Current ecotoxicity testing needs among selected U.S. federal agencies.

U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards associated with substances that may be released into the environment, including but not limited to industrial chemicals, pharmaceuticals, pesticides, food additives, and color additives. These data are used to conduct hazard assessments and evaluate potential risks to aquatic life (e.g., invertebrates, fish), birds, wildlife species, or the environment. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for data from tests utilizing animals must first be clarified. Accordingly, the objective of this review was to identify the ecotoxicity test data relied upon by U.S. federal agencies. The standards, test guidelines, guidance documents, and/or endpoints that are used to address each of the agencies' regulatory and research needs regarding ecotoxicity testing are described in the context of their application to decision-making. Testing and information use, needs, and/or requirements relevant to the regulatory or programmatic mandates of the agencies taking part in the Interagency Coordinating Committee on the Validation of Alternative Methods Ecotoxicology Workgroup are captured. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations.

Biodegradation of pyrene in sand, silt and clay fractions of sediment.

Microbial degradation is the dominant pathway for natural attenuation of PAHs in environmental compartments such as sediments, which in turn depends on the bioavailability of PAHs. The bioavailability of PAHs has seldom been studied at the sediment particle size scale. We evaluated biodegradation of pyrene by Mycobacterium vanbaalenii PYR-1 as a function of sediment particle sizes, and investigated the relationship between the rate of degradation on sand, silt and clay particles with their individual desorption kinetics measured with the Tenax extraction method. Regression analysis showed that the total organic carbon (TOC), black carbon (BC), and specific surface area (SSA) of the specific particle size fractions, instead of the particle size scale itself, were closely related (P<0.01) with the mineralization rate. While the fraction in the rapid desorption pool (F (rapid)) ranged from 0.11 to 0.38 for the whole sediments and different size groups, the fractions mineralized after 336-h incubation (0.52 to 0.72) greatly surpassed the F (rapid) values, suggesting utilization of pyrene in the slow desorption pool (F (slow)). A biodegradation model was modified by imbedding a two-phase desorption relationship describing sequential Tenax extractions. Model analysis showed that pyrene sorbed on silt and clay aggregates was directly utilized by the degrading bacteria. The enhanced bioavailability may be attributed to the higher chemical concentration, higher TOC or larger SSA in the silt and clay fractions, which appeared to overcome the reduced bioavailability of pyrene due to sorption, making pyrene on the silt and clay particles readily available to degrading microbes. This conjecture merits further investigation.

Bioavailability of sorbed phenanthrene and permethrin in sediments to Chironomus tentans.

The availability of sorbed hydrophobic organic contaminants (HOCs) to benthic organisms is important for characterizing sediment toxicity. While many studies show a correlation between the rapid desorption HOC pool and bioavailability to benthic organisms, bioavailability of the slow or very slow desorption fraction is still poorly understood. In this study, Chironomus tentans were exposed to phenanthrene (PHE) or permethrin (PM) to derive biota-sediment accumulation factors (BSAFs) in a sediment that was sequentially desorbed with Tenax extraction or amended with a charcoal to modify the distribution of PHE and PM among the rapid (f(rapid)), slow (f(slow)) and very slow (f(vslow)) desorption pools. As the desorption interval was increased, the f(rapid) quickly decreased to zero and became negligible after 12h desorption for PHE and 48h desorption for PM. However, in samples with depleted f(rapid), BSAF values were substantially greater than zero, suggesting availability of f(slow) and f(vslow). A multivariate linear regression model was further used to estimate BSAFs specific to the different desorption pools, i.e., BSAF(rapid), BSAF(slow) or BSAF(vslow). The slow desorption pool was found to be readily available to C. tentans, with BSAF(slow) values ranging from 25.3 to 73.9% of BSAF(rapid). In comparison, BSAF(vslow) ranged from 0 to 5.9% of BSAF(rapid), suggesting a lack of availability. Therefore, the kinetically slow desorption fraction is relatively bioavailable and should not be ignored in sediment toxicity assessment.

Effect of activated carbon on microbial bioavailability of phenanthrene in soils.

