Acute and chronic toxicity of pesticide formulations (atrazine, chlorpyrifos, and permethrin) to glochidia and juveniles of Lampsilis siliquoidea

Freshwater mussels are among the most imperiled faunal groups in North America; approximately 67% of the nearly 300 native freshwater mussel species (family Unionidae) are listed as endangered, threatened, or of special concern. Despite evidence that glochidia and juvenile life stages are highly sensitive to some chemical contaminants, the effects of pesticides on early life stages of unionid mussels are largely unknown. In the United States, pesticide registration is based on toxicity data of the active ingredient, not formulations as they are sold and applied. Some pesticide formulations, however, are more toxic than their active ingredient (technical-grade pesticide) alone because of the presence of surfactants, adjuvants, or other ingredients in the formulation. The objective of the present study was to compare the toxicity of active ingredients of several current-use pesticides (atrazine, chlorpyrifos, and permethrin) to the toxicity of pesticide formulations to glochidia and juvenile life stages of a freshwater mussel (Lampsilis siliquoidea). The atrazine formulation (Aatrex) was more toxic than technical-grade atrazine in chronic tests with juvenile L. siliquoidea. For other pesticides, acute and chronic toxicity of technical-grade pesticides were similar to the toxicity of pesticide formulations. Median effective concentrations for chlorpyrifos were 0.43 mg/L for glochidia at 48 h, 0.25 mg/L for juveniles at 96 h, and 0.06 mg/L for juveniles at 21 d. Atrazine and permethrin as well as their formulations did not cause significant acute toxicity in glochidia or juveniles at exposure concentrations approaching water-solubility limits. Additional research is needed on other pesticides with different modes of action, on the role of different routes of exposure, and with other species of unionid mussels to evaluate similarities of toxic response.     

Acute and chronic toxicity of glyphosate compounds to glochidia and juveniles of Lampsilis siliquoidea (Unionidae)

Native freshwater mussels (family Unionidae) are among the most imperiled faunal groups in the world. Factors contributing to the decline of mussel populations likely include pesticides and other aquatic contaminants; however, there is a paucity of data regarding the toxicity of even the most globally distributed pesticides, including glyphosate, to mussels. Therefore, the toxicity of several forms of glyphosate, its formulations, and a surfactant (MON 0818) used in several glyphosate formulations was determined for early life stages of Lampsilis siliquoidea, a native freshwater mussel. Acute and chronic toxicity tests were performed with a newly established American Society of Testing and Materials (ASTM) standard guide for conducting toxicity tests with freshwater mussels. Roundup, its active ingredient, the technical-grade isopropylamine (IPA) salt of glyphosate, IPA alone, and MON 0818 (the surfactant in Roundup formulations) were each acutely toxic to L. siliquoidea glochidia. MON 0818 was most toxic of the compounds tested and the 48-h median effective concentration (0.5 mg/L) for L. siliquoidea glochidia is the lowest reported for any aquatic organism tested to date. Juvenile L. siliquoidea were also acutely sensitive to MON 0818, Roundup, glyphosate IPA salt, and IPA alone. Technical-grade glyphosate and Aqua Star were not acutely toxic to glochidia or juveniles. Ranking of relative chronic toxicity of the glyphosate-related compounds to juvenile mussels was similar to the ranking of relative acute toxicity to juveniles. Growth data from chronic tests was largely inconclusive. In summary, these results indicate that L. siliquoidea, a representative of the nearly 300 freshwater mussel taxa in North America, is among the most sensitive aquatic organisms tested to date with glyphosate-based chemicals and the surfactant MON 0818.     

