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. 2026 May 15;49(11):e70207. doi: 10.1111/jfd.70207

Melanoma and Other Melanistic Lesions in Brown Bullhead Ameiurus nebulosus From Waterbodies in the Northeastern United States and Canada: Identification of Risk Factors

Vicki S Blazer 1,✉, Peter Emerson 2, Matthew Bodnar 2, Thomas Jones 2, David R Russell 3, Matthew Pehrson 4, Cheyenne R Smith 1, Danielle Cleveland 5, Mark J Henderson 6, Patricia M Mazik 7
PMCID: PMC13629460  PMID: 42141503

ABSTRACT

Melanistic lesions, including non‐raised black areas due to proliferations of melanocytes and melanomacrophages in the dermis and epidermis, as well as raised black areas consistent with melanoma, are described in brown bullhead (BBH) Ameiurus nebulosus from three water bodies in the northeastern United States and Quebec, Canada. First observed in the Vermont portion of Lake Memphremagog, Vermont, USA and Quebec, Canada, the prevalence of melanistic lesions during 2014–2020 was greater than 30% in BBH 200 mm and longer. In 2023, seven sites throughout the lake were assessed, and prevalence ranged from 18% to 42%. In Hermon Pond, Maine, the prevalence was 29% in 2024, and in Village Pond, New Hampshire, lesions occurred in 22% of BBH in 2025. Compared to skin from visibly normal BBH, skin with melanistic lesions had significantly higher concentrations of seven metals, including arsenic, a known carcinogen and zinc. Lesions associated with oxidative damage, such as the accumulation of ceroid/lipofuscin, were also observed in the gill, spleen and kidney tissue of both affected and visibly normal BBH. The progression of lesions, observed by histopathology, ranged from inflammation, signs of oxidative damage, proliferation and necrosis of club cells, and the presence of melanomacrophages and melanocytes in the epidermis to invasive melanoma and suggests chronic exposure of BBH to environmental initiators and promoters of carcinogenesis.

Keywords: arsenic, brown bullhead, chronic exposure, melanistic lesions, melanoma, zinc

1. Introduction

In 2012, the Vermont Fish and Wildlife Department (VFWD) began to receive reports from anglers observing raised black growths on the body surface, mouth and fins of adult brown bullhead (BBH) Ameiurus nebulosus from Lake Memphremagog, Vermont/Quebec. Initial electrofishing surveys conducted in 2014–2017 in two bays within the Vermont boundaries of the lake, South Bay and Scott's Cove (SC) (Figure 1), documented a prevalence of melanistic lesions (raised and non‐raised) from 23% to 37% of BBH 200 mm and greater in length; younger fish were not affected. Histopathological analyses confirmed that the raised melanistic lesions were malignant melanoma, with occasional metastases observed in the gill, ovary and intestine. Liver neoplasia (cholangiocarcinoma and hepatic cell carcinoma) was also observed in 8% of the BBH sampled in 2015 (Blazer, Shaw, et al. 2020).

FIGURE 1.

FIGURE 1

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(A) Lake Memphremagog sites sampled in 2023. Sites in Vermont included South Bay (JR), South Main Lake (SML), Scott's Cove (SC) and North Main Lake (HC). Sites in Quebec included Fitch Bay (FB), Sargent's Bay (SB) and Magog (MA). Modified from mapZ.com—map data: OpenStreetMapOdbl. (B) Sites sampled in 2023–2025 in Vermont, New Hampshire and Maine, USA and Quebec, Canada. Credits: CHS, Esri, Garman, Naturalvue.

BBH has been used extensively as an indicator species throughout the Great Lakes (Baumann et al. 1996; Pyron et al. 2001; Blazer et al. 2009; Lauren et al. 2010; Pinheiro et al. 2020), Chesapeake Bay drainage (Pinkney et al. 2009, 2019) and elsewhere (Poulet et al. 1994). Skin neoplasms, primarily papilloma and squamous cell carcinoma, have been associated with exposure to legacy contaminants (Baumann et al. 1987, 2008; Pinkney et al. 2004). Putative melanomas have been described in BBH as early as the 1850s (Osburn 1925). However, in tumour surveys, a low incidence (< 5%) of melanoma is generally observed, although the incidence of other skin neoplasms can be high. For instance, in a summary of six surveys conducted during 1992–2006, within the Chesapeake Bay drainage, no melanomas were reported among 647 adult BBH, despite the incidence of other skin tumours ranging from 0% to 53% (Pinkney et al. 2009). Similar results were found in the Anacostia River between 2014 and 2016, where 5%–16% of BBH had skin tumours, but no melanomas were reported (Pinkney et al. 2019). In tumour surveys at eight Great Lakes Areas of Concern and three reference sites between 2011 and 2013, the external tumour prevalence ranged from 0% to 61.5% at individual sites. Of the 878 BBH collected, 6.6% had external neoplasms, with only one melanoma observed (Blazer et al. 2014).

