidant enzymes and between the enzyme activities and TBARS concentration, suggesting a complex response to the oxidative stress. No significant changes in PCC were recorded in any of the treatments. Furthermore, the negative effect of hypoxia on DNA integrity was significant; with frequency of DNA-SB increasing in animals exposed to hypoxia in contaminated sediment. Despite clear effect at the cellular and biochemical levels, no responses at the organism level were observed. Multivariate analyses of the dataset have allowed us to link exposure factors to individual biomarker responses. Of the potential biomarkers assessed in this study, CAT activity was found to be associated with hypoxia, while SOD, GST and AChE activities appear to predict best the effects of exposure to sediments containing several contaminants (e.g. heavy metals, PCBs and PAHs), and TBARS concentration is particularly indicative of combined effects of hypoxia and contamination. In addition to providing new knowledge on the combined effects of multiple stressors on estuarine organisms, the findings of the present study are also important to understand data from biomonitoring studies in the Baltic Sea and in other regions where multiple stress factors co-occur.

Contrary to the suggestion by Rocke and Barker (1), we do not want to exclude botulism as a part of the investigated wildlife disorder (2), because botulism may be secondary to the thiamine deficiency syndrome. This possibility and the investigations by Neimanis et al. (3) are discussed in Text S5 in our SI Appendix (2).

Adult and young herring gulls ( Larus argentatus ) in Sweden and Iceland were investigated with respect to DNA adducts, analysed with the nuclease-P1 version of the 32 P-postlabelling method, and micronucleated erythrocytes. Three important aims were: (1) to estimate the degree of exposure to genotoxic environmental pollutants in the Baltic Sea area and Iceland, (2) to evaluate the utility of the investigated biomarkers in birds, and (3) to investigate if there was any relationship between genotoxic effects and thiamine deficiency. The results demonstrate that both Swedish and Icelandic herring gulls are exposed to genotoxic pollution. Urban specimens have higher levels of DNA adducts than rural specimens, but background exposure to genotoxic environmental pollutants, such as PAHs, is also significant. In the herring gull the general level of DNA adducts in the liver seems to be higher than in fish. DNA adducts were most abundant in the liver, followed by the kidney, intestinal mucosa, and whole blood, in decreasing order. The frequency of micronucleated erythrocytes was probably slightly elevated in all the investigated sites, reflecting a significant background exposure. The level of DNA adducts was unrelated to the frequency of micronucleated erythrocytes, and both these variables were unrelated to symptoms of thiamine deficiency. The investigation confirmed the utility of DNA adducts, and probably also micronucleated erythrocytes, as biomarkers of genotoxicity in birds.

Wild birds of several species are dying in large numbers from an idiopathic paralytic disease in the Baltic Sea area. Here, we demonstrate strong relationships between this disease, breeding failure, and thiamine (vitamin B1) deficiency in eggs, pulli, and full-grown individuals. Thiamine is essential for vertebrates, and its diphosphorylated form functions as a cofactor for several life sustaining enzymes, whereas the triphosphorylated form is necessary for the functioning of neuronal membranes. Paralyzed individuals were remedied by thiamine treatment. Moreover, thiamine deficiency and detrimental effects on thiamine-dependent enzymes were demonstrated in the yolk, liver, and brain. We propose that the mortality and breeding failure are part of a thiamine deficiency syndrome, which may have contributed significantly to declines in many bird populations during the last decades.

By measuring a battery of basic physiological biomarkers and the concentration of SigmaDDT in adult female perch (Perca fluviatilis), an assumed aquatic pollution gradient was confirmed, with the city of Stockholm (Sweden) as a point source of anthropogenic substances. The investigation included an upstream gradient, westwards through Lake Malaren (46 km), and a downstream gradient, eastwards through the Stockholm archipelago (84 km). The results indicated a severe pollution situation in central Stockholm, with poor health status of the perch: retarded growth, increased frequency of sexually immature females, low gonadosomatic index, and disturbed visceral fat metabolism. SigmaDDT, measured as a pollution indicator, was 10-28 times higher than the background in perch from the Baltic Proper. Besides the main gradient other sources of pollution also influenced the response pattern of the measured biomarkers. In particular, there were strong indications of pollution coming from the Baltic Sea.