Acute Toxicity of Six Freshwater Mussel Species (Glochidia) to Six Chemicals: Implications for Daphnids and Utterbackia imbecillis as Surrogates for Protection of Freshwater Mussels (Unionidae)

Acute (24-h) toxicity tests were used in this study to compare lethality responses in early life stages (glochidia) of six freshwater mussel species, Leptodea fragilis, U. imbecillis, Lampsilis cardium, Lampsilis siliquoidea, Megalonaias nervosa, and Ligumia subrostrata, and two standard test organisms, Ceriodaphnia dubia and Daphnia magna. Concentrations of carbaryl, copper, 4-nonylphenol, pentachlorophenol, permethrin, and 2,4-D were used in acute exposures to represent different chemical classes and modes of action. The relative sensitivities of species were evaluated by ranking their LC₅₀ values for each chemical. We used these ranks to determine the extent to which U. imbecillis (one of the most commonly used unionids in toxicity tests) was representative of the tolerances of other mussels. We also calculated geometric mean LC₅₀s for the families Unionidae and Daphnidae. Rankings of these data were used to assess the extent to which Daphnidae can be used as surrogates for freshwater mussels relative to chemical sensitivity. While no single chemical elicited consistently high or low toxicity estimates, carbaryl and 2,4-D were generally the least toxic to all species tested. No species was always the most sensitive, and Daphnidae were generally protective of Unionidae. Utterbackia imbecillis, while often proposed as a standard unionid mussel test species, did not always qualify as a sufficient surrogate (i.e., a substitute organism that often elicits similar sensitivity responses to the same contaminant exposure) for other species of mussels, since it was usually one of the more tolerant species in our rankings. U. imbecillis should be used as a surrogate species only with this caution on its relative insensitivity.     

Partial life-cycle and acute toxicity of perfluoroalkyl acids to freshwater mussels

Freshwater mussels are among the most sensitive aquatic organisms to many contaminants and have complex life-cycles that include several distinct life stages with unique contaminant exposure pathways. Standard acute (24-96 h) and chronic (28 d) toxicity tests with free larva (glochidia) and juvenile mussels are effective at generating data on contaminant effects at two discrete life stages but do not incorporate effects on brooded glochidia. We developed a novel partial life-cycle assay that incorporates exposures to brooding adult female mussels and used this method in combination with acute toxicity tests to assess adverse effects of perfluoroctanesulfonic acid (PFOS) and perfluoroctanoic acid (PFOA) on freshwater mussels. Fatmucket (Lampsilis siliquoidea) were exposed to PFOS at two life stages: brooding glochidia (in marsupia) for 36 d and free glochidia in water for 24 h. In standard acute tests with glochidia (24-48 h exposures) and juveniles (48-96 h exposures) of fatmucket and black sandshell (Ligumia recta), glochidia were 8 to 25 times more sensitive than juveniles. Perfluoroctanesulfonic acid significantly reduced the duration of glochidia viability and reduced probability of metamorphosis at concentrations 3,000 times lower than the most sensitive acute endpoint (24-h EC50). The partial life-cycle test is adaptable to a variety of endpoints and research objectives and is useful for identifying adverse effects at contaminant concentrations below those required for an acute lethal response.     

Intra- and interlaboratory variability in acute toxicity tests with glochidia and juveniles of freshwater mussels (Unionidae)

The present study evaluated the performance and variability in acute toxicity tests with glochidia and newly transformed juvenile mussels using the standard methods outlined in American Society for Testing and Materials (ASTM). Multiple 48-h toxicity tests with glochidia and 96-h tests with juvenile mussels were conducted within a single laboratory and among five laboratories. All tests met the test acceptability requirements (e.g., >or=90% control survival). Intralaboratory tests were conducted over two consecutive mussel-spawning seasons with mucket (Actinonaias ligamentina) or fatmucket (Lampsilis siliquoidea) using copper, ammonia, or chlorine as a toxicant. For the glochidia of both species, the variability of intralaboratory median effective concentrations (EC50s) for the three toxicants, expressed as the coefficient of variation (CV), ranged from 14 to 27% in 24-h exposures and from 13 to 36% in 48-h exposures. The intralaboratory CV of copper EC50s for juvenile fatmucket was 24% in 48-h exposures and 13% in 96-h exposures. Interlaboratory tests were conducted with fatmucket glochidia and juveniles by five laboratories using copper as a toxicant. The interlaboratory CV of copper EC50s for glochidia was 13% in 24-h exposures and 24% in 48-h exposures, and the interlaboratory CV for juveniles was 22% in 48-h exposures and 42% in 96-h exposures. The high completion success and the overall low variability in test results indicate that the test methods have acceptable precision and can be performed routinely.     

