Development and application of whole-sediment toxicity test using immobilized freshwater microalgae Pseudokirchneriella subcapitata

A method for a whole-sediment toxicity test using alginate immobilized microalgae Pseudokirchneriella subcapitata was developed using spiked sediments and applied to contaminated field sediment samples. For method development, a growth inhibition test (72 h) with algal beads was conducted for the sediments spiked with Cu or diuron. The method was validated by determining dose-response relationships for Cu and diuron in both fine-grained and coarse-grained sediments. The results of a spiked sediment toxicity test suggested that sediment particle size distribution (clay content) had a significant effect on the growth of P. subcapitata. The developed method using immobilized microalgae P. subcapitata beads was applied successfully in the toxicity test and toxicity identification evaluation (TIE) for the four field sediment samples. After a series of extractions with 0.01 M CaCl(2) solution, acetone, and dichloromethane, the extracted sediment, which was shown to be nontoxic to algae, was used as the control and diluent for the same sediment in the whole-sediment toxicity test. The results showed that all investigated field sediment samples were found to be toxic to the immobilized algae P. subcapitata, with their median effective concentration (EC50) values ranging from 41.4 to 79.0% after 72 h exposure. In the whole sediment TIE, growth of P. subcapitata was improved to varying degrees after adding zeolite, resin, or activated charcoal, suggesting different contributions to toxicity from ammonia, metals, and organic contaminants in the tested sediments.     

Interlaboratory comparison of a reduced volume marine sediment toxicity test method using the amphipod Ampelisca abdita

The U.S. Environmental Protection Agency has standardized methods for performing acute marine amphipod sediment toxicity tests. A test design reducing sediment volume from 200 to 50 ml and overlying water from 600 to 150 ml was recently proposed. An interlaboratory comparison was conducted to evaluate the precision of this reduced sediment volume toxicity test method using the marine amphipod Ampelisca abdita. A negative control and three sediment samples of varying degrees of toxicity ranging from low to high were tested by six laboratories. Complete agreement was reached in rank of relative toxicity for all samples tested by five out of six laboratories. Test acceptability for control survival was achieved by all laboratories, and 69% agreement in classification of the sediments as toxic or nontoxic was documented. Coefficients of variation in all test samples were similar to those reported in other interlaboratory studies using marine amphipods. Results of this study indicate that the reduced sediment volume test using A. abdita is a reliable and precise measure of acute toxicity in marine sediment samples.     

Evaluation of reduced sediment volume toxicity test procedures using the marine amphipod Ampelisca abdita

During the past 20 years, the role of sediment toxicity tests has expanded from testing of primarily dredged material to risk assessment, decontamination technologies, large-scale regional sediment-quality assessments, and toxicity identification evaluations. Sediment toxicity tests are needed that can provide reliable data using less sediment and smaller test chambers, thus utilizing resources more efficiently. We compared survival results from 10-d standard marine amphipod tests at 20 degrees C using 200 ml of sediment to reduced-volume tests containing 20 or 50 ml of sediment and found no significant differences. Similar survival also was observed in tests conducted for 7-d at 23 degrees C versus the standard 10-d exposure at 20 degrees C. However, amphipod sensitivity was significantly less in reduced-volume tests conducted for only 4- or 7-d at 20 degrees C. The 10-d sediment toxicity tests using 50 ml of sediment provided comparable results to the standard protocol, whereas time for sampling, preparing, and sieving sediments for a test was substantially reduced.     

Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae)

A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed. This microalgae showed a good growth rate during the experimental period, even when low enriched media were used. Sediment spiked with heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)] was employed to determine the EC(50) values, using microalgal growth inhibition as the endpoint. The obtained results were as follows: Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests. The EC(50) values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg(-1) for Cd, 26 mg kg(-1) for Cu, and 29 mg kg(-1) for Pb (in experimental sediment). The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment). The diatom C. closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media.     