Non‐raised melanistic lesions have been observed more often at certain sites. In the Great Lakes tumour surveys (Blazer et al. 2014), 18.8% of the 878 BBH had non‐raised melanistic lesions. At the Ashtabula River Area of Concern on Lake Erie in 2016, 18% (of 150) had non‐raised melanistic lesions with similar findings (13.3% of 150 sampled) at the reference site. Although 26.7% had skin neoplasms at Ashtabula, only one melanoma was observed; 23.3% had skin neoplasms at the reference site, and no melanomas were documented (Blazer et al. 2018). Similarly, in 2002, at the contaminated Ashumet Pond on Cape Cod, 70% of BBH (58 sampled) had non‐raised melanistic lesions, but no melanomas were detected. At a nearby reference pond, < 10% of BBH collected had melanistic lesions and again, no melanomas were observed (Baumann et al. 2008). At these sites, the non‐raised melanistic areas were characterised microscopically by the increased melanocytes in the epidermis. They did not appear to progress to neoplasia, and their aetiology remains unclear. It is interesting to note that non‐raised melanistic areas have also been documented in bass Micropterus spp. related to Adomavirus infections and are characterised by increased melanocytes in the epidermis (Blazer, Young, et al. 2020; Iwanowicz et al. 2025). The above findings highlight the rarity of melanomas in other wild BBH populations, making the recent observations of malignant melanoma in Vermont and other parts of the northeastern United States and Canada particularly noteworthy.

Melanoma incidence in humans is increasing worldwide at a greater rate than other types of cancer, and both genetics and environmental exposures are known to be risk factors (Conforti and Zalaudek 2021). Vermont has the third‐highest age‐adjusted incidence rate of human melanoma cases, and this rate has been increasing over the past 5 years (State Cancer Profiles, National Cancer Institute; https://statecancerprofiles.cancer.gov). In mammalian species, ultraviolet radiation (UVR) exposure and genetics are primary risk factors, and incidence increases with age. However, other environmental factors, such as metal exposure, may play a role. For example, epidemiological studies show a higher incidence of melanoma in occupations associated with electronic and chemical industries. This finding and subsequent research have suggested that increases in reactive oxygen species (ROS) are significant in the pathogenesis of melanocyte transformation and melanoma progression, acting as signalling molecules that promote cancer growth and cell death (Meyskens Jr et al. 2004; Liu‐Smith et al. 2014). Little is known about the risk factors associated with melanoma in wild fish species or the role of carcinogen uptake in fish skin. Melanoma and melanosis in wild coral trout Plectropamus leopardus at sites in the Great Barrier Reef Marine Park were attributed to UV radiation exposure (Sweet et al. 2012), and melanoma has been induced in Xiphophorus hybrids by exposure to UV radiation (Setlow et al. 1989). An association between environmental chemicals and hyperplasia or neoplasia of pigmented cells has been suggested in studies of other fish species (Kimura et al. 1989; Okihiro et al. 1993; Ramos et al. 2013), although no specific contaminants were identified.

Lake Memphremagog is a 1780 km2 natural lake that lies within northern Vermont, USA and southern Quebec, Canada. Three major Vermont rivers, the Clyde, Barton and Black, flow into the lake and provide 70% of the lake's drainage basin. The outflow is the Magog River in Quebec, which ultimately flows into the Saint Lawrence River. The lake is an important recreational and subsistence fishing resource as well as a drinking water supply for some Canadian communities; however, it is listed as impaired due to phosphorus and other water quality issues (https://dec.vermont.gov/sites/dec/files/wsm/Basin172015.pdf; Gerhardt 2018). Melanistic lesions on BBH have continued to be observed in Lake Memphremagog. Additionally, they have now been observed in a few ponds in New Hampshire and Maine (this study). Consequently, the public and management agencies are concerned about causative and contributing factors to these lesions, which may also contribute to adverse human or ecosystem health outcomes.