For reliable monitoring of environmental improvement, the PCB contaminated bay Orserumsviken on Sweden's Baltic coast was investigated prior to remediation. We examined PCB congener patterns and the relative toxic potential of PCBs in extracts of three matrices: bottom sediment, settling particulate matter and the dissolved phase (obtained from semi-permeable membrane devices). Congener patterns were similar in all matrices. Three fractions were isolated from the extracts: (1) aliphatic and monocyclic aromatic compounds (MAC-fraction), (2) dicyclic aromatic compounds (DAC-fraction), including PCBs, and (3) polycyclic aromatic compounds (PAC-fraction), including PAHs. Total extracts and fractions were injected into newly fertilised rainbow trout eggs. At larval stage, hepatic EROD activities were quantified. Though high, the PCB contamination could explain only an estimated 1-5% of the total EROD induction. The order of EROD induction potential was: total extract > PAC-fraction > DAC-fraction > MAC-fraction in all matrices, suggesting that in Orserumsviken PACs made a larger contribution to the EROD induction potential than PCBs and other DACs.

The major aim of this study was to characterize toxic organic compounds in bottom sediments from a PCB polluted bay. To overcome difficulties in pinpointing toxicants in complex environmental samples we applied a bio-effect directed (BED) fractionation approach and investigated the relationships between aromaticity, teratogenicity, and aryl hydrocarbon receptor (AhR) mediated toxicity. Hepatic ethoxyresorufin O-deethylase (EROD) activities and malformations were investigated in rainbow trout (Oncorhynchus mykiss) larvae exposed by injecting sediment extract and fractions (separated by their degree of aromaticity) thereof into newly fertilized eggs. Our results imply that non-additive effects get more pronounced the more complex the exposure. The fraction mainly composed of dicyclic aromatic compounds (DACs), including PCBs, was surprisingly less teratogenic than the fraction mainly composed of polycyclic aromatic compounds (PACs). A major part of the latter potential was isolated in a subfraction mainly composed of three- and four-ring compounds (including alkylated and sulphur-heterocyclic compounds). Though no clear relationship between aromaticity and EROD induction was observed, both the DAC- and the PAC-fractions contributed equally to the EROD induction potential. A major part of the PAC-fraction's induction potential came from a subfraction containing compounds with more than five rings. No clear relationship between teratogenicity and EROD induction was observed, underlining the need for a battery of biomarkers in estimating environmental risk. Two specific malformations not previously described in literature-asymmetric yolk sac and fin edema-could be tracked through the fractionation steps, suggesting that this BED-fractionation strategy is a reliable tool for pinpointing toxic compounds in the environment.

Possible xenobiotic interactions with thiamine were studied in salmonid fish, by repeatedly injecting two model substances, paraquat and menadione, into juvenile rainbow trout (Oncorhynchus mykiss). These two substances were chosen because of their well-known ability to redox-cycle and cause depletion of NADPH in several biological systems. Depletion of NADPH increases metabolism through the pentose-phosphate shunt and may thereby increase the need for thiamine diphosphate by heightened transketolase activity. A special food was produced with lower thiamine content than commercial food, usually enriched with thiamine, which could mask an effect on the thiamine level. After 9 weeks of exposure, glucose-6-phosphate dehydrogenase, transketolase, glutathione reductase and ethoxyresorufin O-deethylase were analysed in liver and kidney cellular sub-fractions as well as analysis of total thiamine concentrations in liver, kidney and muscle. The results showed that paraquat caused a large increase in hepatic glutathione reductase activity and induced hepatic glucose-6-phosphate dehydrogenase activity, i.e., the rate-limiting enzyme in the oxidative part of the pentose-phosphate shunt. Despite this paraquat exposure did not affect transketolase activity and total thiamine concentration.

A two-year study on perch (Perca fluviatilis) in Lake Molnbyggen, Sweden, located in a pristine area but with a public refuse dump in the vicinity, has been conducted. The mechanistic approach through a set of biomarkers during the first year included age, condition, somatic growth, liver, gonad, and spleen weights, and a number of other physiological variables, in addition to ethoxyresorufin O-deethylase, glutathione-S-transferase, glutathione reductase, catalase, and the formation of DNA adducts in the liver. Perch from the uncontaminated Lake Djursjön, located in a neighboring drainage area, were used as reference fish. The most pronounced effect was a 80% reduction in the gonadosomatic index (GSI) for females and a corresponding 36% reduction in males. Fin erosion and shallow open sores were also frequently observed. Biomarkers and later chemical analysis employed indicated that exposure to well-known environmental pollutants was low, suggesting that less well-known antrophogenic substances are responsible for the effects observed in perch from Lake Molnbyggen. During the second year, roach (Rutilus rutilus) of both sexes were also included in this study. In addition, aromatase (P450arom) activity in the brain and testosterone and 17beta-estradiol levels in blood plasma were analyzed. Only one-fourth of the female perch were found to be sexually mature, which was associated with decreased GSI, lower P450arom activity, and reduced circulating levels of steroids. The reproductive disorders observed indicates disturbed endocrine function(s), arresting the majority of the female perch in a sexually nonreproducible immature stage. This novel study is the first to report evidence for endocrine disruption in wild populations of fish living in a lake exposed to leakage water from a public refuse dump.

