Douglas Holdway

Pulp and paper mill effluent treatments have differential endocrine‐disrupting effects on rainbow trout

Endocrine disruption (ED) effects due to pulp and paper mill effluents extracts involving different industrial procedures and effluent treatments (nontreated, primary, and secondary treated) were evaluated using immature triploid rainbow trout in a pulse-exposure toxicity experiment. The protocol involved the use of intraperitoneal injection of mill extracts (solid-phase extraction [SPE]) corrected for individual fish weight and included several laboratory standards (steroidal hormones and phytosterols). Biological endpoints at two different levels of biological organization were analyzed (molecular and individual organism). Results indicated that nonsignificant changes were observed in the individual physiological indices represented by condition factor, liver somatic index, and gonad somatic index during the experiment. Significant induction of liver ethoxyresorufin-O-deethylase activity was observed between different effluent treatments and experimental controls. Significant endocrine-disrupting effects at the reproductive level were observed in all effluent treatments involving significant increments in plasma vitellogenin (VTG) levels. Fish exposed to untreated effluent extracts had significantly higher VTG levels compared to fish exposed to primary and secondary treatment effluent extracts, indicating a decrease of the estrogenic effect due to the effluent treatment. The present study has shown that for the Chilean pulp and paper mill SPE extracts evaluated, an endocrine disruption effect was induced in immature triploid rainbow, reaffirming the significant estrogenic effects demonstrated previously in laboratory and field experiments.

Estrogenic and anti-estrogenic effects of wood extractives present in pulp and paper mill effluents on rainbow trout

Wood extractives present in pulp and paper mill effluents may cause reproductive disturbances in fish. A chronic-exposure toxicity experiment using immature rainbow trout (Oncorhynchus mykiss) was conducted in order to assess the endocrine disrupting effects of two Chilean pulp and paper mill specific extracts (solid phase extraction, SPE) obtained from primary and secondary treated effluents. The (anti)estrogenic potencies and toxicity of the wood extractives regularly present in pulp mill effluent such as dehydroabietic acid (DHAA), beta-sitosterol (BS), and model estrogen 17beta-estradiol (E2) were evaluated by analysis of plasma vitellogenin (VTG) levels, gonadal somatic index (GSI) and liver ethoxyresorufin-O-deethylase (EROD) activity, respectively. The protocol involved the use of multiple intra-peritoneal injections (1 injection every 7 days for a total exposure period of 28 days). Analysis of variance/covariance, demonstrated no differences associated with fish gender other than GSI. The phytosterol BS, E2 and both pulp mill effluent extracts showed significant inductions of EROD and increased VTG levels after 4, 7, 14, 21 and 28 of exposure. While fish injected with secondary treated effluent extract showed a delayed induction in VTG levels compared to primary effluent injected fish, no effects on VTG and EROD levels were observed in DHAA injected fish. Moreover simultaneous injection of DHAA+E2 reduced the VTG levels found in E2 injected fish, indicating a potential indirect anti-estrogenic effect of this resin acid. The results of this study indicate that Chilean pulp and paper mill effluent extracts are estrogenic in rainbow trout males and females.

Pharmaceuticals and personal care products: A critical review of the impacts on fish reproduction

Abstract Research in environmental toxicology involving pharmaceuticals and personal care products (PPCPs) has increased greatly over the last 10–15 years. Much research has been focused on the endocrine-disrupting potential of PPCPs, as they relate to negative population impacts of aquatic organisms. This review assesses the current data on the reported effects of PPCPs on fish reproduction with an emphasis on fecundity, a predictor of population effects. Studies of both individual PPCPs and PPCP mixtures are presented. As the majority of individual PPCP studies reviewed demonstrate negative effects on fish fecundity, we relate these findings to detected surface water concentrations of these compounds. Very few studies involving PPCP mixtures have been conducted; however, the need for these types of studies is warranted as fish are most likely exposed to mixtures of PPCPs in the wild. In addition, laboratory and field assessments of wastewater treatment plant (WWTP) effluents, a major source of PPCPs, are reviewed. Much of the data provided from these assessments are variable and do not generally demonstrate negative impacts on reproduction, or the studies are unable to directly associate observed effects with WWTP effluents. Finally, future research considerations are outlined to provide an avenue into understanding how wild populations of fish are affected by PPCPs. These considerations are aimed at determining the adaptation potential of fish exposed to mixtures of PPCPs over multiple generations. As global use of PPCPs continually rises, the need to discern the effects of chronic exposure to PPCPs is greatly increased.