Chronic toxicity of copper and ammonia to juvenile freshwater mussels (Unionidae)

The objectives of the present study were to develop methods for conducting chronic toxicity tests with juvenile mussels under flow-through conditions and to determine the chronic toxicity of copper and ammonia to juvenile mussels using these methods. In two feeding tests, two-month-old fatmucket (Lampsilis siliquoidea) and rainbow mussel (Villosa iris) were fed various live algae or nonviable algal mixture for 28 d. The algal mixture was the best food resulting in high survival (>or=90%) and growth. Multiple copper and ammonia toxicity tests were conducted for 28 d starting with two-month-old mussels. Six toxicity tests using the algal mixture were successfully completed with a control survival of 88 to 100%. Among copper tests with rainbow mussel, fatmucket, and oyster mussel (Epioblasma capsaeformis), chronic value ([ChV], geometric mean of the no-observed-effect concentration and the lowest-observed-effect concentration) ranged from 8.5 to 9.8 microg Cu/L for survival and from 4.6 to 8.5 microg Cu/L for growth. Among ammonia tests with rainbow mussel, fatmucket, and wavy-rayed lampmussel (L. fasciola), the ChV ranged from 0.37 to 1.2 mg total ammonia N/L for survival and from 0.37 to 0.67 mg N/L for growth. These ChVs were below the U.S. Environmental Protection Agency 1996 chronic water quality criterion (WQC) for copper (15 microg/L; hardness 170 mg/L) and 1999 WQC for total ammonia (1.26 mg N/L; pH 8.2 and 20 degrees C). Results indicate that toxicity tests with two-month-old mussels can be conducted for 28 d with >80% control survival; growth was frequently a more sensitive endpoint compared to survival; and the 1996 chronic WQC for copper and the 1999 chronic WQC for total ammonia might not be adequately protective of the mussel species tested. However, a recently revised 2007 chronic WQC for copper based on the biotic ligand model may be more protective in the water tested.     

Lethal and sublethal effects of ammonia to juvenile Lampsilis mussels (Unionidae) in sediment and water-only exposures

We compared the sensitivity of two juvenile unionid mussels (Lampsilis cardium and Lampsilis higginsii) to ammonia in 96-h water-only and sediment tests by use of mortality and growth measurements. Twenty mussels were placed in chambers buried 2.5 cm into reference sediments to approximate pore-water exposure (sediment tests) or elevated above the bottom of the experimental units (water-only tests). In the sediment tests, a pH gradient existed between the overlying water (mean 8.0), sediment- water interface (mean 7.7), and 2.5 cm depth (mean 7.4). We assumed that mussels were exposed to ammonia in pore water and report effect concentrations in pore water, but if they were exposed to the higher pH water, more of the ammonia would be in the toxic un-ionized (NH(3)) form. The only differences in toxicity and growth between mussel species occurred in some of the water-only tests. In sediment tests, median lethal concentrations (LC50s) ranged from 124 to 125 microg NH(3)-N/L. In water-only tests, LC50s ranged from 157 to 372 microg NH(3)-N/L. In sediment tests, median effective concentrations (EC50s based on growth) ranged from 30 to 32 microg NH(3)-N/L. Juvenile mussels in the water-only tests grew poorly and did not exhibit a dose-response relation. These data demonstrate that growth is a sensitive and valuable endpoint for studies on ammonia toxicity with juvenile freshwater mussels and that growth should be measured via sediment tests.     

