Toxicity of forest fire retardant chemicals to stream-type chinook salmon undergoing parr–smolt...

TOXICITY OF FOREST FIRE RETARDANT CHEMICALS TO STREAM-TYPE CHINOOKSALMON UNDERGOING PARR–SMOLT TRANSFORMATION

JOSEPH P. DIETRICH,*y MARK S. MYERS,z STACY A. STRICKLAND,y AHNA VAN GAEST,y and MARY R. ARKOOSHyyEnvironmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service,

National Oceanic and Atmospheric Administration, Newport, Oregon

zEnvironmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service,

National Oceanic and Atmospheric Administration, Seattle, Washington

(Submitted 4 May 2012; Returned for Revision 14 June 2012; Accepted 9 September 2012)

Abstract—Long-term fire retardants are used to prevent the spread of wildland fires. These products are normally applied by aircraft andare intended specifically for terrestrial application, but fire retardants have entered aquatic habitats by misapplication and/or accidentalspills and have resulted in fish mortalities. The authors examined the toxicity of two fire retardant products, PHOS-CHEK 259F andLC-95A, to salmon undergoing parr–smolt transformation. Yearling stream-type chinook salmon at the smolt stage were exposed toeight concentrations of each retardant in freshwater and a no-PHOS-CHEK control for 96 h to determine acute toxicity. Concentrationsof the products that caused 50% mortality were 140.5 and 339.8mg/L for 259F and LC-95A, respectively, and could occur duringaccidental drops into aquatic habitats. Damage to gill tissues seen in histopathological sections was attributed to fire retardant exposure.Un-ionized ammonia levels, from 259F, were sufficient to cause acute mortality; but additional factors, indicated by increasedphagosome prevalence in the gills, might have contributed to mortality during LC-95A exposure. Seawater and disease challenges wereperformed to determine sublethal effects of product exposures on fish health. Although PHOS-CHEK exposure did not adversely affectchinook salmon’s susceptibility to Listonella anguillarum, exposure did significantly reduce seawater survival. Reduced salmon survivalresulting from prior fire retardant exposure during their transition from freshwater rearing environments to seawater may decrease theabundance of salmon populations. Environ. Toxicol. Chem. 2013;32:236–247. # 2012 SETAC

Keywords—Salmon Wildfire Toxicity PHOS-CHEK Smolt

INTRODUCTION

Long-term fire retardants are used to prevent the spread ofwildland fires. These products are normally applied by aircraftto create containment perimeters or long-term combustionbarriers around the fire and are intended specifically for terres-trial application. Between the years 2000 and 2010, 36,148aerial loads were dropped in the United States to combatwildfires, averaging over 31 million liters of fire retardantper year [1]. The U.S. Forest Service attempts to avoid intro-duction of fire retardants into aquatic systems by using a 91.4-mbuffer [1]. Nevertheless, fire retardant chemicals may enteraquatic ecosystems that provide habitat for endangered andthreatened species either directly through intentional applica-tions within the buffer or unintentionally via accidental drop orsurface runoff [2]. In fact, between the years 2008 and 2010,there were 49 accidental fire retardant drops within the buffer,and another 54 fire retardant drops were granted exceptions tothe buffer guideline by the U.S. Forest Service for fires thatposed greater threat to life, property, or natural resources [1].Although data are limited, fire retardants have caused fish kills;for example, significant trout mortality occurred in the FallRiver, Oregon, USA, as a result of misapplication while fightinga fire in 2002 [2,3].

During application, fire retardants are essentially 85% waterand 10% inorganic salts (fertilizers), with 5% additives such asgum thickeners, coloring agents, corrosion inhibitors, stabil-izers, and bactericidal agents [2]. Older product formulations

contained sodium ferrocyanide as a corrosion inhibitor, butwere found to be severely and acutely toxic when exposed toultraviolet radiation [3]. The use of fire retardants containingsodium ferrocyanide was discontinued in 2007 [1]. Approvedfire retardants contain diammonium phosphates (DAP;[NH4]2HPO4) and ammonium polyphosphate salts. The primaryrisk associatedwith their use is the potential for ammonia toxicity[3–6], which is also influenced by pH and temperature [7].

Twenty-eight Pacific salmonid Evolutionarily SignificantUnits (ESUs) in Washington, Oregon, Idaho, and California,USA, are currently listed as either threatened or endangered(www.nwr.noaa.gov/ESA-Salmon-Listings/upload/1-pgr-8-11.pdf).A stock of Pacific salmon is considered an ESU underthe Endangered Species Act if it is reproductively isolated fromother stocks and ‘‘represent[s] an important component in theevolutionary legacy of the species’’ [8]. Each ESU is considereda distinct population segment, or species, under the EndangeredSpecies Act [8]. Studies have examined the toxicity of fireretardants on various life stages of salmonids. Among early lifestages, swim-up fry and salmonids that are 60 to 90 d posthatchare more sensitive than eyed eggs [4,6]. However, no data areavailable regarding the sensitivity of fish to fire retardantsundergoing the parr–smolt transformation. The parr–smolttransformation is an important period for salmonids whereinthey undergo physiological and behavior changes in advance oftransitioning to a marine environment [9,10]. Several of thephysiological and behavior changes during the transformation(e.g., decreased total body lipids, increased oxygen consump-tion, changes in endocrine activity, increased ammonia produc-tion, and increased growth rate [11]) may also increase fishstress. Not only have salmonids been found to be more sensitiveto contaminants during the parr–smolt transformation relative to

Environmental Toxicology and Chemistry, Vol. 32, No. 1, pp. 236–247, 2013# 2012 SETAC

Printed in the USADOI: 10.1002/etc.2052

* To whom correspondence may be addressed([email protected]).

Published online 19 November 2012 in Wiley Online Library(wileyonlinelibrary.com).

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other life stages [12,13] but the success of parr–smolt trans-formation can also be affected by exposure to contaminants atearlier life stages [14–16].

The objectives of the present study were to determine theacute toxicity and sublethal effects of two fire retardants,PHOS-CHEK 259F and LC-95A, on yearling chinook salmon(Oncorhynchus tshawytscha) at the smolt stage. These formu-lations are currently approved by the U. S. Forest Service forfighting wildland fires. Acute toxicity was determined in 96-hstatic exposures. Separately, histopathology examinations andanalyses were completed on tissues collected from salmonexposed to fire retardants for up to 96-h under static conditions.Sublethal effects on seawater tolerance and disease suscepti-bility were assessed with separate challenges conducted onsurvivors of previous fire retardant exposures.

