Introduction - California
Hyper-Salinity Toxicity Thresholds for Nine California Ocean Plan Toxicity Test ProtocolsPreliminary Draft Final ReportPrepared by:Bryn M. Phillips, Brian S. Anderson, Katie Siegler, Jennifer P. Voorhees, Scott Katz, Lydia Jennings, and Ron S. TjeerdemaUniversity of California, Davis, Department of Environmental ToxicologyPrepared for:California State Water Resources Control BoardAgreement Number 11-133-250July 16, 2012IntroductionA growing number of coastal cities in California are the exploring the potential for ocean desalination as a means to augment freshwater supplies. There are currently 21 desalination facilities proposed or operating in California. The process of ocean desalination utilizes reverse osmosis to remove salts from groundwater, reclaimed water, and seawater. This process results in production of hypersaline reverse osmosis (RO) reject brine that is discharged into the marine environment, usually in combination with a sewage treatment plant or power plant effluent. Typical RO reject brine produced using 100% seawater can have approximately twice the original salt content of seawater. In order to ensure that brine discharges do not pose risk to coastal receiving waters, it is anticipated that desalination plants will be required to monitor effluent toxicity. Monitoring requirements will likely include testing for effluent toxicity using some combination of the short-term chronic toxicity test protocols listed in the California Ocean Plan ADDIN EN.CITE <EndNote><Cite><Author>SWRCB</Author><Year>2005</Year><RecNum>266</RecNum><DisplayText>(SWRCB, 2005)</DisplayText><record><rec-number>266</rec-number><foreign-keys><key app="EN" db-id="sd5dtpzxlpapr1e5w2fpdv2odvw522st052v">266</key></foreign-keys><ref-type name="Government Document">46</ref-type><contributors><authors><author>SWRCB</author></authors></contributors><titles><title>Water Quality Control Plan for Ocean Waters of California - California Ocean Plan</title></titles><dates><year>2005</year></dates><publisher>State Water Resources Control Board, Sacramento, CA</publisher><urls></urls></record></Cite></EndNote>(SWRCB, 2005). While the tests listed in the Ocean Plan have been the subject of ongoing research, no comprehensive studies of the effects of hyper-salinity have been conducted with all seven toxicity test organisms (nine protocols when including sand dollar and urchin fertilization endpoints). The results from these tests will allow determination of the toxicity of hypersaline brine in the absence of any additional toxic constituents that may be produced during the desalination process. These results provide resource managers with background information to facilitate interpretation of toxicity test results from desalination plant effluent testing. MethodsHyper-saline Brine Toxicity TestsThe seven organisms assessed in this project are listed in the California Ocean Plan ADDIN EN.CITE <EndNote><Cite><Author>SWRCB</Author><Year>2005</Year><RecNum>266</RecNum><DisplayText>(SWRCB, 2005)</DisplayText><record><rec-number>266</rec-number><foreign-keys><key app="EN" db-id="sd5dtpzxlpapr1e5w2fpdv2odvw522st052v">266</key></foreign-keys><ref-type name="Government Document">46</ref-type><contributors><authors><author>SWRCB</author></authors></contributors><titles><title>Water Quality Control Plan for Ocean Waters of California - California Ocean Plan</title></titles><dates><year>2005</year></dates><publisher>State Water Resources Control Board, Sacramento, CA</publisher><urls></urls></record></Cite></EndNote>(SWRCB, 2005). All toxicity tests followed U.S. EPA west coast methods ADDIN EN.CITE <EndNote><Cite><Author>USEPA</Author><Year>1995</Year><RecNum>190</RecNum><DisplayText>(USEPA, 1995)</DisplayText><record><rec-number>190</rec-number><foreign-keys><key app="EN" db-id="sd5dtpzxlpapr1e5w2fpdv2odvw522st052v">190</key></foreign-keys><ref-type name="Government Document">46</ref-type><contributors><authors><author>USEPA</author></authors></contributors><titles><title>Short-term methods for estimating the chronic toxicity of effluents and receiving waters to west coast marine and estuarine