Investigation on Endocrine Disruption of the Larval Lampricide 3-Trifluoromethyl-4-Nitrophenol: Short-Term Reproduction Assay with Fathead Minnow (Pimephales promelas).

3-Trifluoromethyl-4-nitrophenol (TFM) has been used for more than 60 yr to control the invasive parasitic sea lamprey (Petromyzon marinus) in the Great Lakes Basin (USA/Canada). In the early 1990s, researchers reported that TFM induced vitellogenin in fish and that TFM was an agonist for the rainbow trout estrogen receptor. To support continued registration of TFM for sea lamprey control, regulatory agencies required further testing to evaluate potential endocrine disruption effects. Fathead minnow (Pimephales promelas) were exposed to TFM at measured concentrations of 0.0659, 0.181, 0.594, 1.79, and 5.11 mg active ingredient (a.i.)/L for 21 d. No-observable- and lowest-observable-effect concentrations (NOEC and LOEC, respectively) were determined to be 1.79 mg/L or greater for each endpoint. Male survival in the highest treatment group was reduced relative to the controls. Percentage of egg fertility was reduced in the highest treatment group, resulting in an estimated NOEC of 1.79 mg/L. Whereas no effect on the gonadosomatic index (GSI) was observed for males, female GSI was increased in the 5.11-mg/L treatment. Vitellogenin production was not altered relative to the controls for all TFM treatment groups. However, female testosterone was elevated in the 5.11-mg/L treatment. The results suggest that prolonged exposure to TFM at concentrations exceeding 1.79 mg/L has the potential to disrupt endocrine function. Biologically relevant effects were found at the highest exposure concentration following a 21-d exposure. However, the duration of exposure in our study is not consistent with typical treatment durations (12 h) for sea lamprey control. Environ Toxicol Chem 2020;39:1599-1607. © 2020 SETAC.

Control of invasive sea lampreys using the piscicides TFM and niclosamide: Toxicology, successes & future prospects.

The invasion of the Laurentian Great Lakes of North America by sea lampreys (Petromyzon marinus) in the early 20th century contributed to the depletion of commercial, recreational and culturally important fish populations, devastating the economies of communities that relied on the fishery. Sea lamprey populations were subsequently controlled using an aggressive integrated pest-management program which employed barriers and traps to prevent sea lamprey from migrating to their spawning grounds and the use of the piscicides (lampricides) 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide to eliminate larval sea lampreys from their nursery streams. Although sea lampreys have not been eradicated from the Great Lakes, populations have been suppressed to less than 10% of their peak numbers in the mid-1900s. The ongoing use of lampricides provides the foundation for sea lamprey control in the Great Lakes, one of the most successful invasive species control programs in the world. Yet, significant gaps remain in our understanding of how lampricides are taken-up and handled by sea lampreys, how lampricides exert their toxic effects, and how they adversely affect non-target invertebrate and vertebrates species. In this review we examine what has been learned about the uptake, handling and elimination, and the mode of TFM and niclosamide toxicity in lampreys and in non-target animals, particularly in the last 10 years. It is now clear that the mode of TFM toxicity is the same in non-target fishes and lampreys, in which TFM interferes with oxidative phosphorylation by the mitochondria leading to decreased ATP production. Vulnerability to TFM is related to abiotic factors such as water pH and alkalinity, which we propose changes the relative amounts of the bioavailable un-ionized form of TFM in the gill microenvironment. Niclosamide, which is also a molluscicide used to control snails in areas prone to schistosomiasis infections of humans, also likely works by uncoupling oxidative phosphorylation, but less is known about other aspects of its toxicology. The effects of TFM include reductions in energy stores, particularly glycogen and high energy phosphagens. However, non-target fishes readily recover from sub-lethal TFM exposure as demonstrated by the rapid restoration of energy stores and clearance of TFM. Although both TFM and niclosamide are non-persistent in the environment and critical for sea lamprey control, increasing public and institutional concerns about pesticides in the environment makes it imperative to explore other means of sea lamprey control. Accordingly, we also address possible "next-generation" strategies of sea lamprey control including genetic tools such as RNA interference and CRISPR-Cas9 to impair critical physiological processes (e.g. reproduction, digestion, metamorphosis) in lamprey, and the use of green chemistry to develop more environmentally benign chemical methods of sea lamprey control.

Using silver and bighead carp cell lines for the identification of a unique metabolite fingerprint from thiram-specific chemical exposure.