MATERIALS AND METHODS

Fire retardants

Both PHOS-CHEK LC-95A and 259F (ICL PerformanceProducts LP) are proprietary compounds provided by the U.S.Department of Agriculture Forest Service Wildland Fire Chem-ical Systems Program of the Missoula Technology Develop-ment Center (Missoula, MT, USA). Product information forboth retardants is provided by the Forest Service and themanufacturer. In brief, PHOS-CHEK 259F is a diammoniumphosphate-based dry powder formulation with a manufacturer-recommended mix ratio of 136.6 g per liter of water (1.14 lbper U.S. gallon). At the approved mix ratio, 259F has a specificweight of 1.066 kg/L (8.90 lb/gal), has a low viscosity of 75-259 cP, and is 10.9% (w/w) DAP, (NH4)2HPO4. The formula-tion contains a red fugitive dye agent that fades over time insunlight. PHOS-CHEK LC-95A is an ammonium polyphos-phate-based liquid concentrate formulation with a specificgravity of 1.473 kg/L (12.29 lbs/gal) and a manufacturer-rec-ommended mix ratio of 5.5 parts concentrate to 1 part water. Atthe approved mix ratio, LC-95A has a specific weight of1.074 kg/L (8.97 lb/gal), has a low viscosity of 75 to 225 cP,and is 7.6% (w/w) P2O5 equivalent.

Experimental animals and facility

Yearling stream-type chinook salmon (O. tshawytscha) wereraised from embryos in the Fish Disease Laboratory at theNational Oceanic and Atmospheric Administration’s NewportResearch Station (Newport, OR, USA). The Fish DiseaseLaboratory is supplied with two water sources: municipalfreshwater that is dechlorinated by carbon filtration prior touse and seawater pumped from Yaquina River bay at high tidethat is sand-filtered and UV-irradiated prior to use. The exper-imental fish represent the 2007 brood year at Rapid RiverHatchery (Riggins, ID, USA), which would have been releasedto begin their outmigration to the ocean in February and March2009 and would characteristically enter the ocean in Maythrough June 2009. The 96-h PHOS-CHEK exposures andsubsequent seawater and disease challenges commenced oncethe experimental stock fish had transitioned sufficiently fromparr to smolts to ensure high survival when seawater wasintroduced. At the time of the fire retardant exposures, themean fish weight was 12.2 g, and mean fork length was105.8mm.

Parr–smolt development

The status of the physiological transition from parr to smoltof the laboratory-raised fish was monitored weekly from

February 11 to April 16, 2009. Monitoring involved determin-ing the Naþ-Kþ-ATPase (hereafter ATPase) activity in the gillsof 10 stock fish per week held in freshwater. From February 26to April 16, 2009, an additional 10 stock fish per week wereplaced in full-strength seawater for 24 h. At the conclusion ofthe 24-h challenge, the numbers of survivors were tallied andcompared with the previous week’s challenge.

ATPase activity

To determine ATPase activity, the first gill arch on the leftside of each fish was collected and placed in a 0.5-ml micro-centrifuge tube filled with SEI buffer (250mM sucrose,10mM ethylenediaminetetraacetic acid [EDTA]-Na2, 50mMimidazole, pH 7.3 [17]) and flash frozen in the gas phase ofliquid nitrogen. Samples were then stored at �808C and ana-lyzed within six to eight months. Fish-gill ATPase activitieswere determined as the difference in the linear rate of ATPhydrolysis in the absence and presence of ouabain in micro-meters ADP/mg protein/h as per McCormick [17]. ATPaseactivities in the gill homogenates were corrected for proteincontent following the bicinchoninic acid (BCA) protein assay(Pierce; part of Thermo Fisher Scientific). Mean ATPase activ-ities among sample groups (exposure concentration or weeklysamples) were compared by one-way analysis of variance(ANOVA), followed by Tukey’s honestly significant differencepost hoc test in SYSTAT 12 (Systat Software), with significancelevel (a) set at 0.05.

Acute PHOS-CHEK exposures

Acute toxicity of either PHOS-CHEK 259F or LC-95A wasevaluated by exposing yearling chinook salmon to eight con-centrations of each formulation together with an unexposedcontrol group (PHOS-CHEK control) for 96 h under staticfreshwater holding conditions (Table 1). The salmon werenot fed during the exposure period. The exposures were initiatedon April 23, 2009. Concentrations of the fire retardant wereprepared on a PHOS-CHEK formulation mass-to-exposurevolume basis (i.e., milligrams of PHOS-CHEK 259F dry pow-der or milligrams of PHOS-CHEK LC-95A liquid concentrate).For each PHOS-CHEK treatment, volumes of concentratedstock solutions, prepared with a magnetic mixer, were added

Table 1. Concentrations of PHOS-CHEK fire retardants used during theacute 96-h toxicity test and mean cumulative mortalities

PHOS-CHEK LC-95A PHOS-CHEK 259F

Dose(mg/L)

96-h Mortalitya

(standarddeviation, %)

Dose(mg/L)

96-h Mortalityb

(standarddeviation, %)

115.5 0 (0) 34.3 0 (0)225.3 4.0 (4.0) 68.5 2.7 (4.6)340.8 50.7 (48.0) 102.8 32.0 (48.7)453.4 96.0 (4.0) 137.0 47.3 (32.3)566.0 100 (0) 164.4 66.0 (24.0)681.5 100 (0) 205.5 98.7 (2.3)906.8 100 (0) 274.0 100 (0)1,132.1 100 (0) 685.0 100 (0)

aMean cumulative mortality observed after 96 h of exposure to PHOS-CHEK LC-95A in three replicate tanks per fire retardant dose. The medianlethal concentration (LC50) calculated from logistic regression was339.8mg/L.

bMean cumulative mortality observed after 96 h of exposure to PHOS-CHEK 259F in three replicate tanks per fire retardant dose. The LC50calculated from logistic regression was 140.5mg/L.

Toxicity of forest fire retardants to chinook salmon Environ. Toxicol. Chem. 32, 2013 237

to 30-L, polyethylene exposure vessels and thoroughly mixedwith 25.5 L freshwater to achieve the desired exposure con-centration immediately prior to the addition of fish. The con-centrated fire retardant was manually mixed within the exposurevessel with a stirrer rod for roughly 10 to 20 s in each direction.Three replicate exposure vessels were used for each PHOS-CHEK treatment. The exposure vessels were placed withinlarger tanks with a continuously refreshed water jacket sur-rounding the tanks to maintain a uniform water temperature(�11–128C). Each of the tanks contained a porous air-stonewith constant air supply. Once the exposure vessels had beenthoroughly mixed, 25 fish were added to each of the vessels,with a mean loading density of approximately 12.0 g fish/L.During the 96-h exposure period, dead fish were removed fromthe exposure vessels at approximately 5, 24, 30, 48, 52, 72, and78 h, with the exact time of collection recorded. The lengths andweights of all fish were obtained at the time of collection(mortalities) or conclusion of the saltwater exposure (survi-vors). The lethal concentration (LC) curves and estimates of theconcentration at which 50% of the fish would experiencemortality (LC50) when exposed to PHOS-CHEK 295F andLC-95A were determined by logistic regression in SYSTAT 12at the conclusion of the 96-h acute exposure.

