Title: Introduction Text: Limited information is available on the toxicity of ordnance compounds to marine organisms and the fate of these chemicals within the marine environment. These compounds may have been released to the marine environment near sites used to detonate, demilitarize, bury, or dispose of explosives. This web data sheet highlights an approach developed by NAVFAC to evaluate potential ecological risks associated with ordnance compounds in coastal sediments. This approach will help the Navy avoid costly and unnecessary remedial actions (such as capping or dredging) by identifying sites where ordnance-impacted sediments do not pose elevated risks, and/or where natural recovery of the sediments is occurring. Visual Direction: Picture of a water body and a dock with old buildings.

Title: Ordnance Compounds Text: Some typical ordnance compounds found at Navy sites include 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (1,3,5-TNB), 2,4-dinitrotoluene (DNT), 1,3-dinitrobenzene (1,3-DNB), 2,6-DNT, tetryl, royal demolition explosive (RDX), and picric acid. Visual Direction: No graphic.

Title: Background Text: In the early to mid-1990s, a number of ordnance compounds were identified in sediments located adjacent to the Jackson Park and Port Hadlock Naval facilities in Puget Sound, WA. The WA Department of Ecology requested that these ordnance compounds be removed to below detectable levels. Toxicity values for ordnance constituents in sediments were not available, which led the Navy to conduct the effort described here. The main goal of the effort was to determine whether the impacted sediments posed an ecological or human-health risk which needed to be removed in order to protect the marine environment. The estimated cost of removing impacted sediments so that concentrations of ordnance constituents are below detectable levels is $9M. Visual Direction: Map of the continental United States. State of Washington advances with locations of Port Hadlock and Jackson Park identified.

Title: Database Study (1 of 2) Text: The first phase of the Navy effort at Puget Sound focused on identifying the most sensitive marine species and the relative toxicity of different ordnance compounds. The five marine species shown at left were selected for the study, and eight ordnance compounds were tested including 2,4,6-TNT, 1,3,5-TNB, 2,4-DNT, 1,3-DNB, 2,6-DNT, tetryl, RDX, and picric acid. This testing involved exposing each species to seawater spiked with each type of ordnance compound. The effects of this exposure were measured using nine toxicity test endpoints. Data from this study was then compiled into a comprehensive database. Roll over the pictures to the left to obtain a species name. Visual Direction: Photos of five marine species used in toxicity testing: sea urchin, red fish larvae, opossum shrimp, macro-algae, and polychaete. Roll over of photo gives common and scientific names of each.

Title: Toxicity Endpoint Text: The toxicity endpoint of an ecotoxicity study is the observable effect of a toxin on the test organism. Typical endpoints are death, genetic effects, behavioral changes, and growth inhibition, but there are many others. Several standards have emerged as the most commonly used endpoints in toxicity testing. The standards used in this study are the LC50, EC50, NOEC, and LOEC. LC50 is defined as the amount of toxin present in aqueous solution that is lethal to 50% of the test organisms within the stated study time. EC50 is the effective concentration of toxin in aqueous solution that produces a specific measurable effect in 50% of the test organisms within the stated study time. NOEC is the "no observed effect concentration”, or the level below which no adverse effects are observed. Note that this concentration depends strongly on the sensitivity of the techniques used to measure the effects. LOEC is the "lowest observed effect concentration”, or the lowest concentration at which adverse effects are observed. Note that this concentration depends strongly on the sensitivity of the techniques used to measure the effects. Visual Direction: No graphic.

Title: Database Study (2 of 2) Text: As a result of this database study, the most toxic ordnance compounds were found to be tetryl and 1,3,5-TNB. In addition, the most sensitive species and endpoints were as follows: Polychaete reproduction Macro-algae germling growth Sea urchin embryological development. Visual Direction: Pictures of tetryl and 1,3,5-TNB chemical structures.

Title: Sediment Toxicity Study (1 of 2) Text: Next, a sediment toxicity study was conducted to assess the degree and extent of toxicity in the vicinity of the two Naval facilities. Sediment samples were collected from over 50 sites in Puget Sound. The sediments were first analyzed for pore water toxicity using the sea urchin fertilization and embryological development tests. Chemical analyses were then run on a subset of sediments from the most contaminated areas to identify the chemicals that might contribute to the toxicity. Chemical analyses included a suite of ordnance compounds, trace metals, polycyclic aromatic hydrocarbons (PAHs), organo-chlorinated pesticides, polychlorinated biphenyls (PCBs), and butyltins. Visual Direction: Photo of people working with samples on a boat.