Enhancing toxicity test performance by using a statistical criterion

Aquatic toxicity tests are laboratory experiments that measure the biological effect (e.g., growth, survival, reproduction) of effluents, receiving waters, or storm water on aquatic organisms. These toxicity tests must be performed using the best laboratory practices, and every effort must be made to enhance repeatability of the test method. We evaluated the generated reference toxicant test data for insurance of a level of quality assurance for tests over time within a laboratory and among laboratories. We recommend the reporting and evaluation of the percent minimum significant difference (PMSD) value for all toxicity test results. The minimum significant difference (MSD) represents the smallest difference between the control mean and a treatment mean that leads to the statistical rejection of the null hypothesis (i.e., no toxicity) at each concentration of the toxicity test dilution series. The MSD provides an indication of within-test variability, and smaller values of MSD are associated with increased power to detect a toxic effect. We recommend upper and lower PMSD bounds for each test method in order to minimize within-test variability and increase statistical power. To ensure that PMSD does not exceed an upper bound, testing laboratories may need to increase replication, decrease variability among replicates, or increase the control mean performance.     

Development of marine toxicity data for ordnance compounds

A toxicity database for ordnance compounds was generated using eight compounds of concern and marine toxicity tests with five species from different phyla. Toxicity tests and endpoints included fertilization success and embryological development with the sea urchin Arbacia punctulata; zoospore germination, germling length, and cell number with the green macroalga Ulva fasciata; survival and reproductive success of the polychaete Dinophilus gyrociliatus; larvae hatching and survival with the redfish Sciaenops ocellatus; and survival of juveniles of the opossum shrimp Americamysis bahia (formerly Mysidopsis bahia). The studied ordnance compounds were 2,4- and 2,6-dinitrotoluene, 2,4,6-trinitrotoluene, 1,3-dinitrobenzene, 1,3,5-trinitrobenzene, 2,4,6-trinitrophenylmethylnitramine (tetryl), 2,4,6-trinitrophenol (picric acid), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The most sensitive toxicity test endpoints overall were the macroalga zoospore germination and the polychaete reproduction tests. The most toxic ordnance compounds overall were tetryl and 1,3,5-trinitrobenzene. These were also the most degradable compounds, often being reduced to very low or below-detection levels at the end of the test exposure. Among the dinitro- and trinitrotoluenes and benzenes, toxicity tended to increase with the level of nitrogenation. Picric acid and RDX were the least toxic chemicals tested overall. Received: 12 December 2000/Accepted: 11 May 2001     

Class III metallothioneins in response to cadmium toxicity in the marine microalga Tetraselmis suecica (Kylin) Butch

Microalgae are one of the most important organisms in our ecosystems being seriously affected by metal pollution. However, the microalgae Tetraselmis suecica (Kylin) Butch is tolerant to cadmium; the concentration of this metal that reduces the population growth to 50% of the control growth level is 5.8 mg/L after 96 h of exposure. In this study, class III metallothioneins were investigated for their involvement as a possible tolerance mechanism in this microalga when exposed to cadmium. A set of these molecules was purified from these microalgal cells after exposure to the metal. These polypeptides were analyzed by capillary zone electrophoresis, which is a technique that allows the length of the metallothioneins synthesized by this microalga to be known. The T. suecica cells were able to synthesize class III metallothioneins of three to six subunits of (gamma-Glu-Cys). The most abundant polypeptide possessed four subunits, and (gamma-Glu-Cys)6-Gly was the largest polypeptide synthesized by this microalga and detected by this technique. Tolerance to cadmium as a function of increasing polypeptide length is also discussed.     

Relationships between acute sediment toxicity in laboratory tests and abundance and diversity of benthic infauna in marine sediments: a review

Acute sediment toxicity tests have become important in regulatory, monitoring, and scientific programs, partly because it has been assumed that they are indicative of ecological damage to benthic infaunal resources. Data from tests of sediment toxicity and measures of benthic community structure were examined from > 1,400 saltwater samples to determine the relationships between acute toxicity and changes in the abundance and diversity of infauna resources. Data were compiled from studies conducted along portions of the Atlantic, Gulf of Mexico, and Pacific coasts of the United States. There was considerable variability among the data sets in the relationships between laboratory results and benthic measures. However, in 92% of the samples classified as toxic, at least one measure of benthic diversity or abundance was < 50% of the average reference value. In 67% of these samples, at least one measure of benthic infauna abundance or diversity was < 10% of average reference conditions. No amphipods were found in 39% of samples that were classified as toxic, whereas amphipods were absent from 28% of the nontoxic samples. In many survey areas, the abundance of crustaceans (notably the amphipods) decreased in the infauna as amphipod survival decreased in the laboratory tests. There appeared to be a break point in the data indicating that, generally, amphipod abundance in the field was lowest when survival in the laboratory tests dropped below 50% of controls. Based on the weight of evidence from all the data analyses, we conclude that ecologically relevant losses in the abundance and diversity of the benthic infauna frequently corresponded with reduced amphipod survival in the laboratory tests.     