Water quality measurements

Water quality was assessed during each of the 96-h staticexposures from one randomly selected tank per replicate con-centration at the onset of exposure and at 24-h intervals duringthe 96-h exposure. Water quality measurements included dis-solved oxygen, temperature, combined nitrate and nitrite, pH,ammonia, and water hardness. Temperature and dissolvedoxygen were determined with a multiprobe (YSI) suspendedin the water column of individual tanks or exposure vessels.Individual 125-ml grab samples were used for the remainingparameters.

Nitrate and nitrite. Combined nitrate and nitrite (nitrate–nitrite) concentration in water samples was determined by firstreducing all nitrate to nitrite. The combined nitrite concentra-tion was then determined with the Griess reaction [18]. Allwater samples were preserved with 250ml concentrated sulfuricacid (95–98% H2SO4) and stored at 48C prior to analysis [19].The samples were then serially filtered through glass fiberprefilters (Millipore and Corning), intermediate cellulose fiberfilters (Whatman), and 0.45-mm HAWP final filters (Millipore)to remove colored PHOS-CHEK particles before reducing thesample nitrate to nitrite. As per method 4500-NO3-E of Eatonet al. [19], 25ml filtered sample was mixed with 75ml ammo-nium chloride-EDTA solution at pH 8.5 [19] and passedthrough a column filled with copper–cadmium granules. Thefirst 25ml of the reduced sample to exit the column wasdiscarded. The reduced nitrate plus the nitrite originally presentin the sample (nitrate–nitrite) was then determined for eachwater sample following method 4500-NO3-E [19]. In brief, a50-ml aliquot of the reduced sample was incubated with 2mlcolor reagent (sulfanilamide/N-[1–naphthyl]ethylenediaminedihydrochloride solution; Ricca Chemical). Samples were incu-bated at room temperature for 10min to allow color develop-ment, followed by an absorbance reading on a Genesys 10spectrophotometer at 543 nm (Thermo Fisher Scientific). Thenitrate–nitrite concentration was calculated from a standardcurve generated by the reduction of a serial dilution ofa 0.01M nitrate calibration standard (Sigma-Aldrich). Todetermine the maximum nitrite contribution of PHOS-CHEKformulations in the nitrate–nitrite measurements, nitrite was

determined by method 4500-NO3-E of Eaton et al. [19], withoutnitrate reduction. The nitrite contribution analyses were con-ducted only for the highest PHOS-CHEK concentrations(1,132mg LC-95A/L and 685mg 259F/L) and controls andin the absence of fish.

pH. The pH of water samples was determined by using aRoss Sure-Flow pH electrode 8172 (Thermo Fisher Scientific).Three standards (pH 4.0, 7.0, and 10.0; Fisher Scientific) wereused to calibrate the electrode each day prior to readingsamples. Samples were stored at 48C for less than one weekbefore analysis. Samples were brought to room temperature andmixed well, and 100ml was aliquoted into a glass beaker on astirrer plate.

Ammonia. Water samples were analyzed for ammonia bymethod 4500-NH3-D [19] using an Orion ion-selective ammo-nia electrode 9512HP (Thermo Fisher Scientific). Samples werepreserved with 250ml concentrated sulfuric acid (95–98%H2SO4) and stored at 48C for less than 30 d before analysis.Five ammonium chloride standards (0.5, 5.0, 50.0, 100.0, and200.0mg/L) were made from a 0.1M ammonium chloridestandard solution (Thermo Fisher Scientific) and were usedto calibrate the electrode each day prior to reading samples.Samples were brought to room temperature and mixed well, anda 100-ml aliquot was added to a glass beaker on a stirrer plate.Direct readings were taken after 2ml of ionic strength adjustor(5N sodium hydroxide; Thermo Fisher Scientific) had beenadded to the sample. The electrode was rinsed, and a duplicatereading was taken of each sample; the average of the tworeadings was recorded as the result. Un-ionized ammoniaconcentrations were then determined from tables presentedby Thurston et al. [20] based on the calculations of Emersonet al. [21] using temperature, pH, and total ammonia measure-ments obtained from each tank.

Water hardness. Measurements of water hardness wereattempted with a water hardness test kit (model AG-3, code4766; LaMotte). However, the red dye in the fugitive PHOS-CHEK fire retardant formulations interfered with the colori-metric assay’s ability to measure hardness. Consequently, hard-ness values could not be determined. Water hardness of thelaboratory freshwater was 42mg/L prior to any PHOS-CHEKaddition.

Seawater challenge

Seawater challenge tests were conducted as a measure ofsmolt readiness for seawater entry [22]. Immediately after the96-h acute toxicity test, fish in exposure vessels that had 10 ormore survivors were transferred to tanks (�300L) with full-strength, flow-through seawater (34 ppt) for 24 h. In addition,three replicates of 25 fish were transferred from a freshwaterholding tank to individual flow-through seawater tanks to beincluded as naive fish that had not experienced 96 h of staticholding. Immediately after the 24-h seawater challenge, themortalities were recorded to assess survival to the challenge.Chi-square tests of association (Systat Software) were used tocompare the mean survival among the controls and the PHOS-CHEK concentrations. In addition, up to five survivors perseawater exposure tank were necropsied for an evaluation oftheir parr–smolt development status by measuring the gillATPase levels.

Disease challenge

The impact of fire retardant exposure on chinooksalmon disease susceptibility was evaluated with a diseasechallenge assay. OnMay 4, 2009, fish were exposed to sublethal

238 Environ. Toxicol. Chem. 32, 2013 J.P. Dietrich et al.

concentrations of PHOS-CHEK 259F (34.3 and 13.7mg/L),LC-95A (113.3 and 22.7mg/L), and a nonchemical control(PHOS-CHEK control) in static, 96-h exposures. The twosublethal concentrations of fire retardant represented the lowestconcentrations with no observed mortality during the 96-h acuteexposure and a less than 1% lethal concentration of PHOS-CHEK LC-95A and 259F (LC0.01 and LC0.70, respectively)based on the logistic regression of the acute exposure data. Foreach PHOS-CHEK treatment, volumes of concentrated stocksolutions were added to six replicate 100-L, polyethyleneexposure vessels and thoroughly mixed with 60 L freshwaterto achieve the desired exposure concentration immediatelyprior to the addition of fish. The 100-L tanks were held withinlarger tanks and surrounded by a continuously refreshed waterjacket, as described above. Fifty fish were held in each of theexposure vessels for 96 h at a mean loading density of approx-imately 9.8 g fish/L. After the exposure, fish from each of thetanks were transferred to larger tanks with approximately 300 Lflow-through freshwater for a 24-h recovery period. In addition,six replicates of 50 fish were transferred from a freshwaterholding tank to individual flow-through freshwater tanks to beincluded as naive fish that had not experienced 96 h of staticholding. Each tank was then transitioned to full-strength sea-water (34 ppt) over 6 d. During this period, the proportion ofseawater was incrementally increased. The fish remained onfull-strength seawater for 1 d prior to the disease challenge.