organisms. EPA/600/R-95/136</title></titles><dates><year>1995</year></dates><publisher>Office of Research and Development. Washington DC, USA</publisher><urls></urls></record></Cite></EndNote>(USEPA, 1995), with the exception of the mysid test ADDIN EN.CITE <EndNote><Cite><Author>USEPA</Author><Year>2002</Year><RecNum>191</RecNum><DisplayText>(USEPA, 2002)</DisplayText><record><rec-number>191</rec-number><foreign-keys><key app="EN" db-id="sd5dtpzxlpapr1e5w2fpdv2odvw522st052v">191</key></foreign-keys><ref-type name="Government Document">46</ref-type><contributors><authors><author>USEPA</author></authors></contributors><titles><title>Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms. EPA-821-R-02-014</title></titles><dates><year>2002</year></dates><publisher>Office of Water, Washington DC, USA</publisher><urls></urls></record></Cite></EndNote>(USEPA, 2002). Short-term chronic larval development tests were conducted with the bay mussel (Mytilus galloprovincialis, Carlsbad Aquafarms), the purple sea urchin (Strongylocentrotus purpuratus, Marine Pollution Studies Laboratory), the sand dollar (Dendraster excentricus, San Diego population supplied by Dave Gutoff, and local population supplied by MPSL), and the red abalone (Haliotis rufescens, MPSL and Monterey Abalone Company). The purple sea urchin and the sand dollar were also tested with the fertilization endpoint. Giant kelp was (Macrocystis pyrifera, MPSL) was tested with the germination and germ tube growth endpoints. Topsmelt (Atherinops affinis, Aquatic Bio Systems) was tested with the survival and biomass endpoints, and mysid shrimp (Americamysis bahia, Aquatic Bio Systems) was tested with the survival and growth endpoints. All organisms were acclimated to ambient salinity prior to test initiation. Brief descriptions of the toxicity tests are below.Larval development tests were conducted in 20 mL scintillation vials containing 10 mL of test solution. Abalone and mussel tests were conducted for 48 hours, whereas urchin and sand dollar tests were conducted for 72 hours. Fertilization tests were conducted for 40 minutes in scintillation vials containing 5 mL of test solution. Giant kelp tests were conducted in 250 mL crystallizing dishes containing 200 mL of solution. Topsmelt tests were conducted in one-liter beakers containing 200 mL of tests solution and mysid tests were conducted in 600 mL beakers containing 200 mL of test solution.Testing for two organisms is currently not complete because the timing of the project did not coincide with the spawning seasons. Red abalone are usually ready to spawn by February, but as of May 2012, abalone with gonads that appear mature are still not spawning. Efforts have been made to obtain viable brood stock from other locations, but the current conditions exist statewide. Sand dollars generally spawn during the summer months. In order to get sand dollars to spawn within the project timeline, two populations were obtained from different parts of the state and held under a summertime lighting regime to bring the organisms into spawning condition. Five of the six tests are complete, and data from three of these tests are presented in the results.Sample PreparationBrine was prepared by freezing pristine natural Marine Pollution Studies Laboratory-Granite Canyon (MPSL-Granite Canyon) seawater that was filtered to 1 ?m. Brine produced using this method is recommended for effluent salinity adjustments in the U.S. EPA Guidance document (U.S. EPA 1995). Seawater was frozen overnight and the brine was separated from the frozen freshwater component. Resulting brine had a salinity of approximately 70‰. Range-finder tests consisted of six salinities ranging from 34‰ (ambient) to as high as 70‰ depending on the organism (Table x). Definitive tests were organism specific and were designed based on the result of the range-finding tests.Three tests were also conducted with Reverse Osmosis reject brine discharge from the desalination facility at the Monterey Bay Aquarium. Brine discharge from this facility ranged in salinity from 51-55‰. The brine discharge was tested in a dilution series with the larval topsmelt, mussel development, and giant kelp protocols. Test concentrations were 