effects of maternal versus direct exposure to pulp and ...

Journal of Toxicology and Environmental Health, Part A, 68:369–387, 2005 Copyright© Taylor & Francis Inc. ISSN: 1528–7394 print / 1087–2620 online DOI: 10.1080/15287390590900787

EFFECTS OF MATERNAL VERSUS DIRECT EXPOSURE TO PULP AND PAPER MILL EFFLUENT ON RAINBOW TROUT EARLY LIFE STAGES Rosanne J. Ellis,1 Michael R. van den Heuvel,1 Murray A. Smith,1 Nicholas Ling2 1

Forest Research, Rotorua, New Zealand, and University of Waikato, Hamilton, New Zealand

2

The acute and chronic effects of secondary-treated effluent from a New Zealand pulp and paper mill were assessed using both long-term adult and early life stage (ELS) laboratory exposures of rainbow trout. The relative impact of maternal exposure versus ELS exposure was assessed by a comparison of directly exposed eggs and larvae with the eggs and larvae of exposed adult trout that were reared in reference water. Rainbow trout were exposed to a secondary-treated mixed thermomechanical/bleached kraft mill effluent at a concentration of 15% or to reference water from the egg through to 320-d-old juveniles. The 2 adult rainbow trout exposures were undertaken with nominal concentrations of 10% and 12% treated effluent, respectively. There was no marked effect of water hardening with 15% effluent on fertility or survival of eggs to 16 d. In a subsequent exposure (with hardening in reference water), no significant effects were found on mortality to hatch, time to hatch, length at hatch, mortality to swim-up, mortality to 320 d, or deformity rate at hatch. At experimental termination (320 d), direct-exposed juveniles had smaller livers and reduced condition factor, likely due to differences in food consumption. In 2 subsequent consecutive experiments, exposure of adult trout to 10% and 12% effluent for 2 mo, followed by incubation of the fertilized eggs in reference water, produced no impact on fertility, survival to hatch, survival to swim-up, or length and weight of fry at swim-up. Exposure of adult trout to 12% treated effluent for 8 mo prior to egg fertilization also did not result in differing rates of fertility, mortality to hatch or mortality to swim-up. However, the 8-mo maternal exposure did result in swim-up fry that were significantly shorter and weighed less than the reference swim-up fry. This difference was directly attributable to smaller eggs in the 8-mo-exposed female trout. These results demonstrate that this pulp and paper mill effluent is more likely to elicit indirect impacts on progeny size through chronic exposure of adults to effluent during gonadal recrudescence rather than through direct exposure of early life stages to effluent.

Funding for this research was provided by the Arthur and Aenne Feindt Foundation of Germany, The Foundation for Research Science and Technology of New Zealand and by the Norske-Skog/Carter Holt Harvey Tasman Mill. The authors acknowledge the assistance of Megan Harris, Luca Chiaroni, Suzanne Lambie, Murray Smith, Nicola Marvin, Rob Hunter, Robert Donald, and Errol Cudby. Current address for Rosanne J. Ellis is URS New Zealand Limited, Level 6, URS Centre, PO Box 821, 13–15 College Hill, Auckland, NZ. Address correspondence to M. R. van den Heuvel, Forest Research, Private Bag 3020, Sala Street, Rotorua, New Zealand. E-mail: [email protected] 369

