Fish Bioassays Research Articles

Cell-specific biotransformation of benzophenone-2 and bisphenol-s in zebrafish and human in vitro models used for toxicity and estrogenicity screening.

Several human and fish bioassays have been designed to characterize the toxicity and the estrogenic activity of chemicals. However, their biotransformation capability (bioactivation/detoxification processes) is rarely reported, although this can influence the estrogenic potency of test compounds. The fate of two estrogenic chemicals, the UV filter benzophenone-2 (BP2) and the bisphenol A substitute bisphenol S (BPS) was deciphered in eight human and zebrafish in vitro cell models, encompassing hepatic and mammary cellular contexts. BP2 and BPS were metabolized into a variety of gluco- and sulfo-conjugated metabolites. Similar patterns of BP2 and BPS biotransformation were observed among zebrafish models (primary hepatocytes, ZFL and ZELH-zfER cell lines). Interestingly, metabolic patterns in zebrafish models and in the human hepatic cell line HepaRG shared many similarities, while biotransformation rates in cell lines widely used for estrogenicity testing (MELN and T47D-KBLuc) were quantitatively low and qualitatively different. This study provides new data on the comparative metabolism of BP2 and BPS in human and fish cellular models that will help characterize their metabolic capabilities, and underlines the relevance of using in vitro zebrafish-based bioassays when screening for endocrine disrupting chemicals.

Efficacy of phytoremediation technology in decontaminating the toxic effluent released during recovery of metals from polymetallic sea nodules

Efficacy of phytoremediation using two macrophytes Azolla pinnata and Lemna minor in decontaminating the toxic effluent released during recovery of metals from polymetallic sea nodules was analysed by applying fish bioassay. The economically important fish, L. rohita, was exposed to both, the Azolla-phytoremediated effluent (APE) and Lemna-phytoremediated effluent (LPE) for assessment of metal bioaccumulation (Fe, Mn, Zn, Cu, Pb, Cr and Cd) and alterations in biochemical (proteins, lipids, glycogen, cholesterol, AST (aspartate amino transferase), ALT (alanine amino transferase) and ALP (alkaline phosphatase) composition of various tissues. Accumulation of metals (e.g. Mn, Zn, Cu and Fe) decreased in most of the tissues exposed to both the phytoremediated effluents perhaps due to decontamination of metals by the two macrophytes. The significantly recovered concentrations of different biomolecules included glycogen, lipids, cholesterol and proteins. The activities of three marker enzymes (AST, ALT and ALP) in phytoremediated effluent-exposed fish also decreased due to lowering of the toxicity of the decontaminated effluents achieved by phytoremediation. The improvement in different biomolecules and reduction in metal concentration in the fish tissues were better in APE exposed fish. However, their concentrations in both the phytoremediated effluent-exposed fish failed to reach the levels of control fish. This study points towards the efficacy of phytoremediation in detoxification of metal-contaminated effluents often released following industrial activities.

Hematological changes and cytogenotoxicity in the tilapia Oreochromis niloticus caused by sub-chronic exposures to mercury and selenium.

Fish bioassays are valuable tools that can be used to elucidate the toxicological potential of numerous substances that are present in the aquatic environment. In this study, we assessed the antagonistic action of selenium (Se) against the toxicity of mercury (Hg) in fish (Oreochromis niloticus). Six experimental groups with six fish each were defined as follows: (1) control, (2) mercury (HgCl(2)), (3) sodium selenite (Na(2)Se(4)O(3)), (4) sodium selenate (Na(2)Se(6)O(4)), (5) mercury + sodium selenite (HgCl(2) + Na(2)Se(4)O(3)), and (6) mercury + sodium selenate (HgCl(2) + Na(2)Se(6)O(4)). Hematological parameters [red blood cells (RBC), white blood cells (WBC), and erythroblasts (ERB)] in combination with cytogenotoxicity biomarkers [nuclear abnormalities (NAs) and micronuclei (MN)] were examined after three, seven, ten, and fourteen days. After 7 days of exposure, cytogenotoxic effects and increased erythroblasts caused by mercury, leukocytosis triggered by mercury + sodium selenite, leukopenia associated with sodium selenate, and anemia triggered by mercury + sodium selenate were observed. Positive correlations that were independent of time were observed between WBC and RBC, ERB and MN, and NA and MN. The results suggest that short-term exposure to chemical contaminants elicited changes in blood parameters and produced cytogenotoxic effects. Moreover, NAs are the primary manifestations of MN formation and should be included in a class characterized as NA only. Lastly, the staining techniques used can be applied to both hematological characterization and the measurement of cytogenotoxicity biomarkers.

