The Toxicity of Glyphosate and Several Glyphosate Formulations to Four Species of Southwestern Australian Frogs

The acute toxicity of technical-grade glyphosate acid, glyphosate isopropylamine, and three glyphosate formulations was determined for adults of one species and tadpoles of four species of southwestern Australian frogs in 48-h static/renewal tests. The 48-h LC₅₀ values for Roundup? Herbicide (MON 2139) tested against tadpoles of Crinia insignifera, Heleioporus eyrei, Limnodynastes dorsalis, and Litoria moorei ranged between 8.1 and 32.2 mg/L (2.9 and 11.6 mg/L glyphosate acid equivalent [AE]), while the 48-h LC₅₀ values for Roundup? Herbicide tested against adult and newly metamorphosed C. insignifera ranged from 137–144 mg/L (49.4–51.8 mg/L AE). Touchdown? Herbicide (4 LC-E) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis, and L. moorei was slightly less toxic than Roundup? with 48-h LC₅₀ values ranging between 27.3 and 48.7 mg/L (9.0 and 16.1 mg/L AE). Roundup? Biactive (MON 77920) was practically nontoxic to tadpoles of the same four species producing 48-h LC₅₀ values of 911 mg/L (328 mg/L AE) for L. moorei and >1,000 mg/L (>360mg/L AE) for C. insignifera, H. eyrei, and L. dorsalis. Glyphosate isopropylamine was practically nontoxic, producing no mortality among tadpoles of any of the four species over 48 h, at concentrations between 503 and 684 mg/L (343 and 466 mg/L AE). The toxicity of technical-grade glyphosate acid (48-h LC₅₀, 81.2–121 mg/L) is likely to be due to acid intolerance. Slight differences in species sensitivity were evident, with L. moorei tadpoles showing greater sensitivity than tadpoles of the other four species. Adult and newly emergent metamorphs were less sensitive than tadpoles. Received: 19 February 1998/Accepted: 16 August 1998     

Experimental studies of rainbow trout populations exposed to field applications of Roundup® herbicide

This paper reports the effects of Roundup^®3 herbicide (MON 02139) on rainbow trout viability and behavior in several field experiments at the University of British Columbia Research Forest. Laboratory and field 96-hr LC50 values were similar: 54.8 and 52.0 mg/L. Avoidance-preference data indicated that fish would avoid lethal levels of Roundup. Operational application of Roundup at the recommended field dose of (2.2 kg a.e./ha), as well as 10× and 100× field dose resulted in no mortality to rainbow trout in field streams. Results indicate that operational spraying with this herbicide for weed control should not be detrimental to rainbow trout populations. Improper use or accidental spills of Roundup could be avoided by rainbow trout and should not be lethal if diluted in a moderately-flowing stream.     

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.     

An Ecological Risk Assessment of the Exposure and Effects of 2,4-D Acid to Rainbow Trout (<Emphasis Type="Italic">Onchorhyncus mykiss</Emphasis>)

Numerous state and federal agencies are increasingly concerned with the rapid expansion of invasive, noxious weeds across the United States. Herbicides are frequently applied as weed control measures in forest and rangeland ecosystems that frequently overlap with critical habitats of threatened and endangered fish species. However, there is little published chronic toxicity data for herbicides and fish that can be used to assess ecological risk of herbicides in aquatic environments. We conducted 96-h flowthrough acute and 30-day chronic toxicity studies with swim-up larvae and juvenile rainbow trout (Onchorhyncus mykiss) exposed to the free acid form of 2,4-D. Juvenile rainbow trout were acutely sensitive to 2,4-D acid equivalent at 494 mg/L (95% confidence interval [CI] 334–668 mg/L; 96-h ALC₅₀). Accelerated life-testing procedures, used to estimate chronic mortality from acute data, predicted that a 30-day exposure of juvenile rainbow trout to 2,4-D would result in 1% and 10% mortality at 260 and 343 mg/L, respectively. Swim-up larvae were chronically more sensitive than juveniles using growth as the measurement end point. The 30-day lowest observable effect concentration (LOEC) of 2,4-D on growth of swim-up larvae was 108 mg/L, whereas the 30-day no observable effect concentration (NOEC) was 54 mg/L. The 30-day maximum acceptable toxicant concentration (MATC) of 2,4-D for rainbow trout, determined as the geometric mean of the NOEC and the LOEC, was 76 mg/L. The acute:chronic ratio was 6.5 (i.e., 494/76). We observed no chronic effects on growth of juvenile rainbow trout at the highest concentration tested (108 mg/L). Worst-case aquatic exposures to 2,4-D (4 mg/L) occur when the herbicide is directly applied to aquatic ecosystems for aquatic weed control and resulted in a 30-day safety factor of 19 based on the MATC for growth (i.e., 76/4). Highest nontarget aquatic exposures to 2,4-D applied following terrestrial use is calculated at 0.136 mg/L and resulted in a 30-day safety factor of 559 (e.g., 76/0.163). Assessment of the exposure and response data presented herein indicates that use of 2,4-D acid for invasive weed control in aquatic and terrestrial habitats poses no substantial risk to growth or survival of rainbow trout or other salmonids, including the threatened bull trout (Salvelinus confluentus).     

