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Chronic Toxicity of Chloroform to Japanese Medaka Fish Author(s): Margaret W. Toussaint, Alan B. Rosencrance, Linda M. Brennan, Joseph R. Beaman, Marilyn J. Wolfe, Florence J. Hoffmann, Henry S. Gardner, Jr. Reviewed work(s): Source: Environmental Health Perspectives, Vol. 109, No. 1 (Jan., 2001), pp. 35-40 Published by: Brogan & Partners Stable URL: http://www.jstor.org/stable/3434918 . Accessed: 21/02/2012 01:17
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Articles

Chronic Toxicity of Chloroform to Japanese Medaka Fish

Margaret W. Toussaint,1 Alan B. Rosencrance,2 Linda M. Brennan,1 Joseph R. Beaman,1 Marilyn J. Wolfe,3 Florence J. Hoffmann,2 and Henry S. Gardner, Jr2
INC., 1GEO-CENTERS, Fort Detrick,Maryland,USA; 2U.S. Army Center for EnvironmentalHealth Research, Fort Detrick,Maryland,USA; 3ExperimentalPathology Laboratory,Herndon,Virginia,USA Under the continuous flow-through dosing regimen, sufficient chloroform remained in solution during testing to maintain dosage levels. Approximately 10 L of 100 mg/L chloroform stock were prepared each day. Processed well water was the diluent used. The chloroformstocks were stirredfor 24 ? 2 hr in a sealed glass container, and then pumped into a diluterdosing bottle. BrdU (CAS No. 59-14-3) of 99% purity was obtained from Sigma Chemical Co. (St. Louis, MO). BrdU is water soluble, not volatile, and stable under test conditions. A in stocksolutionof 10 g/L BrdUwas prepared ASTM Type I wateron the day of exposure. Animal care. Researchwas conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals,and adheresto principlesstated in the Guide for the Care and Use of Animals (27). The facilities are Laboratory fully accredited by the Association for the Assessmentand Accreditationof Laboratory Animal Care,International. Japanese medaka fish (Oryzias latipes) were supplied from the U.S. Army Center for Environmental Health Research (USACEHR) in-house cultures and were reared according to USACEHR standing operating procedures. Fish were fed flake food (Aquatox Certified Diet, Ziegler Brothers,Gardners,PA) and live 24-hr brine shrimp daily. Fish were fasted 24 hr before the 6- and 9-month histopathologysacrifices
to Addresscorrespondence M.W. Toussaint,GEOCENTERS, INC., @ USACEHR, 568 Doughten Drive, Fort Detrick, MD 21702-5010. Telephone: (301) 619-7209. Fax: (301) 619-2569. E-mail: Margaret.Toussaint@amedd.army.mil We thank R. Miller, R. Bishoff, M. Hennessey, and W. Dennis for technicalassistance. This projectwas sponsoredby the NIEHS under InteragencyAgreementY1-ES-8051-02 (formerly Y1-ES-7096-02), the U.S. Army Medical Research and MaterielCommand,and the U.S. Army Corps of Engineers. The views, opinions, and/or findings contained in this reportare those of the authors and should not be construed as official Department of the Army position, policy, or decision, unlessso designated by other officialdocumentation.Citationsof commercial organizations or trade names in this reportdo not constitute an official Departmentof the Army endorsementor approval the products of or services these organizations. of Received 2 June 2000; accepted 7 September 2000.