Comparison and validation of fish bioassays for effluent toxicity testing.

Comparison and validation of fish bioassays for effluent toxicity testing.

Effects of 17α-ethinylestradiol (EE2) on reproductive endocrine status in mummichog (Fundulus heteroclitus) under differing salinity and temperature conditions

Exposure to 17α-ethinylestradiol (EE2), a synthetic estrogen, has previously been shown to decrease reproductive endocrine status and egg production in northern mummichog (Fundulus heteroclitus macrolepidotus). The objective of this study was to evaluate if variations in salinity or temperature conditions of EE2-exposed mummichog modify the effect on whole organism reproductive endocrine status and gonadal steroidogenesis. Mummichog were exposed in vivo for 14 days to 0, 50 and 250ng/L EE2 in 0, 16 and 32ppt salinity at 18°C and to 0 and 250ng/L EE2 at 10, 18 and 26°C at 16ppt. There was a little overall effect of salinity on measured endpoints. In the salinity exposure, 250ng/L EE2-exposed females had significantly reduced 17β-estradiol (E2) levels. Increased temperature triggered gonadal growth in both sexes and increased plasma E2 and E2 production and decreased 11-KT (11-ketotestosterone) production. EE2 counteracted the effect of temperature by depressing gonadal growth in males. In both exposures, EE2 effects on testosterone (T) production were variable. The use of steroidogenic precursors (25-OH-cholesterol, and/or pregnenolone and/or testosterone) in the in vitro gonadal incubations indicated decreased E2 production in females and 11-KT production in males were predominately due to suppression of the terminal conversion step between T and E2 or 11-KT. Ovarian aromatase A (cyp19a) gene expression at 16ppt and 18°C was not affected by 250ng/L EE2 (the only treatment combinations tested). Overall, temperature is a factor regulating northern mummichog reproduction; EE2 overrides its effects and disrupts the terminal step of steroidogenesis. Our results should be considered in designing future estuarine fish bioassays and in understanding effects of estrogenic endocrine disruptors in estuaries.

Assessment of toxicity of two nitroaromatic compounds in the freshwater fish Cyprinus carpio

This study was conducted to evaluate the toxicological response of p-nitrotoluene and p-nitroaniline to the key fish species, Cyprinus carpio. A freshwater fish bioassay based on the 96 h LC 50 was used to estimate the single and joint toxicity of the two chemicals. The toxicity of p-nitrotoluene was greater than that of p-nitroaniline based on 96 h LC 50 values of 40.74 mg·L−1 and 48.99 mg·L−1, respectively. Both compounds had moderate toxicity toward Cyprinus carpio, and this toxicity increased with the exposure duration and concentration. Binary mixtures of the compounds were more toxic than the individual compounds at 96 h, and they acted upon partial addition. When the exposure time was longer, the toxicity increased for mixtures of compounds with the same concentration or toxicity. The results of this study suggest that exposure to a combination of these chemicals would result in a higher environmental risk in aquatic systems than exposure to either compound alone. Further research is needed to investigate the combined effects and sublethal toxicity of p-nitrotoluene and p-nitroaniline, since they are both still used in China.

REPORT OF A BLOOM-FORMING DINOFLAGELLATE TAKAYAMAACROTROCHA FROM TROPICAL COASTAL WATERS OF SINGAPORE(1).

Four clonal cultures of the unarmored dinoflagellate Takayama acrotrocha (J. Larsen) de Salas, Bolch et Hallegraeff were established from Singapore coastal water on October 20, 2004, and January 1, 2007, for a HAB monitoring project. LM and SEM observations demonstrated that the isolates were not consistent with the other five species within this genus in position of nucleus, shape of the apical groove, and number and shape of chloroplasts. New morphological observations of the Singapore isolates that were not in the type description of T. acrotrocha include a narrow and shallow slit located above the entire anterior edge of the cingulum, a tube-like structure in the sulcus, numerous multilateral plate-like surface vesicles, a sulcal intrusion into the epicone, and possibly a peduncle in between the two emerging points of flagella. The presence of sulcal intrusion into the epicone was not consistent with the type description but is prominent in SEM micrographs. Phylogenetic analysis of the partial LSU rDNA sequences indicated Singapore strains of T. acrotrocha are conspecific with two isolates from Italy, but less homologous to T. helix, T. tasmanica, and T. tuberculata. Laboratory fish bioassays using Asian sea bass (Lates calcarifer) and sheepshead minnows (Cyprinodon variegates) did not indicate fish-killing activity by this species, and to our knowledge, there were no reports of fish-kills occurring during blooms of this species in Singapore and Italy. This is the first report of T. acrotrocha from tropical waters and indicates a likely cosmopolitan distribution of the species.