To identify risk factors and potential contributing factors for these lesions, a comprehensive set of endpoints was developed by a multidisciplinary team. Temporal and spatial prevalence data; associations of prevalence with age, sex and morphometric endpoints; chemical analyses and relationships to microscopic pathology and the progression of lesions are presented here.

2. Materials and Methods

2.1. Field Methods

Electrofishing surveys were conducted by VFWD in the Scott's Bay and South Bay portions of Lake Memphremagog, Vermont, during 2014 to 2020 (May–August) to determine prevalence and associated morphometric parameters. All BBHs collected were measured (total length, mm), and melanistic lesions were documented. In May 2023, to assess the spatial distribution, prevalence and potential risk factors, electrofishing surveys were conducted at seven sites (Figure 1A) throughout the lake. The four sites in Vermont were SC, South Main Lake (SML), South Bay (JR), North Main Lake (HC) and three sites in Quebec were Sargent's Bay (SB), Fitch Bay (FB) and Magog at the mouth of Cherry River (MA). All BBHs captured were measured, and lesions were documented. In addition, complete necropsies were conducted on 10 clinically normal and 10 BBH with melanistic lesions. Fish were held in aerated buckets and necropsied following approved US Geological Survey Eastern Ecological Science Center Institutional Animal Care and Use protocols for wild fish health surveys (EESC 2021‐18L). Fish were euthanised with buffered tricaine methanesulfonate (Syndel USA, 350 mg/L and sodium bicarbonate at a 1:1 ratio) diluted in lake water, weighed (gm), measured (mm) and blood‐sampled from the caudal vessel using a sodium heparinised 3 cc syringe with a 22‐gauge needle. Blood was placed in a Vacutainer with sodium heparin (Thermo Fisher Scientific, Rockville, MD, USA) and stored on wet ice until centrifuged (within 4 h). Plasma was collected, aliquoted into cryovials and stored on dry ice until placed into a −80°C freezer. External abnormalities were documented and photographed. Small pieces (approximately 30 mg) of normal skin, lesioned skin and liver tissues were preserved in RNAlater Stabilizer Solution (ThermoFisher) for molecular analysis. Pieces of normal and lesioned skin, liver, anterior and posterior kidney, spleen, gill and gonad tissues were preserved in Z‐fix (Anatech Ltd., Battle Creek, MI, USA) for histopathology. Brain tissue was removed and placed on dry ice for genetic analyses, and sagittal otoliths were removed and stored dry in coin envelopes for age analysis. The remaining carcasses were frozen. The condition factor (KtL) was calculated using the formula KtL = 100 × (body weight/total length3), where weight is in gm and length in cm.

In May 2024, BBH was surveyed in Hermon Pond, Maine (Figure 1B), a 187 ha, shallow (maximum depth = 5.2 m), warm water, recreational fishing pond. A total of 62 BBH were captured, weighed, and examined for melanistic lesions. Five normal and five lesioned individuals were collected for necropsies as described above. In June 2025, 73 BBHs from Village Pond, New Hampshire (Figure 1B) were weighed and examined; 9 normal and 16 with melanistic lesions were necropsied. Village Pond is an artificial 14‐ha pond in the Alshuelot River drainage.

2.2. Laboratory Analyses

Tissues preserved for histology were trimmed into cassettes, processed through graded alcohols and embedded in paraffin. Blocks were sectioned at 5 μm, and slides were routinely stained with haematoxylin and eosin (Luna 1992) for evaluation by light microscopy.

Otoliths were embedded using West System 105 epoxy resin and West System 205 hardener (West System, Gougeon Brothers Inc., Bay City, MI, USA) and left to set for 24 h. The set epoxy was then cut with a hacksaw before further sectioning using an Isomet Low Speed precision cutter equipped with a diamond‐tooth blade (Buehler, Lake Bluff, IL, USA). The resulting sections were sanded and polished using 30‐μm lapping film (Precision Surfaces International, Houston, TX, USA), keeping the film wet throughout the process. The polishing process was intermittently paused to examine the otoliths. Polished otoliths were immersed in mineral oil and examined under a Richter Optica U‐2D biological microscope at 10× magnification. The annuli were counted to estimate the age of each BBH. Two VFWD fisheries biologists independently reviewed each specimen to ensure accuracy. For 91.3% of the samples, age determinations matched, while 16 specimens (8.2%) had age differences of 1 year and were reread for consensus.

