Human activity is resulting in unprecedented amounts of carbon dioxide (CO₂) being released into the atmosphere. Since the Industrial Revolution, atmospheric CO₂ levels have increased by over 45%, from approximately 280 ppm (Joos and Spahni, 2008) to over 410 ppm today (Dlugokencky and Tans, 2019), higher than any time in the past several million years (Masson-Delmotte et al., 2013). In the worst case scenario, following the business-as-usual representative concentration pathway (RCP) 8.5, atmospheric CO₂ levels will increase to over 900 ppm by the end of this century. Even if substantial efforts are made to curb global CO₂ emissions to keep warming below 2°C, atmospheric CO₂ levels will still likely exceed 600 ppm by 2100 (Betts and McNeall, 2018). The ocean has absorbed 20–30% of anthropogenic CO₂ emissions since the mid-1980s (Bindoff et al., 2019), causing a reduction in seawater pH referred to as ocean acidification. Furthermore, CO₂ in the surface ocean is increasing at the same rate as in the atmosphere (Bindoff et al., 2019); therefore, marine organisms will need to cope with higher CO₂ levels as well as declining seawater pH. Finally, due to a decrease in the ocean’s buffering capacity as CO₂ content rises, natural CO₂ fluctuations in the ocean are projected to amplify dramatically at future higher CO₂ levels (Shaw et al., 2013; McNeil and Sasse, 2016). Natural diel (Hofmann et al., 2011; Santos et al., 2011; Shaw et al., 2012) and seasonal CO₂ fluctuations (McNeil et al., 2007; Feely et al., 2008) will be amplified by up to three times in the future (McNeil and Sasse, 2016; Gallego et al., 2018), meaning that marine organisms will experience elevated CO₂ levels for certain periods of time (daily or seasonally) much earlier than predictions based on atmospheric CO₂ alone.

Elevated CO₂ has been found to affect a range of processes in marine organisms, including altering calcification (Ries et al., 2009; Kroeker et al., 2013), growth and survival (Fabry et al., 2008; Kurihara et al., 2008), and behavior (Briffa et al., 2012; Clements and Hunt, 2015; Nagelkerken and Munday, 2015). Projected future CO₂ levels were first found to alter animal behavior in orange clownfish Amphiprion percula larvae reared in a partial pressure of CO₂ (pCO₂) of ∼1,050 μatm (Munday et al., 2009). In laboratory experiments, the olfactory discriminatory abilities of 11-day-old clownfish larvae were tested in a two-channel flume. Most strikingly, larvae reared in control seawater (∼390 μatm pCO₂) avoided the side of the flume with chemical cues from pungent tree leaves compared to the seawater control side. However, larvae reared in elevated CO₂ spent nearly all their time in the side with these odors. Larval clownfish reared in ∼1,050 μatm CO₂ were also unable to discriminate between the odor of parents and non-parents, whereas control larvae avoided the odor of their parents (Munday et al., 2009). Elevated CO₂ has since been found to affect a variety of behavioral traits in a wide spectrum of fishes, including tropical and temperate reef species, eels, salmon and sharks (Munday et al., 2019). Behavioral alterations at elevated CO₂ have also been demonstrated in a variety of marine invertebrates, including cnidaria, polychaetes, echinoderms, arthropods and mollusks, from a range of environments, including the intertidal zone, coastal and offshore waters and the deep-sea (Clements and Hunt, 2015; Nagelkerken and Munday, 2015; Wang and Wang, 2019). Marine invertebrates exhibit alterations in a range of behaviors at elevated CO₂, including activity levels (Rosa and Seibel, 2008; Ellis et al., 2009; Spady et al., 2014), feeding rates (Saba et al., 2012; Vargas et al., 2014), settlement and metamorphosis behaviors (Albright et al., 2010; Doropoulos et al., 2012; Guo et al., 2015), burrowing behaviors (Green et al., 2013; Clements and Hunt, 2014), shelter selection (de la Haye et al., 2011), predatory behaviors (behaviors related to finding and eating prey) (Kim et al., 2015; Queirós et al., 2015; Spady et al., 2018) and predator avoidance (Bibby et al., 2007; Manríquez et al., 2013, 2014a; Spady et al., 2014; Watson et al., 2014). Behavioral categorization is often ambiguous as one behavior may actually include multiple behaviors or decision-making processes. For example, predator avoidance behaviors include multiple decisions such as mode of avoidance (including crypticity versus escape), flight-initiation distance and mode of escape (Lima and Dill, 1990). In this review, we use the behavioral category that was reported in the corresponding research paper.