Acute toxicity of hydrogen cyanide to freshwater fishes

Acute toxicity of hydrogen cyanide was determined at various temperatures from 4° to 30°C and oxygen concentrations of 3.36 to 9.26 mg/L on different life history stages of five species of fish: fathead minnow,Pimephales promelas Refinesque; bluegill,Lepomis macrochirus Rafinesque yellow perch,Perca flavescens (Mitchill); brook trout,Salvelinus fontinalis (Mitchill); and rainbow trout,Salmo gairdneri Richardson. Median lethal threshold concentrations and 96-hr LC50's were established by flow-through type bioassays. Acute toxicity varied from 57μg/L for juvenile rainbow trout to 191μg/L for field stocks of juvenile fathead minnows. Juvenile fish were more sensitive at lower temperatures and at oxygen levels below 5 mg/L. For most species juveniles were most sensitive and eggs more resistant. Paper No. 9954, Scientific Journal Series,Minnesota Agricultural Experiment Station, St. Paul, Minnesota. Research supported by theU.S. Environmental Protection Agency, Environmental Research Laboratory, Duluth, Minnesota, under Grant No. R802914.     

Comparative Toxicity of Glyphosate-Based Herbicides: Aqueous and Sediment Porewater Exposures

Glyphosate-based herbicides are widely used for aquatic weed control. However, their aquatic toxicity data, especially those on sediment, are relatively scarce. In this study, the water-only acute toxicity of three formulations based on glyphosate (Rodeo, Roundup Biactive, and Roundup) were compared using a water-column organism (cladoceran: Ceriodaphnia dubia) and a benthic organism (amphipod: Hyalella azteca). In addition, Roundup Biactive and Roundup were spiked into a clean sediment which was amended with appropriate amounts of peat moss to study the effect of different organic carbon levels (0, 0.4, 1.2, and 2.1%) on their sediment toxicity, with C. dubia exposed to overlying water or porewater prepared from the contaminated sediments. Results showed that the toxicity based on 48-h LC50s for the three herbicides in the water-only tests was Roundup (1.5–5.7 mg L^-1) > Roundup Biactive (82–120 mg L^-1) > Rodeo (225–415 mg L^-1), and H. azteca was generally more sensitive than C. dubia to these herbicides. Toxicity differences between formulations were due to the different surfactant components in these herbicides. From the porewater toxicity tests, Roundup Biactive (340 mg kg^-1) and Roundup (244 mg kg^-1) were similarly toxic in the sediment tests at 0% organic carbon, indicating that the surfactants in Roundup were considerably more adsorptive than those in Roundup Biactive to the sediment of the same organic carbon. Also, an increase in organic carbon significantly decreased the toxicity of Roundup in sediment, but not for Roundup Biactive. Sediment–porewater partitioning of glyphosate was found to be influenced by sediment organic carbon (i.e., glyphosate adsorption increased with sediment organic carbon).     

Copper,cadmium, and zinc concentrations in aquatic food chains from the upper sacramento river (california) and selected tributaries

Metals enter the Upper Sacramento River above Redding, California, primarily through Spring Creek, a tributary that receives acid-mine drainage from a US EPA Superfund site known locally as Iron Mountain Mine. Waterweed (Elodea canadensis) and aquatic insects (midge larvae, Chironomidae; and mayfly nymphs, Ephemeroptera) from the Sacramento River downstream from Spring Creek contained much higher concentrations of copper (Cu), cadmium (Cd), and zinc (Zn) than did similar taxa from nearby reference tributaries not exposed to acid-mine drainage. Aquatic insects from the Sacramento River contained especially high maximum concentrations of Cu (200 mg/kg dry weight in midge larvae), Cd (23 mg/kg dry weight in mayfly nymphs), and Zn (1,700 mg/kg dry weight in mayfly nymphs). Although not always statistically significant, whole-body concentrations of Cu, Cd, and Zn in fishes (threespine stickleback, Gasterosteus aculeatus; Sacramento sucker, Catostomus occidentalis; Sacramento squawfish, Ptychocheilus grandis; and chinook salmon, Oncorhynchus tshawytasch) from the Sacramento River were generally higher than in fishes from the reference tributaries.     