Toxicity of sewage sludge toRhepoxynius abronius,a marine benthic amphipod

Relative toxicity of sewage sludges from six treatment plants was determined by the LC50 of sludge-sediment mixtures to the infaunal marine amphipod,Rhepoxynius abronius. LC50s were measured as the increase in the percent total volatile solids (TVS) of the mixture due to the addition of sludge required to kill 50% of the amphipods during a 10-day exposure. LC50s ranged from 2.83% TVS addition for sludge from the small, domestic community of Waldport, Oregon to <0.1% TVS addition for metropolitan, more industrialized sources in Los Angeles, California. The toxicity of the sludge-sediment mixtures is attributed primarily to chemical contamination rather than organic enrichment. Rank correlations between toxicity and specific chemicals were usually not statistically significant, indicating that different combinations of stresses involving multiple or unmeasured factors were probably responsible for observed effects. Integrative measures of contamination (oil/grease concentration; consensus ranking of contamination based on 15 parameters) were significantly correlated withR. abronius survival indicating that the more toxic sludges had a higher overall level of contamination.     

A 48-h larval development toxicity test using the marine polychaete Galeolaria caespitosa Lamarck (Fam. Serpulidae)

Assessing the risk of effluents and other anthropogenic inputs to the receiving environment is ultimately best done on a site-specific basis, which often requires toxicity tests using organisms relevant to that environment. Additionally, the test species or life stage needs to be available for a reasonable portion of the year to allow temporal fluctuations to be assessed. A 48-h larval development toxicity test using the marine polychaete Galeolaria caespitosa was developed. This test was developed as G. caespitosa releases viable gametes year-round, and the test species is environmentally relevant to the marine system receiving the liquid effluent being evaluated. Toxicity tests were conducted using G. caespitosa from different locations and evaluating the gamete response to copper. All population responses were comparable, with EC50 values ranging from 16 to 40 μg/L copper (as CuCl₂· 2H₂O). Toxicity tests were also conducted using G. caespitosa gametes with an effluent produced by a lead smelting operation. The response of the G. caespitosa test with this effluent was compared with three other test methods using two microalgal species, Isochrysis sp. and Nitzschia closterium, and gametes from the mussel Mytilus edulis. The G. caespitosa larval development toxicity test was the most sensitive test to the effluent, with EC50 values ranging from 1–23% effluent, while it ranked second in sensitivity to copper. This test could be applied to other common serpulids worldwide. Received: 31 May 2000/Accepted: 20 November 2000     

Toxicity of bisphenol A and its bioaccumulation and removal by a marine microalga Stephanodiscus hantzschii

The toxicity of bisphenol A (BPA) to Stephanodiscus hantzschii, a diatom isolated from tidal water of Futian Mangrove Nature Reserve, China, and the bioaccumulation and removal capability of the marine microalga to BPA were investigated in the present study. Toxicity experiments showed that the 96-h EC50 of BPA was 8.65+/-0.26 mg/L, and the cell number and chlorophyll a content of S. hantzschii decreased significantly with increases in BPA at concentrations higher than 3.00 mg/L. S. hantzschii had high removal capability at low BPA concentrations as BPA was bioaccumulated and biodegraded by cells. After 16-day treatment, 88%, 99%, 92%, 61%, 48%, 28% and 26% of BPA were removed by the diatom in the media supplemented with 0.01, 0.10, 1.00, 3.00, 5.00, 7.00 and 9.00 mg/L BPA, respectively. The present study demonstrated that S. hantzschii was a tolerant isolate that could be used to remove BPA from contaminated waters.     