On May 15, 2009, a disease challenge was performed as perArkoosh et al. [23]. Briefly, three of the six replicate tanks perPHOS-CHEK concentration, PHOS-CHEK control, and naivefish were exposed to 2.9� 104 cfu/ml of Listonella anguillarumin trypticase soy broth (TSB) supplemented with 1.5% NaCl.The fish were exposed for 1 h in 38-L containers with 18.75 Lstatic seawater and aeration at an approximate density of 50 gfish/L. The remaining three replicate tanks per treatment wereused as no-pathogen controls and were exposed to sterile TSBsupplemented with 1.5% NaCl for 1 h under identical challengeconditions. After the 1-h bath exposure, all the fish werereturned to their original flow-through seawater tanks(�300L) for a 9-d observational period. Mortalities werecollected twice daily (at �9:00 and �16:00) during the obser-vation period. Approximately one fish was necropsied for everythree dead fish (39.9% of mortalities necropsied) collected,to confirm that the deaths were associated with L. anguillaruminfection. Immediately after the dead fish had been removedfrom the tank, they were sprayed with 75% ethanol, andthe kidneys were surgically exposed and aseptically struck ontoa trypticase soy agar (TSA) plate supplemented with 0.5%NaCl. If colonies formed on the TSA plates after 48 h at268C, L. anguillarum growth was confirmed with a Mono-Vaagglutination test kit (Bionor, A/S). Of the mortalities thatoccurred in the pathogen-exposed tanks, 276 (or 92.3% ofthe fish struck) had growth on TSA, with 98.9% that aggluti-nated. Four of the mortalities that occurred in the no-pathogencontrols (or 5.8%) had growth on TSA, with only one colonythat agglutinated.

Survival curves were generated using the nonparametricKaplan–Meier method across all replicate tanks for fish in eachpathogen-exposed treatment. Survival curves were assessed forthe potential impact of both fire retardant exposures on diseasesusceptibility and tank effects using the Mantel method for thelog-rank chi-square test with the null hypothesis of a commonsurvival curve. Any replicate tanks identified as outliers, by log-rank chi-square tests, within a common treatment were removedfrom later analysis. SYSTAT 12 was used to generate all

survival curves and to perform log-rank chi-square tests, withthe significance level (a) set at 0.05 in all comparisons.

Pathology

To investigate pathological effects on specific tissues, addi-tional stream-type chinook were exposed to PHOS-CHEKformulations. Fish were exposed to five different concentrationsof PHOS-CHEK 259F and LC-95A and a nonchemical control(PHOS-CHEK control) in static exposures for periods up to 96 h(Table 2). The durations of exposures were selected based onacute toxicity results, with fish exposed to the three highestdoses of 259F and LC-95A for no longer than 24 h because ofthe expected rapid mortality (data not shown). At 24-h intervals(and one 6-h sample point), 10 fish were sacrificed, and the gillsand viscera were dissected from each fish and placed intoseparate tissue cassettes with Davidson’s fixative (at a minimumvolume of 1 part tissue to 20 parts Davidson’s; [24]). Tissueswere fixed for a minimum of 48 h and then stored in 70%ethanol. Subsequent histological processing included paraffininfiltration and embedding, sectioning at 5mm, and stainingwith hematoxylin and eosin reagents [25,26]. Gill, liver, endo-crine and exocrine pancreas, esophagus, stomach, pyloric caeca,upper intestine, lower intestine, heart, anterior and posteriorkidney, spleen, gonad, skin, and lateral line system from eachfish were all evaluated for histopathological lesions and con-ditions that could result from fire retardant exposure. Differ-ences in diagnosis prevalence across treatments for the sameexposure duration were determined with the Fisher’s exact test.Lesion severity was classified on an ordinal scale (0–7), andstatistical comparisons were made by ANOVA and Fisher’sprotected least significant difference post hoc test, and awas setat 0.05 in all statistical comparisons of pathology results.

RESULTS

Parr–smolt development

Monitoring of gill ATPase and seawater survival indicatedthat the stock fish were smolting and thus were prepared forseawater entry at the time of PHOS-CHEK exposure (April 23).During the 10-week monitoring period, ATPase activity levelsin fish gills significantly increased (Fig. 1). Each of the first fourweeks of monitoring had significantly less mean ATPase activ-ity than each of the last four weeks (p� 0.001). Weeks 5 and 6indicated a potential transitional period (Fig. 1). In weeks 5 and6, ATPase activity levels were not significantly different fromeach other (p¼ 1.0), but the levels in each week were signifi-cantly greater than in each of the first four weeks (p� 0.05) andsignificantly less than in each of the last four weeks (p� 0.038).In addition, 70 to 100% of the fish survived weekly seawaterchallenges after week 3. The results are consistent with a stockpopulation that is undergoing the physiological changes neces-sary for seawater entry. There is no fixed ATPase activity levelthat equates to seawater preparedness; rather, ATPase activity isgenerally low at the hatcheries, increases at the time when fishare released into the river system, and continues to increase asoutmigration progresses [11].

Acute PHOS-CHEK exposures

Yearling chinook salmon had different sensitivities to acuteexposures of the two PHOS-CHEK formulations. The lowestno-effect concentrations (no mortalities) were observed in tanksexposed to 34.3 and 115.5mg/L 259F and LC-95A, respec-tively, with 100% mortality observed in tanks exposed to 274.0and 566.0mg/L 259F and LC-95A, respectively (Table 1). The


logistic regression estimates of the LC50s for 259F and LC-95Awere 140.5mg/L and 339.8mg/L, respectively. The PHOS-CHEK control and eight PHOS-CHEK doses for each of theformulations were well described by logistic regression(LOGIT) models. The predicted LOGIT models of survival

probabilities following 96 h of exposure to 259F and LC-95Aare presented in Figure 2.

High significance and classification strength of the modelswere indicated from various assessment metrics accompanyingthe logistic regression. The estimates of the parameters in both

Fig. 1. ATPase activity measured in the gills of the experimental yearlingchinook salmon stock from February 11 (week 1) to April 16 (week 10).

Fig. 2. LOGIT regression models of observed fish survival following theacute 96-h exposure to PHOS-CHEK LC-95A (gray lines) and 259F (blacklines).

