Copper toxicity to blue mussel embryos (Mytilus galloprovincialis)

Highlights

• DOC-quality and salinity alter Cu-speciation in estuarine waters.

• [Cu’], together with [L], DOC-quality, and salinity are good proxies to predict 48-h-EC₅₀ Cu-values to mussel embryos.

• Embryos actively produce ligands to complex inorganically bound Cu and free Cu.

• Cu-speciation including [L] needs to be considered in the BLM and future aquatic risk assessments of copper.

Abstract

Copper (Cu) is a naturally occurring micronutrient of eco-toxicological concern in aquatic ecosystems. Current knowledge of Cu-speciation and bioavailability in natural saline environments is insufficient to adequately inform environmental protection policy for estuarine systems. We assessed the combined effect of two of the main drivers of metal bioavailability, salinity and natural dissolved organic carbon (DOC), on Cu-speciation and associated Cu-toxicity to blue mussel (Mytilus galloprovincialis) embryos in a standard 48-h bioassay. We placed special emphasis on measurement of Cu-speciation rather than modelling. Cu-toxicity was found to be a function of DOC and salinity. The varying protective effect of different DOC-types suggests that estuarine DOC is more protective against Cu-toxicity than oceanic DOC. Salinity was negatively correlated with [Cu_48-h-EC50], indicating a salinity-induced alteration in the physiology of the exposed mussel embryos and/or Cu-DOC-reactivity. These two assumptions were supported by (1) the relative uniformity of bioavailable copper ([Cu′]) across similar salinity treatments despite considerable variation in [Cu_48-h-EC50] and DOC-concentrations, and (2) the fact that Cu-toxicity and [Cu′] were slightly higher in the 35 salinity treatment compared to the 25 salinity treatment. Stripping voltammetry studies determined the presence of only one strong Cu-binding ligand class (i.e., L₁), either actively or passively released by the exposed embryos. [L₁] was found to be proportional to the total dissolved Cu-concentration ([Cu_T]), suggesting a protective effect of Cu-binding-ligands, in addition to the protective effect of DOC. There was also a strong positive correlation between [L₁] and [Cu_48-h-EC50], implying that electrochemically defined ligand concentrations along with measurements of [Cu′], DOC-quality, and salinity can be used as proxies for 48-h-EC₅₀ Cu-values in estuarine waters, which may result in a significant improvement to risk assessments of Cu in estuarine systems.

Introduction

Estuarine environments are productive ecosystems with high ecological, economic and cultural value (Williams and Hall, 1999). The health of these ecosystems is, however, subjected to constant deterioration by influences such as pollution from urbanisation, industrialization and agricultural intensification (Williams and Hall, 1999), which makes it important to assess and quantify associated risks to aquatic organisms. Of special concern is the increase in chemical pollutants such as trace metals in estuaries (Buck and Bruland, 2005). Copper is one example. Copper serves as either an essential micronutrient to marine organisms, or as a toxicant when exceeding a critical concentration (Brand et al., 1986). The window between sufficiency and toxicity can be narrow and early life stages are usually the most vulnerable to Cu-toxicity (Fitzpatrick et al., 2008; Jakimska et al., 2011).

Chemical speciation is the driving factor for understanding Cu-bioavailability (i.e., fraction of copper in the aquatic system bioavailable for uptake by biota) and toxicity to aquatic organisms (Van Briesen et al., 2010). Cu-toxicity is directly related to the bioavailable Cu-concentration ([Cu′]) (Huang et al., 2003; Hassler et al., 2004, Hassler et al., 2007), which according to the biotic ligand model (BLM) is the concentration of free hydrated Cu-ions ([Cu_f]) and Cu weakly complexed by inorganic ligands ([CuX_IN]), such as Cl⁻, OH⁻ and SO₄⁻² (Luoma, 1983; Campbell et al., 2002; Semeniuk et al., 2015), rather than the total dissolved Cu-concentration ([Cu_T]) present in solution (Luoma, 1983; Huang et al., 2003; Hassler et al., 2004, Hassler et al., 2007). The BLM integrates the concept of bioavailability to evaluate actual metal-toxicity risks (lethal and sub-lethal effects) to exposed aqueous biota (Niyogi and Wood, 2004; Smith et al., 2015) and, therefore, greatly improves the ability to generate site-specific ambient water quality criteria (AWQC) for metals in the natural environment (US EPA, 2002; Niyogi and Wood, 2004; Smith et al., 2015). The most mature BLM, and recently incorporated into the updated AWQC, is that for the toxicity of Cu (US EPA, 2007). The BLM also recognises that bioavailable metal concentrations in the water column are strongly mediated by geochemical conditions (Sander et al., 2015), which are highly variable both temporally and spatially within tide-dominated estuarine systems (Cloern and Nichols, 1985).