werecontinually dilutersystemfor 9 medaka exposedin a flow-through Japanese (Oryzias latipes) of evalchloroform concentrations 0.017, 0.151, or 1.463 mg/L.Parameters monthsto measured uated were hepatocarcinogenicity, proliferation,hematology,and intrahepatic hepatocellular was at was chloroform concentration. Histopathology evaluated 6 and9 months.Chloroform not at at tested.Chronictoxicitywas evidenced to hepatocarcinogenic the medaka the concentrations lesionsand bile duct these time points by statistically (a significant = 0.05) levelsof gallbladder We in treatedwith 1.463 mg/L chloroform. assessed abnormalities medaka hepatocellular prolifin waterfor 72 hr after4 erationby exposingtest fish to 5-bromo-2'-deoxyuridine the aquarium we indicesof the liversusing and 20 daysof chloroform area-labeling exposure; then quantified We no in increases cellular proliferimageanalysis. observed treatment-related computer-assisted after6 monthsof chloroform cells in circulating blood in medaka ation.We analyzed exposure. different cell leukocrit, viability,and cell countsof treatedfishwerenot significantly Hematocrit, concentrafromthoseof controlfish. Using gas chromatography (GC),we evaluated intrahepatic in tions of chloroform fish after9 months of exposure.Liversfrom the 0.151 and 1.463 mg/L amountsof chloroform, theselevelswerealwayslower fish but chloroform-treated had detectable of that concentrations chloroform. did thanthe aquaria Thus, it appeared chloroform not bioacin metabolite cumulate the liver.Unidentified peakswerefoundin the GC tracings presumptive cell of these fish livers.Keywords: carcinogenicity, aquatictoxicology,5-bromo-2'-deoxyuridine, chloroform,fish, hematology,histopathology,medaka,Oryziaslatipes.Environ proliferation, HealthPerspect 109:35-40 (2001). [Online12 December 2000] niehs. http://ehpnetl. nih.gov/docs/2001/109p35-40toussaint/abstract.html Chloroform is a common drinking water disinfection by-product. Recently, present U.S. Environmental Protection Agency (U.S. EPA) drinking water standards for chloroformhave been scrutinized(1,2): "Are today'sstandardsbased on rodent megadose studies relevant to real world exposures?" Carcinogenicresults in these rodent studies were found at concentrationsseveralorders of magnitude higher than the chloroform maximum contaminant level goal (MCLG) maxof 0 mg/L and the total trihalomethane imum contaminant level (MCL) of 0.08 mg/L (3). Nontraditional models such as fish can be used to study chloroform-inducedtoxicity at exposurescloserto concentrationlevels found in drinking water. Fish have been shown to be sensitiveto tracelevels of contaminants in aquatic media (4-8). As waterdwelling organisms, fish receive dermal exposurethroughwhole-body immersion in the exposuresolution. Intakeof test material also occursthroughfeedingand respiration. Japanese medaka have been studied extensivelyboth in the United States and in Japan (9-13). Their hardiness, small size, ease of culturing, and relativelyshort timeto-tumor response make the medaka an attractivetest model. Sections of the entire animal will fit on one microscope slide so that examinationof everytissue is possiblein Environmental HealthPerspectives * its anatomical context. The low rate of spontaneous neoplasms in medaka aids in the interpretation bioassayresults(14). of is Increased cellular proliferation necessary to transform normaltissueto neoplastictissue (15). 5-Bromo-2'-deoxyuridine (BrdU), a thymidine analogthat labelscells in S-phase, has been used extensively to study cellular proliferation in rodents (16- 18) and fish (19,20). BrdU cell labelinghas been accomplished previouslyvia intraperitonealinjection, implantationof an osmotic pump, and aqueous exposure (19-24). Previous BrdU studieswith medakain this labrange-finding oratoryhave demonstratedan effective cellof labelingconcentration BrdU in the ambient waterto be 75 mg/L for 72 hr (25,26). Our purposewas to determinethe effect of chloroform on Japanese medaka after 9 months of continuousexposure.Specificend were severityand prevalence points evaluated of neoplasms, hepatocellular proliferation, hematology, intrahepatic chloroform concentration,fish growth,and fish survival.

Materials and Methods

Test materials. Chloroform (CAS No. 6766-3) of 99.8% purity was obtained from Aldrich Chemical Company (Milwaukee, WI). Weekly chemical analyses of aquaria water demonstratedthat chloroformdid not break down to form other compounds.

