Water Qual. Res. J. Canada, 2005 • Volume 40, No. 3, 334–346 Copyright © 2005, CAWQ

Overview of Methodology and Endpoints in Fathead Lifecycle Tests Assessing Pulp and Paper Mill Effluents

Joanne L. Parrott*

National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario L7R 4A6

Fathead minnow ( promelas) lifecycle tests have been used to assess the effects of many North American pulp mill effluents. are exposed under controlled laboratory conditions to final effluent, from the egg stage through hatching, juvenile stage and mature adult stage. Outlined here are methods for the lifecycle test (which takes from 4 to 5 months to complete) with sampling of juvenile fish at 1, 2 and 3 months of age, and sampling of mature adults after breeding. The results of most fathead minnow lifecycle studies have shown that pulp mill effluents cause metabolic and reproductive dis- ruption (enlarged livers, reduced egg production, smaller gonads, decreased sex hormones and reduced secondary sex charac- teristics). Thus, the fathead minnow lifecycle assay is able to mimic the most commonly observed changes seen in wild fish exposed to pulp mill effluents. Sensitive indicators of reproductive effects in fathead exposed for a lifecycle to pulp mill effluents include secondary sex characteristics, time to first reproduction and number of eggs laid. Egg production is often the most sensitive response to pulp mill effluents. Because of the length and cost of full lifecycle tests, a shortened assay using adult fish was developed as a screen for endocrine-disrupting compounds. This assay, the terminal reproduction test, has been used with success on a few pulp mill effluents. The assay exposes fathead minnow breeding pairs for three weeks to effluent, and compares egg production, sex characteristics and of reproductive performance in pre-exposure versus post-exposure fish. For assessment of pulp mill effluents, it appears that the sensitivity of the shortened terminal reproductive fathead minnow assay may be improved by assessing bioindicators of reproductive performance (such as circu- lating levels of sex hormones, and circulating or hepatic vitellogenin) along with changes in secondary sex characteristics and egg production.

Key words: pulp mill effluent, fathead minnow, lifecycle tests, egg production, sex characteristics

Introduction predict whether effluents will be acutely toxic to fish, but they do not predict the observed reproductive impacts in Wild fish exposed to North American pulp mill effluents wild fish. Survival and growth of fathead minnows (PMEs) often have reduced gonadal weights, decreased exposed for 7 d to final effluents from Canadian pulp mills steroids and increased growth and liver size. Across did not predict the reproductive impacts on wild fish in the Canada, surveys of wild fish health done as part of the receiving environment (Munkittrick et al. 1994; Robinson Environmental Effects Monitoring (EEM) Program have et al. 1994; Walker et al. 2004). These acute tests assess shown that increases in fish growth (increases in fish the ability of the pulp mill effluents to cause overt toxicity length, weight and condition factor) are related to nutrient or growth changes in larval fish. These short-term fish tests enrichment from the effluent. However, the increased were never designed to predict long-term changes in wild growth is often not accompanied by increased gonad fish, such as reproductive changes and metabolic alter- sizes, but instead decreases in ovary and testes size are ations. Instead, the acute lethality and growth tests provide often seen, along with liver enlargement. These signs are an indication of the toxicity of the effluent, and so these classified as metabolic disruption. Thus nutrient enrich- tests have been useful in tracking the improvements in final ment and metabolic disruption are the most commonly PMEs (such as installation of secondary effluent treatment) observed responses in fish downstream of pulp mills. This over the past decade (Environment Canada 1997; Scrog- pattern of response in wild fish was seen at approximately gins and Miller 2000; Lowell et al. 2003, 2004). However, 40% of Canadian pulp mills during 1999 to 2002 (Lowell these acute lethality and growth tests are unable to detect et al. 2003, 2004). the subtle reproductive effects of some PMEs. Current assessments of laboratory bioassays con- In an effort to predict reproductive changes in fish ducted alongside the EEM adult fish health assessments exposed to PMEs, tests of fish steroids have been used suc- have shown that sublethal short-term acute toxicity tests cessfully (McMaster et al. 1996; Parrott et al. 2000; Trem- blay and Van Der Kraak 1999; Dubé and MacLatchy * [email protected] 2000, 2001a,b, 2002). These tests showed decreased steroid