Since the review by Clements and Hunt (2015) at least 61 additional papers have assessed the impact of elevated CO₂ on marine invertebrate behaviors (Supplementary Table S1). Research has continued to focus on mollusks, arthropods and echinoderms, however, cnidarian settlement and metamorphosis (Foster et al., 2015; Olsen et al., 2015; Viyakarn et al., 2015; Fabricius et al., 2017; Yuan et al., 2018), the settlement behavior and swimming activity of a bryozoan (Pecquet et al., 2017), and settlement of an annelid (Nelson et al., 2020) have also been studied. In addition to continuing to assess the impact of elevated CO₂ on the range of behaviors previously studied (above), a few new behaviors have also been investigated. For example, the first study assessing the effect of elevated CO₂ on marine invertebrate reproductive behavior was recently published (Borges et al., 2018). Exposure of male amphipods Gammarus locusta to elevated CO₂ (800 μatm pCO₂) for two generations disrupted the chemosensory detection of potential mates (Borges et al., 2018). A light/dark test on swimming crabs Portunus trituberculatus exposed to control (485 μatm pCO₂) or elevated (750 μatm and 1,500 μatm pCO₂) CO₂ was the first to assess the impact of elevated CO₂ on anxiety-like behavior in a marine invertebrate. Crabs exposed to elevated CO₂ levels spent significantly more time in the dark zone (Ren et al., 2018).

There appears to be large variability in behavioral responses to elevated CO₂; across taxonomic groups, the same behavior can respond differently to elevated CO₂, and within a species, some behaviors but not others can be affected (Nagelkerken and Munday, 2015) (Supplementary Table S1). As the phylogenetic variation among invertebrate taxa is enormous, it may account for a large component of the variability in behavioral responses across taxonomic groups; some taxa may be more tolerant to elevated CO₂ than others. At the same time, various behaviors are likely associated with different processes, such as specific circuits in the nervous system. These processes may be affected differently by elevated CO₂, accounting for the effects of elevated CO₂ on some, but not all, behaviors within a species. It must also be noted that variability may be due to differences in experimental techniques and conditions.

Animal behavior is, to a large extent, the functional output of the nervous system (Simmons and Young, 1999); therefore, behavioral changes induced by elevated CO₂ are likely caused by neurobiological mechanisms. In the nervous system, simplistically, the pathway that produces behavior involves sensory receptors that detect environmental stimuli (e.g., chemical cues, light waves) and internal stimuli (e.g., spatial orientation of the body). The received information is transduced into electrical impulses and neurotransmission relays these electrical impulses between neurons. Neurotransmission must be rapid to produce timely behavioral responses, and this is achieved via ligand-gated ion channel (LGIC) mediated neurotransmission (Dent, 2010). LGICs are transmembrane protein complexes that, upon binding of a specific neurotransmitter, allow ion flow which results in excitation or inhibition of neuronal firing depending on the ion charge and direction of flow (Tovar and Westbrook, 2012). When the sensory information arrives at higher centers of the nervous system, this information is processed and a behavioral output is produced (Blom, 1978; Kreher et al., 2008) (Figure 1).

Elevated CO₂ could induce behavioral alterations through a range of mechanisms in the nervous system that disturb different components of the pathway that produces behavior [Briffa et al. (2012), as described in Figure 1]. (1) Sensation may be disrupted via changes to sensory stimuli at elevated CO₂, such as structural alteration of chemical cues, disturbed transmission of acoustic cues and altered sensory output from animals experiencing behavioral changes (Roggatz et al., 2016; Nagelkerken et al., 2019). (2) Alternatively, elevated CO₂ may disrupt sensation via physical change to sensory organs or structural alteration of sensory receptors (Maneja et al., 2011; Briffa et al., 2012; Bignami et al., 2013). (3) Morphological and respiratory changes at elevated CO₂ may alter the context within which decision making is carried out, influencing behavioral responses (Bibby et al., 2007; Chan et al., 2011; Peng et al., 2017; Rich et al., 2018). (4) Elevated CO₂ may induce change in ion gradients across neuronal membranes, due to acid–base regulation to prevent acidosis at elevated CO₂, which may disrupt LGIC-mediated neurotransmission via the γ-aminobutyric acid (GABA) type A receptor (GABA_A R) (Nilsson et al., 2012). These mechanisms are not necessarily mutually exclusive and may interact to alter behavior at elevated CO₂.