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and the BrdU exposures. Durotest Optima Choice fluorescent bulbs with a color rendering index (CRI) of 91 (Durotest Lighting, Fairfield, NJ) provided the light for the 16-hr light/8-hr darkcycle of light. Test design. We chose the amount of chloroformroutinelyfound in dechlorinated tap water as the lowest concentrationtested. In 96-hr range-findingstudies with juvenile medaka,a length NOEL (no-observed-effect level) of 21-25 mg/L and a weight NOEL of 22-26 mg/L were establishedfor chloroform (28). We chose a log scale for the three concentrations of chloroform, which ensured that all concentrationstested were below the NOELs established in acute testing. Four test aquariawere randomlyassignedto each nominal concentration of 0, 0.015, 0.15, and 1.5 mg/L chloroform. The test beganwhen 14-day-oldfry (? 1 day) were randomizedto each 5-gallon test 15 aquarium,with 80 fry and approximately L test solutionper sealedaquarium. The proportionaldiluterwas set to deliver300 ?+15 mL to each aquariumevery 3 min + 15 sec, yielding9-10 tankvolumesper day. During the first month of chloroform exposure,five fish per aquariumfor each of four time points were used for the hepatocellular proliferationassays.After 6 months of chloroform exposure, we evaluated 20 fish per aquarium by histopathology, and an additional five fish per aquarium by hematology.At 9 months of exposure,five fish per aquarium were used for the chloroform intrahepatic concentration analysis. We euthanized all remaining fish to assess the following tissues by histopathology: bone (vertebra),brain, chromaffin tissue, corpuscle of Stannius, esophagus,eye, gallbladder, gill, heart, hematopoietic tissue, interrenal tissue, intestine, kidney, liver, nares, ovary, pancreas, peripheral nerve, pineal organ, pituitarygland, pseudobranch,skeletalmuscle, skin, spinal cord, spleen, stato-acoustic organ, swim bladder,testis, thymus, thyroid tissue,urinarybladder,and grosslesions. Statisticalanalyses.We analyzedlengths, weights, and histopathologydata statistically for the chronic carcinogenicity test. For these and all other test end points, we applied normalizing transformationswhere required,and selected the best-fittingmodel for each test end point. We used analysisof variance (ANOVA) to test for effects, and regressionanalysisfor point estimation. We used SAS PROC GLM computer software (SAS Institute, Inc., Cary, NC) for these analyses(29). Chloroformchemicalanalyses.All exposure tankswere sampledweeklyand analyzed on the day of collection. Daily stock solutions were collected and stored at 5?C until the weekly analysisof the exposuretanks.All

sampleswere collectedin 40 mL borosilicate glass U.S. EPA water-sampling vials with Teflon-linedsiliconerubber septain the cap. To analyze samples we used a Hewlett Packard6890 gas chromatographequipped with an electron capturedetector and interfaced to Hewlett Packardmodel 7694 headspace sampler (Agilent Technologies, Palo Alto, CA). We used a Hewlett Packard ChemStationfor instrumentcontroland data acquisition. The capillary column used throughoutthe study was a Hewlett Packard 25-m HP-1 (cross-linkedmethyl polysiloxane), 0.2 mm i.d. and 0.33 pm film thickness. The oven temperatureof the gas chromatoat graphheld isothermally 40?C, the inlet was set at 250?C, and the electroncapturedetecat tor was maintained 300?C. We chose headspace analysis for this study for its sensitivity,simplicity,and rapid throughput. Headspace analysis has been used elsewhereto analyzeratliver, urine, and blood (30,31) and was applied here to fish tissue and water samples. For the weekly analysis of exposure tanks, three 5 mL aliquots of each sample were placed in 10 mL headspacevials and sealed with Teflonlined crimp caps. The vials were then placed in the headspace sampler with the sampler oven set at 60?C, the 1 mL sample loop at 65?C, and the transferline to the gas chromatographat 70?C. The sample vial equilibration time was 30 min with agitation set on high. Externalcalibrationstandardswere used for this method and preparedfresh on the day of analysis.The technique required no further sample preparation and there were no interfering contaminants from the sample matrix. The detection limit was was 0.003 mg/L and the recovery 95.2%. BrdU chemical analyses. We collected samplesfor chemicalanalysisat the initiation and termination of each exposure. Spiked of recovery 102%. samplesshowedan average We used a Hewlett Packard1050 serieshigh performance liquid chromatograph equipped with a variable wavelengthdetector,autosampler, and Hewlett Packard Chemstation (AgilentTechnologies)to analyzeBrdU samples. Sampleswerefilteredthrougha 0.45 pm membranebeforeanalysis.The solvent delivery system was programmedto deliver 15% methanol/85% water at a flow rate of 1.5 mL/min. The UV detectorwas set at 277 nm to monitor the eluate.A SupelcoTM LC-18 column (25 cm x 0.46 i.d., 5 pm particlesize; Supelco,Bellefonte,PA) was used for the separation. The injectionvolumewas 5 pL. Hepatocellularproliferation assays.Five were removedfrom each of fish per aquarium the 16 test aquariaon test days 2, 4, 7, and 20, exposedto 75 mg/L BrdU for 72 ? 4 hr, and euthanizedwith an overdoseof tricaine methane sulfonate (MS-222) on test days 5,