Bioavailability is a governing factor that controls the rate of biological degradation of hydrophobic organic contaminants in soil. Among the solid phases that can adsorb hydrophobic organic contaminants in soil, black carbon (BC) exerts a particularly significant effect on phase distribution. However, knowledge on the effect of BC on the microbial availability of polycyclic aromatic hydrocarbons in soil is still limited. In the present study, the effect of a coal-derived activated carbon on the bioavailability of phenanthrene (PHE) during its degradation by Mycobacterium vanbaalenii PYR-I was measured in three soils. The freely dissolved concentration of PHE was concurrently determined in soil solutions using disposable polydimethylsiloxane fibers. The results showed that PHE mineralization was significantly inhibited after addition of activated carbon in all test soils. After 216 h, only 5.20, 5.83, and 6.85% of PHE was degraded in the 0.5% BC-amended soils initially containing organic carbon at 0.23, 2.1, and 7.1%, respectively. Significant correlation was found between PHE degradability and freely dissolved concentration, suggesting that BC affected PHE bioavailability by decreasing chemical activity. The effect of activated carbon in the amended soils was attributed to its enhancement of soil surface areas and pore volumes. Results from the present study clearly highlighted the importance of BC for influencing the microbial availability of polycyclic aromatic hydrocarbons in soils.

Microbial availability of different forms of phenanthrene in soils.

Microbial degradation is the most important removal process for hydrophobic organic compounds (HOCs) in soil or sediment, and chemical availability is often a governing factor. However, the availability of HOCs in the sorbed forms is still a topic of debate. In this study, we applied rigorous kinetics analysis to the relationship between the freely dissolved concentration (Cfree) of phenanthrene (PHE) measured by polydimethylsiloxane (PDMS) fibers and its degradation by a PAH degrading bacterium PYR-1 under a range of soil conditions. In solutions of soils with varying organic carbon (OC) contents, Cfree of PHE decreased from 28.63 +/- 2.15 to 0.79 +/- 0.04 microg L(-1) when the soil OC content changed from 0.23 to 7.1%. Correlation analysis between Cfree and PHE mineralization rates revealed that the bacterium quickly exhausted the PHE pool available for equilibrium distribution, including Cfree and the reversibly sorbed fraction, after which the sequestered pool was utilized. In addition, unlike changes in Cfree, degradation rates of total PHE only varied by a factor of 1.6-2.1 over the same soil OC range. Regression analysis using a multivariate relationship showed that soil OC content and porosity properties such as soil surface area had a compounded effect on the microbial availability of PHE in these soils. The kinetics analysis using Cfree, as proposed in this study, may be applied to other HOCs to gain a better understanding of microbial availability under various conditions.

Measuring pyrethroids in sediment pore water using matrix-solid phase microextraction.

Pyrethroids are hydrophobic insecticides commonly used in both agricultural and urban environments. Their high toxicity to aquatic organisms, including benthic invertebrates, and detection in the sediment at many locations in California, U.S.A., have spawned interest in understanding their bioavailability in bed sediments. A recent study showed good correlation between uptake of 14C-permethrin in Chironomus tentans and solid-phase microextraction (SPME) fibers in sediments. The present study was directed at the development of an SPME technique applicable to trace levels of nonlabeled pyrethroids in sediment. Disposable polydimethylsiloxane fibers were used to detect freely dissolved pore-water concentrations of bifenthrin, fenpropathrin, cis-permethrin, trans-permethrin, cyfluthrin, cypermethrin, and esfenvalerate under agitated and static conditions. Partition equilibrium between fiber and sediment was reached in <5 d when the samples were agitated on a shaker at low speed, while much longer times (>23 d) were needed without agitation. Polydimethylsiloxane to water partition ratios (K(PDMS)) of the seven pyrethroids were measured separately and ranged from 2.83 x 10(5) to 1.89 x 10(6). When applied to field-contaminated sediments, agitated matrix-SPME was able to detect pore-water concentrations as low as 0.1 ng/L. The method developed in the present study may be coupled with bioassays to gain mechanistic understanding of factors affecting pyrethroid toxicities, and applied to field samples to better predict sediment toxicities from pyrethroid contamination.

Effects of black carbon on pyrethroid availability in sediment.

Pyrethroids are widely used synthetic insecticides with the characteristics of high hydrophobicity and broad-spectrum aquatic toxicity. Many studies indicate that black carbon (BC) plays an important role in the bioavailability of hydrophobic compounds such as polycyclic aromatic hydrocarbons and polychlorinated biphenyls in soils and sediments. However, the effect of BC on bioavailability of other compounds such as pyrethroids in sediments is less known. In this study, we simultaneously measured pyrethroid uptake into polydimethylsiloxane (PDMS) fibers and 24 h bioaccumulation in Chironomus tentans in a sediment amended with a charcoal at different rates. There were significant negative correlations between the accumulation of pyrethroids in PDMS fibers (C(PDMS)) and the charcoal level in sediment. When the charcoal content was increased from 0 to 1.0%, C(PDMS) decreased by 5.7-9.1%. Amendment of 1.5% charcoal to the original sediment decreased biota sediment accumulation factor (BSAF) of (14)C-permethrin in C. tentans from 2.8 to 1.7. The effect of charcoal was further found to be similar for the different subcellular fractions of C. tentans, including cell debris, organelles and proteins, and granules. The overall effect of charcoal on pyrethroid availability, however, was modest, and adsorption of pyrethroids on pure charcoal was found to be similar to that on sediment organic carbon. The relatively weak sorption on charcoal was likely due to the large molecular weight and sizes of pyrethroids, which might hinder their diffusion into charcoal nanopores.

