Rodrigo Orrego

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.

Patterns of fish community composition along a river affected by agricultural and urban disturbance in south-central Chile

Patterns of fish community composition in a south-central Chile river were investigated along the altitudinal-spatial and environmental gradient and as a function of anthropogenic factors. The spatial pattern of fish communities in different biocoenotic zones of the Chillan River is influenced by both natural factors such a hydrologic features, habitat, and feeding types, and also by water quality variables which can reduce the diversity and abundance of sensitive species. A principal component analysis incorporating both water quality parameters and biomarker responses of representative fish species was used to evaluate the status of fish communities along the spatial gradient of the stream. The abundance and diversity of the fish community changed from a low in the upper reaches where the low pollution-tolerant species such as salmonid dominated, to a reduced diversity in the lower reaches of the river where tolerant browser species such as cypriniformes dominated. Even though the spatial pattern of fish community structure is similar to that found for the Chilean Rivers, the structure of these communities is highly influenced by human disturbance, particularly along the lower reaches of the river.

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.

Effects of pulp and paper mill effluent extractives on aromatase CYP19a gene expression and sex steroid levels in juvenile triploid rainbow trout

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.

Evaluating the potential of effluent extracts from pulp and paper mills in Canada, Brazil and New Zealand to affect fish reproduction: Estrogenic effects in fish

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.