7, 10, and 23, respectively.BrdU exposure, fixation, sectioning, staining, and counting methodshave been describedpreviously(26). We used a Bioquant/True Color Image AnalysisSystem(Bioquant-R&MBiometrics, Inc., Nashville,TN) to evaluateBrdU-labeled slides, with five BrdU-stainedsections from each fish. A count labeling index (CLI) was done to validatethe arealabelingindex (ALI) method and to ensure that hepatic cell size did not changedue to chloroformtreatment. R2valuescomparingthe CLI and ALI for the 4-day and 20-day sacrificepoints were 0.934 and 0.921, respectively. Chronic carcinogenicity. At 6 and 9 months of chloroform exposure, fish were euthanized with an overdose of MS-222. Fish necropsy procedures,fixation, sectioning, and stainingwere done as describedpreviously (32). Fish tissues, listed in "Test Design,"were evaluatedby histopathology. Fish hematology. During the fish chronic wereremovedafter test, five fish per aquarium 6 months of continuous exposureto chloroform. Fish were euthanizedby an overdoseof A MS-222, and then weighed and measured. capillary tube (10-20 pL) of blood was removedfrom each fish, and each fish'santerior kidneys, a major site of hematopoeisis, were excised. Hematocritand leukocritwere measuredfor each blood sample.Cell counts were performedusing a hemacytometer. Cell viabilitywas assessed(trypanblue exclusion) from kidneycell suspensions. Fish intrahepatic chloroformconcentration. At 9 months of chloroform exposure, five fish per aquarium were removed from the test for analysisof chloroform intrahepatic concentration. Fish were euthanized with an overdose of MS-222, weighed, and measured.The liverswere excised, weighed, flash frozen in individual cryovialsin liquid nitrogen, and then stored at -70?C. On the day of analysis,liverswere thawed and transferred to 10 mL glass head space vials containing 5 mL of 2% (w/v) sodium dodecyl sulfate (SDS). Two percent SDS was added to denatureliver enzymes immediately,thus terminating metabolism. The vials were capped and heated at 60?C for 2 hr before headspace analysis by capillary gas chromatography (as described in "Chloroform ChemicalAnalyses"). The detection limit for chloroform in the SDS solution was 0.5 pg/L, which was converted to milligramsof chloroformper gramof livertissue. Recovery was 95.2%.