An Ecological Risk Assessment of the Acute and Chronic Toxicity of the Herbicide Picloram to the Threatened Bull Trout (<Emphasis Type="Italic">Salvelinus confluentus</Emphasis>) and the Rainbow Trout (<Emphasis Type="Italic">Onchorhyncus mykiss</Emphasis>)

We conducted acute and chronic toxicity studies of the effects of picloram acid on the threatened bull trout (Salvelinus confluentus) and the standard coldwater surrogate rainbow trout (Oncorhynchus mykiss). Juvenile fish were chronically exposed for 30 days in a proportional flow-through diluter to measured concentrations of 0, 0.30, 0.60, 1.18, 2.37, and 4.75 mg/L picloram. No mortality of either species was observed at the highest concentration. Bull trout were twofold more sensitive to picloram (30-day maximum acceptable toxic concentration of 0.80 mg/L) compared to rainbow trout (30-day maximum acceptable toxic concentration of 1.67 mg/L) based on the endpoint of growth. Picloram was acutely toxic to rainbow trout at 36 mg/L (96-h ALC50). The acute:chronic ratio for rainbow trout exposed to picloram was 22. The chronic toxicity of picloram was compared to modeled and measured environmental exposure concentrations (EECs) using a four-tiered system. The Tier 1, worst-case exposure estimate, based on a direct application of the current maximum use rate (1.1 kg/ha picloram) to a standardized aquatic ecosystem (water body of 1-ha area and 1-m depth), resulted in an EEC of 0.73 mg/L picloram and chronic risk quotients of 0.91 and 0.44 for bull trout and rainbow trout, respectively. Higher-tiered exposure estimates reduced chronic risk quotients 10-fold. Results of this study indicate that picloram, if properly applied according to the manufacturer’s label, poses little risk to the threatened bull trout or rainbow trout in northwestern rangeland environments on either an acute or a chronic basis.     

An Ecological Risk Assessment of the Acute and Chronic Effects of the Herbicide Clopyralid to Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Clopyralid (3,6-dichloro-2-pyridinecarboxylic acid) is a pyridine herbicide frequently used to control invasive, noxious weeds in the northwestern United States. Clopyralid exhibits low acute toxicity to fish, including the rainbow trout (Oncorhynchus mykiss) and the threatened bull trout (Salvelinus confluentus). However, there are no published chronic toxicity data for clopyralid and fish that can be used in ecological risk assessments. We conducted 30-day chronic toxicity studies with juvenile rainbow trout exposed to the acid form of clopyralid. The 30-day maximum acceptable toxicant concentration (MATC) for growth, calculated as the geometric mean of the no observable effect concentration (68 mg/L) and the lowest observable effect concentration (136 mg/L), was 96 mg/L. No mortality was measured at the highest chronic concentration tested (273 mg/L). The acute:chronic ratio, calculated by dividing the previously published 96-h acutely lethal concentration (96-h ALC₅₀; 700 mg/L) by the MATC was 7.3. Toxicity values were compared to a four-tiered exposure assessment profile assuming an application rate of 1.12 kg/ha. The Tier 1 exposure estimation, based on direct overspray of a 2-m deep pond, was 0.055 mg/L. The Tier 2 maximum exposure estimate, based on the Generic Exposure Estimate Concentration model (GEENEC), was 0.057 mg/L. The Tier 3 maximum exposure estimate, based on previously published results of the Groundwater Loading Effects of Agricultural Management Systems model (GLEAMS), was 0.073 mg/L. The Tier 4 exposure estimate, based on published edge-of-field monitoring data, was estimated at 0.008 mg/L. Comparison of toxicity data to estimated environmental concentrations of clopyralid indicates that the safety factor for rainbow trout exposed to clopyralid at labeled use rates exceeds 1000. Therefore, the herbicide presents little to no risk to rainbow trout or other salmonids such as the threatened bull trout.     