0, 20, 40, 60, 80, and 100% effluent, which roughly equated to 34, 38, 42, 46, 50, and 54‰.All tests consisted of a range of seawater samples adjusted with hypersaline brine to give a range of salinities and a negative control consisting of 1-um filtered seawater. Controls for the brine addition were also tested. These ambient salinity controls consisted of the same volume of brine as the highest salinity concentration. Definitive tests and effluent tests were also accompanied by copper, cadmium, or zinc reference toxicant tests, as appropriate for each protocol. In some cases the volume of brine added to create very high salinity treatments designed to affect the more salinity-tolerant test organisms exceeded the volume needed to create a high-salinity brine control. In these cases, a 50% brine control was created by combining equal parts brine (70‰) and Nanopure water (0‰), resulting in a brine control at approximately ambient salinity (35‰). The salinity of the test solutions was measured with a Fisher Accumet electrode standardized to ambient salinity (34‰). If the dilution series contained samples with salinities greater than 55‰, the electrode could not be used because hypersaline calibration standards were not available. Samples from these dilution series were measured with a refractometer. Accuracy of the refractometer was confirmed at salinities less than 55‰ using the Accumet electrode. Dissolve oxygen concentration and pH were also measured using a Fisher Accumet water quality meter and appropriate electrodes. Temperature was measured at the initiation and termination of the test using a standard thermometer, and throughout the test using a continuous temperature recorder.Data AnalysisDilution series data from the salinity tolerance tests were analyzed using Comprehensive Environmental Toxicity Information System software (CETIS 1.8.4.14, Tidepool Software, McKinleyville, CA). No observed effect concentrations (NOECs), lowest observed effect concentrations (LOECs), and median effect concentrations (EC50s for survival, development and germination) or 25% effect concentrations (EC25s for biomass, growth and germ tube length) were calculated for range-finder and definitive tests. 95% confidence limits (CL) were calculated for EC50s and EC25s. Brine controls were statistically compared to dilution controls using a separate-variance t-test to determine if the brine had a significant effect on the test organism. Dilution series data from the reference toxicant tests were analyzed for the same statistics as the salinity tolerance tests. Median effect concentrations and EC25s were plotted in control charts to determine if organism responses to reference toxicants were within the range of historic tests.ResultsQuality AssuranceControl responses for all tests were greater than U.S. EPA test acceptability criteria, and no brine control responses were significantly different from control responses, indicating that the hypersaline brine did not cause adverse effects beyond altering the salinity of the sample. Reference toxicant tests were conducted with each definitive test, and control charts were constructed for protocols that had at least three historical tests in addition to the two current tests. In cases where at least five reference toxicant tests were available for evaluation, effect concentrations (EC50 and EC25) were all within two standard deviations of a running mean (Appendix A). 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ADDIN EN.CITE.DATA (Lussier et al., 1985; Dinnel et al., 1989; Hunt and Anderson, 1989; Bailey et al., 1995). Salinity ToleranceOcean plan toxicity test protocols demonstrated a wide range of salinity tolerances, with the euryhaline topsmelt and mysid shrimp, and the giant kelp being the most tolerant to elevated salinities (Table 1). The most sensitive protocols were the marine larval development tests. Red abalone, sand dollar, and purple sea urchin were more sensitive than mussels, which also occur in estuarine environments. Although only the red abalone rangefinder test is complete, the EC50 results are highly indicative of the final definitive results, and suggest this protocol