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The exposure of aquatic organisms to pulp and paper mill effluents has been extensively investigated since the 1960s, and linked to a variety of effects including reproductive impairment. Some of the observed reproductive impacts include decreased egg size, smaller gonads, and increased age to maturity (McMaster et al., 1991, 1996; Munkittrick et al., 1992a, 1994; van den Heuvel & Ellis, 2002). These changes have been linked to decreased circulating sex steroid levels due to lower gonadal steroid production (Van Der Kraak et al., 1992; Munkittrick et al., 1992a; McMaster et al., 1995). Most field studies related to pulp mill effluent exposure have exclusively examined the adult fish component of reproduction. To date, there are no compounds that have been conclusively identified as being causative of the observed reproductive impacts on adult fishes (van den Heuvel, 2004). Limited research effort has been directed at elucidating maternally mediated mechanisms of toxicant action on the early life stages (ELSs) of fishes as compared to the volume of research examining impacts of chemicals on the adult components of reproduction. In one study, eggs from mature adult pike exposed to pulp and paper effluent appeared less sensitive to subsequent effluent exposure and to water hardening in effluent as compared to eggs from unexposed adults (Tana & Nikunen, 1986). Embryos of white sucker exposed maternally to bleached kraft mill effluent (BKME) in the field did not show differences in fertilization or hatchability when incubated in laboratory water, but larval growth differences were observed (McMaster et al., 1992). Experiments conducted with an acutely lethal effluent (LC50 6–30% v/v) found reduced hatching success in brown trout eggs directly exposed to a concentration of 2% v/v BKME (Vuorinen & Vuorinen, 1987). Exposure of adult trout to this same effluent (0.5% v/v) resulted in smaller, less viable progeny (Vuorinen & Vuorinen, 1985). Brown trout exposed to a thermomechanical pulping (TMP) treated effluent at 2.5% v/v dilution showed no effluent-related differences in survival of progeny, though some size differences in hatchlings were observed (Johnsen et al., 2000). The same study did not show effluent-related effects of direct exposure of eggs to 2.5% v/v effluent. Exposure of striped bass eggs and larvae to BKME up to a concentration of 20% showed no impacts to hatching, yet some direct toxicity to prolarvae was observed (Burton et al., 1983). Kovacs et al. (1995a, 1995b) performed whole-life-cycle experiments with fathead minnow and found no impacts of a TMP effluent on reproductive parameters, but an increased male gender ratio and lower egg production in adults exposed to a BKME. In recent years, the pulp and paper industry has committed considerable resources to environmental improvement through process modifications and the installation or upgrade of secondary treatment facilities (Kovacs et al., 1997). The benefits of these investments in terms of improved effluent quality and the decreased impact on physiology of receiving water fish populations have been documented (Munkittrick et al., 1997). For example, Kovacs et al. (1996) observed the disappearance of previously observed (Kovacs et al., 1995b) reproductive impacts in fathead minnows following mill modernization. Nonetheless, significant mill specific impacts on reproductive parameters

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continue to be recorded at some sites where process and treatment modifications have been implemented (Ellis et al., 2002; Munkittrick et al., 1992b; van den Heuvel and Ellis, 2002). The aim of the present study was to undertake an assessment of the effects of a mixed BK/TMP effluent on reproductive success of rainbow trout (Oncorhynchus mykiss). Second, this work sought to compare the impacts of maternal exposure of the ovarian follicles with direct exposure of fertilized eggs. Experiments examined early-life-stage viability, growth, and development, as well as other reproductive and physiological parameters. The accumulation of pulp and paper related organic extractives through both maternal and direct effluent exposure was quantified in egg and larval tissue.

METHODS AND MATERIALS Mill Description The mill studied is an integrated bleached kraft and thermomechanical (BK/TMP) pulp and paper mill, producing 760 and 1010 air dried tons per day, respectively at the time of this study. Mill furnish is primarily softwood (Pinus radiata) with occasional eucalypt production, and the bleaching process has been elemental chlorine free (ECF) since April 1998. The wastewater treatment system consists of an in-mill mobile bed bioreactor for the TMP effluent stream only. All mill effluents including the pretreated TMP effluent are collected into a single drain, passed through two bar screens, a clarifier, and a settling pond for solids removal prior to treatment in a threepond aerated stabilization basin system. The ponds have an area of 45 ha with a retention time of 5–6 d. Following treatment in the aerated lagoon system effluent is discharged into the Tarawera River, New Zealand, at a total mean volume of 180,000 m3/d. Dilution of effluent in the river ranges between 5 and 12%. Experimental Design This study consisted of two separate components: (1) the direct exposure of eggs to effluent in the laboratory, and (2) the exposure of sexually mature rainbow trout to effluent in mesocosms, followed by the incubation of eggs from those fish in reference water. The direct exposure of eggs was further subdivided into a preliminary study of the impacts of water hardening in effluent and a subsequent study of the survival of eggs, fry, and juveniles exposed to effluent. The exposure of adult trout was conducted as two experiments over consecutive years. During the first year, exposure was initiated halfway though the vitellogenic period for a total exposure time of 2 mo. During the second year, the study of the first year was duplicated, plus an additional exposure that started 3 mo prior to the initiation of ovary growth was run in parallel (total exposure time of 8 mo).