Toxicity of cigarette butts, and their chemical components, to marine and freshwater fish

BackgroundCigarette butts are the most common form of litter, as an estimated 4.5 trillion cigarette butts are thrown away every year worldwide. Many chemical products are used during the course...

The versatile, changing, and advancing roles of fish in sediment toxicity assessment—a review

Sediments serve as integral and dynamic parts of our aquatic systems. Within the last 15 to 20 years, however, the scientific community has begun noticing deterioration of sediment quality at an alarming rate worldwide. Sediments are now harboring hazardous pollutants that can directly influence water quality, thereby creating very stressful conditions for aquatic life. As a consequence, global efforts were initiated in the early 1970s, to find ways to assess sediment quality. Because of their obvious ecological and economic significance, fish have remained a major taxonomic group for appraising the general quality of aquatic systems. However, for sediment risk assessment, fish have lagged behind invertebrates due to their mobility and generally, pelagic lifestyle. To our knowledge, this is the first paper that comprehensively presents and reviews the versatile role of fish in assessing the state of health of aquatic sediments. Through a literature search of the more relevant and/or more recent studies, this review attempted to trace the development of the various approaches as well as to describe the future prospects of using fish as sentinels for sediment quality assessment. Initially, the use of whole fish (juveniles or adults) bioassays contributed immensely to our understanding of sediment contamination and ecotoxicology. But due to economic as well as ethical issues linked to the use of live vertebrates for toxicity testing, the approach has shifted to using fish cell cultures and fish embryos. Much newer approaches involving receptors and gene arrays in fish cells to elucidate the mode of action of sediment-borne contaminants are very promising. The review paper also lists and explores some of the issues associated with the use of juvenile or adult fish, fish cell cultures, fish embryos, and fish gene expression profiles in sediment toxicity evaluations to stimulate further discussions, and hopefully, to serve as benchmark for future handling of similar or related aspects of fish utilization in sediment risk assessment. Overall, the present review has comprehensively explored the changing and progressing roles of fish in sediment toxicity evaluation. Indeed the usefulness of this taxon as test organisms has provided a significant contribution to the advancement of sediment toxicology. However, despite the quite optimistic and bright future for these current procedures, a number of issues and problems remain. Therefore, efforts to develop new technologies and to refine current methods and approaches continue to challenge many laboratories worldwide.

Evaluation of Short-Term Fish Reproductive Bioassays for Predicting Effects of a Canadian Bleached Kraft Mill Effluent

Abstract Under the Canadian Environmental Effects Monitoring (EEM) program for pulp and paper effluents, the observation of a national response pattern of decreased gonad size and increased fish condition and liver size has triggered a centralized multiagency investigation of cause (IOC) of reproductive impacts in fishes. The purpose of the component of the IOC study presented here is to compare a number of fish bioassays for determining reproductive and reproductive-endocrine effects of a bleached kraft mill effluent. The bleached kraft mill chosen for this study had demonstrated the national response pattern in previous EEM cycles. The bioassays employed to examine reproduction were fathead minnow (Pimephales promelas) 5- and 21-d, mummichog (Fundulus heteroclitus) 25-d, and zebrafish (Danio rerio) 7-d tests, all of which had egg production as the primary reproductive endpoint. Additional bioassays examining reproductive-endocrine endpoints included a 7-d mummichog test, a 7- and a 21-d threespine stickleback (Gasterosteus aculeatus) test, a rainbow trout (Oncorhynchus mykiss) 7-d test, and in vitro sex steroid receptor and plasma protein binding bioassays. The zebrafish and fathead minnow reproductive tests showed significant suppression of egg production at the 100% effluent concentration. Endocrine data derived from the tests showed that this effluent did not impact steroidogenic endpoints at any concentration. Bioassays showed that this effluent i) was capable of eliciting cytochrome P4501A induction at as low as 10% vol/vol effluent, ii) was weakly androgenic at 10% vol/vol, and iii) showed no evidence of in vivo estrogenicity. These results were consistent with in vitro receptor binding assays showing a highly variable level of androgenic equivalents over six months of effluent testing, with little evidence of estrogenic activity. Bioassay results were consistent in that the overall conclusion was that this effluent has only a weak potential to cause reproductive impairment and would likely not do so at environmentally relevant concentrations. Field studies and a fathead minnow lifecycle study conducted concurrently were in agreement with reproductive bioassay results as white sucker exposed in the receiving environment no longer had significantly reduced gonadal development. Overall, this study provided evidence that the laboratory assays evaluated for various reproductive endpoints have potential application for future IOC work.