Using accelerated life testing procedures to compare the relative sensitivity of rainbow trout and the federally listed threatened bull trout to three commonly used rangeland herbicides (picloram, 2,4-D, and clopyralid)

We conducted 96-h static acute toxicity studies to evaluate the relative sensitivity of juveniles of the threatened bull trout (Salvelinus confluentus) and the standard cold-water surrogate rainbow trout (Onchorhyncus mykiss) to three rangeland herbicides commonly used for controlling invasive weeds in the northwestern United States. Relative species sensitivity was compared using three procedures: standard acute toxicity testing, fractional estimates of lethal concentrations, and accelerated life testing chronic estimation procedures. The acutely lethal concentrations (ALC) resulting in 50% mortality at 96 h (96-h ALC50s) were determined using linear regression and indicated that the three herbicides were toxic in the order of picloram acid > 2,4-D acid > clopyralid acid. The 96-h ALC50 values for rainbow trout were as follows: picloram, 41 mg/L; 2.4-D, 707 mg/L; and clopyralid, 700 mg/L. The 96-h ALC50 values for bull trout were as follows: picloram, 24 mg/L; 2.4-D, 398 mg/L; and clopyralid, 802 mg/L. Fractional estimates of safe concentrations, based on 5% of the 96-h ALC50, were conservative (overestimated toxicity) of regression-derived 96-h ALC5 values by an order of magnitude. Accelerated life testing procedures were used to estimate chronic lethal concentrations (CLC) resulting in 1% mortality at 30 d (30-d CLC1) for the three herbicides: picloram (1 mg/L rainbow trout, 5 mg/L bull trout), 2,4-D (56 mg/L rainbow trout, 84 mg/L bull trout), and clopyralid (477 mg/L rainbow trout; 552 mg/L bull trout). Collectively, the results indicated that the standard surrogate rainbow trout is similar in sensitivity to bull trout. Accelerated life testing procedures provided cost-effective, statistically defensible methods for estimating safe chronic concentrations (30-d CLC1s) of herbicides from acute toxicity data because they use statistical models based on the entire mortality:concentration:time data matrix.     

Acute toxicity of cadmium, lead, zinc, and their mixtures to stream-resident fish and invertebrates

The authors conducted 150 tests of the acute toxicity of resident fish and invertebrates to Cd, Pb, and Zn, separately and in mixtures, in waters from the South Fork Coeur d'Alene River watershed, Idaho, USA. Field-collected shorthead sculpin (Cottus confusus), westslope cutthroat trout (Oncorhynchus clarkii lewisi), two mayflies (Baetis tricaudatus and Rhithrogena sp.), a stonefly (Sweltsa sp.), a caddisfly (Arctopsyche sp.), a snail (Gyraulus sp.), and hatchery rainbow trout (Oncorhynchus mykiss), were tested with all three metals. With Pb, the mayflies (Drunella sp., Epeorus sp., and Leptophlebiidae), a Simuliidae black fly, a Chironomidae midge, a Tipula sp. crane fly, a Dytiscidae beetle, and another snail (Physa sp.), were also tested. Adult westslope cutthroat trout were captured to establish a broodstock to provide fry of known ages for testing. With Cd, the range of 96-h median effect concentrations (EC50s) was 0.4 to >5,329 μg/L, and the relative resistances of taxa were westslope cutthroat trout ≈ rainbow trout ≈ sculpin < other taxa; with Pb, EC50s ranged from 47 to 3,323 μg/L, with westslope cutthroat trout < rainbow trout < other taxa; and with Zn, EC50s ranged from 21 to 3,704 μg/L, with rainbow trout < westslope cutthroat trout ≈ sculpin < other taxa. With swim-up trout fry, a pattern of decreasing resistance with increasing fish size was observed. In metal mixtures, the toxicities of the three metals were less than additive on a concentration-addition basis.     