Influence of pH on the acute toxicity of ammonia to juvenile freshwater mussels (fatmucket, Lampsilis siliquoidea)

The objective of the present study was to evaluate the influence of pH on the toxicity of ammonia to juvenile freshwater mussels. Acute 96-h ammonia toxicity tests were conducted with 10-d-old juvenile mussels (fatmucket, Lampsilis siliquoidea) at five pH levels ranging from 6.5 to 9.0 in flow-through diluter systems at 20 degrees C. Acute 48-h tests with amphipods (Hyalella azteca) and 96-h tests with oligochaetes (Lumbriculus variegatus) were conducted concurrently under the same test conditions to determine the sensitivity of mussels relative to these two commonly tested benthic invertebrate species. During the exposure, pH levels were maintained within 0.1 of a pH unit and ammonia concentrations were relatively constant through time (coefficient of variation for ammonia concentrations ranged from 2 to 30% with a median value of 7.9%). The median effective concentrations (EC50s) of total ammonia nitrogen (N) for mussels were at least two to six times lower than the EC50s for amphipods and oligochaetes, and the EC50s for mussels decreased with increasing pH and ranged from 88 mg N/L at pH 6.6 to 0.96 mg N/L at pH 9.0. The EC50s for mussels were at or below the final acute values used to derive the U.S. Environmental Protection Agency's acute water quality criterion (WQC). However, the quantitative relationship between pH and ammonia toxicity to juvenile mussels was similar to the average relationship for other taxa reported in the WQC. These results indicate that including mussel toxicity data in a revision to the WQC would lower the acute criterion but not change the WQC mathematical representation of the relative effect of pH on ammonia toxicity.     

Sensitivity of juvenile freshwater mussels (Lampsilis fasciola, Villosa iris) to total and un-ionized ammonia

This study evaluated the sensitivity of juveniles of two freshwater unionid mussel species (Villosa iris [Lea] and Lampsilis fasciola [Rafinesquel) to un-ionized and total ammonia. Five concentrations of ammonium chloride were tested using 96-h static-renewal toxicity tests at 12 and 20 degrees C. Based on their respective mean 96-h lethal concentration to 50% (LC50s), V. iris (0.11 mg/ L NH3-N) was more sensitive than L. fasciola (0.26 mg/L NH3-N). At 96 h, significant differences in sensitivity to un-ionized ammonia between the two temperatures were not observed for either species. Comparison of LC50s reported for other aquatic organisms to the 96-h LC50s calculated for juvenile L. fasciola and V. iris shows these two mussel species to be among the most sensitive to un-ionized ammonia. Based on reported levels of un-ionized ammonia in the aquatic environment from anthropogenic sources, un-ionized ammonia may be an important limiting toxicological factor to freshwater mussel populations.     

Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mussels (Unionidae)

The objective of the present study was to determine acute toxicity of copper, ammonia, or chlorine to larval (glochidia) and juvenile mussels using the recently published American Society for Testing and Materials (ASTM) Standard guide for conducting laboratory toxicity tests with freshwater mussels. Toxicity tests were conducted with glochidia (24- to 48-h exposures) and juveniles (96-h exposures) of up to 11 mussel species in reconstituted ASTM hard water using copper, ammonia, or chlorine as a toxicant. Copper and ammonia tests also were conducted with five commonly tested species, including cladocerans (Daphnia magna and Ceriodaphnia dubia; 48-h exposures), amphipod (Hyalella azteca; 48-h exposures), rainbow trout (Oncorhynchus mykiss; 96-h exposures), and fathead minnow (Pimephales promelas; 96-h exposures). Median effective concentrations (EC50s) for commonly tested species were >58 microg Cu/L (except 15 microg Cu/L for C. dubia) and >13 mg total ammonia N/L, whereas the EC50s for mussels in most cases were <45 microg Cu/L or <12 mg N/L and were often at or below the final acute values (FAVs) used to derive the U.S. Environmental Protection Agency 1996 acute water quality criterion (WQC) for copper and 1999 acute WQC for ammonia. However, the chlorine EC50s for mussels generally were >40 microg/L and above the FAV in the WQC for chlorine. The results indicate that the early life stages of mussels generally were more sensitive to copper and ammonia than other organisms and that, including mussel toxicity data in a revision to the WQC, would lower the WQC for copper or ammonia. Furthermore, including additional mussel data in 2007 WQC for copper based on biotic ligand model would further lower the WQC.     

