Influence of solids and hydraulic retention times on microbial diversity and removal of estrogens and nonylphenols in a pilot-scale activated sludge plant.

The removal of EDCs in activated sludge processes can be enhanced by increasing solid and hydraulic retention times (SRT and HRT); it has been suggested that the improvement in removal is due to changes in microbial community structure (MCS). Though the influence of SRT and HRT on chemical removal and MCS has been studied in isolation, their synergistic impact on MCS and the removal of estrogens and nonylphenols in activated sludge remains unknown. Hence, we investigated how both parameters influence MCS in activated sludge processes and their ulterior effect on EDC removal. In our study, an activated sludge pilot-plant was fed with domestic sewage fortified with 100 and 1000 ng/L nonylphenols or 2 and 15 ng/L estrogens and operated at 3, 10 and 27 d SRT (constant HRT) and at 8, 16 and 24 h HRT (constant SRT). The MCS was assessed by phospholipid fatty acids (PLFA) analysis, and the archaeal and bacterial diversities were determined by 16S rRNA analysis. From the PLFA, the microbial abundance ranked as follows: Gram-negative > fungi > Gram-positive > actinomycetes whilst 16S rRNA analysis revealed Proteobacteria > Bacteroidetes > Others. Both PLFA and 16S rRNA analysis detected changes in MCS as SRT and HRT were increased. An SRT increment from 3 to 10 d resulted in higher estrone (E1) removal from 19 to 93% and nonylphenol-4-exthoxylate (NP4EO) from 44 to 73%. These findings demonstrate that EDC-removal in activated sludge plants can be optimised where longer SRT (>10 d) and HRT (>8 h) are suitable. We have also demonstrated that PLFA can be used for routine monitoring of changes in MCS in activated sludge plants.

A Comparison of Different Approaches for Characterizing Microplastics in Selected Personal Care Products.

Any uncertainty in determining numbers of microplastics in the environment may be a barrier to assessing their impact and may stem from various aspects of methodologies used to quantify them. We undertook a comparison of approaches to quantify and characterize microplastics in 4 personal care products. The aim was not only to determine how many particles were present but to assess any differences due to the methods used. Counting of extracted microplastics was undertaken using particle size analysis, light microscopy, and imaging flow cytometry. Micro-Fourier transform infrared spectroscopy (µ-FTIR) was used to characterize the particles in each product. The mean size distribution of microplastics differed depending on the method employed, and it was apparent that imaging flow cytometry was affected by high background noise that may require staining of plastics to overcome. The application of µ-FTIR confirmed polyethylene as the microplastic in each product. Methodological challenges encountered in the study and the literature have highlighted the need for standardization of methods for determining microplastics. Environ Toxicol Chem 2022;41:880-887. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Uptake, Elimination and Effects of Cosmetic Microbeads on the Freshwater Gastropod Biomphalaria glabrata.

The presence of plastic cosmetic microbeads in the environment due to their extensive use in society and inevitable dispersal into wastewater is concerning. Therefore, it is vital to understand the processes of microplastic uptake and elimination by aquatic organisms, and to further assess their potential to cause harmful effects and wider impacts. We therefore investigated the short-term (48-h) and long-term (21-d) uptake, elimination, and effects of exposure to polyethylene microbeads (a mixture of fragments and spheres extracted from commercially available facial scrubs) on the freshwater snail, Biomphalaria glabrata. We found fast uptake in the short-term (75 µg/g/h) and the long-term (6.94 µg/g/h) in B. glabrata exposed to 800 particles/200-mL and 80 particles/200-mL, respectively. Irregular fragments were more easily ingested and egested compared to spheres (ANOVA, p < 0.05) in both 48-h and 21-d exposures. The mean size of the fragments in B. glabrata tissues (413 ± 16 µm) after 48-h exposure was significantly larger than that of the standard sample (369 ± 26 µm) (ANOVA, F3,20 = 3.339, p = 0.033), suggesting that aggregation in the gut may occur. Floating feces containing microbeads were observed in the long-term exposure, which could alter the fate, behavior, and bioavailability of egested microbeads. No significant effects on survival and growth were shown within 48-h or 21-d exposure periods. Thus, further studies on the specific features of microplastics (e.g., their shape and size) influencing uptake and elimination, as well as toxic molecular mechanisms, should be explored in future ecotoxicological studies.