Establishing cause-effect relationships in hydrocarbon-contaminated sediments using a sublethal response of the benthic marine alga, Entomoneis cf punctulata

A sublethal whole-sediment toxicity test that uses flow cytometry to measure inhibition of esterase activity in the marine microalga Entomoneis cf punctulata was applied to the assessment of hydrocarbon-contaminated sediments and toxicity identification and evaluation (TIE). Concentration-response relationships were developed, and a 20% effect concentration for total polycyclic aromatic hydrocarbons (PAHs) of 60 mg/kg normalized to 1% total organic carbon was calculated. Relationships between toxic effects and sediment organic carbon concentrations, organic carbon forms (e.g., black carbon), and sediment particle size indicated that further normalization of hydrocarbon concentrations to sediment particle size may improve concentration-response relationships. The algal toxicity test was applied as a rapid whole-sediment TIE procedure that involved the addition to sediment of powdered coconut charcoal (PCC), a hydrophobic, carbon-based material that strongly adsorbs PAHs and decreases the pore-water exposure pathway. Sediments with PCC concentrations of up to 15% (w/w) provided acceptable responses in control sediments. For six sediments with total PAH concentrations of 1,060, 4,060, 5,120, 9,150, 9,900, and 15,900 mg/kg, inhibition of E. cf punctulata esterase activity (% of control) was 75, 97, 94, 93, 100, and 97%, respectively. Following a 15% PCC amendment to these sediments, inhibition of esterase activity was 0, 1, 11, 69, 32, and 68%, respectively, indicating a decrease in toxicity in all sediments. Because the alga E. cf punctulata is exposed to toxicants via both pore water and overlying water, the reduction in toxicity achieved by 15% PCC additions can be related to the efficient removal of dissolved hydrocarbons released from sediment particles. The sediment-PCC manipulations coupled with algal whole-sediment toxicity tests provided an effective and rapid TIE method to determine whether hydrocarbon contaminants are responsible for toxicity in sediments.     

Effects of triclosan on marine benthic and epibenthic organisms

Triclosan is an antimicrobial compound that has been widely used in consumer products such as toothpaste, deodorant, and shampoo. Because of its widespread use, triclosan has been detected in various environmental media, including wastewater, sewage sludge, surface waters, and sediments. Triclosan is acutely toxic to numerous aquatic organisms, but very few studies have been performed on estuarine and marine benthic organisms. For whole sediment toxicity tests, the sediment-dwelling estuarine amphipod, Ampelisca abdita, and the epibenthic mysid shrimp, Americamysis bahia, are commonly used organisms. In the present study, median lethal concentration values (LC50) were obtained for both of these organisms using water-only and whole sediment exposures. Acute 96-h water-only toxicity tests resulted in LC50 values of 73.4 and 74.3 μg/L for the amphipod and mysid, respectively. For the 7-d whole sediment toxicity test, LC50 values were 303 and 257 mg/kg (dry wt) for the amphipod and mysid, respectively. Using equilibrium partitioning theory, these whole sediment values are equivalent to interstitial water LC50 values of 230 and 190 μg/L for the amphipod and mysid, respectively, which are within a threefold difference of the observed 96-h LC50 water-only values. Triclosan was found to accumulate in polychaete tissue in a 28-d bioaccumulation study with a biota-sediment accumulation factor of 0.23 kg organic carbon/kg lipid. These data provide some of the first toxicity data for triclosan with marine benthic and epibenthic species while also indicating a need to better understand the effects of other forms of sediment carbon, triclosan ionization, and organism metabolism of triclosan on the chemical's behavior and toxicity in the aquatic environment.     

Development of a standardized reproduction toxicity test with the earthworm species Eisenia fetida andrei using copper, pentachlorophenol and 2,4-dichloroaniline

This article describes a standardized test method for determining the effect of chemical substances on the reproduction of the earthworm Eisenia fetida andrei. It is based on the existing guidelines for acute toxicity testing with earthworms, and for reasons of standardization the same artificial soil substrate and earthworm species were chosen as prescribed by these guidelines. After being preconditioned for one week in untreated soil, earthworms are exposed to the chemical substances for 3 weeks. The number of cocoons produced is determined, and cocoons are incubated in untreated artificial soil for 5 weeks to assess hatchability. Results are presented from toxicity experiments with pentachlorophenol, copper, and 2,4-dichloroaniline. For these compounds no-effect levels (NEL) for cocoon production were 32, 60-120, and 56 mg.kg-1 dry soil, respectively. Hatching of cocoons was influenced by pentachlorophenol (NEL, 10 mg.kg-1), but not by copper and dichloroaniline. Following exposure, earthworms were incubated in clean soil again to study the possibility of recovery of cocoon production. For copper and dichloroaniline earthworms did recover cocoon production to a level as high as the control level or even higher; in case of pentachlorophenol, cocoon production was still reduced after 3 weeks in clean soil.