Table 2. Prevalence of gill lesions and conditions detected by histopathology in chinook salmon smolts exposed to PHOS-CHEK LC-95A and 259F comparedwith controls, for up to 96 h

TreatmentExposuretime (h)

Gill lesion type (prevalence, % affected)a

Lamellarmicroaneurysms

Respiratoryepithelial hyperplasia

Respiratoryepithelial necrosis

Respiratory epitheliallifting or exfoliation

Respiratoryepithelial hypertrophy Phagosomesb

PHOS-CHEK control 0 22 0 0 0 0 024 20 0 0 0 0 048 10 0 0 0 0 072 30 0 0 0 0 096 50 0 0 0 0 0

PHOS-CHEK LC-95A23.0mg/L 24 10 10 0 0 0 0

48 10 0 0 0 0 072 0 0 0 10 10 096 30 0 0 10 20 0

113.0mg/L 24 10 0 0 10 0 048 10 0 0 0 0 072 10 10 0 0 10 096 0 0 0 0 0 0

227.0mg/L 24 0 0 0 0 0 10340.0mg/L 24 0 0 0 0 0 90c,d

453.0mg/L 24 10 0 0 0 0 100c,d

PHOS-CHEK 259F14.0mg/L 24 11 0 0 0 0 0

48 10 0 0 0 10 072 30 0 0 0 0 096 60 0 0 10 40c,d 0

41.0mg/L 24 11 0 0 0 0 048 10 0 0 0 0 072 30 0 0 0 0 096 20 10 0 10 40c,d 0

103.0mg/L 24 0 0 0 0 0 0137.0mg/L 24 10 0 0 40c,d 20 20206.0mg/L 6 0 0 10 30d 40c,d 0

24 0 0 0 20 30 100c,d

a Nine or ten fish were analyzed by histopathology for all of the control and treatment groups at each exposure time indicated.b Phagosomes were identified in the respiratory epithelium or gill macrophages.c Significantly higher prevalence than in controls at the same exposure time point by the Fisher’s exact test, p< 0.05.d Significantly higher lesion severity (ordinal scale 0–7) than in controls by analysis of variance (ANOVA) and Fisher’s protected least significant difference posthoc test, p< 0.05.


LOGIT models (constant and PHOS-CHEK concentration)were significant (p< 0.001). Two indicators of model fit(Cox and Snell R2 and Naglekerke’s r2; both mimic r2 valuesfor linear regression and are based on log likelihoods andsample size) had maximum values of 1.0 for each of the models(SYSTAT Software). A similar metric, McFadden’s RHO2, hadhigh values of 0.620 and 0.828 for the LOGIT models of the259F and LC-95A data, respectively. Finally, prediction-suc-cess analysis indicated that, overall, 82.9% of the modelpredictions were correct for the 259F LOGIT model and92.7% correct for the LC-95A model.


Exposure conditions were designed to limit variation oftemperature and dissolved oxygen levels in different treatmenttanks. The minimum optimal dissolved oxygen level for main-taining salmon health is 8.8mg/L at 11.38C [27]. Each tank wassupplied with constant aeration throughout the 96-h exposureperiod and resulted in an average of 10.6mg/L dissolved oxy-gen. Dissolved oxygen ranged from 7.2 to 11.2mg/L duringindividual measurements (data not shown). Average dailymeasurements of dissolved oxygen were lowest after the first24 h (7.2–9.4mg/L), but increased in the subsequent days. Thetemperature ranged from 11.2 to 11.68C in most tanks, with twoexceptions in which the water jacket temporarily malfunc-tioned. During the malfunction, one replicate of LC-95A(1132.1mg/L) had a temperature reading of 12.28C immedi-ately before fish were added; and one replicate of LC-95A(340.8mg/L) had a temperature reading of 13.68C at the firstsampling point (24 h). Neither of the temperature anomaliesresulted in tank survivals that were different from their treat-ment replicates.

Nitrate–nitrite. The mean concentration of nitrate–nitritewas 0.89mg/L (range, 0.76–1.04mg/L) in all PHOS-CHEKtreatments prior to fish addition compared with 0.83mg/L in thePHOS-CHEK control. The nitrite contribution was not detect-able in laboratory freshwater or the highest concentration of259F (685mg/L). The nitrite contribution in the highest con-centration of LC-95A (1132mg/L) was just detectable at0.01mg/L. After fish addition and during exposure, the meanconcentration of nitrate–nitrite increased slightly to 0.93mg/L(ranging from 0.84 to 1.02mg/L) in all PHOS-CHEK treat-ments and to 0.86mg/L in the PHOS-CHEK control. Nitriteconcentration less than 0.1mg/L and nitrate levels less than1.0mg/L are considered safe for rearing salmon [27].

pH. The pH of the water in tanks containing LC-95A rangedfrom 6.9 to 7.3, with no observable trend in dose or exposuretime (Fig. 3A). In contrast, the pH of the water in tankscontaining 259F ranged from 6.9 to 7.6 and seemed to increaseslightly with increasing 259F concentration (Fig. 3B). ThePHOS-CHEK control had an initial pH of 7.6 prior to theaddition of fish but ranged from 6.9 to 7.2 thereafter.

Ammonia. In contrast to pHmeasurements, total ammonia inthe water increased with increasing concentrations of bothPHOS-CHEK formulations (Fig. 3C and D). Total ammoniaalso increased with exposure time in lower doses of 259F andLC-95A and in the PHOS-CHEK control. However, the major-ity of total ammonia was detected immediately after PHOS-CHEK addition. Given that total ammonia was not detected inthe PHOS-CHEK control prior to fish addition, the steadyincrease most likely is due to accumulation of fish excretaduring the static holding conditions. This increase with time wasnot observed with higher doses because of the high mortalitiesin these tanks, presumably reducing the source of ammonia.

Un-ionized ammonia concentrations were calculated basedon the tank temperature, pH, and total ammonia detected in eachof the tanks. Given the relatively constant temperature range forall tanks, variation in pH and total ammonia had the greatestimpact on un-ionized ammonia concentrations. In the LC-95Atreatments, the pH was relatively constant, whereas the totalammonia increased with increasing dose (Fig. 3A and C). Incontrast, in the 259F treatments, pH and total ammoniaincreased with increasing dose (Fig. 3B and D). The resultwas a fairly uniform range of un-ionized ammonia concentra-tions across all LC-95A treatments (0.14–0.49mg/L; Fig. 3E)and increasing un-ionized ammonia concentrations withincreasing 259F treatments (0.10–2.84mg/L; Fig. 3F).

Seawater challenge

Sublethal exposures to PHOS-CHEK 259F and LC-95A for96 h affected the ability of yearling spring chinook salmon tosurvive a subsequent 24-h seawater challenge (Fig. 4). The 96-hstatic experimental design significantly reduced the seawatersurvival of salmon in the PHOS-CHEK control (31%;p< 0.001) relative to the naive salmon that had not experienced96 h of static holding. High survival (97%) among the naive fishdid indicate that the yearling chinook were prepared for sea-water entry at the time of the challenge.