From dishwasher to tap? Xenobiotic substances benzotriazole and tolyltriazole in the environment.

There is increasing evidence that the use of chemicals frequently results in widespread environmental contamination with little understanding of the toxicological implications. Benzotriazoles are used in, among other applications, dishwashing formulations for home use, and are a class of chemicals recently reported to be present in European waters. This study demonstrates their presence in UK wastewaters, rivers, and drinking water. It also estimates that their use as silver polishing agents in dishwasher tablets and powders may account for a significant proportion of inputs to wastewaters. The lack of a complete set of good quality (eco)toxicological data on possible chronic effects of these high use chemicals should caution against using them in a manner which may have contributed to such widespread environmental contamination.

Fate of alkylphenolic compounds during activated sludge treatment: impact of loading and organic composition.

The impact of loading and organic composition on the fate of alkylphenolic compounds in the activated sludge plant (ASP) has been studied. Three ASP designs comprising carbonaceous, carbonaceous/nitrification, and carbonaceous/nitrification/denitrification treatment were examined to demonstrate the impact of increasing levels of process complexity and to incorporate a spectrum of loading conditions. Based on mass balance, overall biodegradation efficiencies for nonylphenol ethoxylates (NPEOs), short chain carboxylates (NP(1-3)EC) and nonylphenol (NP) were 37%, 59%, and 27% for the carbonaceous, carbonaceous/nitrification, and carbonaceous/nitrification/denitrification ASP, respectively. The presence of a rich community of ammonia oxidizing bacteria does not necessarily facilitate effective alkylphenolic compound degradation. However, a clear correlation between alkylphenolic compound loading and long chain ethoxylate compound biodegradation was determined at the three ASPs, indicating that at higher initial alkylphenolic compound concentrations (or load), greater ethoxylate biotransformation can occur. In addition, the impact of settled sewage organic composition on alkylphenolic compound removal was evaluated. A correlation between the ratio of chemical oxygen demand (COD) to alkylphenolic compound concentration and biomass activity was determined, demonstrating the inhibiting effect of bulk organic matter on alkylphenol polyethoxylate transformation activity. At all three ASPs the biodegradation pathway proposed involves the preferential biodegradation of the amphiphilic ethoxylated compounds, after which the preferential attack of the lipophilic akylphenol moiety occurs. The extent of ethoxylate biodegradation is driven by the initial alkylphenolic compound concentration and the proportion of COD constituted by the alkylphenol polyethoxylates (APEOs) and their metabolites relative to the bulk organic concentration of the sewage composed of proteins, acids, fats, and polysaccharides. Secondary effluents from this study are characterized by low bulk organic concentrations and comparatively high micropollutant concentrations. Based on the biodegradation mechanism proposed in this study, application of high rate tertiary biological treatment processes to secondary effluents characterized by low bulk organic concentrations and comparatively high APEO concentrations is predicted to provide a sustainable solution to micropollutant removal.

The fate of steroid estrogens: partitioning during wastewater treatment and onto river sediments.

The partitioning of steroid estrogens in wastewater treatment and receiving waters is likely to influence their discharge to, and persistence in, the environment. This study investigated the partitioning behaviour of steroid estrogens in both laboratory and field studies. Partitioning onto activated sludge from laboratory-scale Husmann units was rapid with equilibrium achieved after 1 h. Sorption isotherms and Kd values decreased in the order 17α-ethinyl estradiol>17α-estradiol>estrone>estriol without a sorption limit being achieved (1/n>1). Samples from a wastewater treatment works indicated no accumulation of steroid estrogens in solids from primary or secondary biological treatment, however, a range of steroid estrogens were identified in sediment samples from the River Thames. This would indicate that partitioning in the environment may play a role in the long-term fate of estrogens, with an indication that they will be recalcitrant in anaerobic conditions.

Testicular dysgenesis syndrome and the estrogen hypothesis: a quantitative meta-analysis.