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Experiment 1: Direct Exposure of Eggs at the Water Hardening Stage of Development In July 1998, rainbow trout eggs were obtained from adult trout captured at the Eastern Region Fish and Game fish trap in Te Wairoa Stream (Lake Tarawera, Rotorua, New Zealand). Eggs and sperm were stripped and pooled from three females and three males, respectively. Approximately 1000 eggs were mixed with sperm, immediately halved into two groups, placed in either Lake Tarawera water or 15% v/v effluent diluted in lake water, and left undisturbed for 15 min. Eggs were transported back to the laboratory (travel time of 20 min) and incubated for 24 h in custom-made flow-through chambers at 12°C before fertility was assessed. Fertility was measured as the proportion of viable eggs after 24 h; dead eggs were identified as those that were opaque white. A subsample of eggs was preserved in Stockards solution 18 h postfertilization (5% formaldehyde, 4% glacial acetic acid, 6% glycerol in distilled water), and percent fertilization was determined by light microscopy in order to validate the method. The preserved eggs showed a 100% rate of fertilization as judged by early embryo cleavage (32 or 64 cells), and this technique was employed to assess fertility in all subsequent experiments. Experiment 2: Direct Exposure From Early Fertilized Embryos Through to 320-d-Old Juveniles The second experiment was a 320-d laboratory exposure that investigated embryonic development, success, and timing of hatch, survival to swim-up, and growth and development of juvenile trout. Eggs were obtained in October 1998 from the Department of Conservation National Trout Hatchery fish trap on the Tongariro River (Turangi, New Zealand). Eggs were stripped and pooled from three ripe females and fertilized on-site with sperm pooled from two males. Eggs were left undisturbed for 15 min while water hardening and fertilization occurred in river water, then were transported on ice back to the laboratory (travel time of 1 h). Approximately 1500 eggs were immediately divided volumetrically among 5 replicates each (300 eggs per pot) of either treated TMP/BKME at 15% (v/v) or reference water. Effluent was obtained from the point immediately before discharge into the Tarawera River, and transported to the laboratory in Rotorua. Approximately 1000 L of 100% effluent was transported on a weekly basis. Dechlorinated Rotorua City tap water was used as reference and diluent water. Sodium thiosulfate (NaS2O3) at a concentration of 1 mg/L was used to remove chlorine from the tap water. The experimental setup consisted of polyvinyl chloride (PVC) pots (15 cm diameter) with a total volume of 2 L. Eggs were housed in fine mesh baskets (5 cm deep), held in each pot. Water flow-through (from bottom to top) was at a flow rate of 400 ml/min in each pot. Final treated effluent was continuously pumped via a peristaltic pump into the effluent exposure mixing tank and mixed with water controlled by a valve for a desired concentration of 15% (v/v).


