Understanding the transformation, speciation, and hazard potential of copper particles in a model septic tank system using zebrafish to monitor the effluent

Abstract

Although copper-containing nanoparticles are used in commercial products such as fungicides and bactericides, we presently do not understand the environmental impact on other organisms that may be inadvertently exposed. In this study, we used the zebrafish embryo as a screening tool to study the potential impact of two nano Cu-based materials, CuPRO and Kocide, in comparison to nanosized and micron-sized Cu and CuO particles in their pristine form (0-10 ppm) as well as following their transformation in an experimental wastewater treatment system. This was accomplished by construction of a modeled domestic septic tank system from which effluents could be retrieved at different stages following particle introduction (10 ppm). The Cu speciation in the effluent was identified as nondissolvable inorganic Cu(H2PO2)2 and nondiffusible organic Cu by X-ray diffraction, inductively coupled plasma mass spectrometry (ICP-MS), diffusive gradients in thin-films (DGT), and Visual MINTEQ software. While the nanoscale materials, including the commercial particles, were clearly more potent (showing 50% hatching interference above 0.5 ppm) than the micron-scale particulates with no effect on hatching up to 10 ppm, the Cu released from the particles in the septic tank underwent transformation into nonbioavailable species that failed to interfere with the function of the zebrafish embryo hatching enzyme. Moreover, we demonstrate that the addition of humic acid, as an organic carbon component, could lead to a dose-dependent decrease in Cu toxicity in our high content zebrafish embryo screening assay. Thus, the use of zebrafish embryo screening, in combination with the effluents obtained from a modeled exposure environment, enables a bioassay approach to follow the change in the speciation and hazard potential of Cu particles instead of difficult-to-perform direct particle tracking.

Keywords: copper particles; high content screening; speciation; transformation; wastewater treatment; zebrafish.

Figure 1

Cu dissolution and hatching interference of as-received Cu particles. (A) Calculation of the weight percent dissolution of nano Cu, Kocide and CuPRO (highly soluble); nano CuO (intermediate solubility); and micro Cu and CuO (minimally soluble). (B) Percent hatching of zebrafish embryos exposed to as-received Cu particles (0 – 10 ppm) for 72 hours, commencing at 4 hours post-fertilization. The dose dependent curve is expressed as % hatching vs. Log [Cu] (ppb).

Figure 2

Combined use of a model septic tank and zebrafish embryo high content screening to study the effects of Cu-containing effluents on embryo hatching. (A) Schematic diagram of the model septic system to generate effluents for testing in zebrafish embryos. (B) Percent hatching of zebrafish embryos exposed to the effluents collected weekly from the nano Cu, CuPRO, and micro Cu groups for 6 weeks. The introduction of 0.5 ppm nano Cu in Holtfreter’s medium was used as a positive control. Symbol * denotes statistical significance at p < 0.05.

Figure 2

Combined use of a model septic tank and zebrafish embryo high content screening to study the effects of Cu-containing effluents on embryo hatching. (A) Schematic diagram of the model septic system to generate effluents for testing in zebrafish embryos. (B) Percent hatching of zebrafish embryos exposed to the effluents collected weekly from the nano Cu, CuPRO, and micro Cu groups for 6 weeks. The introduction of 0.5 ppm nano Cu in Holtfreter’s medium was used as a positive control. Symbol * denotes statistical significance at p < 0.05.

Figure 3

Characterization of the septic effluents. (A) ICP-MS measurements were undertaken to quantify elemental Cu in the effluents collected on a weekly basis following the introduction of the different particle types. The dashed lines represent the as-received Cu²⁺, nano Cu and CuPRO concentrations providing ~50% hatching interference in Holtfreter’s medium. (B) XRD analysis on the as-received particulates as well as the corresponding effluents at week 3. (C) Visual MINTEQ modeling to show Cu speciation in the presence and absence of Dissolved Organic Matter (DOM). Humic acid (HA) was used as a form of DOM to perform Visual MINTEQ modeling. Without the presence of DOM, Cu²⁺ is the dominant species, accounting for 75% of the total Cu. The presence of 100 ppm DOM decreases the Cu²⁺ content precipitously (to 2%) as a result of metal complexation. DOM-bound Cu (DOM₁-Cu(6): 63%, or DOM₂-Cu(6): 27%) accounts for 90% of the total Cu. DOM₁ and DOM₂ are used to describe different humic components. (D) Visual MINTEQ modeling to show the Cu distribution into ionic (Cu²⁺) and organic Cu following the introduction of incremental amounts of humic acid. The grey area indicates the humic acid concentration range (30 – 100 ppm) that is expected in the septic tank effluent.

Figure 3

Characterization of the septic effluents. (A) ICP-MS measurements were undertaken to quantify elemental Cu in the effluents collected on a weekly basis following the introduction of the different particle types. The dashed lines represent the as-received Cu²⁺, nano Cu and CuPRO concentrations providing ~50% hatching interference in Holtfreter’s medium. (B) XRD analysis on the as-received particulates as well as the corresponding effluents at week 3. (C) Visual MINTEQ modeling to show Cu speciation in the presence and absence of Dissolved Organic Matter (DOM). Humic acid (HA) was used as a form of DOM to perform Visual MINTEQ modeling. Without the presence of DOM, Cu²⁺ is the dominant species, accounting for 75% of the total Cu. The presence of 100 ppm DOM decreases the Cu²⁺ content precipitously (to 2%) as a result of metal complexation. DOM-bound Cu (DOM₁-Cu(6): 63%, or DOM₂-Cu(6): 27%) accounts for 90% of the total Cu. DOM₁ and DOM₂ are used to describe different humic components. (D) Visual MINTEQ modeling to show the Cu distribution into ionic (Cu²⁺) and organic Cu following the introduction of incremental amounts of humic acid. The grey area indicates the humic acid concentration range (30 – 100 ppm) that is expected in the septic tank effluent.