Multigeneration assessment of nonylphenol and endosulfan using a model Australian freshwater fish, Melanotaenia fluviatilis

Changes over two generations in offspring and reproductive ability were recorded in crimson‐spotted rainbowfish (Melanotaenia fluviatilis), a model Australasian freshwater fish, following a 24 h exposure to nominal nonylphenol concentrations of 50, 100, 500, 1000, 2250, and 5000 μg/L and following a 4 h exposure to nominal endosulfan concentrations of 1.0, 5.0, 10, 22, 33, and 50 μg/L. There were also four replicated control treatments: control 1 and 2 and solvent control 1 and 2, as well as “positive” female and male controls: 1 μg/L estradiol 1 and 2 and 1 μg/L testosterone 1 and 2. Results suggested that there may be some parental transfer of toxicants to embryos even over this short exposure period. Fertility of M. fluviatilis was reduced by a 24 h pulse exposure of adults to 100 μg/L nonylphenol and a 4 h exposure to 1.0 μg/L endosulfan. Hatch rates were significantly reduced after exposure to nonylphenol, endosulfan, and estradiol control but not in solvent controls and testosterone control. Significant correlations were found between reproductive and physiological parameters for nonylphenol and endosulfan exposed F0 adult rainbowfish. The major reproductive effects were on hatchability of the F1 generation and the gonadosomatic indices of male F1. The respective nominal NOEC and LOECs for nonylphenol were 50 and 100 μg/L, and for endosulfan were <1.0 and 1 μg/L. These observed effects have the potential to significantly impact exposed rainbowfish populations through the observed ∼45% reduction of hatchability and thus larval production.

Physiological changes in reproductively active rainbowfish (Melanotaenia fluviatilis) following exposure to naphthalene

Naphthalene makes up a substantial fraction of polycyclic aromatic hydrocarbons (PAHs) in crude oil and is an important by-product of industry; however, few studies have investigated the toxicity of naphthalene to aquatic organisms. We examined the toxicity of increasing concentrations (0, carrier control, 130, 200 and 400microg/l) of naphthalene to adult rainbowfish (Melanotaenia fluviatilis) for 3 and 14 days to determine its potential to act as an endocrine disruptor. After exposure for 3 days, no changes in sex steroids were measured. After 14 days, a decrease of serum estradiol in females and an increase in serum testosterone in males was observed. These results suggest that naphthalene has the potential to act as an endocrine disruptor, although since no changes in plasma vitellogenin concentrations were observed in either sex, it is unlikely that naphthalene is acting as a xenoestrogen. There was a positive correlation between the incidences of deformities in larval offspring with increasing naphthalene concentrations, suggesting parental transfer of the toxicant. Egg production, hatchability and larval lengths remained unaltered, whilst few changes were measured in gamma-glutamyltranspeptidase (GTP), an enzymatic indicator of spermatogenesis. Contrary to other PAHs, hepatic ethoxyresorufin-O-deethylase (EROD) activities declined with increasing exposure concentration, suggesting that naphthalene was either having a cytotoxic effect or disrupting enzyme synthesis.

Use of the Australian Crimson-Spotted Rainbowfish (Melanotaenia Fluviatilis) as a Model Test Species for Investigating the Effects of Endocrine Disruptors

Few studies have investigated the potential reproductive effects of toxicants on Australian freshwater fish species. The present study uses the Australian rainbowfish (Melanotaenia fluviatilis) as a model for testing the potential effects of 17beta-estradiol. Groups of reproductively active rainbowfish were exposed to waterborne 17beta-estradiol (control, carrier control, and 30, 100, 300, and 1,000 ng/L) for 3- and 14-d periods. Biomarkers of both low ecological relevance (plasma estradiol and testosterone, phosphoprotein, gamma-glutamyltranspeptidase [GTP]) and high ecological relevance (egg counts, hatchability, larval lengths, histopathology) were measured and the relationships between these examined. Relative to the controls, exposed female rainbowfish had a decline in plasma estradiol. An increase in phosphoprotein (vitellogenin) also was observed after exposure to 1,000 ng/L after 3 d, and histological examination of the gonads showed an increase in oocyte atresia at 1,000 ng/L on days 3 and 14. Changes in egg production were observed at 300 and 1,000 ng/L. No changes were detected to egg hatchability and larval lengths of offspring. Although there were no changes in male rainbowfish plasma testosterone or the histological organization of testes, levels of phosphoprotein increased and GTP activities were increased after 3 d of exposure at all test concentrations, but these declined at day 14. The present study demonstrated that, though measurements at the biochemical level were responsive to 17beta-estradiol exposure, fewer changes were observed in markers of higher ecological relevance at the exposures concentrations and durations tested.

The acute and chronic effects of endosulfan pulse-exposure on Jordanella floridae (Florida flagfish) over one complete life-cycle

Endosulfan is an organochlorine pesticide, which is used worldwide and has known toxic effects on non-target organisms including fish. This research investigated the acute and chronic effects of pulse-exposed endosulfan on Florida flagfish (Jordanella floridae). A 4-h pulse-exposure of endosulfan to larval flagfish caused a significant increase in mortality after 96 h at nominal concentrations equal to or greater than 100 μg/L. Some of the acute sub-lethal observations included hyperactivity, convulsions, and axis malformation. Seven-eight day old post-hatch flagfish were pulse-exposed for 4h to endosulfan and then monitored over one full life-cycle for chronic effects on growth, reproduction, and survivability. There were no growth or reproductive effects of endosulfan pulse-exposure up to the highest exposure concentration of 10.8 μg/L. Thus, the life-cycle 4-h pulse-exposure no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were 3.3 and 10.8 μg/L endosulfan, respectively, based on significantly higher larval and juvenile mortality.