Despite the growth in literature demonstrating elevated CO₂-induced behavioral alterations in marine invertebrates, the mechanisms responsible for these behavioral alterations are still poorly understood. Due to the diversity of invertebrate nervous and neurobiological systems, it is likely that a suite of different processes underlie these behavioral changes. Here, we discuss the potential mechanisms by which elevated CO₂ may alter marine invertebrate behavior; (1) disturbed sensation, (2) altered context within which behavioral choices are made, and (3) disrupted LGIC-mediated neurotransmission. Since the prominent hypothesis for altered LGIC-mediated transmission is the GABA hypothesis proposed in fish by Nilsson et al. (2012), here we discuss evidence for the GABA hypothesis in marine invertebrates and propose research to expand our understanding of the GABA hypothesis in marine invertebrates. We demonstrate that the effects of altered GABA_A receptor function are likely to be widespread, including non-behavioral effects. Finally, we identify other neurobiological mechanisms that should be affected if the GABA hypothesis is correct, propose alternative neurobiological mechanisms by which behavior could be altered by elevated CO₂ and suggest techniques to be utilized for future study of elevated CO₂-induced behavioral alterations.

GABA is the main inhibitory neurotransmitter in the invertebrate peripheral and central nervous systems (Lummis, 1990; Lunt, 1991), acting on the ionotropic GABA receptor (iGABA R), which is also permeable to Cl^– and HCO₃^– ions (Kaila and Voipio, 1987). Thus, the GABA hypothesis likely applies to marine invertebrates. The GABA hypothesis has been tested in marine arthropods and mollusks using pharmacological studies (Watson et al., 2014; Charpentier and Cohen, 2016; Clements et al., 2017), measurement of ion concentrations (de la Haye et al., 2012; Charpentier and Cohen, 2016) and molecular studies (Moya et al., 2016; Ren et al., 2018) (Table 1).

One method of assessing the GABA hypothesis involves administering the GABA_A R antagonist gabazine (SR-95531) (Heaulme et al., 1986). If GABA_A R functioning is altered at elevated CO₂, gabazine administration should reverse elevated CO₂-induced behavioral alterations by inhibiting channel opening and thus blocking the altered ion flow (Nilsson et al., 2012). Indeed, impaired escape behavior caused by exposure to 961 μatm pCO₂ was restored to control levels by gabazine in the jumping conch snail Gibberulus gibbosus (Watson et al., 2014). In the soft shell clam Mya arenaria, burrowing behaviors altered by CO₂ sediment levels representing present day variation were restored by gabazine (Clements et al., 2017). By contrast, in Asian shore crab larvae Hemigrapsus sanguineus, the loss of chemical cue-induced photosensitive behavior at elevated CO₂ conditions (1,380 μatm pCO₂) was not restored by gabazine (Charpentier and Cohen, 2016). These contrasting results initially appear to suggest that the GABA hypothesis applies to some, but not other, marine invertebrate taxa. However, crustacean iGABA Rs are commonly insensitive to gabazine (El Manira and Clarac, 1991; Jackel et al., 1994; Pearstein et al., 1996; Barry, 2002), meaning that gabazine may be inadequate for testing the GABA hypothesis in crustaceans.

It is interesting to note that the action of gabazine differed across control animals; gabazine significantly altered the behavior of control crab larvae (Charpentier and Cohen, 2016), had a non-significant trend of altering the behavior of control snails (Watson et al., 2014), and did not alter control clam burrowing behavior (Clements et al., 2017). As gabazine also blocks ion flow under normal conditions, preventing inhibition, over-excitation and behavioral alterations should occur in control animals. As gabazine does not appear to affect crustacean iGABA Rs, the behavioral change observed in crabs held at control CO₂ levels may be through the action of gabazine on a different pathway, such as a different receptor type. Characterizing the pharmacology of gabazine in the studied species and using a range of GABA_A R drugs will be important to confirm the GABA hypothesis is actually being tested (see the section “Pharmacological Considerations”).