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levels in fish exposed to PMEs from 2 to 8 weeks. These 1987, 1996) and Benoit (1982). Modifications to the tests can mimic the steroid reduction that is seen in wild fish lifecycle test for assessment of PMEs are described in exposed to PMEs (reviewed in McMaster et al. In press). Kovacs et al. (1995a,b, 1996) and in NCASI (1996, However, decreases in steroid concentrations have not yet 2000). The following detailed methodology is the one been linked to meaningful effects on fish reproduction. used at National Water Research Institute labs for lifecy- Lifecycle fish tests can be used to provide a link cle testing of effluents and pure compounds. The between laboratory assays and effects of PMEs on wild methodology was developed for the assessment of a fish. Lifecycle tests expose newly fertilized eggs to efflu- bleached sulphite mill effluent over a three-year period ent, and monitor fish as they grow, mature and breed in (1999–2001) at the Fraser Edmundston mill in New the effluent. The fathead minnow lifecycle test takes Brunswick (Parrott et al. 2003, 2004). In addition to from 4 to 5 months to complete, but it is considered the PMEs, the lifecycle exposure of fathead minnows has definitive test for assessment of endocrine-disrupting shown remarkable sensitivity to very low concentrations compounds such as pesticides and industrial chemicals, (1 to 10 ng/L) of synthetic steroids such as ethinylestra- and can provide data that are useful for risk assessments diol and methyltestosterone (Parrott and Wood 2002). (OECD 2000a,b; Parrott et al. 2001). Lifecycle tests with fathead minnows and long-term Exposure System fish exposures assessing fish (fathead minnow or marine mummichog) secondary sex characteristics, sex steroids For lifecycle bioassays, exposures of fathead minnows to and reproduction are useful in the EEM Program at sites pulp and paper mill effluents are conducted using a pro- where the adult fish survey requirements are difficult to portional diluter or series of pumps to deliver the desired achieve. In 2000, caged bivalves and mesocosms were concentration of effluent to each exposure tank. Propor- approved as an alternative approach in the EEM Pro- tional diluters of the Mount-Brungs type, or solenoid gram, if adult fish capture or the field benthic survey valves controlled by a timer are used to deliver selected was difficult because of tidal zones or other confounding concentrations of effluent to aquaria. Each exposure factors (Environment Canada 2000; Porter and Ellis tank is designed as flow-through, with a standpipe at 2000). Full lifecycle fish exposures may be considered as one end of the aquarium, and the incoming tube (con- another alternative for assessing any potential effects of taining fresh exposure solution from the diluter) at the long-term effluent exposure on wild fish, if capture of other end of the aquarium. Flow rate to the aquaria adult wild is problematic at the site. should be over 1 tank volume per day, preferably 3 to 4 Fish lifecycle exposures are one way of assessing the tank volumes per day. The OECD (2002) test methods long-term effects of exposure to pulp mill effluent. Sev- under development recommend 5 volume changes per eral fish species can be used in lifecycle lab studies. Ide- day, however this may not be feasible if PME is collected ally, lifecycle fish test species are small-bodied, are able and shipped to labs off site. to grow and breed in captivity, have a short generation Water for reference aquaria and for dilution of PME time, and are sexually dimorphic. Fathead minnows can be dechlorinated city water, well water, or can be meet these criteria, and are relevant because they are an water taken from the river/lake upstream of where the important forage fish species, found all over North PME discharges. Effluent and dilution water (either America east of the . Because of this, dechlorinated water or upstream river water) are most studies conducted in Canada and the brought to 25ºC, with heaters and chillers in the dilution have traditionally used fathead minnows. water and effluent head tanks. Aquaria should be tem- The following paper overviews fathead minnow lifecy- perature-controlled to 25ºC using aquarium heaters, a cle tests of pulp and paper mill effluents. The review details water bath, or by room air temperature control (if the a generic methodology for the fathead minnow lifecycle exposures are conducted in a bioassay trailer). Exposure test, and reviews the findings of fathead minnow lifecycle solutions are aerated with air stones, and aquaria are tests performed with North American pulp and paper mill covered with Plexiglas lids. Light is 16 h light:8 h dark. effluents. The review examines the endpoints and effects Light intensity is 10 to 20 µE/M2/s, 540 to 1080 lux, or seen in lifecycle exposures of fathead minnows to PMEs, 50 to 100 ft-c (ambient laboratory levels) (OECD 2002). compares results of lifecycle tests to effects seen in wild fish exposed to pulp and paper mill effluents, and assesses Egg Hatching potential new methods for shortening lifecycle tests while still maintaining meaningful reproductive endpoints. For the lifecycle studies, exposures begin with fertilized eggs, and fish are followed as they grow and mature. Overview of Lifecycle Methodology Fertilized eggs are purchased from a bioassay supply company, such as Aquatic Research Organisms (New Methods for lifecycle exposures of fathead minnows to Hampshire, U.S.). Eggs on breeding tiles are collected pure toxicants are described by the U.S. EPA (1982, and shipped overnight. Several tiles of eggs should be

