Contribution of DOC-Bound and Particle-Bound Fractions to Uptake and Toxicity of Very Hydrophobic Organic Contaminants in Sediment Toxicity Tests, Relative to the Freely Dissolved Concentration

In spiked-sediment toxicity tests, exposure of benthic organisms to hydrophobic organic contaminants (HOCs) is increasingly interpreted through the freely dissolved concentration ($C_\mathrm{free}$) in sediment porewater. For very hydrophobic chemicals, however, most of the chemical in porewater is bound to dissolved organic carbon (DOC) rather than freely dissolved: Hiki et al. (2021) measured $C_\mathrm{free}$ and total dissolved concentrations ($C_\mathrm{diss}$) simultaneously in Hyalella azteca sediment tests and found porewater $C_\mathrm{diss}/C_\mathrm{free}$ ratios of 170–220 for benzo[a]pyrene, with indirect evidence that this bound pool may add to bioaccumulation. Whether, and under which conditions of chemical hydrophobicity, organic-matter type, and organism feeding strategy, the DOC-bound and sediment-particle-bound fractions actually contribute to organismal uptake and toxicity relative to $C_\mathrm{free}$ remains unresolved. The question remains open: follow-up experiments give chemical-specific answers in both directions (DOC increased permethrin toxicity but decreased benzo[a]pyrene toxicity; fine particle-bound cypermethrin appears to add to toxicity via ingestion), and no study quantitatively partitions uptake and toxicity among the freely dissolved, DOC-bound, and particle-bound pools in a sediment test system.

Background

Hydrophobic organic contaminants (HOCs) are organic chemicals with high octanol–water partition coefficients (log $K_\mathrm{OW}$ of roughly 3 to well above 6), including polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP), pyrethroid insecticides such as permethrin and cypermethrin, and legacy pesticides such as chlorpyrifos. Because they sorb strongly to organic matter, HOCs accumulate in sediments, and regulatory risk assessment relies on spiked-sediment toxicity tests: standardized assays in which a sediment is spiked with the test chemical and benthic organisms — commonly the freshwater amphipod Hyalella azteca or the midge Chironomus dilutus — are exposed for 10 to 28 days while survival, growth, and bioaccumulation are recorded.

Benthic organisms can take up HOCs through several concurrent routes: passive diffusion from the freely dissolved chemical in porewater and overlying water, uptake of chemical bound to dissolved organic carbon (DOC, the operationally defined organic fraction passing a filter), and ingestion of contaminated sediment particles. The bioavailability literature has long debated how these routes combine. The classical view holds that only the freely dissolved concentration ($C_\mathrm{free}$, the concentration of unbound molecules in water) drives passive uptake, so DOC binding reduces bioavailability; but experiments from the 2000s onward showed that DOM- or protein-associated HOCs can nevertheless reach organism tissues (Akkanen and Kukkonen 2003; Lin et al. 2018), making the contribution of bound species a persistent controversy.

$C_\mathrm{free}$ can be measured in situ by passive sampling, most commonly solid-phase microextraction (SPME), in which a thin polymer fiber (e.g., polydimethylsiloxane) is immersed in the system, accumulates chemical by equilibrium partitioning without measurably depleting the water, and reports $C_\mathrm{free}$ through a polymer–water partition coefficient. The total dissolved concentration ($C_\mathrm{diss}$, free plus DOC-bound, obtained by conventional filtration/extraction) can be measured in parallel, so the ratio $C_\mathrm{diss}/C_\mathrm{free}$ quantifies the DOC-bound pool. International interlaboratory comparisons have advanced ex situ passive sampling of contaminated sediments into a routine risk-assessment tool (Jonker et al. 2018). Underlying this practice is equilibrium partitioning (EqP) theory, which assumes chemical equilibrium among sediment organic carbon, porewater, and organism lipids; on this basis sediment quality benchmarks are derived from water-only toxicity data applied to porewater $C_\mathrm{free}$, and mechanistic reviews support EqP-based predictions for nonionic organic chemicals (Burgess et al. 2024).

Hiki et al. (2021) developed a direct-immersion SPME method with polydimethylsiloxane-coated glass fibers and, for the first time in spiked-sediment tests, measured both $C_\mathrm{free}$ and $C_\mathrm{diss}$ in porewater and overlying water throughout tests with H. azteca exposed to phenanthrene, pyrene, BaP, and chlorpyrifos. They found the test system far from equilibrium (time-varying $C_\mathrm{diss}$ in overlying water and a vertical $C_\mathrm{free}$ gradient at the sediment–water interface); a large DOC-bound pool in porewater ($C_\mathrm{diss}/C_\mathrm{free}$ of 170–220 for BaP); that porewater $C_\mathrm{free}$ was the most representative indicator of chlorpyrifos toxicity (comparing sediment-test and water-only LC50 values, the median lethal concentrations); but, for BaP, that a literature water-only bioconcentration factor fell between the $C_\mathrm{free}$-based and $C_\mathrm{diss}$-based bioconcentration factors measured in the sediment test, suggesting the bound fraction might contribute to bioaccumulation. The authors framed the open question explicitly: it is 'a matter of ongoing debate what role the bound fraction of chemicals can play in the toxicity and bioaccumulation of HOCs,' and the role of bound species in additional uptake and toxicity for very hydrophobic chemicals 'needs to be clarified,' a clarification they suggest will require experiments with more chemicals, sediments of different properties, and species of different habitats and feeding strategies.

Problem Statement

Quantify the contribution of the DOC-bound (porewater) and sediment-particle-bound fractions of very hydrophobic organic chemicals to organism uptake and toxicity in spiked-sediment toxicity tests, relative to the freely dissolved concentration ($C_\mathrm{free}$). Specifically, all three of the following are required, and no one of them alone settles the question: (1) determine, for chemicals spanning high hydrophobicity and sorptive affinity (e.g., benzo[a]pyrene and pyrethroid insecticides with log $K_\mathrm{OW}$ of roughly 5 to above 6), whether uptake (bioconcentration) and effects (survival, growth) are predicted by porewater $C_\mathrm{free}$ alone or additionally by $C_\mathrm{diss}$ (free plus DOC-bound) and sediment/particle concentrations; (2) establish under which conditions of sediment organic-matter type and content, DOC characteristics, water-exchange regime, and test-species habitat and feeding strategy the bound fractions add to uptake and toxicity, and by how much — separating carrier-mediated uptake of bound chemical from confounding physiological effects of DOC and particles themselves; and (3) evaluate whether $C_\mathrm{free}$ is a sufficient unifying exposure metric across sediment and matched water-only exposure scenarios for these chemicals. Separating the uptake and toxicity attributable to each pool requires that $C_\mathrm{free}$ and $C_\mathrm{diss}$ be measured simultaneously.

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