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The authors examined the potential of pulp mill effluent from pulp-producing countries (Canada, Brazil, New Zealand) to affect fish reproduction. Specifically, the estrogenic effects in juvenile rainbow trout (Oncorhynchus mykiss) pulse-exposed to 11 different mill effluent extracts (intraperitoneal injections of solid-phase extraction-dichloromethane nonpolar fraction). The results indicated that effluent extracts were estrogenic in juvenile trout irrespective of the gender, as reflected by increasing level of plasma vitellogenin (VTG; Brazil > New Zealand > Canada). Despite the high variability observed among mills, differences in VTG levels were related to the type of mill process (kraft > elementary chlorine-free kraft > thermomechanical pulping). Moreover, effluent treatments did not appear to significantly decrease VTG induction. A consistent estrogenic effect was observed in those mills that process a combination of feedstocks (softwood and hardwood), with the highest increase in VTG related to eucalyptus feedstock. The results demonstrate significant estrogenic effects of pulp mill effluents on chronically exposed juvenile trout, suggesting that in vivo metabolic activation of precursors is necessary to cause the observed increases in VTG levels. This molecular estrogenic response provides a useful starting point for predicting population-level impacts through the adverse outcome pathway methodology. Environ Toxicol Chem 2017;36:1547-1555. © 2016 SETAC., (© 2016 SETAC.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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., ((c) 2010 Elsevier B.V. All rights reserved.)

We evaluated plasma testosterone (T) and 17beta-estradiol (E2) levels and ovarian aromatase CYP19a gene expression following a single intraperitoneal injection of Chilean pulp and paper mill effluent extracts into juvenile triploid rainbow trout. Fish injected with untreated effluent extracts had increased plasma T after 4 days, while plasma E2 concentration was increased in fish injected with both primary and secondary treated effluent extracts at the same sampling period. Ovarian CYP19a gene expression as measured by qRT-PCR was significantly induced in fish injected with the untreated, primary and secondary treated pulp and paper mill effluent extracts. Similar induction of CYP19a expression was found in fish injected with the androgens androstenedione (ADD) and T. A Principal Component Analysis (PCA) was conducted in order to identify structure in relationships between all measured variables and identifying which factors were most responsible for the variance observed within the plasma steroid levels, upregulation of ovarian CYP19a gene expression and the final estrogenic effect of increased plasma VTG levels. This analysis indicated a cluster correlation between plasma T levels and CYP19a gene expression (Factor 1, explaining 27.2% of total variance), a cluster including condition factor and liver somatic index (Factor 2, explaining 17.3%) and an additional cluster including plasma E2 and vitellogenin levels (Factor 3, explaining an additional 15.8%). The present results indicate that Chilean pulp and paper mill effluent extracts cause estrogenic effects in triploid rainbow trout. These effects could be related to the compounds present in the effluent that act as estrogen receptor agonists, or that induce changes leading to increased amounts of endogenous estrogens, reflected by increased E2 levels and induced aromatase expression/activity., (2010 Elsevier B.V. All rights reserved.)

The responses of cytochrome P4501A1 in the liver, the appearance of fluorescent metabolites in bile, and genotoxic damage in erythrocytes were studied in Oncorhynchus mykiss exposed under laboratory conditions to sediments taken from the Biobio River, central Chile. Samples were taken at four sampling sites in the Biobio River, following a pollution gradient from the discharge, where Stations 2 and 3 are impacted by a petrochemical industrial discharge effluent. Chemical analysis indicates polycyclic aromatic hydrocarbon (PAH) presence in a range of 2000 to 7000 ng g(-1) dry weight in sediments from Stations 2 and 3, respectively. Both the CYP1A1 activity and the bile metabolites of O. mykiss exposed to the PAH-contaminated sediments present statistically significant differences with respect to the other stations. Additionally, the comet assay revealed notable genotoxic damage in trout erythrocytes from Stations 2 and 3, an effect that was not observed at the other stations.

Caging experiments were conducted using hatchery-reared, immature, female rainbow trout (Oncorhynchus mykiss) in three previously defined areas of the Biobio River (south central Chile) representing a pollution gradient from the pulp and paper mill discharges area: a pre-impact area (upstream area, reference location), an impact area (area directly influenced), and a postimpact area (downstream area, less influenced). No significant changes were observed in the physiological index as represented by condition factor (K) and liver somatic index during different sampling times (after 11, 21, and 30 d of exposure). Ethoxyresorufin-O-deethylase activities were significantly higher in trout caged at the impact and postimpact discharges areas (two- to fourfold) compared with the reference (pre-impact) area, and a strong inhibition of acetylcholinesterase activity, reaching 50%, was observed mainly in fish caged at the impact area. A significant endocrine-disrupting effect (reproductive level) was evidenced by significant increments in gonad somatic index and plasma vitellogenin levels combined with an induction of gonad maturation (presence of vitellogenic oocytes) in trout caged at the impact and postimpact areas. These results, generated by an in situ approach, confirmed our group's findings for trout exposed to sediment in the laboratory: discharges of pulp mill effluent in the Biobio River are associated with the effects evaluated at different biological levels.

Four pulp and paper mills discharge their effluents in the same section of the Biobio River in central southern Chile. Pulp mill effluents are a very complex mixture with characteristics that depend on the type of raw material, the process technology, and the effluent treatment. To investigate the effect of pulp mill effluent discharges, immature Oncorhynchus mykiss were exposed to river sediments in the laboratory for 29 d. Three sampling areas were defined in a spatial gradient in the river: Preimpact, impact, and postimpact zones relative to the pulp and paper mill discharge areas. Ethoxyresorufin-O-deethylase activities were significantly higher in fish exposed to impact and postimpact sediments when compared to those exposed to preimpact sediments, and higher levels of vitellogenin were observed in the plasma of female fish exposed to impact and postimpact sediments. Histological analysis of the gonadal tissue showed an induction of gonadal maturation in fish exposed to sediment coming from the impact and postimpact zones (oocytes in a vitellogenic state). No site differences were observed in erythrocytes, although differences were noted in the leukocytes in the exposure areas. Finally, the biomarker approach showed evidence that the sediment associated with pulp mill effluent discharges produces some effects in fish under laboratory conditions.

Cytochrome P450-dependent monooxygenase (MFO) activity and levels of bile PAH metabolites were measured in fish (Oncorhynchus mykiss) exposed to Biobio river (Chile) water. Experiments were performed simultaneously in both the field and laboratory to investigate whether the river water contained notable bioavailable PAH fractions. The field experiment was conducted using fish caged for 41 days at La Mochita (Biobio river mouth) whereas the laboratory experiment involved a 21-day exposure treatment with water collected in the same area. Induction of hepatic MFO, assayed by benzo(a)pyrene monooxygenase activity (BaPMO), and levels of PAH metabolites in the bile were measured in groups of eight (field experiment) and four specimens (laboratory experiment) sampled after 5, 10, 21 and 41 days of exposure. BaPMO was induced by a factor of 23 and 25 in fish caged for 21 and 41 days, respectively. Likewise, a significant BaPMO induction was found in the fish group experimentally exposed to river water for 21 days. Biliary fluorescence measurements, recorded by fixed wavelength fluorescence (FF) and synchronous fluorescence spectroscopy (SFS), revealed significantly high levels of PAH metabolites in bile samples of fish caged for 21 and 41 days. A positive relationship was found between BaPMO activity and biliary PAH metabolites. This study provides evidences that (i) Biobio river water contains a significant bioavailable PAH fraction in terms of marked effects on fish and (ii) the biological indicators MFO activity and bile PAH metabolites represent an excellent screening methodology to assess PAH exposure.