An evaluation of freshwater mussel toxicity data in the derivation of water quality guidance and standards for copper

The state of Oklahoma has designated several areas as freshwater mussel sanctuaries in an attempt to provide freshwater mussel species a degree of protection and to facilitate their reproduction. We evaluated the protection afforded freshwater mussels by the U.S. Environmental Protection Agency (U.S. EPA) hardness-based 1996 ambient copper water quality criteria, the 2007 U.S. EPA water quality criteria based on the biotic ligand model and the 2005 state of Oklahoma copper water quality standards. Both the criterion maximum concentration and criterion continuous concentration were evaluated. Published acute and chronic copper toxicity data that met American Society for Testing and Materials guidance for test acceptability were obtained for exposures conducted with glochidia or juvenile freshwater mussels. We tabulated toxicity data for glochidia and juveniles to calculate 20 species mean acute values for freshwater mussels. Generally, freshwater mussel species mean acute values were similar to those of the more sensitive species included in the U.S. EPA water quality derivation database. When added to the database of genus mean acute values used in deriving 1996 copper water quality criteria, 14 freshwater mussel genus mean acute values included 10 of the lowest 15 genus mean acute values, with three mussel species having the lowest values. Chronic exposure and sublethal effects freshwater mussel data available for four species and acute to chronic ratios were used to evaluate the criterion continuous concentration. On the basis of the freshwater mussel toxicity data used in this assessment, the hardness-based 1996 U.S. EPA water quality criteria, the 2005 Oklahoma water quality standards, and the 2007 U.S. EPA water quality criteria based on the biotic ligand model might need to be revised to afford protection to freshwater mussels.     

Evaluation of Lethality and Genotoxicity in the Freshwater Mussel <Emphasis Type="Italic">Utterbackia imbecillis</Emphasis> (Bivalvia: Unionidae) Exposed Singly and in Combination to Chemicals Used in Lawn Care

Many chemicals, including fertilizers, herbicides, and insecticides, are routinely applied to turf in the care and maintenance of lawns. These chemicals have the potential to leach into nearby surface waters and adversely affect aquatic biota. In this study, we evaluated the lethal and genotoxic effects of chemicals used in lawn care on an early life stage of freshwater mussels (Utterbackia imbecillis). The chemicals tested were copper and commercial formulations of atrazine, glyphosate, carbaryl, and diazinon. Mussel glochidia were exposed to chemicals singly or in combination (equitoxic and environmentally realistic mixtures) for 24 h and toxic interactions were evaluated with Markings additive index. Genotoxicity was quantified with the alkaline single-cell gel electrophoresis assay (Comet assay). In acute tests, copper was the most toxic of all chemicals evaluated (LC50 = 37.4 µg/L) and carbaryl was the most toxic of all pesticides evaluated (LC50 = 7.9 mg/L). In comparison to other aquatic organisms commonly used in toxicity tests (e.g., amphipods, cladocerans, and chironomids), mussel glochidia were as or more sensitive to the chemicals evaluated with the exception of diazinon, where mussels were observed to be less sensitive. The combined toxicity of equitoxic and environmentally realistic mixtures to mussels was additive. Genotoxic responses were observed in mussels exposed to copper, atrazine and diazinon at levels below their respective no-observed-effect concentrations. Together, these data indicate that freshwater mussels are among the most sensitive aquatic organisms tested for some chemicals commonly used in lawn care and that DNA damage may be useful as a screening tool to evaluate potential sublethal effects of lawn care products on non-target aquatic organisms.     