Results
Water quality. Water quality parameters monitored were temperature,pH, dissolved oxygen, conductivity, alkalinity, hardness, and un-ionized ammonia. All water quality parameters were within acceptable limits

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Articles * Chloroform chronic toxicity to fish

based on over 10 years of historical data of our laboratory diluentwater. Chloroform chemical analyses. Over 9 months, we performed 38 weekly chemical analyseson each aquarium.Mean measured concentrations(replicates combined) yielded measured test concentrations of 0, 0.017 ? 0.004, 0.151 ? 0.034, and 1.463 ? 0.242 mg/L. Hepatocellular proliferation assays.Two of the four hepatocellularproliferationtests had unacceptablemortality(> 10%) in control and treatedgroups.In the remainingtwo cell proliferation time points, survival was 100%. Mean measuredBrdU concentration for all treatments was 74.3 ? 2.8 mg/L. Results from the two acceptabletests, time point 2 (4 days of chloroformexposure)and time point 4 (20 days of chloroform exposure),suggestedchloroformtreatment-related effects on hepatocellularproliferationat the concentrations tested (mean values for 20 days of chloroformexposure:0.014, 0.120, and 1.141 mg/L) at time point 2 but not at time point 4. These resultswere not statistihowever. callysignificant, Fish growth and survival. Mortality through the 9-month exposureperiod was < 4% in all control and treated groups. Growth measurements are summarized in Table 1. At 6 months, therewas a suggestion of growth reduction, but these results were not statisticallysignificant. Comparison of estimated means and their 95% confidence limits revealeda reductionin fish length (f= 7.66, p = 0.0059) with the highest test-concentration fish smaller than control fish, 23.8 mm (23.43, 24.18) and 24.8 mm There was also a (24.23, 25.47), respectively. reductionin fish weight (f= 5.41, p = 0.025) overall. At 9 months, no reduction in growth was found for length (f= 3.22, p = 0.0732) or weight (f= 1.58, p = 0.2090). Fish histopathology. 6 months of chloAt roform exposure, the only significant (f= 10.74, p = 0.0055) finding for males at the 1.464 mg/L concentrationwas proliferation, or hyperplasia,of bile ducts of the liver. In contrast, female medaka at the 1.464 mg/L concentration exhibitednine significantfindings in the bile ducts of the liverand the gallbladder.As in males,femaleshad an increase (f= 23.46, p = 0.0003) in bile duct hyperplasia. Additionally, females had bile duct (f= epitheliumhyperplasia 7.84, p = 0.0142), dilatation of the bile ducts (f= 18.93, p = 0.0007), and concretions in the lumen (f= 42.98, p = 0.0001). Granulomatous pericholangitis-an inflammation around bile ducts characterized mainly by macrophages with variablenumbersof lymphocytes-significantly(f= 31.62, p = 0.0001) occurredin as females,presumably a resultof bile leakage into the liver parenchyma.Concretions also Environmental HealthPerspectives *

significantly(f= 35.39, p = 0.0001) occurred in the lumen of the gallbladder. The epithelium of the cysticduct as well as the gallbladder itself exhibitedsignificanthyperplasia (f= 36.17, p = 0.0001 andf= 19.94, p = 0.0005, respectively). Dilatation of the cystic duct was significant (f= 10.60, p = 0.0057) in femalemedaka. After 9 months of exposure to various concentrations chloroform,therewere sigof nificant differences overall between males and femalesin their responsesto chloroform for gallbladder concretions (f= 5.76; p = 0.0231), cystic duct hyperplasia(f= 4.94; p = 0.0341), and bile duct epithelium hyperplasia (f= 6.43, p = 0.0169). These changes occurred with greater frequency among femalesthan among males. At 9 months and with increased measured chloroform concentrations, males demonstrated a significantly (f= 9.57; p = 0.0079) higher incidence of dilatation of the cystic duct of the gallbladderand a tendency toward a significantly (f= 4.12; p = 0.0617) higher incidence of hyperplasiaof the epithelium of the gallbladder. Females