Effects of the Herbicides Roundup and Avans on Euglena gracilis

Glyphosate is the active ingredient in a range of widely used herbicides. The aim of this work is to evaluate the effects of two commercial herbicides, Roundup and Avans, on the motility, velocity, and gravitactic orientation of the aquatic flagellate Euglena gracilis. An early warning system, called ECOTOX, has been used for monitoring the different parameters of movement. The motility was not affected by Roundup and Avans after short period tests (0, 30, and 60 s). However, gravitactic orientation of the cells was affected at concentrations of 1.25 g l⁻¹ and above when treated with Avans, whereas treatments with Roundup showed no specific changes after short period tests. Velocity of the cells was affected by both herbicides, but the effects of Avans were shown to occur at lower concentrations in comparison to Roundup. Avans showed lower no observable effect concentration (NOEC) values in comparison to Roundup for the different parameters after short period tests. After long period (7 days) tests, NOEC values were similar except for the upward swimming, where Avans had a NOEC value of 100 μg l⁻¹ and Roundup 200 μg l⁻¹. The results demonstrate that Avans containing trimethylsulfonium salt of glyphosate is more toxic to E. gracilis than Roundup, which contained isopropylamine salt of glyphosate.     

The toxicity of chlorothalonil to aquatic fauna and the impact of its operational use on a pond ecosystem

Chlorothalonil is a fungicide whose heavy use in eastern Canada gives it the potential for significant aquatic contamination. Laboratory bioassays and field treatments of a pond system were undertaken to determine the toxic effects of Chlorothalonil on aquatic fauna. The 96-h LC50 of technical Chlorothalonil for rainbow trout (Oncorhynchus mykiss) was 76 g/L and was not significantly different (p < 0.05) from that of the formulated product (Bravo^® 500). The 96-h LC50 of Bravo 500 for blue mussels (Mytilus edulis) and clams (Mya arenaria) was 5.9 mg/L and 35.0 mg/L respectively, while its 48-h LC50 toDaphnia magna was between 130 g/L and 200 g/L. Chlorothalonil exposure ofDaphnia to concentrations as low as 32 g/L significantly (p < 0.05) increased the time to production of first young, but there were no delayed effects on number of young produced or growth at concentrations of 180 g/L or less. Chlorothalonil was initially accumulated by blue mussels to concentrations approximately ten times greater than exposure concentrations; however, tissue concentrations returned to the same level as exposure concentrations within 96 h. Spraying of ponds resulted in mortality of caged water boatmen (Sigara alternata) and threespine stickleback (Gasterosteus aculeatus) which could be related to chlorothalonil exposure, however, caddisfly larvae (Limnephilus sp.), freshwater clams (Psidium sp.), water beetles (Haliplus sp.), scud (Gamarus spp.) and midge larvae (Chironomidae) did not suffer substantial chlorothalonil-induced mortality. Changes in endemic benthic invertebrate abundance after sprays were not remarkable or related to treatment. Faunal impacts in the pond were generally of a smaller magnitude than were predicted by bioassay results. Factors such as dilution, adsorption to suspended particles and microbial degradation are thought to have attenuated the initial pond concentrations of Chlorothalonil, thereby reducing their toxicity.     

Comparative toxicity of two glyphosate formulations (original formulation of Roundup® and Roundup WeatherMAX®) to six North American larval anurans

The toxicity of two glyphosate formulations (the original formulation of Roundup? and Roundup WeatherMAX?) to six species of North American larval anurans was evaluated by using 96-h static, nonrenewal aqueous exposures. The 96-h median lethal concentration values (LC50) ranged from 1.80 to 4.22 mg acid equivalent (ae)/L and 1.96 to 3.26 mg ae/L for the original formulation of Roundup and Roundup WeatherMAX, respectively. Judged by LC50 values, four species were more sensitive to Roundup WeatherMAX exposures, and two species were more sensitive to the original formulation. Two of six species, Bufo fowleri (p < 0.05, F = 14.89, degrees of freedom [df] = 1) and Rana clamitans (p < 0.05, F = 18.46, df = 1), had significantly different responses to the two formulations tested. Increased sensitivity to Roundup WeatherMAX likely was due to differences in the surfactants or relative amounts of the surfactants in the two formulations. Potency slopes for exposures of the original formulation ranged from 24.3 to 92.5% mortality/mg ae/L. Thresholds ranged from 1.31 to 3.68 mg ae/L, showing an approximately three times difference in the initiation of response among species tested. For exposures of Roundup WeatherMAX, slopes ranged from 49.3 to 84.2% mortality/mg ae/L. Thresholds ranged from 0.83 to 2.68 mg ae/L. Margins of safety derived from a simulated direct overspray were above 1, except for one species in exposures of Roundup WeatherMAX. Laboratory data based on aqueous exposures are conservative because of the lack of environmental ligands; however, these tests provide information regarding the relative toxicity between these two Roundup formulations.     