Mechanistic support for the GABA hypothesis in marine invertebrates also comes from recent studies indicating changes in ion concentration and altered behavior in the same species at elevated CO₂ (Table 1). Hermit crabs Pagurus bernhardus exhibited impaired chemosensory responses to a food odor and increased hemolymph Cl^– concentration ([Cl^–]) at extremely high (12,061 μatm pCO₂) compared to control (373 μatm pCO₂) conditions (de la Haye et al., 2012). Asian shore crab larvae had altered chemical cue-induced photosensitive behavior and increased extracellular osmolality, but similar extracellular [Cl^–] at elevated CO₂ (1,380 μatm pCO₂) compared to controls (461 μatm pCO₂) (Charpentier and Cohen, 2016). However, the [Cl^–] measurements were very close to the limit of detection, which may be why no difference was observed. The increased extracellular osmolality was suggested to be due to an increase in HCO₃^– concentration ([HCO₃^–]), however, [HCO₃^–] was not directly measured. In a more recent study, sea hares Aplysia californica exposed to elevated CO₂ (1,200 and 3,000 μatm pCO₂) showed a reduced antipredator response and increased hemolymph [HCO₃^–] compared to control (400 μatm pCO₂) (Zlatkin and Heuer, 2019). Together, these studies support the hypothesis of altered [HCO₃^–] and [Cl^–] underlying altered iGABA R function and behavioral change at elevated CO₂.

Molecular studies also provide support for the GABA hypothesis in marine invertebrates (Table 1). Transcriptomic analysis of the Mediterranean pteropod Heliconoides inflatus exposed to elevated CO₂ (617–720 μatm pCO₂) for 3 days showed upregulation of the transcript encoding a GABA_A R subunit (Moya et al., 2016). However, RNA was extracted from the whole animal, potentially masking differential expression within the nervous system, and behavioral assays were not carried out. Ren et al. (2018) isolated the gene encoding the GABA_A R associated-protein (GABARAP) in the crab Portunus trituberculatus. Real-time PCR found that GABARAP mRNA levels were significantly upregulated by 4.34-fold after 6 h at 750 μatm pCO₂ and by 2.89-fold after 3 h at 1,500 μatm pCO₂, compared to control (485 μatm pCO₂). Showing a similar trend, average speed of the crab shoal’s movement was significantly higher after 3 and 6 h at 750 μatm pCO₂, and six and 12 h at 1,500 μatm pCO₂, compared to control. The increase in the GABARAP gene was suggested to assist more GABA_A Rs to cluster on neuronal membranes, which may exaggerate the impaired function of GABA_A Rs at elevated CO₂ and lead to the altered behavior (Ren et al., 2018).

If elevated CO₂ does alter HCO₃^– and Cl^– gradients across neuronal membranes, it is likely that functioning of LGICs other than the iGABA/GABA_A R, that are also permeable to these ions, will also be disrupted (Figure 2). In vertebrates, altered glycine receptor functioning at elevated CO₂ has been suggested due to its similarity to the GABA_A R (Tresguerres and Hamilton, 2017). Many invertebrates lack glycine receptors (Tsang et al., 2006); however, invertebrates possess a larger variety of LGICs than vertebrates (Dent, 2010). Glutamate-gated chloride channels have been found in mollusks (Kehoe and Vulfius, 2000; Kehoe et al., 2009) and crustaceans (Marder and Paupardin-Tritsch, 1978), and are suggested to be the invertebrate equivalent of vertebrate glycine Rs (Vassilatis et al., 1997; Kehoe and Vulfius, 2000). There is also evidence for acetylcholine-gated chloride channels in mollusks (Kehoe, 1972). Moya et al. (2016) found a range of nervous system transcripts differentially expressed at elevated CO₂ in the pteropod Heliconoides inflatus, including genes encoding for the LGICs (and associated proteins) of cholinergic, GABAergic, glutamatergic and glycinergic-like synapses (Moya et al., 2016) (Table 1). Thus, altered [Cl^–] can conceivably disrupt functioning of a range of LGICs (Figure 2). Furthermore, taxa specific differences in the presence of specific LGICs (Dent, 2010) may explain the variability of the effects of elevated CO₂ on marine invertebrate behavior.