Assessing the Chronic Toxicity of Atrazine, Permethrin, and Chlorothalonil to the Cladoceran Ceriodaphnia cf. dubia in Laboratory and Natural River Water

The majority of ecotoxicological data are generated from standard laboratory-based experiments with organisms exposed in nonflowing systems using highly purified water, which contains very low amounts of dissolved organic matter and suspended particulates. However, such experimental conditions are not ecologically relevant. Thus, there is a need to develop more realistic approaches to determining toxicity, including both lethal and sublethal effects. This research provides information on the effect of natural water constituents, such as suspended particulates and dissolved organic matter, in river water (RW) on the chronic toxicity (7-day reproductive impairment) of the pesticides atrazine, chlorothalonil, and permethrin to the freshwater cladoceran Ceriodaphnia cf. dubia. Standard bioassays were conducted under standard laboratory and more environmentally realistic conditions (using RW). The 7-day IC₂₅ (reproduction impairment) values of atrazine, chlorothalonil, and permethrin to C. cf. dubia ranged from 862.4 to >1000, 51.3 to 66.4, and 0.19 to 0.23 μg/L, respectively. Using the Globally Harmonized System of Classification and Labelling of Chemicals, atrazine is classified as moderately to highly toxic, whereas permethrin and chlorothalonil were both highly toxic. The presence of dissolved organic matter and suspended particles in natural RW did not significantly (p > 0.05) change the toxicity of any of the pesticides to C. cf. dubia compared with that tested in laboratory water (LW). For the tested pesticides, toxicity testing in LW provided an adequate estimate of the hazard posed.     

Acute and chronic toxicity of mercury to early life stages of the rainbow mussel, Villosa iris (Bivalvia: Unionidae)

Mercury (Hg) contamination is receiving increased attention globally because of human health and environmental concerns. Few laboratory studies have examined the toxicity of Hg on early life stages of freshwater mussels, despite evidence that glochidia and juvenile life stages are more sensitive to contaminants than adults. Three bioassays (72-h acute glochidia, 96-h acute juvenile, and 21-d chronic juvenile toxicity tests) were conducted by exposing Villosa iris to mercuric chloride salt (HgCl2). Glochidia were more sensitive to acute exposure than were juvenile mussels, as 24-, 48-, and 72-h median lethal concentration values (LC50) for glochidia were >107, 39, and 14 microg Hg/L, respectively. The 24-, 48-, 72-, and 96-h values for juveniles were 162, 135, 114, and 99 microg Hg/L, respectively. In the chronic test, juveniles exposed to Hg treatments > or = 8 microg/L grew significantly less than did control organisms. The substantial difference in juvenile test endpoints emphasizes the importance of assessing chronic exposure and sublethal effects. Overall, our study supports the use of glochidia as a surrogate life stage for juveniles in acute toxicity tests. However, as glochidia may be used only in short-term tests, it is imperative that an integrated approach be taken when assessing risk to freshwater mussels, as their unique life history is atypical of standard test organisms. Therefore, we strongly advocate the use of both glochidia and juvenile life stages for risk assessment.     

Evaluation of influence of sediment on the sensitivity of a unionid mussel (Lampsilis siliquoidea) to ammonia in 28-day water exposures