Effect of Glyphosate on the Development of Pseudosuccinea columella Snails

Glyphosate (Roundup) is one of the most commonly used broad-spectrum herbicides with little to no hazard to animals, man, or the environment. Due to its widespread use, there is continuous contamination of the environment in both soil and water with this herbicide. There is a paucity of long-term exposure studies with sublethal concentrations of glyphosate on aquatic snails. This study was developed to determine the effects of sublethal concentrations of glyphosate on development and survival of Pseudosuccinea columella (intermediate snail host of Fasciola hepatica). This was assessed by continuously exposing three successive generations of snails to varying concentrations (0.1–10 mg/L) of glyphosate. Glyphosate had little effect on the first- and second-generation snails. However, third-generation snail embryos exposed to 1.0 mg/L glyphosate developed much faster than other embryos exposed to 0.1 mg/L, 10 mg/L, and 0 mg/L (control). Hatching was inhibited at 10 mg/L and inhibited slightly at 0.1 mg/L. The egg-laying capacity was increased in snails exposed to 0.1 and 10 mg/L. Abnormalities and polyembryony were observed in snails exposed to 0.1 and 10 mg/L. These results indicate that glyphosate does affect snail reproduction and development. This, in turn, could possibly have an effect on the population dynamics of F. hepatica, which could result in increased infections in animals, including man. Received: 15 November 1996/Accepted: 15 July 1997     

Exposure to a Commercial Glyphosate Formulation (Roundup<Superscript>®</Superscript>) Alters Normal Gill and Liver Histology and Affects Male Sexual Activity of <Emphasis Type="Italic">Jenynsia multidentata</Emphasis> (Anablepidae,Cyprinodontiformes)

Roundup is the most popular commercial glyphosate formulation applied in the cultivation of genetically modified glyphosate-resistant crops. The aim of this study was to evaluate the histological lesions of the neotropical native fish, Jenynsia multidentata, in response to acute and subchronic exposure to Roundup and to determine if subchronic exposure to the herbicide causes changes in male sexual activity of individuals exposed to a sublethal concentration (0.5 mg/l) for 7 and 28 days. The estimated 96-h LC₅₀ was 19.02 mg/l for both male and female fish. Gill and liver histological lesions were evaluated through histopathological indices allowing quantification of the histological damages in fish exposed to different concentrations of the herbicide. Roundup induced different histological alterations in a concentration-dependent manner. In subchronic-exposure tests, Roundup also altered normal histology of the studied organs and caused a significant decrease in the number of copulations and mating success in male fish exposed to the herbicide. It is expected that in natural environments contaminated with Roundup, both general health condition and reproductive success of J. multidenatata could be seriously affected.     

Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats

Background: There is intense debate on whether glyphosate can inhibit the shikimate pathway of gastrointestinal microorganisms, with potential health implications.Objectives: We tested whether glyphosate or its representative EU herbicide formulation Roundup MON 52276 affects the rat gut microbiome.Methods: We combined cecal microbiome shotgun metagenomics with serum and cecum metabolomics to assess the effects of glyphosate [0.5, 50, 175mg/kg body weight (BW) per day] or MON 52276 at the same glyphosate-equivalent doses, in a 90-d toxicity test in rats.Results: Glyphosate and MON 52276 treatment resulted in ceca accumulation of shikimic acid and 3-dehydroshikimic acid, suggesting inhibition of 5-enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway in the gut microbiome. Cysteinylglycine, γ-glutamylglutamine, and valylglycine levels were elevated in the cecal microbiome following glyphosate and MON 52276 treatments. Altered cecum metabolites were not differentially expressed in serum, suggesting that the glyphosate and MON 52276 impact on gut microbial metabolism had limited consequences on physiological biochemistry. Serum metabolites differentially expressed with glyphosate treatment were associated with nicotinamide, branched-chain amino acid, methionine, cysteine, and taurine metabolism, indicative of a response to oxidative stress. MON 52276 had similar, but more pronounced, effects than glyphosate on the serum metabolome. Shotgun metagenomics of the cecum showed that treatment with glyphosate and MON 52276 resulted in higher levels of Eggerthella spp., Shinella zoogleoides, Acinetobacter johnsonii, and Akkermansia muciniphila. Shinella zoogleoides was higher only with MON 52276 exposure. In vitro culture assays with Lacticaseibacillus rhamnosus strains showed that Roundup GT plus inhibited growth at concentrations at which MON 52276 and glyphosate had no effect.Discussion: Our study highlights the power of multi-omics approaches to investigate the toxic effects of pesticides. Multi-omics revealed that glyphosate and MON 52276 inhibited the shikimate pathway in the rat gut microbiome. Our findings could be used to develop biomarkers for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on humans. https://doi.org/10.1289/EHP6990     