Elevated CO₂ may not only disrupt neurotransmission, but also neuronal-glial and glial-glial signaling. Glia are non-neuronal cells present in the vertebrate and invertebrate nervous system (Pentreath, 1989; Laming et al., 2000). Initially thought to be restricted to supporting neurons, the role of glia is now understood to include active participation in nervous system functioning, thus contributing to behavior (Laming et al., 2000; Jackson and Haydon, 2008). Like many other cell types, glial cells regulate pH_i via ion exchange, including HCO₃^–/Cl^– exchange (Deitmer and Rose, 1996). GABA_A Rs are present in vertebrate glial cells (Butt and Jennings, 1994; Fraser et al., 1994). Less research has been carried out on invertebrate glia, with no research on the presence of iGABA Rs on marine invertebrate glial cells. However, leech glial cells reportedly respond to GABA [unpublished work reported in Deitmer and Rose (1996)]. If marine invertebrates are found to express iGABA Rs, elevated CO₂ may also affect information processing through glial cells. Furthermore, fluxes in H⁺ ions have been found to contribute to neuron-glia signaling (Deitmer and Rose, 1996; Laming et al., 2000), which may be disrupted by exposure to elevated CO₂ (and the resultant increase in H⁺ ions).

Changes in [Cl^–] due to acid–base regulatory mechanisms at elevated CO₂ may affect LGIC-mediated neurotransmission through a different mechanism, as well as having alternative effects on the nervous system. Cl^– has a role as a signaling effector, with changes in [Cl^–]_i affecting a range of processes including gene expression, protein activity and cell proliferation (Valdivieso and Santa-Coloma, 2019). Mammalian work has supported the role of Cl^– as a signaling effector in the nervous system, including [Cl^–]_i regulation of GABA_A R expression (Succol et al., 2012) and growth of neuronal processes (Nakajima and Marunaka, 2016). The role of Cl^– as a signaling anion has also been observed in bacterial cells, suggesting a conserved function (Valdivieso and Santa-Coloma, 2019). Thus, invertebrate Cl^– is also likely to act as a signaling effector. If altered [Cl^–]_i has similar effects in marine invertebrates, elevated CO₂ may impact nervous system functioning not only by altered iGABA R function but also changes in iGABA R expression, as well as altered growth of neuronal projections. Thus, altered [Cl^–]_i at elevated CO₂ may have effects additional to altered LGIC function, having widespread consequences.

Mechanistic studies have used targeted approaches to assess the GABA hypotheses in marine invertebrates. These approaches will also be useful to assess potential alternative mechanisms by which elevated CO₂ may alter marine invertebrate behavior (Figure 3). The GABA hypothesis has been pharmacologically assessed by administering the GABA_A R antagonist gabazine to marine invertebrates (Watson et al., 2014; Clements et al., 2017). Likewise, the administration of pharmacological agents targeting different LGICs will be useful to assess whether altered functioning of alternative LGICs may underlie behavioral changes at elevated CO₂ conditions. Due to the diversity of invertebrates and the potential for off-target effects, it will be important to use a range of pharmacological agents, particularly those shown to work in the invertebrate taxa being studied. Furthermore, pilot studies to determine the optimal drug concentration to use will be important. For example, Charpentier and Cohen (2016) tested three gabazine concentrations. Measurement of ion concentrations at control and elevated levels of CO₂ in conjunction with behavioral tests has been carried out in crabs (de la Haye et al., 2012; Charpentier and Cohen, 2016) and a mollusk (Zlatkin and Heuer, 2019). Future studies in other invertebrate taxa, with measurements made within the correct intra- and extra-cellular fluids, as well as determining the HCO₃^–/Cl^– permeability ratio will be important to calculate E_GABA at elevated CO₂ to theoretically assess the GABA hypothesis. Real-time PCR, measuring the expression level of a specific gene, has been employed to assess the GABA hypothesis in a crab (Ren et al., 2018) and will also be useful to assess alternative mechanisms, e.g., measuring the expression of genes encoding for alternative LGIC subunits, and genes involved in neuronal growth and proliferation.