Evaluating the Potential of Effluents and Wood Feedstocks from Pulp and Paper Mills in Brazil, Canada, and New Zealand to Affect Fish Reproduction: Chemical Profiling and In Vitro Assessments

This study investigates factors affecting reproduction in fish exposed to pulp and paper mill effluents by comparing effluents from countries with varying levels of documented effects. To explore the hypothesis of wood as a common source of endocrine disrupting compounds, feedstocks from each country were analyzed. Analyses included in vitro assays for androgenic activity (binding to goldfish testis androgen receptors), estrogenic activity (yeast estrogen screen), and neurotransmitter enzyme inhibition (monoamine oxidase and glutamic acid decarboxylase). Chemical analyses included conventional extractives, known androgens, and gas chromatograph index (GCI) profiles. All effluents and wood contained androgenic activity, particularly in nonpolar fractions, although known androgens were undetected. Effluents with low suspended solids, having undergone conventional biotreatment had lower androgenic activities. Estrogenic activity was only associated with Brazilian effluents and undetected in wood. All effluents and wood inhibited neurotransmitter enzymes, predominantly in polar fractions. Kraft elemental chlorine free mills were associated with the greatest neurotransmitter inhibition. Effluent and wood GCI profiles were correlated with androgenic activity and neurotransmitter enzyme inhibition. Differences in feedstock bioactivities were not reflected in effluents, implying mill factors mitigate bioactive wood components. No differences in bioactivities could be discerned on the basis of country of origin, thus we predict effluents in regions lacking monitoring would affect fish reproduction and therefore recommend implementing such programs.

Comparative embryotoxicity of pulp mill extracts in rainbow trout (Oncorhynchus mykiss), American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes)

This study evaluated the effects of Chilean pulp mill effluent extracts (untreated, primary and secondary treated pulp mill effluents), along with steroid standards (testosterone and 17β-estradiol) and a wood extractive standard (beta-sitosterol) on developing post-fertilized fish embryos. Our study included a cold freshwater species, rainbow trout (Oncorhynchus mykiss), and two warm freshwater species American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes). Embryotoxicity results included delay in time to hatch and decreased hatchability but no significant egg and larvae mortality was observed in the pulp mill extract exposed embryos. By contrast, significant early hatching and increased hatchability were observed in beta-sitosterol exposed embryos, along with high mortality of testosterone exposed embryos across species. Teratogenic responses were observed in medaka embryos in all treatments. Abnormalities were detected starting at development stages 19-20 (2-4 somite stages) and included optical deformities (micro-opthalmia, 1 or 2 eyes) and lack of development of brains and hearts. Additionally, phenotypic sex identification of surviving offspring found female-biased sex-ratios in all treatments except testosterone across species. Overall, our study indicated that Chilean pulp and paper mill extractives caused embryotoxicity (post-fertilized embryos) across species and irrespective of the effluent treatment. The effects were mainly associated with delayed time to hatch, decreased hatchability, and species-specific teratogenesis.

Effects of pulp and paper mill effluent extracts on liver anaerobic and aerobic metabolic enzymes in rainbow trout

This study investigates whether pulse exposure to Chilean pulp and paper mill effluent solid phase extracted (SPE) extracts via intraperitoneal injection (IP), would result in changes in the activities of the respiratory metabolic enzymes citrate synthase (CS) and lactate dehydrogenase (LDH) in rainbow trout livers. It also investigated if an alteration in liver metabolic capacity influenced the liver detoxification processes and estrogenic effects previously reported. Besides, a comparison of those enzymatic activities with fish IP injected with SPE extracts of two model effluents coming from industries that process 100% different type of feedstock (softwood, SW and hardwood, HW) was also evaluated. An initial induction of the anaerobic metabolism (increase in LDH enzymatic activity) was detected in all Chilean pulp mill effluent extracts evaluated, contrary to the initial unaltered aerobic metabolism (CS enzymatic activity) observed. A compensatory relationship in energy metabolism (Pasteur effect) was observed when comparing both enzymatic activities of fish exposed to those effluent extracts. LDH and CS activities observed in fish injected with Chilean extracts seem to be related to the effects observed in fish injected with SW extracts. This study showed that intraperitoneal injection of pulp and paper mill effluent extracts affected the anaerobic and aerobic metabolic capacities in rainbow trout livers, but this metabolic alteration did not affect detoxification capability or estrogenic effect previously reported.