BACKGROUND: Male reproductive tract abnormalities such as hypospadias and cryptorchidism, and testicular cancer have been proposed to comprise a common syndrome together with impaired spermatogenesis with a common etiology resulting from the disruption of gonadal development during fetal life, the testicular dysgenesis syndrome (TDS). The hypothesis that in utero exposure to estrogenic agents could induce these disorders was first proposed in 1993. The only quantitative summary estimate of the association between prenatal exposure to estrogenic agents and testicular cancer was published over 10 years ago, and other systematic reviews of the association between estrogenic compounds, other than the potent pharmaceutical estrogen diethylstilbestrol (DES), and TDS end points have remained inconclusive. OBJECTIVES: We conducted a quantitative meta-analysis of the association between the end points related to TDS and prenatal exposure to estrogenic agents. Inclusion in this analysis was based on mechanistic criteria, and the plausibility of an estrogen receptor (ER)-alpha-mediated mode of action was specifically explored. RESULTS: We included in this meta-analysis eight studies investigating the etiology of hypospadias and/or cryptorchidism that had not been identified in previous systematic reviews. Four additional studies of pharmaceutical estrogens yielded a statistically significant updated summary estimate for testicular cancer. CONCLUSIONS: The doubling of the risk ratios for all three end points investigated after DES exposure is consistent with a shared etiology and the TDS hypothesis but does not constitute evidence of an estrogenic mode of action. Results of the subset analyses point to the existence of unidentified sources of heterogeneity between studies or within the study population.

A sensitive and robust method for the determination of alkylphenol polyethoxylates and their carboxylic acids and their transformation in a trickling filter wastewater treatment plant.

This paper presents a method for the determination of alkylphenols, alkylphenol polyethoxylates (APEO) and alkylphenol ethoxycarboxylates (APEC) in the aqueous and particulate phase of wastewater samples. Quantification was achieved by liquid chromatography-tandem mass spectrometry. The sensitivity of the method is demonstrated by low detection limits, in the dissolved phase 1.2-9.6ngl(-1) for alkylphenol, AP1-3EO and APEC and 0.1-4.1ngl(-1) for longer chain alkylphenol polyethoxylates. The method detection limit for particulate phase samples ranged from 6 to 60ngg(-1) for AP, AP1-3EO and APEC; with the longer chain APEO being from 0.5 to 20ngg(-1). Matrix effects were noted in complex matrix rich samples. There was a distinct change in the distribution of alkylphenol ethoxylates during biological treatment of the wastewater, with the major biotransformation products observed being carboxylated derivatives at concentrations of up to 1768ngl(-1). Shorter chain APEO were present in higher proportions in the suspended solids, due to their higher affinity to particulate matter compared to the long-chain oligomers.

Progesterone potentially degrades to potent androgens in surface waters.

Progesterone is a natural hormone, excreted in higher concentrations than estrogens, and has been detected in the aqueous environment. As with other compounds, it is transformed during wastewater treatment processes and in the environment. However, minor modifications to the structure may result in transformation products which still exhibit biological activity, so understanding what transformation products are formed is of importance. The current study was undertaken to identify putative transformation products resulting from spiking river water with progesterone in a laboratory-based degradation study and hence to follow the metabolic breakdown pathways. On the basis of literature reports and predictions from the EAWAG Biocatalysis/biodegradation database, target putative transformation products were initially monitored under unit resolution mass spectrometry. The identity of these transformation products was confirmed by using accurate-mass quadrupole time-of-flight. The study results highlight that transformation of progesterone can potentially create other classes of steroids, some of which may still be potent, and possess other types of biological activity.

Factors influencing development of management strategies for the Abou Ali River in Lebanon II: seasonal and annual variation.

The water quality of a river at any point reflects several major influences including but are not limited to climatic conditions and anthropogenic inputs. Assessing these influences is essential for managing land and water resources within a particular river catchment. The objectives of this study were to identify the causes of increasing or decreasing trends in the concentrations of various water quality parameters in the Abou Ali River in North Lebanon and to account for the consequential variations both annual and seasonal (low/high flow). The assessment was conducted at the end of the dry season in October 2002 and 2003 and the end of the wet season in March 2003 and 2004. Results established that dissolved oxygen levels were consistently higher at the end of the wet season. The concentrations of biochemical oxygen demand, ammonia nitrogen and ortho-phosphates did not exhibit a clear seasonal or annual variation. While the levels of total dissolved solids and nitrate nitrogen exhibited a decreasing trend in urban catchments, an increasing trend was observed in rural, agricultural and forested areas. The findings of this study reinforce the notion that management of point and non-point sources should be integrated as the combination of both sources connected with land use results in deleterious effects on water quality. The lack of good quality water hinders economic development and the potential for long term sustainability.