Unconstrained and noninvasive measurement of bioelectric signals from small fish

Recently, the technique of fish bioassay has attracted attention as a method for constant monitoring of aquatic contamination. The respiratory rhythms of fish are considered to be an efficient indicator for the monitoring of water quality, since they are sensitive to chemicals and can be measured indirectly from the bioelectric signals generated by their breathing. However, no method has yet been established to measure signals in small free-swimming fish. In this article, we propose a system to measure bioelectric signals in small fish and monitor the frequency component in real time. To cover the large measurement range required in a free-swimming environment, the signals are measured using multiple electrodes. Further, the system focuses on the frequency component of the signal to assess the condition of the fish using frequency analysis and a band-pass filter. Experiments were conducted with the purpose of enabling remote sensing and environment estimation. First, it was verified that the measured signals were synchronized with the breathing of the fish. Then, a remote sensing experiment was performed using medaka (Oryzias latipes) that were allowed to swim freely in a measurement aquarium. The results confirmed that bioelectric signals which were synchronized with breathing could be measured in unconstrained and noninvasive conditions.

The influence of modes of action and physicochemical properties of chemicals on the correlation between in vitro and acute fish toxicity data

New EU legislation is providing an impetus for research aimed at replacing acute fish toxicity testing with in vitro alternatives. In line with such research, the objective of this study was to determine what factors influence the correlation between in vitro and fish toxicity data. Basal cytotoxicity (IC 50) and acute toxicity data from fathead minnow (LC 50) of 82 industrial organic chemicals were obtained from the Halle Registry of Cytotoxicity and the US EPA Fathead Minnow Database. A good correlation between IC 50 with LC 50 data was found ( r 0.84). Yet, IC 50 data were less sensitive than LC 50 data by an order of magnitude. Using multiple regression analysis, the octanol–water partition coefficient ( K OW ) and the Henry’s Law Constant ( H) were found to significantly explain the low absolute sensitivity. The mode of action (MOA) of the chemical was found to significantly explain the general variation in the log IC 50/log LC 50 regression line. These results support the notion that (a) the bioavailability of hydrophobic (high K OW ) and volatile (high H) chemicals is significantly lower in in vitro assays than in the fish bioassay and (b) multiple cell types and endpoints should be included to mimic the modes of action possible in the whole organism.

Identification of euglenophycin – A toxin found in certain euglenoids

Currently cyanoprokaryotic algae, diatoms, haptophytes, dinoflagellates, euglenoids, and rhaphidophytes are known to produce algal toxins. A previous study by the authors reported euglenoid algae producing toxin(s) in aquaculture ponds, with confirmation based on positive fish bioassays following exposure to the isolated clonal algal cultures. Toxicity was observed in euglenoid culture isolates obtained from the pond as well as a clonal, culture collection taxon. Here we provide conclusive evidence for euglenoid toxin production, including HPLC/MS, MS/MS, and NMR analyses of a clonal (non-axenic) isolate of Euglena sanguinea grown in batch culture. Following wet chemical serial fractionation, toxic activity was identified in both the methanol and hexane extracts. These extracts were then purified using HPLC. Bioassay-guided HPLC fractionation of these two extracts demonstrated that a single class of toxic compounds, identical in mass and similar in molecular structure, was produced by this organism. The toxic compounds exhibited a maximal UV absorbance at 238 nm and gave diagnostic mass peaks at 306 (MH +) and 288 (MH +–H 2O). Unambiguous molecular structural determination was carried out by high field NMR analysis operating in 1- and 2-dimensions. Though a predominant isomer represented the bulk of the toxin, several stereo- and structural isomers were evidenced by NMR, and HPLC/MS. This compound is an alkaloid similar in structure to fire ant venom. The compound exhibits ichthyotoxic, herbicidal and anticancer activity at low ppm to ppb dosages.