respondedto increasedmeasuredchloroform concentrations with a higher incidence of of hyperplasia the cystic duct of the gallbladder (f= 25.73, p = 0.0002), hyperplasiaof the gallbladder epithelium (f= 5.94, p = 0.0287), concretions in the lumen of the gallbladder (f= 32.51, p = 0.0001), and granulomatousinflammation in the wall of the gallbladder (granulomatous cholecystitis) (f= 8.30, p = 0.0121) characterizedby the and presenceof macrophages lymphocytes. Few liver neoplasms were observed for control and treated fish at 6 and 9 months. One hepatocellularadenoma, a benign neoplasm of hepatocytes, was observed in a male exposed to 0.151 mg/L chloroform for 6 months, while no malignant hepatocellular neoplasms occurred in any 6month-exposed fish. At 9 months, one hepatocellularcarcinoma (a malignant neoplasm of hepatocytes) was observed in a control female. Two hepatocellular adenomas occurred in medaka examined at 9 months: one in a male treated with 0.017 mg/L chloroform and one in a female treatedwith 0.151 mg/L chloroform.
__

Table 1. Fishchronic chloroform exposure:9-monthsurvivaland growth. Chloroform treatment (mg/L) Replicates Well-water controls 1 2 3
A

Percent survival 100 99 96

Mean wet weight (mg) 296 301 297

Mean standard length (mm) 25.3 24.9 24.8

Treatment mean weight (mg) 297

Treatment mean length (mm) 24.9

Table2. Histopathologymalemedaka of afterchloroform 6 of exposure through and9 months: prevalence

findings. Tissues Gallbladders examined Cholecystitis, granulomatous Concretions duct Cystic concretions ductdilatation Cystic ducthyperplasia Cystic Epithelium hyperplasia Livers examined Bileductconcretions Bileductdilatation Bileductepithelium hyperplasia Bileducthyperplasia Pericholangitis, granulomatous m,months. Control (6 m, 9 m) 46, 53 0,0 0,0 0, 0 0,0 0,0 0, 0 47, 53 0,0 1,4 0,0 0,0 0,0 0.017mg/L (6 m, 9 m) 32, 46 0,0 0, 0 0,0 0,0 0, 0 0,0 34, 46 0,1 0.151mg/L (6m, 9m) 44, 47 0,0 0,1 1,0 1,0 0,0 1,1 46, 47 0, 0 1,11 0,0 0,2 0,0 1.463mg/L (6 m, 9 m) 31,47 0,0 0,3 0,0 1,2 0,0 0,2 34, 49 0,2 1,9 1,0 2,1 0,1

1,5
0,0 0,0 0,0

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A tabularsummaryof non-neoplastictissue alterations in and around the liver in males is shown in Table 2, femalesin Table 3. Trends seen in the occurrenceof gallbladder lesions and bile duct abnormalitiesat 6 months were confirmed at 9 months. Representativephotomicrographs of tissue alterations occurringat the 1.463 mg/L level of chloroformareshown in Figures1 and 2. Table 4 shows relevantfindings at 6 and 9 months where responseswere observedat both time points. The overall trend of the data was to have findings of no significance or marginalsignificanceat 6 months and to havea significantfindingat 9 months, except for cystic duct concretions in 6-month femalesand bile duct hyperplasia 9-month in males,which showedthe oppositetrend. Fish hematology test. The hematology analysis was conducted after 6 months of chloroform exposure. ANOVA performed on data from chloroform-treated fish for hematocrit, leukocrit, cell viability, and cell count demonstratedthat none of these parameters were significantly different from those of controls(p = 0.05). Fish intrahepatic concentration. We detectedno chloroformin any of the 20 fish liversfrom the 0 or the 0.017 mg/L aquaria concentration groups. Of the 20 fish analyzed in the 0.151 mg/L aquariaconcentration group, 2 fish livers had measured concentrations of chloroform (33 and 133 mg chloroform/gof fish liver). At the 1.463 mg/L aquariachloroform concentration, 9 fish livers out of the 20 sampled had detectable amounts of chloroform (23, 26, 35, 41, 128, 144, 159, 194, and 219 mg/g). One or two other unidentifiable peaks wereseen frequentlyon the chloroformchroas matographs, illustratedby a representative chromatograph in Figure 3. One of these peakswas in size equal to or greaterthan the chloroformpeak. Repeatedattemptsto identify these other peaksthrough relatedexperiments havenot yet been successful.