is the most sensitive to elevated salinity. The marine fertilization endpoints (purple urchin and sand dollar) were less sensitive than their corresponding development endpoints. Mysid shrimp survival was less sensitive than the echinoderm embryo development and fertilization protocols. A mysid growth effect was not observed at the same concentrations tested for mysid neonate survival because complete mortality occurred before a growth effect registered. The giant kelp germination endpoint was less sensitive than the mysid shrimp survival endpoint, but the kelp germ tube EC25 was comparable to the mysid EC50. Both topsmelt survival and biomass endpoints produced comparable summary statistics (EC50 for survival or EC25 for biomass) of approximately 60‰. Salinity tolerance values from these experiments are corroborated by results of other researchers. Pillard et al. ADDIN EN.CITE <EndNote><Cite><Author>Pillard</Author><Year>1999</Year><RecNum>490</RecNum><DisplayText>(Pillard et al., 1999)</DisplayText><record><rec-number>490</rec-number><foreign-keys><key app="EN" db-id="sd5dtpzxlpapr1e5w2fpdv2odvw522st052v">490</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Pillard, D.A.</author><author>DuFresne, D.L.</author><author>Tietge, J. E.</author><author>Evans, J.M.</author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Response of mysid shrimp (</style><style face="italic" font="default" size="100%">Mysidopsis bahia</style><style face="normal" font="default" size="100%">), sheepshead minnow (</style><style face="italic" font="default" size="100%">Cyprinodon variegatus</style><style face="normal" font="default" size="100%">), and inland silverside minnow (</style><style face="italic" font="default" size="100%">Menidia beryllina</style><style face="normal" font="default" size="100%">) to changes in artificial seawater salinity. </style></title><secondary-title>Environ Toxicol Chem</secondary-title></titles><periodical><full-title>Environ Toxicol Chem</full-title></periodical><pages>430-435</pages><volume>18</volume><dates><year>1999</year></dates><urls></urls></record></Cite></EndNote>(Pillard et al., 1999) used a modified GP2 artificial sea salt formula to produce hypersaline brine for 48h exposures using Americamysis bahia (formerly M. bahia). The 48h LC50 for A. bahia was 43.03‰. Bay and Greenstein ADDIN EN.CITE <EndNote><Cite><Author>Bay</Author><Year>1994</Year><RecNum>489</RecNum><DisplayText>(Bay and Greenstein, 1994)</DisplayText><record><rec-number>489</rec-number><foreign-keys><key app="EN" db-id="sd5dtpzxlpapr1e5w2fpdv2odvw522st052v">489</key></foreign-keys><ref-type name="Report">27</ref-type><contributors><authors><author>Bay, S.M.</author><author>Greenstein, D.</author></authors></contributors><titles><title>Toxic effects of elevated salinity and desalination waste brine, in: J.N. Cross, C. Francisco and D. Hallock (eds.), Southern California Coastal Water Research Project 1992-93 Annual Report. Southern California Coastal Water Research Project. Westminster, CA. pp. 149-153 </title></titles><dates><year>1994</year></dates><urls></urls></record></Cite></EndNote>(Bay and Greenstein, 1994) assessed salinity tolerance of giant kelp and purple sea urchin development using test solutions produced from frozen seawater brine. Solutions of 33.5 - 43‰ did not affect kelp spore germination or germ tube growth. Sea urchin development was significantly reduced at 36.5‰, a lower value than observed in the current tests. Brine Effluent ToxicitySeawater Reverse Osmosis (RO) desalination discharge effluent from the Monterey Bay Aquarium was tested with mussels, giant kelp and topsmelt protocols. The salinity of the three undiluted effluent samples ranged from 51-55‰ (Table 2). The sensitivities of the topsmelt endpoints were both greater than the salinity of the highest concentration of effluent, so no effect from the effluent was observed on topsmelt survival or growth , (NOEC=50.8; LOEC and EC50 > 50.8‰). These results are within the range of effluent tests reported elsewhere using topsmelt larvae exposed to RO effluent. For example, Weston Solutions (2007) conducted 96h topsmelt larval tests with RO concentrate from a