These targeted approaches, however, may leave potentially relevant information unexplored. Omic technologies, such as transcriptomics and proteomics, provide a non-targeted approach in which a priori hypotheses are not required (Figure 3). Thus, data from omic approaches could unveil patterns leading to the development of novel hypotheses. For example, transcriptomics and proteomics have already been employed in fish nervous tissue, providing support for the GABA hypothesis as well as new avenues to pursue (Schunter et al., 2016, 2018; Porteus et al., 2018; Williams et al., 2019).

In marine invertebrates exposed to control and elevated CO₂ levels, transcriptomic studies so far have analyzed the whole animal and have focused on pteropods (Koh et al., 2015; Maas et al., 2015; Moya et al., 2016; Thabet et al., 2017) and sea urchins (Todgham and Hofmann, 2008; Evans et al., 2013; Padilla-Gamiño et al., 2013; Clark et al., 2019). Likewise, proteomic studies have analyzed the whole body of tubeworm larvae (Mukherjee et al., 2013), oyster larvae (Dineshram et al., 2012; Dineshram et al., 2013), barnacle larvae (Wong et al., 2011), sea snail larvae (Di et al., 2019) and clam larvae (Timmins-Schiffman et al., 2019), and a metabolomics study analyzed the whole body of a crab (Trigg et al., 2019) exposed to control or elevated CO₂. To understand the neurobiological mechanisms underlying behavioral changes, rather than the general molecular response to elevated CO₂, it will be important to carry out omic techniques on the nervous tissue, as measurements at the whole body level may mask differential expression in the nervous system due to the heterogeneity and complexity of gene/protein expression. This is exemplified by Liu et al. (2019) who found region-specific regulation of neuropeptides in the nervous tissue of crabs Callinectes sapidus exposed to elevated CO₂.

Omic technologies will provide a powerful, holistic approach to explore neurobiological mechanisms underlying behavioral change, potentially leading to the development of novel hypotheses. However, omic approaches can only determine correlational, and not causative, links between expression and behavior. Gene knockdown, in which the expression of a specific gene is reduced, will be a promising avenue for future research to determine a causative link between gene expression and behavioral change at elevated CO₂. Gene knockdown is yet to be used in elevated CO₂ behavioral research, however, gene knockdown of a heat shock protein assessed the stress tolerance of the white leg shrimp Litopenaeus vannamei to high CO₂ (Aishi et al., 2019).

There is large variability in the effects of elevated CO₂ on marine invertebrate behavior, which is likely due to the incredible diversity of marine invertebrates. Elevated CO₂ likely alters behavior via a range of mechanisms that disrupt the nervous system pathway producing behavior; from sensory input to behavioral output. These mechanisms are not necessarily mutually exclusive, and interactions between mechanisms may account for the diversity in responses. Many of these mechanisms are based on theory and lack solid experimental evidence. Mechanistic research addressing these gaps will be important, for example linking altered sensation at elevated CO₂ to behavioral change. Mechanistic fish research has focused on altered neurotransmission via disrupted GABA_A R functioning at elevated CO₂. The GABA hypothesis, as well as altered functioning of other LGICs, likely applies to marine invertebrates. Further research into the ionic properties of the iGABA R and other LGICs, including whether they are also permeable to HCO₃^–, and measuring intra- and extra-cellular ion levels in the relevant fluids at near-future CO₂ levels will be beneficial for advancing our understanding of this mechanism. The diversity of LGIC subtypes between invertebrate species, and even between nervous system regions, may explain the variability in behavioral responses. Investigating the presence of LGICs on invertebrate glial cells, other modes of neuronal-glial and glial-glial transmission, and the role of Cl^– as a signaling effector in invertebrates will help us understand the wider impact elevated CO₂ may have on nervous system functioning.

The interconnectivity of the nervous system, such as receptor cross-talk, suggests that even disruption of one component or pathway will have widespread effects. This will make understanding the neurobiological mechanisms underlying elevated CO₂-induced behavioral change extremely complex. Omics approaches will be useful in providing an untargeted, holistic approach to understand the response of the nervous system to elevated CO₂, provide support or opposition for proposed mechanisms, and likely provide new avenues to explore. Exploring the mechanisms underlying behavioral change at elevated CO₂ will help us to understand the variability in behavioral responses to elevated CO₂ and predict which marine invertebrates are likely to be the most vulnerable to rising CO₂ levels.

All authors contributed to the conception and design of the review. JT wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.