Factors influencing development of management strategies for the Abou Ali River in Lebanon I: spatial variation and land use.

Surface water bodies are progressively subject to increasing stress as a result of environmentally degrading processes primarily related to anthropogenic activities. This study assesses and examines the impact of land use and land-based activities on the spatial variation in water quality of the Abou Ali River in North Lebanon. It is the first detailed study of its kind in Lebanon and adds to the existing knowledge by shedding light on a relatively small Mediterranean river in a developing country where there is a paucity of such studies. The assessment was conducted at the end of the dry season in 2002 and 2003 and the end of the wet season in 2003 and 2004. The study has demonstrated the importance of anthropogenic influences on the water quality of the Abou Ali River Basin, as concentrations of most contaminants were higher at locations with greatest human activity. The most adversely affected area was the section of the river that flows through an entirely urbanized and highly populated region, the Tripoli conurbation. Upstream rural sites were enriched by contaminants primarily from non-point sources such as agricultural runoff and poultry litter whereas contaminant concentrations at the urban sites were enriched by a combination of sewage discharge and flow of contaminants from upstream. If the Abou Ali River is to be utilized as a managed water resource and its water quality sustained, point source discharges will require treatment and land use management must be planned to minimize the impact of diffuse source pollution on the river. A high priority should be given to the implementation and enforcement of the precautionary and polluter pays principles. Moreover, an effective legal, economic and institutional framework is required to encourage investment in waste reduction and control and to introduce environmentally sound practices.

Degradation of nonylphenolic surfactants in activated sludge batch tests.

The fate of nonylphenol polyethoxylate surfactants in the activated sludge wastewater treatment process is a concern due to the formation of estrogenic nonlyphenols on degradation and due to the large amounts discharged to the aquatic environment through sewage treatment works. Batch tests using activated sludge from a Husmann apparatus were used to determine the effects of these compounds physico-chemical properties and biological sludge characteristics on biodegradation. Degradation of nonylphenol polyethoxylates with up to 12 ethoxy groups was observed in unacclimated sludge with a concomitant production of nonylphenol and short chain nonylphenol polyethoxylate compounds. Degradation was determined to be a biotic process involving intracellular enzyme activity, which resulted in sludge age being an influential parameter. With increasing sludge age there is an increase in mixed liquor solids concentration in activated sludge which results in greater bacterial numbers and the potential for greater species diversity which therefore increases compound degradation. However, increased degradation of long chain compounds resulted in an accumulation of shorter chain compounds and nonylphenol, which are more resistant to degradation.

The partitioning of alkylphenolic surfactants and polybrominated diphenyl ether flame retardants in activated sludge batch tests.

Polybrominated diphenyl ethers and nonylphenol polyethoxylates have been reported to be estrogenic and may enter the aquatic environment through the discharge of treated sewage effluent. Therefore, their fate during wastewater treatment processes is an important factor in determining their environmental impact. Batch tests with activated sludge from a Husmann apparatus were used to determine the effects of physico-chemical properties and sludge characteristics on the partitioning of polybrominated diphenyl ether flame retardants and nonylphenol polyethoxylate surfactants during biological wastewater treatment. Hydrophobic compounds, those with high logK(ow) values, were sorbed more rapidly and to a greater extent to the solid phase than more soluble compounds. For these hydrophobic compounds sorption may become an increasingly important removal mechanism as sludge age and therefore solids content increase. The initial rate of partitioning was greatest for the most hydrophobic compounds but all rates diminished with time as a result of progressive saturation of sorbent binding sites, a reduction of sorbate availability and as a consequence of the system reaching equilibrium. The sorption of polybrominated diphenyl ethers fit Freundlich adsorption isotherms demonstrating generally increasing adsorption capacity and efficiency with increasing hydrophobic nature. A correlation between increasing logK(ow) and increasing organic matter content was also observed for both polybrominated diphenyl ethers and nonylphenol polyethoxylates indicating the organic content of mixed liquor will also be influential in removing compounds during wastewater treatment.

An approach for prioritizing "down-the-drain" chemicals used in the household.