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The incubation chambers were shielded from light with black plastic sheets for the first 20 d. The average temperature over the experimental period was 12.9°C and 12.8°C for reference and effluent treatments, respectively. Temperature, dissolved oxygen, conductivity, and flow rates were monitored daily, and a photoperiod was set at 12 h light: 12 h dark. Dead eggs were removed daily. Samples of eggs and sac fry were periodically collected for organic residue analysis and stored at −20°C. When trout reached swim-up they were transferred into larger rearing troughs with divided baskets (70 cm x 45 cm x 20 cm). Baskets were divided into two sections with mesh screens and were suspended in the rearing troughs (3 m x 0.5 m x 0.25 m) that had a drain at one end to allow continuation of flow-through exposure. The flow rate was increased to 600 ml/min. In addition, small pumps circulated water about the troughs in order to maintain consistent temperature within the system. Once the juveniles were 6 mo old, the baskets were removed and the 5 replicates per treatment were combined into one large group per treatment. Trout were fed commercial salmon food (Pacific start, 10% lipid, 53% crude protein, NRM Feed Mills, Hope-Nelson, NZ). The size of pellet was adjusted as the fish grew, ranging from well ground (crumbles) at fry size to 2.7-mm-diameter pellets. Trout were fed ad libitum daily. Following 320 d of exposure, fish were sacrificed and measurements of length, weight, and liver and spleen size were taken for calculations of somatic indices and condition factor. Gender was determined by visual examination since females showed the preliminary stages of ovary growth. Pooled bile samples were frozen in liquid nitrogen and stored at −80°C pending chemical analysis. Experiment 3: Maternal Exposure and Maintenance of Progeny Through to Swim-Up Fry Detailed descriptions of the exposure of adult trout to pulp and paper effluent are presented elsewhere (van den Heuvel et al., 2002; van den Heuvel & Ellis, 2002). The exposure systems consisted of 12,000 L flow-through fiberglass tanks that received either Tarawera River water or effluent diluted in Tarawera River water. The daily effluent concentrations as measured by conductivity were 10.2 ± 0.2% v/v in yr 1 and 11.6 ± 0.1% v/v in yr 20. The first experimental exposure was started on April 24, 1999, and the fish were sacrificed June 17, 1999. The second experimental exposures started on September 24, 1999, and March 25, 2000, and the trout were sacrificed by May 31, 2000. Thus, the first experiment consisted of 2 mo of effluent exposure and reference treatments. The second experiment consisted of 2 mo of effluent exposure, 8 mo of effluent exposure, and reference treatments. Shortly after tissue sampling of the majority of adults (van den Heuvel & Ellis, 2002), the remaining adult male and female trout were injected with Ovaprim (Syndel Laboratories, Vancouver, Canada) to synchronize gamete maturation. Eggs were stripped and fertilized 2 wk after ovaprim injection. During the 2-wk interval, trout were maintained in their respective effluent concentrations.

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During the first year, eggs from 10 reference and 10 effluent-exposed females were fertilized with pooled milt from at least 20 reference males. Upstream (reference) Tarawera River water was used for water hardening. Approximately 500 eggs per female were placed in each egg incubation chamber (one chamber per female). Each chamber received 400 ml/min of Tarawera River water. Dead eggs/sac fry were counted after 24 h every second day for the first 2 wk and then every day until the termination of the experiment at 44 d. The experiment was ended when all trout had reached swim-up. At the termination of the experiment, all remaining swim-up fry were counted. During the second year, eggs from 10 females from each treatment were again fertilized with pooled milt from at least 20 reference males. Exactly 30 g of unfertilized eggs was weighed out per female prior to fertilization (corresponding to about 300 eggs). After fertilization, eggs were placed in incubation chambers and the flow rate was maintained at 250 ml/min. Eggs were checked daily and dead eggs were removed. A subsample of 50 eggs per individual fish was removed after 24 h in order to test fertility (of the remaining live eggs). Eggs were preserved in Stockard’s solution and fertility was assessed by examination with light microscopy. The experiment was ended when all remaining fry had reached swim-up. At the termination of the experiment (38 d), all remaining swim-up fry were counted, and a subsample from each incubation chamber was weighed and measured for fork length. Measurement of Pulp and Paper Organics in Effluent Weekly effluent samples were collected during the nearly 2-yr period over which these experiments were conducted. Water samples were immediately filtered using 15-cm Whatman GF/C filters. Filtrate and filter papers were stored at −20°C. Aqueous effluent samples for determination of organic extractives were adjusted to pH 9, spiked with surrogate recovery standards, and extracted by continuous liquid–liquid extraction with methylene chloride. The solvent volume was reduced; samples were dried with anhydrous sodium sulfate and then derivatized (silylation) for analysis by gas chromatography/ mass-selective detection (GC-MSD). The filter papers were ground with sodium sulfate and placed in a Soxhlet extractor overnight with methylene chloride. Sample preparation and analysis were identical to those for the aqueous samples. All organics were corrected for surrogate standard recoveries (D10anthracene for resin acid neutrals, 8(14)-abietenic acid for resin acids, and dihydrocholesterol for sterols) and extraction blanks. Measurement of Pulp and Paper Organics in Tissue and Bile Approximately 1.5 g of ovarian tissue, eggs, or fry was ground with sodium sulfate, spiked with surrogate recovery standards, and extracted using supercritical carbon dioxide on an HP 7680T supercritical fluid extractor. Three 25-min extractions were performed with a supercritical fluid pressure of 227 bar, flow of 3 ml/min, CO2 density of 0.7 g/ml, and a thimble extraction temperature of 71°C. Extractives were removed using a C18 trap that was eluted using three