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used to initiate a lifecycle test to ensure offspring from TABLE 1. Age of fathead minnows (days post-hatch, dph) several sets of parents are used in the lifecycle test. Eggs and amounts of newly hatched brine shrimp (mL/fish) and are pooled and mixed to ensure that each tank contains frozen brine shrimp (mL/fish) fed over the lifecycle study fathead minnows from several breeding pairs. Eggs are gently rolled from tiles and assessed for fertility and Newly hatched Frozen brine brine shrimp shrimp counted under a dissecting microscope. Exposures begin Age of fish (mL/fish) (mL/fish) about 36 to 48 h post-fertilization. Eggs (usually about 30–60 per aquarium) are placed in screen-bottomed 1–8 dph 0.020 — 8–16 dph 0.060 — plastic egg-hatching cups. Eggs are checked daily and 16–22 dph 0.120 — dead eggs are removed to discourage fungal growth. 22–30 dph 0.180 — Hatching success of the eggs is monitored. It is usu- 30–45 dph 0.120 0.060 ally over 90% for control treatments. If hatch is less 45–71 dph 0.050 0.15 than 80% in the controls, the test should be restarted 71–105 dph 0.050 0.20 105–120 dph — 0.30 with a new batch of eggs. Larvae are counted and kept 120 dph and above — 0.60 in cups until 7 days post-hatch (dph), when they are counted and released into the aquaria. Survival to 7 dph should be over 60%, or the test should be restarted. trol of fish loading density, growth (length, weight) of Fish Feeding and Maintenance fish will be variable, and may not allow significant dif- ferences to be statistically identified. If mortality occurs The fathead minnow feeding schedule for the lifecycle in any treatment, it is best to cull all tanks to the lower test is outlined in Table 1. Fish are fed twice daily (7 days fish density, or to remove the replicate where fish mor- per week). Hatched fathead minnow larvae are fed newly tality occurred. In either case the number of fish in each hatched brine shrimp (Artemia nauplii). A slurry of newly aquarium should be maintained ±1 fish/aquarium, from hatched brine shrimp is prepared by decanting settled day 30 to the end of the experiment. brine shrimp (hatched for 48 h in saltwater in an aerated On days 30 and 60, juvenile fish are sampled, anaes- separatory funnel) into a fine-mesh fish-net, washing with thetized and killed in clove oil (10 drops/L) or methane dechlorinated lab water, and reconstituting the brine tricainesulfonate (MS222, 1000 mg/L), and weighed, shrimp in 50 mL dechlorinated lab water. Frozen brine measured and externally examined. Fish bodies are shrimp were purchased from an aquarium supply store, saved in formalin for later histopathology. At day 30, 15 and were thawed, stirred and pipetted into aquaria. randomly selected fish are left in each 12-L aquarium or Accurate counts of the number of fish in each aquarium 30 fish in each 35-L aquarium. At day 60, 12 fish are are kept and posted on each tank, so that the feeding rate left in each 12-L aquarium or 24 fish in each 35-L can be adjusted to the number of fish per aquarium. aquarium. At day 90, fish are reduced to 8 fish per 12-L Fish are checked daily. Dissolved oxygen, tempera- aquarium, or 16 fish per 35-L aquarium. ture, and pH of aquaria are checked every other day. Water flow and performance of the diluter is checked Initiating Breeding of Adult Fish several times a day (weekdays) and daily (weekends). Aquaria are cleaned once or twice weekly. Sides are The breeding phase of the fathead minnow lifecycle tests scraped using a plastic or metal spatula, and tanks are is very important. Selection of the 8 fish (from the 12 fish siphoned of excess food, fish waste and scrapings of the in each aquarium) at 90 dph can either be done randomly algal build-up. or can be done purposely to leave 4 to 5 females and 3 to 4 males. Purposeful selection of the largest males and Juvenile Fish Sampling most mature females (Kovacs et al. 1995a,b) ensures the best sex ratio for the breeding stage of the experiment. Juvenile fish are sampled to reduce the number of fish Random selection of fish for the breeding phase (Parrott per aquarium. Some lifecycle-test methods do not per- et al. 2004) allows breeding performance of the average form these culls (Kovacs et al. 1995a,b, 1996), but fish to be assessed, not just the breeding performance of unequal fish density (from 23 to 50 fish per 10-L aquar- the most mature and “best” fish of that exposure group. ium in Kovacs et al. [1995a]) may contribute to variabil- At 90 dph, tile breeding substrates (clay drainage or ity in fish growth and maturation. During the entire life- plastic plumbing pipes that have been cut in half) are cycle study, fish loading density (number of fish per placed in each aquarium. In some cases, breeding tiles aquarium) should be controlled. It is important to con- may be placed in aquaria sooner, if fish are growing and trol fish loading density among control and effluent con- maturing rapidly. Control fathead minnows and those centrations, to ensure that crowding or food availability exposed to 1 and 3% bleached sulphite mill effluent does not influence growth or mask effects. Without con- began breeding at 70 dph (Parrott et al. 2004). For

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breeding, a group of 8 fish and two tiles is preferred. 150 dph, while other experiments required longer expo- Large aquaria (over 15 L) should be subdivided into 2 sures (up to 180 dph) if maturation or breeding of the compartments using a perforated opaque plastic screen fish was slightly slower. Fish are anaesthetized using (that allows water to flow through). Eight fish with two clove oil or MS222 (as above) and blood is sampled breeding tiles are placed on each side of the divider. This from the caudal vein and caudal artery via heparinized limits fish competition and aggression and promotes capillary tube. Blood is spun and blood plasma is frozen breeding in larger exposure tanks. in liquid nitrogen for later measurement of sex steroids. Fish are killed by cervical dislocation. Gonads are exam- Egg Production ined to determine the internal sex of the fish. Internal sex is classified as mature male (M), immature male Breeding is monitored from tile placement until the end (Mi), mature female (F), immature female (Fi) or sex of the exposure. Breeding tiles are checked daily, and undetermined, immature (I). Testes and ovaries are dis- date of spawning and number of eggs recorded for each sected and weighed for calculation of the gonadosomatic aquarium. Fathead minnows spawn on average every 3 index (GSI). The production of sex steroids in ovaries to 4 days, and the mean number of eggs per spawn is 85 and testes is assessed using the method of McMaster et (Jensen et al. 2001). It is important to monitor egg pro- al. (1995). Cut pieces of fresh ovaries and testes (approx. duction from the addition of the tiles (day 90) until the 20 mg) from mature fish are placed in tissue culture cells end of the exposure. Early fathead minnow lifecycle tests containing 1 mL of cell media. Briefly, incubations are sometimes ignored the data from the initial stages of egg 20 h at 18ºC, in media with and without added human production (day 110–170), and began to count and chorionic gonadotropin (hCG, to stimulate the gonad to assess eggs only after day 170 (Kovacs et al. 1995b). produce hormones). After 20 h, media is removed and Assessment of the full capacity for egg production in the frozen in liquid nitrogen for later analysis of testosterone early stages of breeding is recommended. and by radioimmunoassay (McMaster et al. Eggs are assessed for fertilization and hatch. Eggs 1995, 1996). Testosterone and estradiol are expressed as deposited on tiles are removed every morning, rolled pg/g ovary or testis, either basal or hCG stimulated. In from tiles, counted and assessed for fertilization. Eyed addition, various tissues can be preserved for histological eggs are placed in either clean water or the exposure examination. Livers are dissected, weighed for calcula- solution (in screen-bottomed egg cups in aquaria), aer- tion of the liver-somatic index (LSI) and frozen in liquid ated and observed until hatch. Egg-hatching aquaria are nitrogen for later analyses of vitellogenin. temperature-controlled at 25°C. Eggs hatch in approxi- mately five days. Date of hatch should be recorded for Sex Characteristics time-to-hatch comparisons. Fry abnormalities (such as spinal deformities or edema) are noted. Data on egg pro- Fathead minnows are sexually dimorphic, with clear male duction, % fertilization, % hatch, % deformed and % and female secondary sex characteristics (Fig. 1). Males dead fry are collected. Some lifecycle methods perform hatching assessments on several (about 10) batches of eggs from each exposure concentration (Borton et al. 1997; NCASI 2000), instead of on every batch of eggs produced. Limiting the numbers of eggs hatched and assessed reduces the labour involved in the breeding stage of the test. Since hatching success is usually not a very sensitive endpoint in PME exposures of fathead minnows, limiting the number of egg batches assessed for % hatch is possible. However, for other types of endocrine-disrupting substances, such as ethinylestra- diol, egg fertilization proved to be one of the most sensi- tive indicators of effect. For full sensitivity, and when testing pure compounds and other types of effluents, fer- tilization and hatching success of each and every batch of eggs produced in all aquaria should be assessed.