Many chemicals are present in cleaning and personal care products, which after use are washed down the drain and find their way into water bodies, where they may impact the environment. This study surveyed individuals to determine what products were used most in the home, in an attempt to prioritize which compounds may be of most concern. The survey resulted in the identification of 14 categories of products consisting of 315 specific brands. The survey estimated that individuals each discharge almost 33 L of products per year down the drain. Dishwashing liquids and hand wash gels, which accounted for 40% of this volume, were selected for identification of specific ingredients. Ingredients were classified as surfactants, preservatives, fragrances or miscellaneous, with hand wash gels having a wider range of ingredients than dishwashing liquids. A review of the literature suggested that preservatives, which are designed to be toxic, and fragrances, where data on toxicity are limited, should be prioritized. The approach undertaken has successfully estimated use and provisionally identified some classes of chemicals which may be of most concern when used in cleaning and personal care products.

Future water quality monitoring--adapting tools to deal with mixtures of pollutants in water resource management.

Environmental quality monitoring of water resources is challenged with providing the basis for safeguarding the environment against adverse biological effects of anthropogenic chemical contamination from diffuse and point sources. While current regulatory efforts focus on monitoring and assessing a few legacy chemicals, many more anthropogenic chemicals can be detected simultaneously in our aquatic resources. However, exposure to chemical mixtures does not necessarily translate into adverse biological effects nor clearly shows whether mitigation measures are needed. Thus, the question which mixtures are present and which have associated combined effects becomes central for defining adequate monitoring and assessment strategies. Here we describe the vision of the international, EU-funded project SOLUTIONS, where three routes are explored to link the occurrence of chemical mixtures at specific sites to the assessment of adverse biological combination effects. First of all, multi-residue target and non-target screening techniques covering a broader range of anticipated chemicals co-occurring in the environment are being developed. By improving sensitivity and detection limits for known bioactive compounds of concern, new analytical chemistry data for multiple components can be obtained and used to characterise priority mixtures. This information on chemical occurrence will be used to predict mixture toxicity and to derive combined effect estimates suitable for advancing environmental quality standards. Secondly, bioanalytical tools will be explored to provide aggregate bioactivity measures integrating all components that produce common (adverse) outcomes even for mixtures of varying compositions. The ambition is to provide comprehensive arrays of effect-based tools and trait-based field observations that link multiple chemical exposures to various environmental protection goals more directly and to provide improved in situ observations for impact assessment of mixtures. Thirdly, effect-directed analysis (EDA) will be applied to identify major drivers of mixture toxicity. Refinements of EDA include the use of statistical approaches with monitoring information for guidance of experimental EDA studies. These three approaches will be explored using case studies at the Danube and Rhine river basins as well as rivers of the Iberian Peninsula. The synthesis of findings will be organised to provide guidance for future solution-oriented environmental monitoring and explore more systematic ways to assess mixture exposures and combination effects in future water quality monitoring.

The SOLUTIONS project: challenges and responses for present and future emerging pollutants in land and water resources management.

SOLUTIONS (2013 to 2018) is a European Union Seventh Framework Programme Project (EU-FP7). The project aims to deliver a conceptual framework to support the evidence-based development of environmental policies with regard to water quality. SOLUTIONS will develop the tools for the identification, prioritisation and assessment of those water contaminants that may pose a risk to ecosystems and human health. To this end, a new generation of chemical and effect-based monitoring tools is developed and integrated with a full set of exposure, effect and risk assessment models. SOLUTIONS attempts to address legacy, present and future contamination by integrating monitoring and modelling based approaches with scenarios on future developments in society, economy and technology and thus in contamination. The project follows a solutions-oriented approach by addressing major problems of water and chemicals management and by assessing abatement options. SOLUTIONS takes advantage of the access to the infrastructure necessary to investigate the large basins of the Danube and Rhine as well as relevant Mediterranean basins as case studies, and puts major efforts on stakeholder dialogue and support. Particularly, the EU Water Framework Directive (WFD) Common Implementation Strategy (CIS) working groups, International River Commissions, and water works associations are directly supported with consistent guidance for the early detection, identification, prioritisation, and abatement of chemicals in the water cycle. SOLUTIONS will give a specific emphasis on concepts and tools for the impact and risk assessment of complex mixtures of emerging pollutants, their metabolites and transformation products. Analytical and effect-based screening tools will be applied together with ecological assessment tools for the identification of toxicants and their impacts. The SOLUTIONS approach is expected to provide transparent and evidence-based candidates or River Basin Specific Pollutants in the case study basins and to assist future review of priority pollutants under the WFD as well as potential abatement options.