desalination plant. In this experiment, 15 day-old topsmelt larvae were exposed to a series of RO dilutions with salinities ranging from 36 to 60‰. The NOEC, LOEC, and LC50 from these experiments were 42‰, 44‰, and 58.6‰, respectively.The sensitivity of the giant kelp germination endpoint was also greater than the salinity of the highest effluent concentration, but the germ tube growth endpoint was not. The germ tube growth EC25 (based on effluent salinity) was 51.8, whereas the mean EC25 from the tolerance tests was 47.3. These results demonstrate that the effluent did not have any greater effect on the growth of germ tubes beyond that exerted by salinity. Bay and Greenstein (1992/1993) also assessed toxicity of a RO reject brine (Diablo Canyon Power Plant), and found no effects on kelp germination and growth, and no effect on sea urchin fertilization in solutions of 10% RO brine mixed with seawater.The mussel salinity tolerance was lower than the salinity of the undiluted effluent, and a significant effect was observed in the effluent test, but the effluent EC50 (calculated from the salinity of the effluent concentrations) was the same as the mean EC50 from the salinity tolerance experiments (43.3‰). This indicates that salinity was the only component of the effluent that contributed to the observed toxicity. Table 1. No observed effect (NOEC), lowest observed effect (LOEC), and median effect concentration (EC50) or 25% effect concentration (EC25) for range-finder and definitive tests. 95% confidence limits (CL) are presented for the EC50 and EC25. Mean EC is the average of the two definitive tests. ProtocolEndpointTestMeasured Test Solution SalinitiesNOECLOECEC5095% CLMean ECRed Abalone DevelopmentRange Finder35, 42, 49, 56, 63, 7034>3437.8NA?Definitive 1?Definitive 2Sand DollarDevelopmentRange Finder34, 42, 49, 56, 63, 70<434337.8NA?Definitive 1Test completed, data not analyzed?Definitive 2Purple UrchinDevelopmentRange Finder34, 40, 47, 55, 62, 70 344036.936.9-36.9?Definitive 134, 35, 36, 37, 38, 39, 40, 41, 4235.536.837.937.8-37.9?Definitive 234, 35, 36, 37, 38, 39, 40, 41, 4237.438.638.438.4-38.438.1Sand Dollar FertilizationRange Finder34, 42, 49, 56, 63, 70<434339.038.7-39.3?Definitive 135, 38, 39, 41, 43, 45, 47, 48, 50383941.241.1-41.3?Definitive 2Test completed, data not analyzedMussel DevelopmentRange Finder34, 36, 37, 39, 41, 43, 44, 46, 48414242.342.3-42.4?Definitive 134, 40, 41, 42, 43, 44, 45, 46, 47<40.240.242.242.1-42.3?Definitive 235, 40, 41, 42, 44, 45, 46, 47, 4842.243.944.344.3-44.443.3Purple Urchin FertilizationRange Finder34, 40, 47, 55, 62, 70404743.343.3-43.4?Definitive 134, 36, 38, 39, 41, 43, 45, 46, 4841.143.044.444.3-44.5?Definitive 234, 38, 41, 42, 43, 44, 45, 46, 4741.641.944.043.9-44.144.2Mysid ShrimpSurvivalRange Finder35, 43, 49, 56, 64, 70434950.148.8-51.4?Definitive 135, 41, 45, 50, 56, 6144.950.248.047.1-48.8?Definitive 237, 42, 45, 49, 53, 5645.849.247.746.9-48.447.8EC25GrowthRange Finder35, 43, 49, 56, 64, 7049>49>49NA?Definitive 135, 41, 45, 50, 56, 6150.2>50.2>50.2NA?Definitive 237, 42, 45, 49, 53, 5649.2>49.2>49.2NA>49.7Giant KelpGerminationRange Finder35, 42, 48, 55, 63, 70495759.158.8-59.5?Definitive 134, 45, 49, 54, 59, 64495455.855.4-56.1?Definitive 235, 44, 49, 54, 59, 65444955.254.8-55.655.5EC25GrowthRange Finder35, 42, 48, 55, 63, 70495752.750.2-54.4?Definitive 134, 45, 49, 54, 59, 64<454548.344.6-51.2?Definitive 235, 44, 49, 54, 59, 65<444446.343.7-48.247.3TopsmeltSurvivalRange Finder34, 42, 48, 55, 62, 69566360.258.5-62.0Definitive 135, 45, 50, 55, 60, 65, 70556060.458.8-62.1Definitive 235, 44, 50, 54, 60, 65, 70606563.462.0-64.961.9EC25BiomassRange Finder34, 42, 48, 55, 62, 69566357.334-59.7?Definitive 135, 45, 50, 55, 60, 65, 70556057.353.3-58.7?Definitive 235, 44, 50, 54, 60, 65, 70606561.256.1-63.159.3Table 2. No observed effect (NOEC), lowest observed effect (LOEC), and median effect concentration (EC50) or 25% effect concentration (EC25) for Monterey Bay Aquarium seawater RO brine effluent tests. 