Sampling of Mature Fish at the End of the Exposure

The lifecycle experiment ends after maturation and suffi- Fig. 1. Male and female mature fathead minnows showing cient reproduction. Some experiments terminated at secondary sex characteristics.

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are larger and darker than females, with nuptial tubercles, possibility of equipment malfunction or unexplained a dorsal fatpad (an enlarged pad of skin on the head, used mortalities, resulting in the loss of several or all fish to tend the eggs), a dark dorsal fin dot, and a vertical from one or more aquaria. Because of this, four repli- banding pattern (dark head, and dark vertical bands on cates are preferred, so that parametric statistics may be the body, anterior and posterior to the dorsal fin). Nuptial completed. Parametric and non-parametric statistics tubercles are assessed and counted under a dissecting using a program such as SYSTAT (Evanston, Ill., U.S.) microscope. The dorsal fatpad is noted and subjectively are used to compare effects at each effluent dilution to graded (none, small, medium, large). The dorsal fin dot is controls. Fish are grouped by internal sex, and males noted as absent/present. Banding pattern is noted and and females are analyzed separately. Immature fish are graded (none, light, medium, dark prominent banding). left out of the analysis. Analysis of variance (ANOVA) is Female fathead minnows have a more pointed head, a used to assess differences among treatment means (for rounded abdomen, and are silvery-beige in colour. The fish weight, length, condition factor [CF], % hatch, main female secondary sex characteristic is the ovipositor liver-somatic index [LSI] and gonadosomatic index which is used to deposit eggs. Ovipositors can be subjec- [GSI]). Individual exposure concentrations are compared tively graded (none, small, medium, large, huge) on live to controls by using Tukey’s test or paired t-tests (Bon- fish held individually in beakers or can be measured under feroni’s) after ANOVA had detected significant treat- a dissecting microscope after fish have been sampled. ment differences. Subjective ratings of ovipositor index Abnormal secondary sex characteristics that have and male sex index are compared to controls using the been seen in fathead minnows exposed to PMEs include non-parametric Kolmogorov-Smirnov Two Sample Test. ovipositor development on male fish, male secondary sex characteristics on female fish, and changes in gender bal- Fathead Minnow Lifecycle Studies with Pulp ance (Kovacs et al. 1995b; Parrott and Wood 2002; Par- and Paper Mill Effluents rott et al. 2003, 2004). Many types of PME have been evaluated using fathead Statistics minnow lifecycle tests (Tables 2 and 3). Most effluents from kraft process and bleach-using pulp mills have the A minimum of three, preferably four, replicates is ability to reduce reproduction in fathead minnows. Only required. During the long exposures there is always the three PMEs (two thermomechanical mills and one recy-

TABLE 2. Fathead minnow lifecycle tests conducted with bleached kraft (BK) and unbleached kraft mill effluents from North Americaa

EC25 LOEC Mill typeb Endpoint (%) (%) Reference

BK + Cl/ClO2 Total eggs 14 NCASI (1985) % Hatch >100

BK + OD + Cl/ClO2 Eggs/female 12 Robinson (1995) % Hatch >50 Sex steroids 50

BK + OD + Cl/ClO2 Eggs/female 1.7 Kovacs et al. (1995b) % Hatch >10

BK + OD + ClO2 Eggs/female N/D >20 Kovacs et al. (1996) % Hatch N/D >20

BK + OD + ClO2 Eggs/female/day 61 NCASI (1996) % Hatch 88

BK + ClO2 Eggs/female/day 18 NCASI (1997) % Hatch >50

BK + Cl/ClO2 Eggs/female/day 27 NCASI (2000) % Hatch >50 Male sex 12 characteristics Unbleached kraft Eggs/female/day 38 Borton et al. (1997, 2001) % Hatch >100

aEndpoints include total eggs, % egg hatch, sex steroids and male sex characteristics. EC25s and LOECs show that egg production is in most cases more sensitive than % egg hatch. b BK (bleached kraft); OD (oxygen delignification); Cl (elemental chlorine used for bleaching); ClO2 (chlorine dioxide used for bleaching).