Exploring Fish Bioassay of Textile Dye Wastewaters and Their Selected Constituents in Terms of Mortality and Erythrocyte Disorders

Acute (4 day) and short-term (7 day) toxicity studies (at 1/5th and 1/10th of LC(50)) of textile dye wastewaters and their selected ingredients (azo dye methyl red and heavy metals Cd, Cu, Ni and Zn) were made on a freshwater fish Gambusia affinis under laboratory conditions. LC(50) value was found to be the lowest in four cases, and the EC(50) value for reduction in erythrocyte counts in the remaining four tests. Thus, the reduction in erythrocyte counts to the 50% level was similar in sensitivity to fish mortality. The short-term toxicity studies revealed significant disorders in erythrocyte morphology (poikilocytosis) and its counts to be the better indices for toxicity monitoring in the absence of fish mortality.

Morphological Variation and Phylogenetic Analysis of the Dinoflagellate Gymnodinium aureolum from a Tributary of Chesapeake Bay

Cultures of four strains of the dinoflagellate Gymnodinium aureolum (Hulburt) G. Hansen were established from the Elizabeth River, a tidal tributary of the Chesapeake Bay, USA. Light microscopy, scanning electron microscopy, nuclear-encoded large sub-unit rDNA sequencing, and culturing observations were conducted to further characterize this species. Observations of morphology included: a multiple structured apical groove; a peduncle located between the emerging points of the two flagella; pentagonal and hexagonal vesicles on the amphiesma; production and germination of resting cysts; variation in the location of the nucleus within the center of the cell; a longitudinal ventral concavity; and considerable variation in cell width/length and overall cell size. A fish bioassay using juvenile sheepshead minnows detected no ichthyotoxicity from any of the strains over a 48-h period. Molecular analysis confirmed the dinoflagellate was conspecific with G. aureolum strains from around the world, and formed a cluster along with several other Gymnodinium species. Morphological evidence suggests that further research is necessary to examine the relationship between G. aureolum and a possibly closely related species Gymnodinium maguelonnense.

Characterization of a novel ψ-conotoxin from Conus parius Reeve

The M-superfamily of conotoxins currently comprises three major groups of peptides (the μ-, κΜ-, and ψ-families) that share a key structural characteristic, the six-cysteine motif CC–C–C–CC, but differ with respect to their molecular targets. The ψ-family consists of M-superfamily conotoxins that are nicotinic acetylcholine receptor (nAChR) antagonists. To date, only two ψ-conotoxins, PIIIE and PIIIF, are known, both of which were isolated from a single Conus species, Conus purpurascens. In this paper, we report the discovery and initial characterization of a ψ-conotoxin from another Conus species, Conus parius, which we designated as PrIIIE. Its amino acid sequence, inferred from a cloned cDNA, differed significantly from those of PIIIE and PIIIF. Its bioactivity was investigated by using the synthetic form of the peptide in mice and fish bioassays. At 2.5 nmol, the synthetic peptide induced flaccid paralysis in goldfish in ca. 4 min but did not induce any remarkable behavior in mice (after i.c. and i.p. injection of up to 10 nmol of peptide) and did not block action potential in directly stimulated frog muscle preparations. Electrophysiological experiments carried out to measure inhibition of ion currents through mouse nAChR receptors expressed in oocytes revealed that PrIIIE (IC 50∼250 nM) was significantly more potent than PIIIE (IC 50∼7000 nM) and that PrIIIE showed higher inhibition potency against the adult-type than the fetal-type nAChR. In similar electrophysiological assays, PrIIIE showed no inhibitory effects against the mouse muscle subtype Na + channel isoform Na v 1.4. The discovery of this ψ-conotoxin from a Conus species that belongs to the subgenus Phasmoconus, which is distinct from and larger than the clade in which C. purpurascens belongs, suggests that greater structural and functional diversity of ψ-conotoxins remains to be discovered from the members of this subgenus.