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rinses of methylene chloride. Samples were dried over sodium sulfate, concentrated, silylated, and analyzed using a GC-MSD with a SIM method. Samples were corrected for surrogate recoveries and blank determinations. Bile samples were hydrolyzed with ethanolic potassium hydroxide. Hydrolyzed bile was extracted at pH 9 with methyl t-butyl ether and the extract was dried with sodium sulfate. Extracts were derivatized and analyzed by GC-MSD as already described for effluent samples. Samples were corrected for surrogate recoveries and blank determinations. Statistical Analyses Comparisons of fork length and weight were made using a nested analysis of variance (ANOVA). In the case of the direct egg exposure, which was a balanced design, type I sum of squares (SS) were used. For eggs from the adult exposures, type III SS were used, as it was an unbalanced design. In the latter experiment, weight and length were only assessed in replicates containing more than three surviving individuals. Comparisons of liver, spleen, and body weight were analyzed by analysis of covariance (ANCOVA) with weight as the covariate for liver and spleen weights and length as the covariate for body weight. These results are presented as liver and spleen somatic indices (LSI, SSI) and condition factor, respectively. Data were log-transformed prior to ANCOVAs. Comparisons using data that did not conform to the assumptions of parametric statistics, such as rates of fertility, deformity, mortality to hatch, and mortality to swim-up, were analyzed using a Kruskal–Wallis nonparametric test. Deformity data was only assessed for replicates that had at least 25 surviving individuals. The critical level of statistical difference for all analyses was α = .05. RESULTS Effluent and Tissue Chemistry Effluent quality (Table 1) and chemistry (Table 2) reflect that this is a welltreated softwood effluent with ECF bleaching. As such, the effluent was repeatedly found nonlethal to rainbow trout at 100% concentration. As the treatment system is quite shallow (4 m), relatively high levels of suspended solids are

TABLE 1. Mean (SEM, n) Bulk Chemistry Parameters for 100% Treated Effluent Over the Period Encompassed by the Experiments Parameter

100% Effluent

pH Conductivity (µS/cm) Absorbable organic halide (AOX, mg/L) BOD (mg/L) TSS (mg/L) Rainbow trout 96-h LC50

7.4 (0.03, 277) 1056 (1.0, 5928) 1.2 (0.2, 10) 26.7 (0.5, 398) 38.2 (0.8, 397) >100% (—, 4)

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TABLE 2. Total Organics Concentrations in 100% Effluent (µg/L) over the 2-yr Period (n = 41) During Which These Experiments Were Performed and Bile Organics (µg/g Dry Weight) for Juvenile Trout Sacrificed After 320-d Exposure to 15% Effluent Bile organics Compound Resin acid neutrals Fichtelite Dehydroabietin Tetrahydroretene Retene Methyldehydroretene Total resin acid neutrals

Organics in 100% effluent

15% Effluent

Reference

15.0 0.6 9.3 7.7 0.5 33.0