Title: Sediment Toxicity Study (2 of 2) Text: Results from the sediment toxicity studies indicated that, although pore water toxicity was observed in the majority of sediments taken from the Jackson Park and Port Hadlock areas, very few samples had detectable levels of ordnance constituents. Picric acid and 2,6-DNT were detected in several samples from various locations, but only at very low concentrations. These results provided the first indication that non-ordnance related chemicals at the site were contributing to elevated sediment toxicity levels. Visual Direction: Photos of a laboratory set up and people collecting samples in the field.

Title: Toxicity Identification Evaluation (TIE) Study Text: Next, an effort was made to identify exactly which chemicals were responsible for toxicity in the Jackson Park and Port Hadlock sediments, using the U.S. EPA's Toxicity Identification Evaluation (TIE) technology. Results indicated that ordnance compounds were not responsible for toxicity at the site. Instead, toxicity was likely due to unidentified organics, metals, and dissolved ammonia in the porewater. More information about TIE studies is available at: Guide for Planning and Conducting Pore Water Toxicity Identification Evaluations (TIE) Visual Direction: Photos of people analyzing samples in a lab.

Title: Toxicity Identification Evaluation Text: A TIE separates contaminants based on their reaction to various chemical and physical manipulations. In Phase I of a TIE, sediment porewater is subjected to a series of manipulations before being retested for residual toxicity. The data are interpreted by comparing the results of the manipulated sample tests to the results of the baseline test, conducted with unaltered porewater. For example, if solid-phase extraction removes toxicity, then non-polar organic toxicants would be expected. Alternatively, if toxicity is removed by alkaline filtration and the addition of EDTA, toxicity can be attributed to one or more cationic metals (e.g., zinc and/or nickel). Through the results of Phase I studies, the general characteristics of the final porewater toxicity are more clearly defined. With this knowledge, Phase II Toxicity Identification studies are conducted, which focus and build on Phase I results to identify specific causative toxicants. For example, if organic toxicants are indicated, isolation and concentration steps (using Solid Phase Extraction [SPE] and High Pressure Liquid Chromatography [HPLC]), followed by chemical analysis, are the typical Phase II procedures. Conversely, if metals are suspected, Phase II may simply involve analyzing the effluent sample for the presence of metals. Another Phase II study that might be conducted involves the use of mock porewaters. These may be particularly useful in situations where toxicity due to total dissolved solids (TDS) is suspected. The final step in a TIE is Phase III, Toxicity Confirmation. The goals of Phase III are to: 1) confirm that the causative contaminants have been correctly identified, and 2) confirm that the causative contaminants are not changing over time. Given these goals, Phase III confirmation could involve many different types of studies. Usually, these studies are designed to quantitatively correlate measured toxicity with the concentration of the suspect toxicant. Visual Direction: No graphic.

Title: Sediment Spiking Studies (1 of 3) Text: In the third phase of the Navy effort at Puget Sound, sediment spiking was used in sediment toxicity and porewater toxicity tests to: Evaluate the impact of different sediment features, such as particle size and organic content, on the toxicity and stability of ordnance compounds. Determine the concentrations of ordnance compounds that would be toxic to the most sensitive marine organisms. Specific marine organisms and ordnance compounds were selected for this study based on previous test results. Visual Direction: Photos of people in a lab working with samples.

Title: Sediment Spiking Studies (2 of 3) Text: Uncontaminated sediment and porewater samples were collected from Puget Sound, WA and Redfish Bay, TX and spiked with tetryl, 2,6-DNT, and picric acid. Puget Sound sediment samples were mostly fine-grained silts and clays, with a total organic content (TOC) of 1.1%. Redfish Bay sediment samples were mostly sand, with a TOC of 0.1%. Amphipods were used to determine sediment toxicity; sea urchins (embryological development), polychaetes (reproduction), and macro-algae (germling growth) were used to determine porewater toxicity. Visual Direction: Photo of amphipod test jars for the picric acid concentration series.

Title: Sediment Spiking Studies (3 of 3) Text: Please roll over the graphs to the left to learn about the results from the sediment spiking studies and about the impact different sediment features have on toxicity. Visual Direction: Two bar graphs for Puget Sound and Redfish Bay: 1) % Amphipod Survival vs. Sediment Concentration for 2,6-DNT at Puget Sound and Redfish Bay and 2) EC50 for Urchin Embryo, Polychaete Reproduction and Aglae Germling Length for Picric Acid.