Given the difference in survival among the naive fish and fishin the PHOS-CHEK control group, logistic regression of PHOS-CHEK concentration and survival was not possible. However,chi-square statistical analysis of the seawater survival didindicate significant differences between the PHOS-CHEK con-trol and chinook salmon with sublethal exposure to the PHOS-CHEK formulations. All three concentrations of PHOS-CHEKLC-95A with sufficient survivors from the 96-h exposure toparticipate in the subsequent seawater challenge exhibitedreduced survival after challenge in seawater (Fig. 4A). Fishfrom the lowest LC-95A treatment (115.5mg/L) experiencedsignificantly reduced survival (35%; p< 0.001) relative toPHOS-CHEK control (p< 0.001). The 340.8mg/L treatmentwas nearly equal to the estimated LC50 value for LC-95Aexposure, and fish in this treatment had significantly lowersurvival (61%; p< 0.001) than the PHOS-CHEK control duringthe seawater challenge.

The 68.5mg/L treatment of 259F was the lowest concen-tration tested that was significantly different from the PHOS-CHEK control (p< 0.001), and fish experienced a 42%decrease in survival relative to the PHOS-CHEK control(Fig. 4B). The mean survival of fish during the seawaterchallenge that had previously been exposed to 102.8mg/L259F was not significantly different from that of the PHOS-CHEK control. Fish in treatments bracketing the estimated259F LC50 (i.e., 137.0 and 164.4mg/L) had 54 and 63% lowersurvival than the PHOS-CHEK control during the seawaterchallenge.

Impact of fire retardant exposure on parr–smolt developmentindicators

Previous PHOS-CHEK exposure did not have any detectableeffect on the gill ATPase activity levels measured in thesurvivors of the acute 96-h exposure test and seawater challenge(data not shown). Although not statistically significant, ATPaseactivities were greatest in naive fish and fish from the PHOS-CHEK controls as well as from the lowest PHOS-CHEK 259Fdose (34.25mg/L). Small sample size and large standard errorsmight have contributed to our inability to determine statisticallysignificant trends or differences.


Disease challenge

Yearling chinook salmon exposed to sublethal doses ofPHOS-CHEK fire retardants were not found to be more suscep-tible to disease from L. anguillarum. The fish were exposed toPHOS-CHEK 259F doses with LC values of 1.5% (34.3mg/L)and 0.7% (13.7mg/L) and LC-95A doses with LC values of0.16% (113.3mg/L) and 0.01% (22.7mg/L) based on theirlogistic models. No mortalities were observed in any of thetreatments during the 96-h sublethal fire retardant exposures orthe gradual transition to full-strength seawater. The survivalrates of fish in the PHOS-CHEK controls and fish with thelowest dose of LC-95A (22.7mg/L), as well as the naive fish,were not significantly different from each other following a bathchallenge with L. anguillarum (p � 0.900); but fish from thesetreatments had significantly reduced survival relative to fishpreviously exposed to 113.3mg/L of LC-95A (p� 0.003;

Fig. 5A). The survival rates of salmon exposed to the twoPHOS-CHEK 259F doses were not statistically different fromeach other (p¼ 0.428; Fig. 5B). However, the survival rates ofnaive and PHOS-CHEK control fish were significantly reducedrelative to fish exposed to either dose of 259F (13.7 and34.3mg/L; p� 0.013).

Pathology

Histopathological examination of chinook salmon indicatedthat gills were the principal tissues affected by increasingconcentrations of PHOS-CHEK LC-95A and 259F. Full exami-nation of other tissues (liver, endocrine and exocrine pancreas,esophagus, stomach, pyloric caeca, upper intestine, lower intes-tine, heart, anterior and posterior kidney, spleen, gonad, skin,and lateral line system) revealed no significant lesionsthat could be related to exposure to either of the fire retardants(data not shown). Forms of gill pathology included (1) frank

Fig. 3. pH (A andB), total ammonia (C andD), and un-ionized ammonia concentration (E andF)measured in exposure vessels containingPHOS-CHEKLC-95A(A,C, andE) and 259F (B,D, andF). Hatched box in (E) and (F) indicates the published range of ammoniamedian lethal concentration (LC50) values for chinooksalmon smolts.


respiratory epithelial necrosis; (2) respiratory epithelial hyper-plasia, exfoliation, and hypertrophy; lamellar microaneurysms;and (3) phagosomes containing basophilic material in bothrespiratory epithelium and macrophages. Respiratory epithelialhyperplasia and lamellar microaneurysms were not attributed toPHOS-CHEK exposure because they were sporadicallydetected or equally detected in controls. Gill tissues from thePHOS-CHEK controls were healthy (Fig. 6A), with no otherlesions or conditions observed at any sampling time (Table 2).

Respiratory epithelial necrosis, epithelial exfoliation, andhypertrophy. Frank respiratory epithelial necrosis was exhib-ited in one fish with the 206mg/L dose of 259F. However, theconcomitant occurrence of respiratory epithelial exfoliationand epithelial hypertrophy prior to mortality at 6 h suggeststhat these conditions are potential precursors of necrosis(259F-206mg/L; Table 2). Respiratory lifting and exfoliation

(Fig. 6B) occurred at 30 and 20% prevalence in the 206mg/Ldose, and respiratory epithelial hypertrophy was 40 and 30%after 6 and 24 h of exposure, respectively (Table 2). Similarly,the next lower dose of 259F (137mg/L) showed a 40% prev-alence of respiratory epithelial exfoliation and 20% prevalenceof respiratory epithelial hypertrophy after 24 h of exposure.After 96 h of exposure, the two lowest doses of 259F (14.0 and41.0mg/L) also exhibited respiratory epithelial exfoliation andepithelial hypertrophy at 10 and 40% prevalence, respectively.

In contrast, the prevalence of respiratory epithelial exfolia-tion and epithelial hypertrophy was low in fish exposed toPHOS-CHEK LC-95A and occurred only after 72 h of exposure(Table 2). These lesions were prevalent (10% and 20%, respec-tively) with the 23.0mg/L dose of LC-95A after 96 h ofexposure. In addition, both lesions were prevalent in 10%of the fish exposed to 113.0mg/L of LC-95A at individual

Fig. 4. Cumulative survival of chinook salmonparr–smolts after 24-h of seawater challenge immediately following96-h sublethal exposure to PHOS-CHEKLC-95A (A) and 259F (B). Bars identified with different letters represent significant differences based on chi-square analysis, with no attempt at comparisons acrossPHOS-CHEK formulations.