A comparative study on characterization of textile wastewaters (untreated and treated) toxicity by chemical and biological tests

Toxicity of textile wastewaters (untreated and treated) and their ingredient chemicals was quantified in terms of their chemical characteristics, fish ( Gambusia affinis) mortality and end point growth responses of duckweed ( Lemna aequinoctialis) in short-term bioassays. Other parameters of fish bioassay were erythrocyte morphology and its counts. Despite of a definite correlation between data of biological tests (LC/EC 50 values) with that of chemical tests, biological tests were found to be relatively more sensitive to both wastewaters and ingredient chemicals. Amongst all the examined parameters of test organisms, fish RBCs (morphology and counts) sensitivity to pollutants in the wastewaters was usually maximum and therefore, their study should be included in the routine fish bioassay. Other advantage of biological test such as on Lemna is even detection of eutrophic potential of wastewaters, as noted at their higher dilutions. The ingredient chemicals (major) contributing maximum toxicity to textile dye wastewater were, acids (HCl and H 2SO 4), alkali (Na 2O SiO 2), salt (NaNO 2) and heavy metal (Cu), whereas dyes (4) were relatively less toxic.

Dioxins, metals, and fish toxicity in ash residue from space heaters burning used motor oil

Ash residue, generated from burning used motor oil, is a complex and ubiquitous waste stream. Ash samples were collected from space heaters and analyzed for dioxins (N=10), expressed as toxic equivalents (TEQ), and heavy metals (N=9). TEQ averaged 148-164 ng kg(-1) (standard deviation [SD] 385-416 ng kg(-1)), depending on methods used for non-detects (NDs) and toxic equivalency factors (TEFs). It is notable that median TEQ (2.89-3.49 ng kg(-1)) was about 50 fold lower, reflecting the influence of several high end values on the mean. The proportion of NDs among 17 polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in each sample averaged 38.2% (range 0-94.1%). Total metals averaged 103,000 mg kg(-1) (SD 26,600 mg kg(-1)), with Zn, Cu, and Pb contributing 89.3%, 6.4%, and 3.0% of the total, respectively. Rainbow trout bioassays resulted in median mortalities of 3.2% and 42.0% (respective SD 25.3% and 43.2%) at ash concentrations of 10 and 100 mg l(-1), respectively. Nominal concentrations of several metals (e.g., Cu, Zn) in the fish bioassay exceeded their reported median lethal concentrations (LC50s) for the test species. Multiple regressions (Bonferroni P<0.05) demonstrated that most of the variance in fish mortality could be accounted for by pH, metals (e.g., As, Cd, Pb), and TEQ. Mean TEQ concentration in used oil ash ranked on the high end of TEQ content in other environmental matrices, including wood ash, cement kiln dust, biosolids, and soils. Overall, these results suggest that suitable disposal methods are needed for ash generated from burning used motor oil.

A Fish Bioassay to Evaluate the Toxicity Associated with the Ingestion of Benzo[a]pyrene-Contaminated Benthic Prey

A Fish Bioassay to Evaluate the Toxicity Associated with the Ingestion of Benzo[a]pyrene-Contaminated Benthic Prey

A Decade of Research on the Environmental Impacts of Pulp and Paper Mill Effluents in Canada: Development and Application of Fish Bioassays

Laboratory tests have been used to assess the regulatory and research questions related to the effects of pulp mill effluents on aquatic biota. Acute, short-term laboratory tests have clearly shown the improvement in final effluent quality following installation of secondary treatment at Canadian pulp mills. In an effort to predict and investigate impacts on wild fish, laboratory bioassays were developed to examine sublethal endpoints: induction of hepatic mixed function oxygenase activity and reduction of sex steroid concentrations. These laboratory assays have been used to assess whole effluents, specific chemicals, and components of pulp mill processes, and to discriminate between historical and present-day effluent discharges. These tests have shown that induction of mixed-function oxygenase activity and reduction of sex steroid concentrations are produced by effluents from a variety of mill types, with and without chlorine bleaching, in hardwood and softwood pulping facilities, and before and after effluent treatment. These short-term bioassays have enabled reductions in sex steroid concentrations to be linked to mill process streams, and have provided information on effective waste stream treatment. Longer term, life-cycle fish bioassays have shown that chronic exposure to pulp mill effluents commonly results in growth enhancement, liver enlargement, and decreases in gonad size, secondary sex characteristics, and fecundity. These long-term laboratory exposures are able to mimic the most commonly observed alterations of wild fish exposed to pulp mill effluents: increases in condition factors, increases in liver-somatic indices, and decreases in gonadosomatic indices. This pattern of response is a combination of nutrient enrichment with metabolic disruption. The most sensitive and biologically meaningful endpoint is decreased reproduction in fish life-cycle exposures. As the laboratory tests move forward into the next decade, attention will focus on the reproductive endpoints and on the possibility of shortening the fish bioassays while still maintaining sensitivity and biological relevance.