dose) and mice (30 ppm chloroform,90-day and 30 ppm, 14 daysof duration,inhalation; dosing with 2-year duration, inhalation), males had a greatersensitivityto chloroform presumably through testosterone receptor mechanisms in the proximal convoluted tubular cells (33-36). In medaka, females demonstrateda greatersensitivityto chloroform at both 6 and 9 months. Chloroform target organs in rodents were kidney, liver,
of findings.

and nasalpassages(33-35), while in medaka only the gallbladderand bile ducts showed tissue abnormalities.In an inhalationstudy, rats that were exposed to 300 mg/L chloroform developed intestinal crypt-like ducts with periductular fibrosis from nonbiliary cells in their livers(37). No such corresponding abnormality was observed during fish pathologyin the currentstudy. Interestingly, the rodent studies revealed an association

Table3. Histopathologyfemalemedaka afterchloroform of 6 exposure through and9 months: prevalence

Control (6m, 9 m) 31,62 0,0 0,1 0,1 0,0 0,0 0,0 33, 62 0, 0 1,4 0,0 0, 0 0,0 0.017mg/L (6m, 9m) 43, 54 0,0 0,1 1, 1 0,0 1,0 0,0 47, 58 0,1 0,4 0, 0 0,1 0, 0 0.151mg/L (6m, 9m) 34, 66 0,0 0,5 0,1 0, 0 0, 0 0,2 34, 67 0,0 0,8 0, 0 0,0 0,0 1.463mg/L (6 m, 9 m) 48, 55 1,3 9,16 2,0 3,1 1,6 5,4 48, 57 5,6 10,12 3,4 2,7 3,4

Tissues Gallbladders examined Cholecystitis, granulomatous Concretions duct Cystic concretions duct Cystic dilatation ducthyperplasia Cystic Epithelium hyperplasia Livers examined Bileductconcretions Bileductdilatation Bileductepithelium hyperplasia Bileducthyperplasia Pericholangitis, granulomatous m,months.

and Figure1. Hematoxylin eosin-stained slides of medakatissue after 9 months of continuous dosing. (A) Liverand gallbladder(arrows) with concretions in chloroform-treatedfish (1.463 mg/Ltank concentration);the gallbladderwall was thickened by a granulomatousinflammation. Controlliverfrom a fish of (B) the same age. Bar= 300 pm.

Discussion
In medakaexposedto chloroformconcentrations ranging from 0.017 to 1.463 mg/L, induction of liver neoplasmsdue to chloroform exposurewas not significantlydifferent (ac= 0.05) in treatedfish when comparedto control fish, after6 or 9 months of exposure. Gallbladder abnormalities and bile duct abnormalities were observedin treatedfish at significantly increased frequencies at the 1.463 mg/L chloroform level. Chloroform was not hepatocarcinogenicto the medaka after6 or 9 months of exposure. Numerous pathology findings were dissimilarbetween chloroform-exposed rodents and the currentfish study. In rats(477 mg/kg 48-hr duration,singlegavage chloroform/day, 38

b+A

and Figure2. Hematoxylin eosin-stained slides of medakaliversamples. (A) Biliaryhyperplasiain liverof a fish treated for 9 months(1.463mg/Lchloroform). Liver a controlfish of the same age. Bar= 300 pm. of (B)