Conservation biology often requires the control of invasive species. One method is the development and use of biocides. Identifying new chemicals as part of the biocide registration approval process can require screening millions of compounds. Traditionally, screening new chemicals has been done in vivo using test organisms. Using in vitro (e.g., cell lines) and in silico (e.g., computer models) methods decrease test organism requirements and increase screening speed and efficiency. These methods, however, would be greatly improved by better understanding how individual fish species metabolize selected compounds. We combined cell assays and metabolomics to create a powerful tool to facilitate the identification of new control chemicals. Specifically, we exposed cell lines established from bighead carp and silver carp larvae to thiram (7 concentrations) then completed metabolite profiling to assess the dose-response of the bighead carp and silver carp metabolome to thiram. Forty one of the 700 metabolomic markers identified in bighead carp exhibited a dose-response to thiram exposure compared to silver carp in which 205 of 1590 metabolomic markers exhibited a dose-response. Additionally, we identified 11 statistically significant metabolomic markers based upon volcano plot analysis common between both species. This smaller subset of metabolites formed a thiram-specific metabolomic fingerprint which allowed for the creation of a toxicant specific, rather than a species-specific, metabolomic fingerprint. Metabolomic fingerprints may be used in biocide development and improve our understanding of ecologically significant events, such as mass fish kills.

Direct Photolysis Rates and Transformation Pathways of the Lampricides TFM and Niclosamide in Simulated Sunlight.

The lampricides 3-trifluoromethyl-4-nitrophenol (TFM) and 2',5-dichloro-4'-nitrosalicylanilide (niclosamide) are directly added to many tributaries of the Great Lakes that harbor the invasive parasitic sea lamprey. Despite their long history of use, the fate of lampricides is not well understood. This study evaluates the rate and pathway of direct photodegradation of both lampricides under simulated sunlight. The estimated half-lives of TFM range from 16.6 ± 0.2 h (pH 9) to 32.9 ± 1.0 h (pH 6), while the half-lives of niclosamide range from 8.88 ± 0.52 days (pH 6) to 382 ± 83 days (pH 9) assuming continuous irradiation over a water depth of 55 cm. Both compounds degrade to form a series of aromatic intermediates, simple organic acids, ring cleavage products, and inorganic ions. Experimental data were used to construct a kinetic model which demonstrates that the aromatic products of TFM undergo rapid photolysis and emphasizes that niclosamide degradation is the rate-limiting step to dehalogenation and mineralization of the lampricide. This study demonstrates that TFM photodegradation is likely to occur on the time scale of lampricide applications (2-5 days), while niclosamide, the less selective lampricide, will undergo minimal direct photodegradation during its passage to the Great Lakes.

Evaluation of the short term 12 hour toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) to multiple life stages of Venustaconcha ellipsiformis and Epioblasma triquetra and its host fish (Percina caprodes).

The present study evaluated the risk of 12-h exposures of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to multiple life stages of the federally endangered snuffbox (Epioblasma triquetra) and its primary host fish the common logperch (Percina caprodes) as well as a surrogate to the snuffbox, the ellipse (Venustaconcha ellipsiformis). Life stages examined included free glochidia, 1-wk juveniles, and adults of the ellipse; free glochidia, glochidia on host fish, and 1-wk juveniles of the snuffbox; and adult logperch. Larval sea lampreys were also tested alongside adult ellipse and logperch for direct comparison. Survival exceeded 82% among all life stages in both mussel species at levels up to 1.8 times what would be applied during treatments, suggesting that routine sea lamprey control operations would not adversely affect mussels. However, substantial mortality of adult logperch was observed at TFM concentrations typically applied to streams, and loss of host fish could adversely affect snuffbox reproduction. In addition, TFM had no significant effect on the number of glochidia that metamorphosed on adult logperch. Although the snuffbox is not likely to be acutely affected from sea lamprey control operations, mitigation efforts to minimize impacts to the host fish should be considered.

Determination of antimycin-A in water by liquid chromatographic/mass spectrometry: single-laboratory validation.

An LC/MS method was developed and validated for the quantitative determination and confirmation of antimycin-A (ANT-A) in water from lakes or streams. Three different water sample volumes (25, 50, and 250 mL) were evaluated. ANT-A was stabilized in the field by immediately extracting it from water into anhydrous acetone using SPE. The stabilized concentrated samples were then transported to a laboratory and analyzed by LC/MS using negative electrospray ionization. The method was determined to have adequate accuracy (78 to 113% recovery), precision (0.77 to 7.5% RSD with samples > or = 500 ng/L and 4.8 to 17% RSD with samples < or = 100 ng/L), linearity, and robustness over an LOQ range from 8 to 51 600 ng/L.

Residues of the lampricides 3-trifluoromethyl-4-nitrophenol and niclosamide in muscle tissue of rainbow trout.