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TABLE 3. Fathead minnow lifecycle tests conducted with various types of pulp and paper mill effluents from North Americaa

Mill type Endpoint EC25 (%) LOEC (%) Reference Thermomechanical Total eggs No effects N/Db >20 Kovacs et al. (1995a) % Hatch No effects N/D >20 Recycled fibre (de-inked Eggs/female/day No effects >100 Borton et al. (2001) paper furnish) Thermomechanical Eggs/female/day No effects >100 Borton et al. (2001) Bleached sulphite Eggs/female 10 Parrott et al. (2004) % Hatch >10 N/Dc

aEndpoints include total eggs, eggs per female per day, % egg hatch. EC25s and LOECs show that egg production is in most cases more sen- sitive than % egg hatch. bN/D; Not determined, PME was tested up to 20% concentration and no effects were seen. cN/D; Not determined, no eggs were laid in effluent concentrations above 10%, so egg hatchability could not be assessed.

cled fibre mill) caused no effects. Every bleached kraft at 2.5 and 5% effluent (Kovacs et al. 1995b). This effect mill effluent (BKME) (except one) tested thus far has was not seen in similar lifecycle tests performed 2 years been able to reduce reproduction in lifecycle exposures later after changes were made to the BKME process and of fathead minnows (Table 2). This is true whether ele- treatment (Kovacs et al. 1996). mental chlorine (Cl), chlorine dioxide (ClO2) or combi- Bleached sulphite mill effluent (Parrott et al. 2004) nations of the two are used for bleaching. Unbleached and several other mill effluents (Borton et al. 2000) have kraft mills also reduce reproduction (Table 2; Borton et been tested using fathead minnow lifecycle assays con- al. 1997, 2001). One BKME did not affect reproduction ducted in situ in a bioassay trailer at the mill secondary when retested after improvement in mill processes and treatment ponds. This allowed the assessment of fresh effluent treatment (Kovacs et al. 1996). However, the effluent rather than effluent that had been batch-col- PME was tested up to concentrations of only 20%, so lected every 1 to 2 weeks and shipped to laboratories for may still have had effects on fathead minnow reproduc- fish exposures. tion at higher, but untested, concentrations. Pulp mills using processes other than kraft (such as a bleached sul- Specific Endpoints phite mill) are able to reduce reproduction in lifecycle exposures of fathead minnows (Table 3). Hatching success. Hatching success of fathead minnow Robinson (1994) found that exposure to 12.5% eggs exposed to PMEs is not a very sensitive endpoint or BKME reduced reproduction. Borton et al. (2001) evalu- predictive indicator of effects. Often, exposure to high ated effluent from eleven pulp mills: eight kraft mills PME concentrations (greater than 50%) will not affect (two of which had no bleaching, two with Cl bleaching, egg hatching success (Table 2). Concentrations that one with elemental-chlorine-free [ECF] bleaching, and affect hatch are usually severalfold above concentrations two with oxygen delignification [OD] and 70% CLO2 that cause decreases in reproduction (Table 2). If egg bleaching), two recycled fibre furnish mills, and one hatch is affected, the effluent is usually overtly toxic, and thermomechanical mill. No effects were found in fish 7-d fathead minnow acute tests may confirm this. It is exposed for a lifecycle to effluent from the thermome- recommended that the fathead minnow lifecycle test be chanical pulp mill and from one of the recycled fibre run at several (at least 5) concentrations, most of which mills (that used de-inked office paper as furnish) up to should be well below the threshold for toxic effects on 100% effluent. All other (nine of eleven) mill effluents egg or larval survival. significantly decreased fish reproduction, with effective concentrations for 25% reduction in reproduction Growth effects. Lifecycle tests of fathead minnows (EC25s) ranging from 17% to >100% final effluent. exposed to PMEs have shown increased or decreased Final effluent from an unbleached mill reduced repro- growth. NCASI (1985) and Robinson (1994) saw duction in fathead minnows (EC25 = 38%) and delayed decreased growth in fathead minnows exposed for a life- reproduction by 12 days (at the EC25 effluent concen- cycle to high concentrations of BKME. Other studies tration) (Borton et al. 1997, 2001). have shown no effects on growth of fathead minnows Kovacs et al. have performed three lifecycle studies exposed to BKME (Kovacs et al. 1995a,b), although fish with PMEs (Table 2). Effluent from a thermomechanical loading was not consistent among treatments/replicates. mill had no effect on fathead minnows when tested at Most studies have reported increased growth of fat- concentrations up to 20%. Exposure to BKME signifi- head minnows exposed to PMEs. Female fathead min- cantly affected fish reproduction and sex characteristics nows exposed to 100% BKME for a lifecycle (178 d)