Fig. 5. Survival of chinook salmon that had been exposed to sublethal concentrations of PHOS-CHEK LC-95A (A) and 259F (B) for 96 h, followed by animmersion challengewith Listonella anguillarum. Naive fish and fish among the PHOS-CHEKcontrols and fish receiving the lowest dose of LC-95A (22.7mg/L)had significantly reduced survival relative to fish previously exposed to 113.3mg/L LC-95A (p� 0.003;A). Naive fish and fish in the PHOS-CHEK control grouphad significantly reduced survival relative to fish exposed to either dose of 259F (p� 0.013; B).


sampling times prior to 96-h. No fish receiving the three highestdoses of LC-95A (227.0–453.0mg/L) were diagnosed withrespiratory epithelial exfoliation or hypertrophy after 24 h ofexposure (Table 2).

Phagosomes in respiratory epithelium and macrophages.Fish exposed to both PHOS-CHEK formulations exhibited

phagosomes containing basophilic material (potentiallyPHOS-CHECK formulation) in the respiratory epitheliumand in subepithelial macrophages after 24 h of exposure. Thevariably sized phagosomes or cytoplasmic lysosomes werepresent in both respiratory epithelium and subepithelial macro-phages in the gill filaments and gill lamellae, most commonlybetween adjacent gill filaments, indicating intracellular uptake,phagocytosis, and digestion of the fire retardant compounds ordyes (illustrated in Fig. 6C). The prevalence of this conditionshowed a dose–response behavior, with 100% of the fishdiagnosed at the highest doses of LC-95A (453.0mg/L) and259F (206.0mg/L), progressively decreasing to 10% and 0% at227.0mg/L and 103.0mg/L of LC-95A and 259F, respectively(Table 2). No fish among the controls or receiving lower dosesof 259F or LC-95A were diagnosed with this condition duringany of the exposure periods.

DISCUSSION

Our results indicate that two current-use fire retardants,PHOS-CHEK 259F and LC-95A, are acutely toxic to smoltingchinook salmon at concentrations below suggested mix ratios.According to the manufacturer’s mix ratios, the suggestedworking concentrations of LC-95A and 259F are roughly226,590 and 136,600mg/L, respectively. These are the con-centrations that would initially enter a water body during anaccidental drop, prior to any dilution. The estimated 96-h LC50concentrations for yearling chinook salmon were 339.8mg/Land 140.5mg/L for LC-95A and 259F, respectively, or roughly0.15 and 0.10% of the suggested working concentrations forLC-95A and 259F, respectively. In other words, misapplied LC-95A or 259F fire retardant must be diluted roughly 670 or 1,000times, respectively, from its working concentration to reach theobserved LC50 concentration and roughly 2,000 or 4,000 times,respectively, to reach the no-mortality concentrations observedin the present study. The level of actual mortality and dilutionthat could occur in the environment would depend on thephysical characteristics (e.g., water volume and flow rate)and biological characteristics (e.g., species density, movement)of the water body that received the accidental drop, as well as onthe volume of fire retardant received. Consider a scenario inwhich 10% of a full single-engine tanker (i.e., 300 L) wasaccidentally dropped into a small rectangular river channel witha depth of 1.5m and width of 3m. In this simplified example,65m of river channel would be needed to dilute 259F to itsLC50 concentration, or 270 meters of river channel would beneeded to dilute 259F to the no-mortality concentrationobserved in the present study, assuming an instant and homo-geneous dilution. The LC50 of LC-95A was roughly 2.5 timesgreater than 259F and requires diluting with roughly two-thirdsthe water volume of 259F to achieve LC50 concentrations ifapplied at the manufacturer-suggested mix ratios.


Greater un-ionized ammonia concentrations in the 259Ftreatments may have resulted in more ammonia-associatedmortalities with the 259F PHOS-CHEK formulation than withthe LC-95A formulation. Once fire retardants are introduced towater, the ammonia within the formulation equilibrates betweenionized and un-ionized forms depending on water pH andtemperature [2]. The un-ionized ammonia form is more toxicto fish [28]. Increased pH with increasing 259F concentrationsresulted in a greater ratio of un-ionized ammonia to totalammonia than in the LC-95A treatments. Acute toxicity tests

Fig. 6. Histological sections of gill from juvenile chinook salmon that wereunexposed controls (A), exposed to 206mg/Lof PHOS-CHEK259F (B), andexposed to 340mg/L of PHOS-CHEK LC-95A (C) sampled at 24 h afterinitiation of the experiment. Individual micrographs illustrate the normalmorphology of the gill filaments and lamellae (A), multiple areas of the gilllamellae that are affected with respiratory epithelial lifting/exfoliation(arrows; B), and basophilic phagosomes present within the respiratoryepithelium and in subepithelial macrophages, primarily in the basal,interfilamental regionof thegill (arrows;C).Hematoxylin andeosin staining.


of ammonia on juvenile chinook salmon performed by otherresearchers determined LC50 values ranging from 0.399 to0.476 for water pHs between 7.8 and 8.0 [29,30]. The rangeof these LC50 values is indicated in Figure 3E and F with ahatched box. The hatched box clearly indicates that the majorityof LC-95A treatments had un-ionized ammonia concentrationsbelow the ammonia LC50 value, whereas half of the 259Ftreatments had un-ionized ammonia concentrations at or abovethe ammonia LC50 values. Two exposure concentrations (164.4and 205.5mg/L) of 259F had un-ionized ammonia concentra-tions that overlapped the unionized ammonia LC50 valuesduring the 96-h exposure and had 66 and 98% mortality,respectively. This close relationship between reported ammoniatoxicity and observed 259F toxicity suggests that ammoniasignificantly contributed to the observed mortality.

In contrast, an additional factor contributed to mortality offish exposed to PHOS-CHEK LC-95A. Un-ionized ammoniawas below the LC50 value for all but the highest LC-95A dose(1,132mg/L), but 96% mortality was observed during the 96-hexposure period at doses greater than or equal to 453mg/L. Atthe estimated LC50 value of LC-95A, the maximum un-ionizedammonia concentration was 0.28mg/L.

Pathology

Gill tissues were the only tissues identified with histopatho-logical lesions and conditions that could be attributed to fireretardant exposure. Frank respiratory epithelial necrosis wasobserved in one fish at the highest 259F concentration, but theprevalence pattern of respiratory epithelial hypertrophy andexfoliation suggests that they are precursor lesions to necrosis.These diagnoses may be due to ammonia present in the PHOS-CHEK formulations and waste produced during the exposure.Ammonia has been shown to cause gill damage such as hyper-trophy of gill epithelium as well as gill exfoliation in rainbowtrout [31,32]. Respiratory epithelial exfoliation and epithelialhypertrophy were associated primarily with 259F toxicity,because of their rapid occurrence at the high doses, where 50to 100% mortality was observed during acute exposure, as wellas their occurrence within 96 h in fish exposed to low doses.These conditions may also be related to exposure to LC-95A,but only at later exposure periods. Epithelial lifting is consid-ered to be an initial gill response to exposure to a number ofcontaminants [33] and indicative of the edema component of theacute inflammatory response [34].