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between hepatocyte labeling indices and prevalenceof aberrantducts (23,33-35). In the fish, we saw no concurrent increase in hepatocytelabelingand tissue abnormalities. Some of these differences be attributed can to route of exposureand applied concentration levels,while othersare undoubtedlyrelatedto choice of animalmodel. The reasonfor the occurrenceof concretions in the gallbladder and the bile ducts of the medaka is unknown. Concretions are rare in animals (38,39). Previously, biliary concretions have been identified in monkeys, cattle, and pigs (39). The principal constituents of these concretions were cholesterol, precipitatedbilirubin, and calcium carbonate, respectively. When concretions occur, it is not unusualfor them to be composites of these three materials (39). The causeof these concretionsis usuallyan infection. In sheep choleliths (concretions), has Pseudomonas aeruginosa been identified as the infectiousagent (39). The mechanism for formation is usually solid particles of dead organismsservingas a nidus for crystallization. Additionally, disturbances in the resorptiveactivities in the gallbladder may promote the development of concretions (38). Furtherinvestigationsare necessaryto determine the origin and makeup of these concretionsin chloroform-treated medaka. Hepatocellular proliferation in chloroform-treatedfish livers was not significantly differentfrom proliferationratesobservedin control fish liversat 4 and 20 days of chloroform exposure.The exact cause of fish death in the other cell proliferation time points was not determined,but we suspect that the high mortalityresultedfrom fungal and bacterial contamination in the stagnant processed well water delivery line used to makethe BrdU exposuresolutions. Rodent studies have shown a positive associationbetween increasedcell proliferation and the hepatocarcinogenicity a comof pound (2,40,41). In a previous study (26), between carcinogenicity and this relationship

cell proliferation also seen in the medaka was aftera 48-hr exposureto the livercarcinogen, diethylnitrosamine. Intrahepatic chloroform concentration was less than external aquaria concentrations, with a higher number of fish livers containing chloroform at the 1.463 mg/L concentration. Phosgene is a known mammalian metabolite of chloroform (42), but we did not confirm its presencein fish livers exposedto chloroform.Chloroformdoes not appearto have bioconcentratedin fish liver; the chloroform intrahepatic concentration was alwayslower than the externalaquarium concentration. Chloroformwas not acutelytoxic to fish at concentrations two or three orders of magnitudeabove median drinkingwaterlevels. Chronic toxicity effects of chloroform were demonstratedby statistically significant findings in the gallbladderand bile ducts of fish treatedwith 1.463 mg/L chloroform. Given the significant biliary findings observedat the high concentrations without a it similarearlyinduction of cell proliferation, is intriguing to hypothesize that in initiated populationsof cells (43), chronicexposureto the high concentration of chloroform may It promotebiliary carcinogenesis. is alsosignificant that althoughwe observedno evidence of earlyhepatocellular necrosisand compenthe satoryhyperplasia, highest concentration

of chloroform appeared to cause a chronic hyperplasia in animals sacrificed after 9 months of exposure.Furtherstudies are warranted with this nonmammalian vertebrate model to add to the weight of evidence in public health decisions about balancing potentialdisinfectionby-producttoxicityand diseaseriskfrom microbialcontamination. REFERENCES AND NOTES
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Time(min) of Figure3. Chromatogram a fish liversample used to determine intrahepaticchloroformconcentration in medakafish exposed to 1.463mg/Lchloroform for 9 months. Peaks include the injection peak, two unknown peaks, and the chloroform detection limitsof peak, respectively.Instrumental chloroform were 0.001mg/L.

Table 4. Chloroform concentrationeffectsa on fish histopathologyend points at 6 and 9 months,by sex. Males Endpoint Gallbladder duct Cystic dilatation Epithelium hyperplasia Granulomatous cholecystitis Concretions duct Cystic concretions Liver Bileductconcretions Bileductdilatation Bileductepithelium hyperplasia Bileducthyperplasia Granulomatous pericholangitis 6 months No No NF NF NF Marginal No NF Yes NF 9 months Marginal Marginal NF NF NF No No NF No NF 6 months NF NF Marginal Yes No Yes Yes Yes Yes Yes Females 9 months NF NF Yes Yes Yes Yes Yes Yes Yes Yes

Abbreviations: NF, No, Yes, Marginal,> 0.05< 0.10; nonefound; p> 0.10; p < 0.05. p 'ANOVA level significance = 0.05.

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