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were 92% heavier than control females, and males from of estradiol was about half that of control fish at all 100% BKME were 44% heavier than control males BKME concentrations tested (3–100%), but no dose (NCASI 2000). Similarly, length and weight of female response was noted. The EC25 for reduced estradiol fish were greater in bleached sulphite mill effluent con- production was 1.6% BKME, which was far lower than centrations of 10% and above, with a doubling of fish the EC25 for lowered egg production (eggs/female/day) weight in fish exposed to 30% effluent compared to con- (NCASI 2000). Testosterone production by excised trol fish (Parrott et al. 2004). testes was not as sensitive, and showed reductions in These increases in fathead minnow growth may be testosterone produced by excised testes from male fish caused by nutrient enrichment from the carbon, nitrogen exposed to 100% BKME compared to testes from con- and phosphorus in the effluent, which can increase bac- trol males. Production of testosterone was about half of terial and algal growth (Culp et al. 1996; Glozier et al. that of control fish, with an EC25 estimated to be 64% 2002). The increased algal and bacterial biomass can BKME (NCASI 2000). Robinson (1994) saw steroid result in increases in the numbers of . Nutri- production in excised testes and ovaries was affected at ent addition alone caused increases in the BKME concentrations two to three times those that neg- and zooplankton abundance and resulted in increases in atively affected fecundity and egg production. the growth and reproduction of fathead minnows (Grant and Tonn 2002). However, the nutrient enrichment Secondary sex characteristics from PMEs causes increased growth, but does not enhance reproduction (see discussion on metabolic dis- Lifecycle exposures of fish to pulp and paper mill efflu- ruption, below). ents have shown changes in sexual development and decreases in or delays in the expression of secondary sex Sex steroids. Concentrations of sex steroids in blood characteristics. Delayed development of sex characteris- plasma collected from fathead minnows exposed to tics and de-masculinization of male fish have been seen in pulp and paper mill effluents can be a useful indicator fathead minnows exposed to PMEs for a lifecycle. Male of effects. There is considerable variability in blood fathead minnows exposed to 50% BKME had a delay in steroid concentrations over the reproductive cycle of appearance of secondary sex characteristics (Robinson male and female adult fathead minnows (Jensen et al. 1994). NCASI (2000) saw a delay in the development of 2001). In control female fathead minnows, blood estra- secondary sex characteristics in fathead minnows diol peaks (concentrations double) one day after spawn- exposed to a bleached kraft mill effluent. Delays in the ing (Jensen et al. 2001). Testosterone concentrations in development of secondary sex characteristics occurred in blood of females show similar trends, while concentra- fish exposed to concentrations of 12% BKME and above. tions of testosterone and 11-ketotestosterone in blood This concentration was less than that required to cause of control male fathead minnows rise prior to spawning significant decreases in the time to first spawn and the (Jensen et al. 2001). Because of this variability, plasma number of spawns produced (NCASI 2000). steroids are thought to be less reliable than in vitro pro- Masculinization of female fish and a shift of sex duction of steroids by ovaries and testes (Dubé and ratios towards more male fish have been seen after expo- MacLatchy 2000). sure of fathead minnows to PMEs. Fathead minnows In small fish species such as the fathead minnow, exposed to concentrations of 2.5% secondary-treated sex steroids are usually measured in vitro (or ex vivo) in BKME and above for 275 d had increased male sec- excised ovaries or testes placed in cell culture media for ondary sexual characteristics (Kovacs et al. 1995b). 18 h at controlled temperature. Gonadal production of Masculinization has been seen in adult female mosqui- and is measured under basal condi- tofish (Ellis et al. 2000), sticklebacks (Gasterosteus tion and under stimulated condition with added human aculeatus) (Katsiadaki et al. 2002) and guppies (Larsson chorionic gonadotropin (hCG). The method is outlined et al. 2002) exposed to PMEs. in McMaster et al. (1995). Feminization of male fish has also been seen in life- Exposure to PME usually causes decreases in the cycle exposures of fathead minnows to pulp and paper production of gonadal sex steroids. In most cases con- mill effluents. Fathead minnows exposed to bleached centrations of sex hormones in blood and the production sulphite mill effluent from a Canadian mill for 5 months of sex hormones by excised testes and ovaries are less showed an increasing proportion of fish with ovaries sensitive indicators of effect than reproductive output. (Parrott et al. 2003). Exposure also resulted in prema- Borton et al. (1997) saw decreased in vitro steroid pro- ture development of ovipositors (an extension of the duction by excised ovaries and testes (but concentrations urogenital papillae of female fathead minnows used for of PME were two times higher than those affecting laying eggs) in juvenile fathead minnows, and develop- reproduction). Excised testes from male fathead min- ment of ovipositors in mature male fathead minnows nows exposed to BKME for a lifecycle (178 d) produced (Parrott et al. 2003, 2004). Feminization has also been less estradiol than testes of control male fish. Production seen with rainbow trout (Tremblay and Van Der Kraak