Exposure to either 259F or LC-95A at concentrations equalto or greater than 137 and 227mg/L, respectively, resulted in theappearance of variably sized phagosomes or cytoplasmic lyso-somes in the respiratory epithelium and in subepithelial macro-phages. The presence of phagosomes indicated intracellularuptake, phagocytosis, and digestion of the fire retardant com-pounds or dyes. No fish among the controls or with lower dosesof 259F or LC-95A were diagnosed with phagosomes duringany of the exposure periods. The presence of phagosomessuggests that an additional factor contributed, or was a pre-cursor, to PHOS-CHEK toxicity. At their LC50 concentrations,90% of the fish exposed to LC-95A were diagnosed withphagosomes, compared with 20% of the fish exposed to259F. The increased occurrence of phagosomes suggests adifferential source of toxicity in LC-95A relative to 259F.

Seawater challenge

In addition to acute mortality, 96-h of PHOS-CHEKLC-95Aand 259F exposure resulted in delayed effects to smoltingchinook salmon. Chinook salmon smolts that experienced less

than 5% mortality during the 96-h PHOS-CHEK exposure hadsignificantly reduced survival (35–40%) during the subsequentseawater challenge, compared with controls. Seawater chal-lenge results indicated a general sensitivity to subsequent stressand a specific sensitivity to seawater. No disruptions tospecific parr–smolt indicators were found, suggesting that theseawater sensitivity might have been due to physical damageduring PHOS-CHEK exposure. The histopathological changesobserved in the gills after exposure to fire retardants might havethe potential to cause reduced oxygen diffusion capacity of thegills, ultimately resulting in reduced survival [32].

Disease challenge

Both PHOS-CHEK LC-95A and 259F exposure did notadversely affect the juvenile chinook salmon’s disease suscept-ibility during challenge with L. anguillarum. In fact, previousexposure to 259F resulted in significantly increased survivalrelative to the PHOS-CHEK control. Gills are a critical portal ofentry for the L. anguillarum pathogen [35]. Damaged gill tissue,possibly from high ammonia levels in the fire retardant expo-sures, might have contributed to reduced L. anguillarum infec-tions in fish exposed to the fire retardants. Morris et al. [36]demonstrated that survival of juvenile Lost River suckers(Deltistes luxatus) challenged with Flavobacterium columnareincreased with prior exposure to increasing concentrations ofun-ionized ammonia (0.125, 0.25, and 0.5mg NH3/L). How-ever, ammonia can be an immunosuppressant for pathogens thatdo not rely on gill entry for pathogenesis or if the gills arebypassed during exposure. For example, Saprolengia parasiticais a pathogen that infects and breaks down the superficial tissueof the fish [37,38]. Rainbow trout exposed to S. parasitica,following a 1- or 10-d exposure to un-ionized ammonia (0.50 or0.05mg/L, respectively), were more susceptible [37,38].Finally, L. anguillarum mortality was significantly higher inchinook salmon pre-exposed to 10mg/L total ammonia nitrogenwhen the pathogen was injected [39].

CONCLUSIONS

This is the first study to determine the sensitivity of chinooksalmon to fire retardant chemicals at the peak of the parr–smolttransition. In summary, PHOS-CHEK 259F and LC-95A for-mulations were acutely toxic to chinook salmon physiologicallyprepared for smoltification at doses significantly diluted frommanufacturer-suggested mix ratios and likely to occur duringmisapplication in salmon habitat. Full strength PHOS-CHEK259F or LC-95A would have to be diluted 4,000 or 2,000 times,respectively, in order to reach the concentrations at which nomortalities were observed in the present study. Chinook salmonengaged in the parr–smolt transition were more sensitive toPHOS-CHEK 259F than LC-95A. The sensitivity of earlierchinook salmon life stages to 259F and LC-95A is not known. InLC50 comparisons with other salmonids, chinook salmonexposed at the smolt stage were similarly or slightly moresensitive to 259F than swim-up rainbow trout (148mg/L [1],168mg/L [5], 94–165mg/L [5]) and coho salmon (170–250mg/L [5]). Chinook salmon do appear to be more sensitive to LC-95A exposure at the smolt stage than rainbow trout (435mg/L[1]) at 60 d posthatch.

Exposed salmon were diagnosed with histopathologicallesions or conditions in gill tissues that were attributed toPHOS-CHEK exposure. Gills are the respiratory organs of fishand also represent the primary site of osmoregulation andammonia excretion in anadromous salmon [40]. Ammonia


may be significantly contributing to the toxicity of 259F, basedon un-ionized ammonia concentrations at doses near the 259FLC50 and the histopathology typically associated with ammo-nia exposure. However, some additional source of mortalitylikely exists in LC-95A. The increased presence of phagosomesin the respiratory epithelium and subepithelial macrophages inthe gill filaments and lamellae of fish exposed to LC-95Arelative to 259F did suggest a differential mode of toxicity.A further possibility is an adverse reaction to unknown(proprietary) compounds within the fire retardants. ThePHOS-CHEK exposure concentrations that did not result inacute mortality were found to impair seawater survival signifi-cantly but not susceptibility to L. anguillarum. AlthoughL. anguillarum infection may have been reduced by damagedgill tissues, ammonia toxicity can increase susceptibility toL. anguillarum when the gills are not the portal of entry[39]. Ammonia exposure can also increase the susceptibilityof fish to other pathogens [37,38]. The same gill damage thatmight have prevented pathogen entry may have also impairedchloride cells in the gills. Chloride cells have been shown to besites of critical molecular functions during changes in salinity[14,16]. Reduced osmoregulation by the gills may explain theobserved inability of fire-retardant-exposed salmon to cope withimmediate seawater entry.

The cumulative adverse impact of fire retardants on chinooksalmon abundance includes not only the acute mortality imme-diately following a misapplication but also the delayed mortal-ity once the exposed salmon enter seawater. Movement fromfreshwater rearing environments to seawater is critical in the lifecycle of Pacific salmon. However, this active outmigration tothe sea takes time. Delayed mortality in seawater was observedimmediately after exposure to the PHOS-CHEK formulations.The permanence of this seawater sensitivity or time to recoveryfrom previous PHOS-CHEK exposure is currently unknown.

Acknowledgement—We appreciate the technical efforts of D. Boylen, J.Osborn, and G. Hutchinson.We are also grateful for the insights provided byE. Little, U.S. Geological Survey, Columbia, Missouri, and the thoughtfulcomments of N. Scholz, K. Peck-Miller, and K. MacNeal of the NationalMarine Fisheries Service during manuscript review. The present study wasfinancially supported by the U.S. Department of Agriculture-Forest ServiceWildland Fire Chemical Systems Program.

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