Influence of exposure duration on the toxicity of triclopyr ester to fish and aquatic insects

Flow-through toxicity tests were conducted to determine the effects of exposure time on the toxicity of triclopyr butoxyethyl ester (Garlon^® 4) to fish (rainbow trout and chinook salmon) and stream insects (Hydropsyche sp. and Isonychia sp.). The toxicity of triclopyr ester to fish increased with exposure time, but the rate of increase in toxicity declined with increasing exposure duration. Median lethal concentrations for rainbow trout exposed for 1, 6, or 24 h were 22.5, 1.95, and 0.79 mg/L triclopyr ester (expressed as acid equivalent, nominal concentrations), respectively. Comparable values for chinook salmon were 34.6, 4.7, and 1.76 mg/L. The toxicity of triclopyr ester to aquatic insects also increased with increasing exposure time, but was considerably less than the toxicity to fish. There was no significant mortality (chi-square p>0.05) of insects following 3-h exposures to the maximum test concentration of approximately 110 mg/L. Median lethal concentrations following 9- and 24-h exposures were 14.9 and 4.0 ml/L for Hydropsyche sp., and 37.0 and 8.8 mg/L for Isonychia sp., respectively. At each exposure time in the toxicity tests, there was a sharp increase in mortality over relatively small increases in concentration, resulting in extremely steep slopes of the probit lines (6.3–33.8), and indicating an apparent response threshold. The herbicide exhibited delayed lethal effects, particularly in fish, but only at short term exposures to higher concentrations. The risk of adverse effects on fish and aquatic insects from triclopyr ester contamination, based on the results of these time-toxicity tests, is discussed.     

Acute and embryo-larval toxicity of phenolic compounds to aquatic biota

Because of the prevalence of phenolic compounds in various types of effluents, both acute and embryo-larval bioassays were performed on eight phenolic compounds with rainbow trout, fathead minnows andDaphnia pulicaria. In flow-through bioassays, the 96-hr LC₅₀ values for rainbow trout and fathead minnows ranged from <0.1 mg/L for hydroquinone to >100 mg/L for resorcinol.Daphnia pulicaria was consistently the least sensitive species tested as measured in 48-hr bioassays, while fathead minnows and rainbow trout varied in their relative sensitivity to phenolics as measured in 96-hr tests. Fathead minnows were more sensitive to phenol at 25°C than at 14°C.In embryo-larval bioassays with phenol, fathead minnow growth was significantly reduced by 2.5 mg/L phenol, while rainbow trout growth was significantly reduced by 0.20 mg/L phenol. For both species the embryolarval effects concentration was 1.1% of the 96-hr LC₅₀. Another embryolarval bioassay was attempted withp-benzoquinone, a highly toxic phenolic compound found in fossil fuel processing wastewaters, which was discontinued because the compound was rapidly degraded chemically or biologically in the headtank and aquaria.Work funded under an Interagency Agreement between the U.S. Department of Energy and the U.S. Environmental Protection Agency under Contract No. DE-AS20-79 LC 01761 to the Rocky Mountain Institute of Energy and Environment, University of Wyoming.     

Use of Behavioral Responses of Rainbow Trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> in Identifying Sublethal Exposure to Hexavalent Chromium

Laboratory tests were conducted in a flow-through apparatus on 1-year-old rainbow trout Oncorhynchus mykiss to evaluate the sensitivity of a number of their behavioral responses to hexavalent chromium (Cr⁶⁺). Test fish were exposed to Cr⁶⁺ concentrations corresponding to 0.001–1 parts of the rainbow trout 96-h LC50 (0.029–28.5 mg Cr/L, respectively) in short-term (15 min) tests. Sensitivity parameter responses could be arranged into the following sequence: latent period of detection response = locomotor activity > gill ventilation frequency > coughing rate. All the rainbow trout responses were sensitive behavioral indicators of sublethal exposure. Behavioral responses meet the criteria as rapid tools for bioassay testing and could be easily standardized using Cr⁶⁺ as a reference toxicant.