Relationships between desorption intervals and availability of sediment-associated hydrophobic contaminants.

Availability is an important factor regulating the fate and toxic effects of hydrophobic organic compounds (HOCs) in soil and sediment. Many methods have been proposed for measuring HOC availability, but ambiguity exists in the selection of methods or method conditions. In this study, using pyrethroid insecticides as model HOCs, we measured their desorption kinetics from black carbon (BC)-amended sediments and used comprehensive statistical analysis to understand the dependence of the derived parameters on desorption intervals. Fitting of data from Tenax-aided depletive desorption to a three-phase model gave estimates of 11-13, 28-33, and 57-60% pyrethroid distribution in the rapid (F(rapid)), slow (F(s)), and very slow (F(vs)) desorption fractions, respectively. The desorbed fraction after 24 h, or F(24h), essentially equaled to F(rapid), while the desorbed fraction after 6 h (F(6h)) was only about half of F(rapid), suggesting that the practice of using F(6h) in lieu of F(rapid) would lead to inaccurate assessment of availability. In contrast, Pearson correlation coefficients for the desorbed fractions and uptake into polydimethylsiloxane (PDMS) fibers decreased with increasing desorption intervals, with F(6h) giving the most agreeable measurements. Therefore, while F(rapid) estimated from depletive desorption reflects the total chemical accessibility, desorbed fractions after short intervals likely provide a measure for the immediate availability, much like PDMS fibers. The use of desorbed fractions after short intervals (e.g., F(6h)) to approximate F(rapid) may give estimates substantially different from F(rapid) and therefore should be avoided.

Using disposable polydimethylsiloxane fibers to assess the bioavailability of permethrin in sediment.

Pyrethroid insecticides are widely used in both agricultural and urban environments. Pyrethroids have been frequently detected in California, USA, stream bed sediments. Pyrethroids are strongly hydrophobic so their bioavailability is determined by their sorption to sediment. In the present study, we used disposable polydimethylsiloxane (PDMS) fibers to sample from the freely dissolved (effective) permethrin concentration that governs bioaccumulation and toxicity, and tested the correlation of those measurements with uptake by Chironomus tentans. In sediments that were incrementally diluted with silica sand, both PDMS fiber and organic carbon (OC) normalized sediment concentrations were highly correlated with C. tentans permethrin uptake. However, for multiple sediments with OC ranging from 1.4 to 27%, C. tentans permethrin uptake showed a better correlation with PDMS fiber concentrations than sediment OC-normalized concentrations. We conclude that the qualitative properties of sediment OC influence permethrin phase distribution and therefore the bioavailability of permethrin in sediment-water systems. Consequently selective methods such as PDMS fibers yield improved estimates of bioaccumulation and toxicity as such methods detect freely dissolved permethrin concentrations in the sediment.

Effect of suspended solids on bioavailability of pyrethroid insecticides.

Runoff and surface-water effluents commonly contain suspended solids. Adsorption to suspended particles and the associated dissolved organic matter (DOM) may significantly decrease the freely dissolved concentration of a hydrophobic compound and, hence, its availability to aquatic organisms. In the present study, we evaluated phase distribution and bioaccumulation of two synthetic pyrethroids, bifenthrin and permethrin, in water samples containing suspended solids from different source sediments. Uptake of [14C]bifenthrin or [14C] permethrin by Daphnia magna after 24 h consistently decreased with increasing levels of suspended solids in the range of 0 to 200 mg/L. The trend of decrease was closely mimicked by pesticide accumulation on polydimethylsiloxane (PDMS) fibers exposed under the same conditions, and the ratio of body residues in D. magna to the concentration detected in the PDMS fiber was consistently around 2.4. Regression analysis showed that the pesticide adsorbed on particles or DOM was completely unavailable to D. magna for uptake during the 24-h exposure. The relative contribution of particles and DOM to the reduced bioavailability depended on the organic matter content and the texture of the source sediment. The influence from particles was predominant for sandy sediments, but contribution from DOM became comparable to or even greater than particles when the organic matter content of the source sediment was 1% or greater. The inhibitory effects of suspended solids on bioavailability should be considered when monitoring runoff and surface-water effluents for synthetic pyrethroids. The proposed PDMS method is simple and inexpensive, and it may serve as an effective option for obtaining ecotoxicologically relevant concentrations.