|
Introduction
Exposures of fish to pulp and paper mill effluents have been associated with several alterations in reproductive indicators/biomarkers, including reductions in gonad size, delayed sexual maturation, and reduced production of sex steroids (Marshall et al. 1992, Munkittrick et al. 1994, McMaster et al. 1996, Sepúlveda et al. 1998 & 1999). However, understanding how these changes can lead to alterations in reproductive success of populations of free-ranging fish has received little attention.
Developing fish embryos and larvae are often considered the most sensitive stages in the life cycle of a teleost. The effects caused at these and other stages of development by contaminants may be very subtle and go unrecognized at the individual level, but can have detrimental effects at the population level (Westernhagen 1988).
Objectives
The primary objective of this study was to assess the potential effects of paper mill effluent exposure on reproductive success of largemouth bass.
Reproductive success endpoints measured included: fecundity, egg size, egg viability and hatchability, fry growth, and fry survival.
Materials and Methods
In Vivo Exposures
At the end of a 56-day in vivo exposure, 15 males and 20 females were collected from each of five treatment tanks (0, 10, 20, 40, and 80% paper mill effluent exposures) and transported to five 0.1 acre spawning ponds that had 20 spawning mats. Ponds were monitored daily for any signs of spawning activity. Immediately after the detection of spawning behavior (10 days after transfer to ponds males exhibited territorial behavior), mats were checked every other day for the presence of eggs. Half of the mats with eggs were collected and moved to the laboratory for controlled hatchability studies (see below), while the remaining mats were left in the ponds for future monitoring of fry numbers.
Indoor Hatchability Studies
In the laboratory, eggs were recovered from mats after immersion in a 1.5% sodium sulfite solution for 5 min. Eggs were then rinsed with tap water and counted volumetrically. Egg diameter was determined from each batch after measuring 30 eggs under a dissecting scope. Only viable eggs were left in fish hatching jars for a total of five days. Jars received well water at a flow rate of approximately 3 L/min. Throughout the study, dissolved oxygen, temperature, and pH averaged 7.7 mg/l, 21°C, and 7.6 respectively. Jars were treated daily with hydrogen peroxide to prevent fungal growth. At day five, fry were collected (approximately 3 days of age) from each jar and counted using an automatic fry counter. Hatchability was defined as the number of fry produced as a percentage of viable eggs.
Outdoor Fry Production Studies
Approximately half of the mats with eggs were left in the ponds to hatch under natural conditions. Fry were first seen schooling on top of the mats at about 7 days of age, but were not collected until they were at least 9 days of age. Since it was difficult to collect the whole school at once, nests were visited every other day for up to three times. The range of fry ages collected were 9 to 19 days. In the laboratory, smaller fry were counted using an automatic fry counter, whereas numbers of larger fry were estimated manually.
Fry Measurements
Complete batches of largemouth bass fry collected from both hatching jars and spawning mats were saved in 10% formalin for future measurements. From all batches, total length was measured in 30 fry/batch, while fry weights were estimated by weighing four groups of 25 fry each after tapping them in paper towel to remove excess water. Finally, the frequency of gross abnormalities to the head, vertebral column, and yolk sac were quantified by evaluating up to 500 fry/batch.
Statistical Analysis
Pairwise comparisons were conducted using one-way ANOVA followed by a Tukey's multiple comparison test to test whether treatment effluent concentration caused significant differences in any of the parameters measured. The frequency distributions of the different developmental abnormalities were compared between treatments using a X2 Test. Statistically significance was assessed at p 0.05.
Effluent Characteristics
The effluent tested in this study comes from a paper mill that has two bleached (40% product) and one unbleached line (60% product), which together release an estimated 36 million gallons of effluent/day. At the time this study was conducted, the bleaching sequences for the bleach line were CEHD and C90d10EopHDp. The bleaching lines manufacture paper towels and tissue paper, whereas the unbleached line produces mainly kraft bag and linerboard. The wood furnish of this mill consists typically of 50% softwood (slash, sand, loblolly) and 50% hardwood (gums, tupelo, magnolia, and water oaks) species. Effluents receive secondary treatment, which consist of both anaerobic followed by aerobic biological degradation after a retention period of 40 days.
Results
In this study, effluent exposure was verified by measuring resin acids (isopimaric, abietic, and dehydroabietic) in bile. Exposed bass had increased concentrations of resin acids in bile in comparison to controls (Figure 1).
In vivo exposure of female bass to  20% effluents resulted in declines of plasma concentrations of E2 and VTG (Figure 2). In males, abnormal patterns of 11-KT and E2 were also observed (Figure 2). These alterations, however, did not result in reductions for several egg parameters (fecundity, egg sizes, viability, and hatchability determined at 3 days post-hatch) (Figure 3) measured under indoor controlled conditions (hatching jars). Although fry lengths did not differ across treatments, fry weights and frequency of fry abnormalities were increased after exposures to high effluent concentrations ( 40%) (Figure 4).
Results from the outdoor pond study are presented in Figures 5 and 6. The number of fry produced per spawned female (determined at an average fry age of 14 days), was significantly reduced in ponds that were stocked with bass exposed to 10% effluent concentration (Figure 4). In contrast to what was observed in the indoor study, exposure of adult bass to paper mill effluents (> 20%) resulted in decreased fry weights and lengths, but did not increase the frequency of abnormalities (Figure 6).
|
U.S. Department of the Interior |
U.S. Geological Survey
-estradiol (E2), 11-ketotestosterone (11-KT), and vitellogenin (VTG). At the end of the 56-day period, bass were moved to hatchery ponds for an evaluation of spawning success. Spawning mats with eggs were either brought indoors for evaluation of fecundities, hatchabilities, and egg and fry size (measured at age 3 days), or left in ponds and fry number and size recorded (average age of 14 days). Effluent exposure was verified by measuring resin acids (isopimaric, abietic, and dehydroabietic) in bile. Exposed bass had increased concentrations of resin acids in bile in comparison to controls. In general, exposed females had decreased concentrations of E2 and VTG, whereas males had lower concentrations of 11-KT (
The reproductive changes observed in the laboratory exposure were not sufficient to interfere with reproductive performance (i.e. reproductive behavior, ovoposition, spermiation, fertilization, hatchability), at least under indoor controlled conditions.