A draft update of the U.S. Environmental Protection Agency ambient water quality criteria (AWQC) for ammonia substantially lowers the ammonia AWQC, primarily due to the inclusion of toxicity data for freshwater mussels. However, most of the mussel data used in the updated AWQC were generated from water-only exposures and limited information is available on the potential influence of the presence of a substrate on the response of mussels in laboratory toxicity tests. Our recent study demonstrated that the acute sensitivity of mussels to ammonia was not influenced by the presence of substrate in 4-d laboratory toxicity tests. The objective of the current study was to determine the sensitivity of mussels to ammonia in chronic 28-d water exposures with the sediment present (sediment treatment) or absent (water-only treatment). The chronic toxicity test was conducted starting with two-month-old juvenile mussels (fatmucket, Lampsilis siliquoidea) in a flow-through diluter system, which maintained consistent pH (≈8.3) and six concentrations of total ammonia nitrogen (N) in overlying water and in sediment pore water. The chronic value (ChV, geometric mean of the no-observed-effect concentration and the lowest-observed-effect concentration) was 0.36 mg N/L for survival or biomass in the water-only treatment, and was 0.66 mg N/L for survival and 0.20 mg N/L for biomass in the sediment treatment. The 20% effect concentration (EC20) for survival was 0.63 mg N/L in the water-only treatment and was 0.86 mg N/L in the sediment treatment (with overlapping 95% confidence intervals; no EC20 for biomass was estimated because the data did not meet the conditions for any logistic regression analysis). The similar ChVs or EC20s between the water-only treatment and the sediment treatment indicate that the presence of sediment did not substantially influence the sensitivity of juvenile mussels to ammonia in the 28-d chronic laboratory water exposures.     

Effects of ammonia on juvenile unionid mussels (Lampsilis cardium) in laboratory sediment toxicity tests

Ammonia is a relatively toxic compound generated in water and sediments by heterotrophic bacteria and accumulates in sediments and pore water. Recent data suggest that unionid mussels are sensitive to un-ionized ammonia (NH3) relative to other organisms. Existing sediment exposure systems are not suitable for ammonia toxicity studies with juvenile unionids; thus, we modified a system to expose juveniles to ammonia that was continuously infused into sediments. This system maintained consistent concentrations of ammonia in pore water up to 10 d. Juvenile Lampsilis cardium mussels were exposed to NH3 in pore water in replicate 96-h and 10-d sediment toxicity tests. The 96-h median lethal concentrations (LC50s) were 127 and 165 microg NH3-N/L, and the 10-d LC50s were 93 and 140 microg NH3-N/L. The median effective concentrations (EC50s) (based on the proportion affected, including dead and inactive mussels) were 73 and 119 microg NH3-N/L in the 96-h tests and 71 and 99 microg NH3-N/L in the 10-d tests. Growth rate was substantially reduced at concentrations between 31 and 76 microg NH3-N/L. The lethality results (when expressed as total ammonia) are about one-half the acute national water quality criteria for total ammonia, suggesting that existing criteria may not protect juvenile unionids.     

Acute effects of road salts and associated cyanide compounds on the early life stages of the unionid mussel Villosa iris

The toxicity of cyanide to the early life stages of freshwater mussels (order Unionida) has remained unexplored. Cyanide is known to be acutely toxic to other aquatic organisms. Cyanide-containing compounds, such as sodium ferrocyanide and ferric ferrocyanide, are commonly added to road deicing salts as anticaking agents. The purpose of the present study was to assess the acute toxicity of three cyanide compounds (sodium cyanide, sodium ferrocyanide, and ferric ferrocyanide), two road salts containing cyanide anticaking agents (Morton and Cargill brands), a brine deicing solution (Liquidow brand), and a reference salt (sodium chloride) on glochidia (larvae) and juveniles of the freshwater mussel Villosa iris. Sodium ferrocyanide and ferric ferrocyanide were not acutely toxic to glochidia and juvenile mussels at concentrations up to 1,000 mg/L and 100 mg/L, respectively. Lowest observed effect concentrations (LOECs) for these two chemicals ranged from 10 to >1,000 mg/L. Sodium cyanide was acutely toxic to juvenile mussels, with a 96-h median effective concentration (EC50) of 1.10 mg/L, although glochidia tolerated concentrations up to 10 mg/L. The EC50s for sodium chloride, Liquidow brine, Morton road salt, and Cargill road salt were not significantly different for tests within the same life stage and test duration (range, 1.66-4.92 g/L). These results indicate that cyanide-containing anticaking agents do not exacerbate the toxicity of road salts, but that the use of road salts and brine solutions for deicing or dust control on roads may warrant further investigation.     