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1999), zebrafish (Örn et al. 2001) and Chinook salmon (NACSI 1985, 1996; Robinson 1994; Kovacs et al. (Oncorhynchus tshawytscha) (Afonso et al. 2002) 1995b; Borton et al. 1997, 2000; Parrott et al. 2003, exposed to PMEs. 2004). One of the earliest reported pulp mill fathead minnow lifecycle tests assessed secondary treated efflu- Liver-somatic index. Liver-somatic index (LSI) may be ent from a bleached kraft mill (Robinson 1994). After 6 increased in fathead minnows exposed to pulp and paper months of exposure to 3 to 50% BKME, fathead min- mill effluents. Enlarged livers usually occur in fathead nows had significantly reduced egg production and sig- minnows exposed to high PME concentrations (greater nificantly delayed spawning compared to control fish. than 50% effluent). Increases in LSI usually require Male minnows had decreases in secondary sexual char- higher PME-exposure concentrations than those acteristics, but these were significant only at 50% BKME required to reduce fathead minnow reproduction (Bor- (Robinson 1994). This mill had historically been associ- ton et al. 1997; Parrott et al. 2004). Female and male ated with alterations in fish growth and reproductive fathead minnow LSIs were increased with exposure to parameters (Munkittrick et al. 1994). bleached sulphite mill effluent. Livers of fathead min- Fathead minnows exposed to four pulp mill efflu- nows from 50% effluent were two to three times the size ents had average spawning delays of 10 to 14 days at the of livers in control fish (Parrott and Wood 2002). EC25 effluent concentration (Borton et al. 1997). Egg Enlarged livers and increased LSIs were seen in fathead production (measured as number of eggs per female per minnows exposed for an entire lifecycle to 50 and 100% day) correlated most strongly (R2 = 0.65) to time to first BKME (NCASI 2000). spawning, in a study of five lifecycle tests of four differ- ent PMEs (Borton et al. 1997). Gonadosomatic index. Fathead minnow GSIs can be Fathead minnows exposed to bleached sulphite mill reduced or increased by exposure to PMEs, but impacts effluent for a lifecycle had dramatic decreases in egg pro- on GSI occur at higher concentrations than impacts on duction at exposures of 10% effluent. A surprising finding reproduction. Female fathead minnows exposed to an was that females exposed to 10% effluent had GSIs that unbleached kraft mill effluent, elemental-chlorine-free were similar to control fish GSI, even though egg produc- kraft mill effluent, and a bleached (70% ClO2) kraft mill tion was one-tenth of control fish and fish exposed to 1 to effluent for a lifecycle had GSIs that were larger than 3% bleached sulphite mill effluent (Parrott et al. 2004). control fish (Borton et al. 1997). GSIs of female fathead Egg production was the most sensitive indicator of effect, minnows exposed for a lifecycle to an elemental-chlo- and was severely impacted even though female ovary size rine-free kraft mill effluent were reduced (Borton et al. was normal. 1997). However, all changes in GSIs of females occurred Effects of PME on fish reproduction are dramatic. at higher concentrations than reductions in reproduction Fish exposed to 100% BKME produced 0.1 egg/female/ (Borton et al. 1997). GSIs of male fish were unaffected day, and exposure to 50% BKME resulted in egg produc- by exposure to these same PMEs, except for increases in tion of 6.8 eggs/female/day (compared to 14.5 eggs/female/ GSI of males exposed to the bleached (70% ClO2) kraft day in control fish) (NCASI 2000). Exposure of fathead mill effluent (Borton et al. 1997). Borton et al. (2004) minnows for a lifecycle to 10% bleached sulphite mill efflu- saw no significant differences in GSI among fathead ent caused an 80% decrease in reproduction compared to minnows exposed to 100% BKME, even though expo- control fish, and exposure of fish to 30% effluent and sure completely halted egg production. above resulted in no reproduction (Parrott et al. 2004).

Fathead minnow reproduction. Reproductive success of Multi-generation Fathead Minnow Exposures fathead minnows exposed to PMEs can be measured as age at first spawn, total number of eggs produced, num- Multi-generation tests of PMEs have found that effects ber of eggs per female, or number of eggs per female per between generations are fairly consistent, and there day. Percent fertilization and percent hatching can also appear to be no increasing effects over generations. The be measured, but changes in percent fertilization are not expansion of fathead minnow lifecycle tests to multi- usually affected by pulp and paper mill effluents. generation tests is laborious, expensive and time con- Decreases in percent hatch were seen with some pulp suming; however, it has been done for several pulp mill and paper mill effluents (Borton et al. 2000; Rickwood effluents. Borton et al. (2000) examined fathead minnow et al. 2003), but only at the highest PME exposure con- reproduction and health through four generations of centrations. Decreases in percent hatch are usually exposure to various PMEs in the U.S. Egg production caused by overt toxicity of the effluent on the very sensi- was consistently decreased by exposure to both bleached tive developing/hatching larvae. and unbleached kraft effluents, but egg hatchability was Lifecycle exposures of fathead minnows to PMEs not affected. Egg production was reduced to one-third of have usually found that egg production and time to first that of control fish at BKME concentrations as low as spawning are the most sensitive parameters studied 10% (Borton et al. 2000).