In estuarine waters, metal speciation is mainly influenced by changes in salinity and dissolved organic carbon (DOC) quantity and quality (Cooper et al., 2014; Deruytter et al., 2015). Alteration in these three parameters, typical for tidal environments, can alter metal complexation processes and bioavailability to aquatic organisms (Huang et al., 2003; Buck and Bruland, 2005; Grosell et al., 2007; Cooper et al., 2014). At present, the effects of rapid fluctuations in salinity, DOC-concentration and/or the nature of DOC on Cu-speciation in estuaries have not been assessed (Grosell et al., 2007). In constant conditions, several experiments have demonstrated (1) nonlinear and potentiating effects of increasing salinity on Cu-toxicity to various aquatic organisms instead of the toxicity decrease that is expected based on speciation processes (i.e., complexation and competition with cations) (Grosell et al., 2007; Deruytter et al., 2015), and (2) a proportionality between DOC-concentration and Cu-toxicity (Arnold et al., 2009, Arnold et al., 2010; Deruytter et al., 2013, Deruytter et al., 2015). These studies (Rosen et al., 2005; Arnold et al., 2009, Arnold et al., 2010; Deruytter et al., 2013, Deruytter et al., 2015; Bosse et al., 2014) illustrate, however, substantial differences in Cu-sensitivity for M. galloprovincialis embryos with [Cu_48-h-EC50] (mortality and malformation as the half-maximal response of the embryos) between 65 and 547 nM. Changes with salinity in embryo osmoregulatory physiology as well as differences in DOC-sources and/or DOC-Cu-reactivities with salinity are thought to be responsible for these ambiguous Cu-toxicity results (Grosell et al., 2007; De Polo and Scrimshaw, 2012; Cooper et al., 2014; Deruytter et al., 2013, Deruytter et al., 2015).

It is well established that Cu has a high affinity for binding to some constituents that make up DOC, a process which prevents or attenuates Cu-uptake by, and Cu-toxicity to, aquatic organisms (Cooper et al., 2014). Cu-binding constituents of natural DOC are mainly humic substances (HS) from allochthonous (i.e., terrestrial input including stormwater and wastewater (DePalma et al., 2011; Mahmood, 2017)) and autochthonous (i.e., organic carbon formed in the aquatic environment) sources (Arnold et al., 2009; Cooper et al., 2014; Abualhaija et al., 2015; Nogueira et al., 2017) with allochthonous DOC being more protective against Cu-toxicity than autochthonous DOC (Cooper et al., 2014; Mahmood, 2017; Nogueira et al., 2017). The different Cu-binding affinities of the different Cu-binding species of DOC render DOC-source and related DOC-quality more important in determining Cu-complexation processes in the natural aquatic environment than DOC-concentration (Cooper et al., 2014; Deruytter et al., 2015). Thiols, known to emanate from reducing marine sediments or produced by marine organisms, may represent a second component of the Cu-binding ligand pool in estuarine and oceanic waters (Whitby and Van den Berg, 2015; Whitby et al., 2017). All in all, little is known regarding the effect of (1) different DOC-fractions on Cu-speciation, or (2) salinity on DOC-Cu-reactivity, which impedes comparison between eco-toxicological studies and prevents precise risk assessments for Cu in saline environments.

The focus of the present study was thus to assess the combined effect of natural DOC and salinity on Cu-toxicity in estuarine waters using a 48-h bioassay of blue mussel embryos (Mytilus galloprovincialis). Mytilus galloprovincialis is common and widely distributed internationally and is found in the intertidal zone of New Zealand (Gardner, 2004). Although not an estuarine species, M. galloprovincialis is known to be tolerant of a range of salinities (12 to 38 (Bayne, 1976)), and is used internationally for toxicity testing (ASTM, 1997). Given the lack of explicit Cu-speciation measurements in estuarine eco-toxicological studies, special emphasis was put on the measurement of Cu-speciation rather than a modelling approach. Cu-speciation parameters were determined using adsorptive cathodic stripping voltammetry (AdCSV) with salicylaldoxime (SA) as the complexing agent or anodic stripping voltammetry (ASV). Consequently, the present study builds on earlier research by Nadella et al. (2009), Arnold et al. (2010), and Deruytter et al. (2013 and 2015), thereby contributing to an integrated picture of Cu-speciation processes in estuarine environments to improve the BLM and associated Cu-risk assessments.