Chronic Toxicity of 4-Nonylphenol to Two Unionid Mussels in Water-Only Exposures

Limited studies indicate that mussels are generally insensitive to organic chemicals; however, these studies were conducted in acute or short-term exposures, and little is known about the chronic sensitivity of mussels to organic chemicals. We evaluated the chronic (28 days) toxicity of 4-nonylphenol (4-NP) to two commonly tested species of mussels: fatmucket (Lampsilis siliquoidea) and rainbow mussel (Villosa iris). By the end of the 28 days chronic exposures, mean survival was ≥93% in all treatments, but the mean dry weight and biomass of mussels at the highest exposure concentrations were significantly reduced relative to the control. The 20% effect concentrations were similar between the two species. When compared to all other tested species, fatmucket and rainbow mussels are among the top four most sensitive species to 4-NP. However, U.S. Environmental Protection Agency chronic water quality criterion of 6.6 μg 4-NP/L should protect the two mussel species.     

Toxicity of Synthetic Musks to Early Life Stages of the Freshwater Mussel Lampsilis cardium

Polycyclic musk fragrances are common additives to many consumer products. As a result of their widespread use and slow degradation rates, they are widely found in aquatic environments. This study reports on the lethal and sublethal toxicity of the polycyclic musks AHTN (Tonalide) and HHCB (Galaxolide) to glochidial (larval) and juvenile life stages of the freshwater mussel Lampsilis cardium (Rafinesque, 1820). In glochidia, 24-h median lethal concentrations (LC50s) ranged from 454 to 850 microg AHTN/L and from 1000 to >1750 microg HHCB/L (water solubility). Results for 48-h tests were similar to the 24-h tests. In 96-h tests with juveniles, we did not observe a dose-response relation between mortality and either musk. However, the growth rate was reduced by musk exposure. The median effective concentrations (EC50s, based on growth) were highly variable and ranged from 108 to 1034 microg AHTN/L and 153 to 831 microg HHCB/L. While all adverse effects occurred at concentrations that are much greater than those reported in natural waters (low microg/L to ng/L), these results indicate the potential for adverse effects on these long-lived organisms from exposure to synthetic musk fragrances.     

Changes in juvenile coho salmon electro-olfactogram during and after short-term exposure to current-use pesticides

For anadromous salmonids, olfaction is a critical sense, enabling return migration. In recent years, several pesticides have been identified that interfere with salmonid olfaction at concentrations in the microg/L range; thus, they may pose a risk to species longevity. In the present study, we investigated the acute effects of five agricultural pesticides on juvenile coho salmon (Oncorhynchus kisutch) olfaction using the electro-olfactogram (EOG), a measure of odorant-evoked field potentials. Electro-olfactogram responses to the odorant L-serine were measured during and following a 30-min exposure of the left olfactory rosette to chlorothalonil, endosulfan, glyphosate acid, iodocarb (IPBC), trifluralin, and 2,4-dichlorophenoxyacetic acid. With the relatively insoluble pesticides endosulfan and trifluralin, decreases in EOG amplitude were only apparent at relatively high concentrations (100 and 300 microg/L, respectively) following 20 min of exposure and were absent for chlorothalonil (1 mg/L). With the water-soluble herbicide glyphosate, significant EOG reductions occurred within 10 min of exposure to 1 mg/L and more rapidly with higher concentrations. Recovery of EOG post-glyphosate exposure was concentration-dependent, and complete recovery was not observed with some concentrations at 60 min postexposure. Dichlorophenoxyacetic acid only affected EOG at high concentration (100 mg/L), where it eliminated EOG within 2 min of exposure. With IPBC, EOG was decreased at 25 min of exposure to 1 microg/L; higher concentrations caused decreases to occur more rapidly. Excluding IPBC and glyphosate, all EOG reductions occurred at concentrations greater than the current Canadian water-quality guidelines and reported 96-h lethality values. Our results show that olfactory neurons can be impaired rapidly by some current-use pesticides, even at exposures in the low-microg/L range.