Comparative environmental assessment of biocides used in antifouling paints.

In response to increasing scientific evidence on the toxicity and persistence of organotin residues from antifouling paints in the aquatic environment, the use of triorganotin antifouling products was banned on boats of less than 25 m length in many countries during 1987. Alternatives to tributyltin (TBT) paint are mainly copper based coatings containing organic booster biocides to improve the efficacy of the formulation, and have been utilised on small boats for the last 10 years. With policies encouraging a total ban on TBT, it is expected that these biocides will be used to a greater extent in the future. Limited data and information are available on the environmental occurrence, fate, toxicity, and persistence of these biocides, and thus any decisions on policies regulating antifoulants cannot be fully informed. In this study, a multicriteria comparison of alternative biocides, based on a general assessment of available information in the literature, provided support for the use of the precautionary principle with respect to policies on antifouling products. This assessment was validated by a more detailed comparison of four selected biocides and TBT. Results indicate that TCMS pyridine and TCMTB demonstrate environmental characteristics similar to TBT and thus detail risk assessments are needed before their use is permitted. The widespread use of the other biocides should be allowed only after research to fill the gaps in knowledge with respect to their toxicity and persistence in aquatic environments.

An assessment of the bioaccumulation of estrone in Daphnia magna.

The bioaccumulation of estrone by Daphnia magna was determined. Direct uptake via the aqueous medium occurred within the first 16 h. A bioconcentration factor of 228 was established over all temporal periods. Ingestion via Chlorella vulgaris gave a partitioning factor of 24, which may approximate to a biomagnification factor assuming steady state conditions. These preliminary results indicate that the partitioning to Daphnia magna via the food source, C. vulgaris is less significant than bioconcentration.

Transport of mecoprop from agricultural soils to an adjacent salt marsh.

Salt marshes are important ecological areas and play a significant role in coastal flood defence schemes. In many areas of the UK they are adjacent to agricultural areas utilised for the growth of cereal crops, for which mecoprop is used as a selective herbicide in the control of broad-leaf weeds. This study measured concentrations of mecoprop in soils, drainage ditch waters and sediments and salt marsh sediments over a period of 138 days following spring application. Soil concentrations of up to 1827 microg/g were recorded after application, which demonstrated a half life for mecoprop of from 9 to 12 days, with first order kinetics. However, a major rainfall event 9 days after application resulted in significant transport of herbicide to the salt marsh via subsurface field drains, drainage ditches and discharge sluice. Mecoprop concentrations of up to 386 microg/l observed in water samples were above UK guidelines.

A new role for carbonic anhydrase 2 in the response of fish to copper and osmotic stress: implications for multi-stressor studies.

The majority of ecotoxicological studies are performed under stable and optimal conditions, whereas in reality the complexity of the natural environment faces organisms with multiple stressors of different type and origin, which can activate pathways of response often difficult to interpret. In particular, aquatic organisms living in estuarine zones already impacted by metal contamination can be exposed to more severe salinity variations under a forecasted scenario of global change. In this context, the present study aimed to investigate the effect of copper exposure on the response of fish to osmotic stress by mimicking in laboratory conditions the salinity changes occurring in natural estuaries. We hypothesized that copper-exposed individuals are more sensitive to osmotic stresses, as copper affects their osmoregulatory system by acting on a number of osmotic effector proteins, among which the isoform two of the enzyme carbonic anhydrase (CA2) was identified as a novel factor linking the physiological responses to both copper and osmotic stress. To test this hypothesis, two in vivo studies were performed using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) as test species and applying different rates of salinity transition as a controlled way of dosing osmotic stress. Measured endpoints included plasma ions concentrations and gene expression of CA2 and the α1a-subunit of the enzyme Na+/K+ ATPase. Results showed that plasma ions concentrations changed after the salinity transition, but notably the magnitude of change was greater in the copper-exposed groups, suggesting a sensitizing effect of copper on the responses to osmotic stress. Gene expression results demonstrated that CA2 is affected by copper at the transcriptional level and that this enzyme might play a role in the observed combined effects of copper and osmotic stress on ion homeostasis.