Rainbow trout (Oncorhyncus mykiss) were exposed to the (14)C-labeled lampricide 3-trifluoromethyl-4-nitrophenol (TFM) (2.1 mg/L) or niclosamide (0.055 mg/L) in an aerated static water bath for 24 h. Fish were sacrificed immediately after exposure. Subsamples of skin-on muscle tissue were analyzed for residues of the lampricides. The primary residues in muscle tissue from fish exposed to TFM were parent TFM (1.08 +/- 0.82 nmol/g) and TFM-glucuronide (0.44 +/- 0.24 nmol/g). Muscle tissue from fish exposed to niclosamide contained niclosamide (1.42 +/- 0.51 nmol/g), niclosamide-glucuronide (0.0644 +/- 0.0276 nmol/g), and a metabolite not previously reported, niclosamide sulfate ester (1.12 +/- 0.33 nmol/g).

Metabolism of niclosamide in sediment and water systems.

A series of experiments analyzed the kinetics and mechanisms of [(14)C]niclosamide degradation. The aerobic aquatic metabolism of [(14)C]niclosamide was studied in nonsterile river water/sediment mixtures. Test systems, maintained under aerobic conditions, were treated with niclosamide and incubated in the dark at 25.0 +/- 1.0 degrees C for 30 days. Half-lives of 4.9 and 5.4 days were calculated for the chlorosalicylic acid- and chloronitroaniline-labeled test systems, respectively. From 0 to 21 days after treatment (DAT), the only metabolism product observed in either test system was aminoniclosamide. At the final sampling interval, five peaks were resolved from the chlorosalicylic acid label, and three peaks were resolved from the chloronitroaniline label test substance. By 30 DAT, sediment-bound residues represented approximately 70% of the observed radioactivity. For the anaerobic aquatic metabolism of [(14)C]niclosamide, test systems were incubated under anaerobic conditions for 365 days. Half-lives of 0.65 day for the chlorosalicylic acid label and 2.79 days for the chloronitroaniline label were calculated. From 0 to 3 DAT, niclosamide was first transformed into aminoniclosamide. Aminoniclosamide is readily formed, as it was observed in the chlorosalicylic acid label 0 DAT sampling. Several minor metabolites were observed in the water and sediment extracts. None of these metabolites were formed to a significant amount until the parent niclosamide dissipated below the detection limit. Two of the byproducts from these metabolism studies are polar unknowns eluting at 3 and 5 min by HPLC, similar to the unknowns observed in aqueous photolysis studies.

Aqueous photolysis of niclosamide.

The photodegradation of [(14)C]niclosamide was studied in sterile, pH 5, 7, and 9 buffered aqueous solutions under artificial sunlight at 25.0 +/- 1.0 degrees C. Photolysis in pH 5 buffer is 4.3 times faster than in pH 9 buffer and 1.5 times faster than in pH 7 buffer. In the dark controls, niclosamide degraded only in the pH 5 buffer. After 360 h of continuous irradiation in pH 9 buffer, the chromatographic pattern of the degradates was the same regardless of which ring contained the radiolabel. An HPLC method was developed that confirmed these degradates to be carbon dioxide and two- and four-carbon aliphatic acids formed by cleavage of both aromatic rings. Carbon dioxide was the major degradate, comprising approximately 40% of the initial radioactivity in the 360 h samples from both labels. The other degradates formed were oxalic acid, maleic acid, glyoxylic acid, and glyoxal. In addition, in the chloronitroaniline-labeled irradiated test solution, 2-chloro-4-nitroaniline was observed and identified after 48 h of irradiation but was not detected thereafter. No other aromatic compounds were isolated or observed in either labeled test system.

Relatively rapid loss of lampricide residues from fillet tissue of fish after routine treatment.

The selective sea lamprey (Petromyzon marinus) larvicide 3-trifluoromethyl-4-nitrophenol (TFM) is currently used to control parasitic sea lampreys in tributaries to the Great Lakes basin. The concentration and persistence of TFM and its major metabolite, TFM glucuronide (TFM-glu), was determined in fillet tissue of fish after a typical stream application. Rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) were exposed to a nominal concentration of 12.6 nmol/mL TFM for about 12 h during a sea lamprey control treatment of the Ford River in Michigan. Concentrations of TFM and TFM-glu were greatest in the fillet tissues during the exposure period, with greater residues in channel catfish (wet wt; mean, 6.95 nmol/g TFM; mean, 2.40 nmol/g TFM-glu) than in rainbow trout (wet wt; mean, 1.45 nmol/g TFM; mean, 0.93 nmol/g TFM-glu). After the exposure period, residues in both species decreased by 90-99% within 6-12 h and were less than the quantitation limit (<0.03 nmol/g) within 36 h.