95% confidence limits (CL) are presented for the EC50 and EC25. Statistics were calculated based on measured effluent salinities.ProtocolEndpointMeasured Test Solution SalinitiesNOECLOECEC5095% CLMussel Development35, 39, 43, 45, 49, 5338.842.743.343.2-43.3??Giant KelpGermination36, 41, 44, 48, 52, 5553.0>53.0>53.0NAGrowth36, 41, 44, 48, 52, 5553.0>53.051.848.3-NATopsmeltSurvival34, 37, 40, 45, 49, 5150.8>50.8>50.8NABiomass34, 37, 40, 45, 49, 5150.8>50.8>50.8NAFurther ResearchThe current project determined the tolerance of Ocean Plan organisms to hypersaline brine as measured by salinity. The seawater used to prepare the brine was from a pristine source, and the brine was generated by freezing the seawater. Further research topics could include testing brine prepared from other sources of water. For ocean disposal, discharge from some desalinization facilities will likely be combined with effluent discharges from POTWs and power plants to facilitate dilution. Additional testing should be conducted with various brine effluent mixtures. The WET protocols used in the current research were designed to provide short-term indications of chronic toxicity. Because there is some concern over the chronic effects of brine effluent on marine receiving systems, longer-term chronic toxicity studies should be conducted to confirm the WET protocols are adequately protective of ocean receiving systems impacted by hypersalinity. Of the most sensitive organisms in the current results, the red abalone is the most amenable to chronic effects evaluation. The red abalone larval development test can be extended to a settling and metamorphosis endpoint, which provides a more sensitive assessment of chronic effects. The giant kelp and topsmelt protocols can also be extended to longer-term exposures to assess chronic effects. In addition to concerns about elevated salinity in ocean receiving systems, studies are also needed to assess brine discharges in estuarine receiving systems. Because of the potential for reduced brine dilution and dispersal in these systems there is likely a greater potential for impacts on estuarine water column and benthic species in estuaries. Additional toxicity assessments could be conducted using estuarine species sensitive to salinity gradients (e.g., migrating salmonids), and using estuarine benthic infaunal species which may be impacted by negatively buoyant brine discharge plumes. References ADDIN EN.REFLIST Bailey, H.C., Miller, J.L., Miller, M.J., Dhaliwal, B.S., 1995. Application of Toxicity Identification Procedures to the Echinoderm Fertilization Assay to Identify Toxicity in a Municipal Effluent. Environ Toxicol Chem 14, 2181-2186.Bay, S.M., Greenstein, D., 1994. Toxic effects of elevated salinity and desalination waste brine, in: J.N. Cross, C. Francisco and D. Hallock (eds.), Southern California Coastal Water Research Project 1992-93 Annual Report. Southern California Coastal Water Research Project. Westminster, CA. pp. 149-153 Dinnel, P.A., Link, J.M., Stober, Q.J., Letourneau, M.W., Roberts, W.E., 1989. Comparative Sensitivity of Sea Urchin Sperm Bioassays to Metals and Pesticides. Arch Environ Contam Toxicol 18, 748-755.Hunt, J.W., Anderson, B.S., 1989. Sublethal effects o zinc and municipal effluents on larvae of the red abalone Haliotis rufescens. Marine Biology 101, 545-552.Lussier, S.M., Gentile, J.H., Walker, J., 1985. Acute and Chronic Effects of Heavy Metals and Cyanide on Mysidopsis bahia (Crustacea: Mysidacea). Aquat Toxicol 7, 25-35.Pillard, D.A., DuFresne, D.L., Tietge, J.E., Evans, J.M., 1999. Response of mysid shrimp (Mysidopsis bahia), sheepshead minnow (Cyprinodon variegatus), and inland silverside minnow (Menidia beryllina) to changes in artificial seawater salinity. . Environ Toxicol Chem 18, 430-435.SWRCB, 2005. Water Quality Control Plan for Ocean Waters of California - California Ocean Plan. State Water Resources Control Board, Sacramento, CA.USEPA, 1995. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to west coast marine and estuarine organisms. EPA/600/R-95/136. Office of Research and Development. Washington DC, USA.USEPA, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms. EPA-821-R-02-014. Office of Water, Washington DC, USA.Appendix A – Reference Toxicant Control Charts ................
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