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Predictive Ability of Lifecycle Bioassays ductive impairment due to this BKME was immediate, and that compounds that may have been accumulated Long-term lifecycle exposures of fish provide some of the during the first 75 days (prior to pairing and breeding) most convincing evidence linking PME exposure to repro- were not responsible for the reproductive effects. ductive effects in fish. Lifecycle tests of fathead minnows Other research has shown that effects on reproduc- have examined growth, maturation and reproduction. tion are permanent, and that fish cannot recover when Similar to the effects seen in wild fish exposed to PME, placed in clean water. Kovacs et al. (1995b) saw egg lifecycle exposures of small laboratory species have shown production drop from 90 eggs/female to 10 eggs/female growth increases, alterations in sexual development, in 10% BKME. Adult fathead minnows from 10% delays in time to first spawning, reductions in the numbers BKME were removed to clean water (from 206 to 270 of eggs laid, or decreases in levels of sex steroids. dph), but failed to show any increase in egg production Lifecycle assays were able to mimic many of the compared to fish kept in 10% BKME. responses of wild fish exposed to PMEs. Lifecycle and The common finding of reproduction as the most long-term fish exposures have shown increased body size sensitive response to PME and the observation that ces- (length and weight) and increased liver size. Liver-somatic sation of reproduction in PME-exposed adult fish can be indices (LSIs) were increased in fathead minnows exposed a sensitive and immediate response holds promise for to PMEs (NCASI 2000; Parrott and Wood 2002). These shortening the lifecycle tests, while still maintaining this changes are similar to the enlarged livers found in fish important and meaningful endpoint. captured at sites downstream of pulp and paper mill dis- The fathead minnow adult terminal reproductive charges (reviewed in McMaster et al. In press). assay, outlined in Harries et al. (2000) and Ankley et al. Decreases in gonad weight and increases in liver (2001) has recently been tested with PMEs. The test weight, condition factor and age were the most common monitors breeding success (egg production, secondary response patterns seen in the EEM surveys of wild fish sex characteristics) of mature fathead minnows in con- exposed to Canadian PMEs (Environment Canada 2002; trol water for 3 weeks. Then, exposures of PME begin, Lowell et al. 2003, 2004). This pattern of effects in wild breeding and sex characteristics are tracked, and fish are fish exposed to PMEs was classified as ‘a form of meta- sampled to measure bioindicators such as vitellogenin, bolic disruption in combination with a nutrient enrich- sex steroids and gonad histopathology. ment effect’ (Lowell et al. 2003, 2004). Fathead minnows exposed to 50 and 100% BKME for 3 weeks had decreased egg production (Rickwood et Limitations of the Fathead Minnow Lifecycle Test al. 2003). It should be noted that this was a particularly potent BKME which was toxic to nearly all exposed The limitations of the fathead minnow lifecycle test are eggs: hatching success of eggs in 100% BKME was the length of time it takes to complete the test and the reduced to 15% hatch, while control eggs had 80 to logistics of effluent delivery. A fathead minnow lifecycle 90% hatch (Rickwood et al. 2003). test is expensive, and requires at least 2 people for 5 Adding more endpoints to the adult terminal repro- months. Effluent exposure requires either setting up the ductive assay may enhance the sensitivity to PMEs. test in situ, or shipping large quantities of effluent (600 When reproductive bioindicators (such as sex steroids to 5000 L/week, depending on concentrations of effluent and vitellogenin) are used along with assessment of tested) (Kovacs et al. 1995a,b, 1996; NCASI 2000; Par- reproduction and sex characteristics, the adult terminal rott et al. 2003, 2004). Because the test is meaningful, reproductive assay with fathead minnows may show but costly, there are several attempts to shorten or mod- more promise. When mature, breeding fathead minnows ify the fathead minnow lifecycle test while still retaining were exposed to six PMEs (at 20% dilution) for 4 the biologically relevant endpoints. weeks, only one of the six PMEs tested caused a cessa- tion in egg production (Martel et al. 2004). However, Shortening the Fathead Minnow Full Lifecycle when reproductive biomarkers were examined in the Test: The Terminal Reproduction Assay PME-exposed fish, nearly all of the effluents had some effect. Five of the six PMEs significantly affected one (or Recent research has shown that there is an immediate more) of the reproductive indicators: whole body sex decrease in egg production of fish exposed to BKME, steroids, male secondary sex characteristics and vitel- regardless of previous exposure. Borton et al. (2004) logenin (Martel et al. 2004). exposed fathead minnows to 100% BKME or control These shortened reproductive assays may be a fast water from the egg stage to 75 days post-hatch. When fish and meaningful way to assess the effects of PMEs. Lifecy- were mated and held in control water or 100% BKME, all cle and long-term fish exposures are laborious, time con- pairs in the 100% BKME had significantly lower egg pro- suming and expensive. The terminal reproductive assay is duction, regardless of their past exposure (control or much shorter and less expensive, as it assesses a sensitive BKME) (Borton et al. 2004). This showed that the repro- and meaningful response of fish (reproduction) over a

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6-week period. Exposure to effluent is for 3 weeks. It is Conclusions hoped that the 6-week terminal reproduction assay (3 weeks of pre-exposure, and 3 weeks of PME exposure) Fathead minnow lifecycle tests have been successfully will be a faster and meaningful fish test, the results of conducted on a variety of pulp and paper mill effluents. which will be predictive of wild fish health in the PME The paper has overviewed a generic methodology for the receiving environment. fathead minnow lifecycle test in its application to testing It is expected that the shorter test will lose sensitiv- of pulp mill effluents, and the endpoints and effects seen ity compared to the full lifecycle. Preliminary evidence in lifecycle exposures of fathead minnows to PMEs. The suggests there is some loss of sensitivity when the tests results of fathead minnow lifecycle tests performed with are shortened to 3 weeks of exposure, compared to a North American pulp and paper mill effluents suggest lifecycle or longer exposure. Rickwood et al. (2003) saw that egg production is one of the most sensitive indica- no response to 10 ng/L (a synthetic tors of effect. Exposing mature, breeding adult fish may , used as a positive control compound to cause shorten the costs of the assay, while still maintaining endocrine disruption in long-term fish tests) in the adult meaningful reproductive endpoints. terminal reproductive assay. In contrast, lifecycle expo- sures to 1 ng/L ethinylestradiol dramatically decreased References the fertilization success of male fathead minnows (Par- rott and Blunt 2005), and 3.5 ng/L ethinylestradiol femi- Afonso LOB, Smith JL, Ikonomou MG, Devlin RH. 2002. nized fathead minnows in 60-day exposures, from the Y-chromosomal DNA markers for discrimination of egg stage to juvenile stage (Parrott and Wood 2002). As chemical substance and effluent effects on sexual differ- well, exposure to 5 to 6 ng/L ethinylestradiol in an entiation in salmon. Environ. Health Perspect. experimental lake system caused a complete crash of the 110:881–887. fathead minnow population within 1 year (Kidd et al. Ankley GT, Jensen KJ, Kahl MD, Korte JJ, Makynen EA. 2003). These changes were not picked up in the short 2001. 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