The first part of this thesis is about the development of a method to inject various substances into the yolk of fish eggs. The method was named nanoinjection and enabled the injection of volumes as small as 10-9 L into the yolk of single fish eggs directly after fertilization, with insignificant mortality due to the injection. The nanoinjection method has been shown to be useful in studies of the toxic effects of environmental contaminants in early life stages and is considered to mimic maternal transfer of anthropogenic and natural substances to the egg. The subsequent parts in this thesis concern thiamine (vitamin B1) and the consequences of thiamine deficiency. Since 1995, it is known that the Baltic salmon (Salmo salar) females transfer insufficient amounts of thiamine into their eggs for their offspring to survive, a condition known as the M74 syndrome. The embryonic and larval development of salmon from the river Dalalven (Sweden) was studied during the seasons of 1994–1995 and 1995–1996. Disorders at hatch and symptoms of thiamine deficiency were described. A parallel study compared early development in cod (Gadus morhua) from the Baltic Sea to cod from the Barents Sea. Both salmon and cod from the Baltic Sea showed a number of disorders at hatch. Nanoinjections of thiamine solutions into newly fertilized eggs from Baltic salmon protected the larvae from the M74 syndrome. Larvae with the M74 syndrome had low hepatic activities of the thiamine diphosphate-dependent enzymes transketolase (TK) and the α-ketoglutarate dehydrogenase complex (KGDHC). Also, apparently healthy Baltic salmon yolk-sac larvae showed reduced activities of TK and KGDHC, compared to the offspring from intraperitoneal thiamine-injected females. The result suggested a sublethal thiamine deficiency in the salmon population. The hypothesis that redox-cycling substances affect thiamine metabolism, was tested in rainbow trout (Oncorhynchus mykiss) juveniles. Exposure to paraquat caused a ten-fold increase in the activity of glutathione reductase and elevated the activity of glucose-6-phosphate dehydrogenase, the rate limiting enzyme in the pentose-phosphate shunt. Despite the nine week period of oxidative stress, neither the TK activity nor thiamine concentrations were affected. The underlying cause(s) of the low thiamine concentration in the Baltic salmon is still unknown.

Since 1974, Baltic salmon, Salmo salar , alevins have been affected by the M74 syndrome, a putative thiamine deficiency with associated high mortality levels that may be reduced by thiamine treatment. This study demonstrates that alevins with M74 suffer from lowered activities of the thiamine-dependent enzymes transketolase and α-ketoglutarate dehydrogenase compared to control fish. In addition, cytochrome P4501A-activity (CYP1A; measured as EROD) is lower in M74-affected alevins. Thiamine treatments administered after hatching resulted in increased activities of transketolase, α-ketoglutarate dehydrogenase and CYP1A in the M74 groups, restoring the thiamine-dependent enzyme activities to levels comparable to those of controls. We suggest that low activities of transketolase and α-ketoglutarate dehydrogenase may be responsible for M74-associated mortality. Low transketolase activities may reduce the intracellular availability of ribose 5-phosphate and NADPH. Ribose 5-phosphate is necessary for protein synthesis and NADPH is the cofactor for NADPH-cytochrome P450 reductase. Reduced cellular availability of these compounds may result in the reduced CYP1A-activities observed in this study.

The rate of extractive alkylation of 24 structurally related sulphonamides has been studied with respect to the alkylating agent and organic solvent. The reaction rate is given by the time for quantitative alkylation calculated from the observed first-order rate constant with 0.1 M tetrabutylammonium (pH 10) as the aqueous phase. The highest reaction rate were obtained with methyl iodide and pentafluorobenzyl bromide as reagents and methylene chloride and methyl isobutyl ketone as organic solvents. The minimum detectable concentration of the N1-methyl derivatives with electron-capture detection was about 10−15 mole/sec.