Title: Text: This figure shows the effects of 2,6-DNT in sandy versus fine-grained sediments. All three ordnance compounds behaved differently in sediments of varying particle size. Tetryl, 2,6-DNT, and picric acid in fine-grained sediment had negligible effect on the amphipods, suggesting that the compounds were either degraded or irreversibly bound in the sediment. Visual Direction: Rollover of graph 1.

Title: Text: Nearly all Puget Sound porewater samples were more toxic than porewater samples from Redfish Bay, regardless of the ordnance compound tested. This is probably due to the toxicity of microbial degradation products in the Puget Sound samples. Most of the Redfish Bay porewater samples were less toxic than filtered seawater spiked with the same compounds. This graph shows the effects of picric acid on porewaters from different locations. Visual Direction: Rollover of graph 2.

Title: Degradation Study (1 of 2) Text: Next, a degradation study was conducted to assess the microbial transformation and degradation of 2,6-DNT and picric acid. The effects of temperature, solar/UV radiation, and sediment type on transformation were examined, as well as the toxicity of daughter products. The study also explored whether the degradation of ordnance compounds would proceed through mineralization given appropriate time and conditions. Visual Direction: Chemical structures of picric acid and 2,6-DNT.

Title: Degradation Study (2 of 2) Text: Results from the degradation study indicated that the toxicity of biotransformation products of ordnance compounds is chemical- and species-specific. Roll over the graphs to the left to learn more about specific results from the degradation studies. Visual Direction: Three graphics: 1) DNT and 2-A-6-NT conc. over time for sandy and fine-grained sediment at 10 and 20 deg C and 2) picric acid conc. over time for fine grained and sandy soils at 10 and 20 deg C, 3) Conc. vs. Hours of SSR for 2,6-DNT and picric acid.

Title: Text: A comparison between the spectra of natural sunlight and the solar simulator indicated that the experiments were conducted at approximately 58% of the summertime solar maximum at a latitude of approximately 39ºN (approximate latitude of Denver, CO, Washington DC, Topeka, KS). Visual Direction: No graphic.

Title: Text: The biotransformation of 2,6-DNT created a chemical (2-amino-6-nitrotoluene) that was less toxic than 2,6-DNT to algae and polychaetes, but more toxic to copeopods. These graphs illustrate that the rate of biotransformation of 2,6-DNT is dependent on both sediment type and temperature. 2,6-DNT degraded faster at 20ºC than at 10ºC and faster in fine-grained sediment than sandy sediment. Visual Direction: Rollover of graph 1.

Title: Text: This set of graphs illustrates that the rate of biotransformation of picric acid is dependent on both sediment type and temperature. Biotransformation occurred more rapidly in the 20ºC, fine-grained material compared to the 10ºC, sandy sediment. Some stages in the biotransformation of picric acid produced very toxic pore waters, but at other stages toxicity was not increased. Visual Direction: Rollover of graph 2.

Title: Text: This set of graphs shows that exposure to simulated solar radiation (SSR) caused the transformation of 2,6-DNT within 72 hours, while concentrations of picric acid remained stable after 42 hours of exposure. Visual Direction: Rollover of graph 3.

Title: Summary Text: The following are key conclusions from the four-part study: There was a large range in toxicity among the eight ordnance compounds tested. The most toxic compounds (tetryl and 1,3,5-TNB) were also the most rapidly degraded under normal conditions. Sediment characteristics such as particle size and organic content played a major role in toxicity and rate of degradation. Certain ordnance compounds can biodegrade rapidly, but the rate is a function of temperature and other factors. TIE results indicated that organic compounds (PAHs, PCBs, pesticides), metals, and ammonia were likely responsible for toxic effects observed in sediments from Jackson Park and Port Hadlock. Ordnance compound concentrations in Puget Sound sediments were not toxic to marine organisms and there was no need to remove them to non-detectable levels. These study results will help the Navy to prevent the unnecessary expenditure of approximately $9M in estimated capping/dredging costs. Visual Direction: No graphic.

Title: Feedback Text: Visual Direction: Picture of a feedback form.

Title: References Text: More information on this project is available in the following references: Toxicity of Marine Sediments and Pore Water Spiked with Ordnance Compounds Development of Marine Sediment Toxicity for Ordnance Compounds and Toxicity Identification Evaluation Studies at Naval Facilities More information on toxicity studies in marine environments can be found here: More Technical Reports from USGS Marine Ecotoxicology Research Station Visual Direction: No graphic.

Title: Contact Text: For more information about this project, please contact: NFESC POC (805) 982-1656 PRTH_NFESCT2@navy.mil Visual Direction: No graphic.