Figure 3

Characterization of the septic effluents. (A) ICP-MS measurements were undertaken to quantify elemental Cu in the effluents collected on a weekly basis following the introduction of the different particle types. The dashed lines represent the as-received Cu²⁺, nano Cu and CuPRO concentrations providing ~50% hatching interference in Holtfreter’s medium. (B) XRD analysis on the as-received particulates as well as the corresponding effluents at week 3. (C) Visual MINTEQ modeling to show Cu speciation in the presence and absence of Dissolved Organic Matter (DOM). Humic acid (HA) was used as a form of DOM to perform Visual MINTEQ modeling. Without the presence of DOM, Cu²⁺ is the dominant species, accounting for 75% of the total Cu. The presence of 100 ppm DOM decreases the Cu²⁺ content precipitously (to 2%) as a result of metal complexation. DOM-bound Cu (DOM₁-Cu(6): 63%, or DOM₂-Cu(6): 27%) accounts for 90% of the total Cu. DOM₁ and DOM₂ are used to describe different humic components. (D) Visual MINTEQ modeling to show the Cu distribution into ionic (Cu²⁺) and organic Cu following the introduction of incremental amounts of humic acid. The grey area indicates the humic acid concentration range (30 – 100 ppm) that is expected in the septic tank effluent.

Figure 3

Characterization of the septic effluents. (A) ICP-MS measurements were undertaken to quantify elemental Cu in the effluents collected on a weekly basis following the introduction of the different particle types. The dashed lines represent the as-received Cu²⁺, nano Cu and CuPRO concentrations providing ~50% hatching interference in Holtfreter’s medium. (B) XRD analysis on the as-received particulates as well as the corresponding effluents at week 3. (C) Visual MINTEQ modeling to show Cu speciation in the presence and absence of Dissolved Organic Matter (DOM). Humic acid (HA) was used as a form of DOM to perform Visual MINTEQ modeling. Without the presence of DOM, Cu²⁺ is the dominant species, accounting for 75% of the total Cu. The presence of 100 ppm DOM decreases the Cu²⁺ content precipitously (to 2%) as a result of metal complexation. DOM-bound Cu (DOM₁-Cu(6): 63%, or DOM₂-Cu(6): 27%) accounts for 90% of the total Cu. DOM₁ and DOM₂ are used to describe different humic components. (D) Visual MINTEQ modeling to show the Cu distribution into ionic (Cu²⁺) and organic Cu following the introduction of incremental amounts of humic acid. The grey area indicates the humic acid concentration range (30 – 100 ppm) that is expected in the septic tank effluent.

Figure 4

Use of the methodology for diffusive gradients in thin-films (DGT) to quantify diffusible Cu²⁺ in the effluents. Schematic diagram of a DGT unit comprised of a nitrocellulose membrane filter, a gel for diffusion, and a resin gel layer (insert). Separation of the diffusible Cu²⁺ and organic Cu in the effluent collected during week 3 (post introduction of micro Cu, nano Cu and CuPRO) was undertaken by incubation the DGT unit in the effluents for 3 days at 28 °C. The resin gel was retrieved and digested in nitric acid, before performance of ICP-OES.

Figure 5

The addition of humic acid (HA) decreased Cu toxicity. (A) The effect of humic acid (0 – 500 ppm) on hatching interference by 0.5 and 1 ppm Cu²⁺ in Holtfreter’s medium. (B) Comparison of the effect of Suwannee River NOM (100 ppm) with humic acid (100 ppm) and “background” effluent for their effects on embryo hatching in the presence of 0.5 and 1 ppm Cu²⁺ in Holtfreter’s medium. The data in the 3D bar chart represent the average of 3 individual experiments in which the standard deviation varied less than 6%. (C) Comparison of the effect of known concentrations (0.125, 0.25, 0.5 and 1 ppm) of Cu²⁺ directly spiked into Holtfreter’s medium or into “background” effluent. (D) Comparison of the effect of zebrafish embryos exposed to known concentrations (0.125, 0.25, 0.5 and 1 ppm) of nano Cu directly spiked into Holtfreter’s medium or into “background” effluent. The * and # symbols denote statistical significance at p < 0.05.

Figure 5

The addition of humic acid (HA) decreased Cu toxicity. (A) The effect of humic acid (0 – 500 ppm) on hatching interference by 0.5 and 1 ppm Cu²⁺ in Holtfreter’s medium. (B) Comparison of the effect of Suwannee River NOM (100 ppm) with humic acid (100 ppm) and “background” effluent for their effects on embryo hatching in the presence of 0.5 and 1 ppm Cu²⁺ in Holtfreter’s medium. The data in the 3D bar chart represent the average of 3 individual experiments in which the standard deviation varied less than 6%. (C) Comparison of the effect of known concentrations (0.125, 0.25, 0.5 and 1 ppm) of Cu²⁺ directly spiked into Holtfreter’s medium or into “background” effluent. (D) Comparison of the effect of zebrafish embryos exposed to known concentrations (0.125, 0.25, 0.5 and 1 ppm) of nano Cu directly spiked into Holtfreter’s medium or into “background” effluent. The * and # symbols denote statistical significance at p < 0.05.