Elemental (metal and metalloid) uptake and exposure were characterised by quantifying total recoverable concentrations of 23 elements in BBH skin. Frozen carcasses were partially thawed, and approximately 1 g of skin was removed from 17 clinically normal fish (4 each from SML, SC, MA and 5 from FB) and 17 from melanistic lesions (3 from SML and SC, 1 from HC, 4 from MA and 6 from FB). In addition, skin from four clinically normal fish and four fish with melanistic lesions from Hermon Pond were also analysed. Tissue was placed in 10 mL polyethylene tubes and shipped overnight on dry ice to the US Geological Survey Columbia Environmental Research Center, Columbia, Missouri, for analyses. Tissues were manually homogenised with cut‐glass rods and lyophilised (VirTis EL‐35; Scientific Products, Westminster, PA, USA) for analysis on a dry weight (dw) basis. Tissues were then microwave‐digested in high‐purity, house‐distilled, concentrated nitric acid and 30% hydrogen peroxide (Optima grade) using a ramp‐to‐temperature method (MARS6; CEM Corporation, Matthews, NC, USA). This total recoverable method is intended to access the bioavailable fraction of metal (e.g., EPA Method 3050B; US EPA 1996). Concentrations of the elements in the digests were then quantified using inductively coupled plasma‐mass spectrometry (ICP‐MS) using a method similar to EPA Method 6020B (US EPA 2014). Quality control measures included the use of NIST‐traceable calibration standards, second‐source initial and continuing calibration verification standards, laboratory control solutions, certified reference materials, method and analysis spikes and replicates and interference checks. All quality control results were acceptable for the methods. Results were censored at the estimated method limits of quantification (LOQ; estimated using the propagation of error method; Long and Winefordner 1983). Analytes and detection limits are listed in Table S1.

2.3. Statistical Analyses

Data were analysed using GraphPad Prism version 10.1.2 (GraphPad Software Inc., La Jolla, CA, USA). Differences between visibly normal and lesioned BBH at each site were compared using the Mann–Whitney test, because data did not pass the Shapiro–Wilk normality test. A p value of 0.05 was used to indicate significance in all tests.

3. Results

3.1. Visual Observations

Melanistic lesions ranged from small, non‐raised spots to slightly raised and large, raised areas, sometimes covering more than 50% of the body. Melanistic lesions were observed on the body surfaces, fins, lips and within the mouth (Figure 2). Some melanistic areas, potentially metastases, were noted in the gills and internal organs, including the brain, intestine and ovary (Figure 3).

FIGURE 2.

FIGURE 2

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Types of melanistic lesions observed on brown bullhead collected in Lake Memphremagog. (A) Non‐raised black discolouration on the lip (arrow). (B) Small, non‐raised black area on the body surface (arrow). (C) Multiple, non‐raised black spots (black arrows) and larger, raised areas (white arrow). (D) Large, raised coalescing black lesions (white arrows). (E) Large, raised black lesion in the mouth (white arrow). (F) Large, raised black lesion covering most of the anterior body surface (white arrows).

FIGURE 3.

FIGURE 3

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Examples of melanistic lesions in brown bullhead internal tissues. (A) Black areas within the gill tissue (arrow). (B) Focal black area within the brain tissue (arrow). (C) Black area within the ovary (arrow).

3.2. Prevalence of Melanistic Lesions in Lake Memphremagog

Melanistic lesions were only observed in BBH 200 mm or greater during surveys from 2014 to 2020 at JR and SC. A total of 61 BBH < 200 mm was examined and found to have no lesions. At JR, 283 BBH 200 mm or greater was collected, and 33.6% had black lesions. At SC, 33.5% of 576 BBH 200 mm or greater had black lesions. Individuals were binned into length categories of 24 mm to compare the prevalence by length category. Data were compared by combining all years at both sites (Figure 4). Lesion prevalence generally increased by fish length to 324 mm and then decreased; however, sample sizes in each length group differed, precluding definitive comparisons.

FIGURE 4.

FIGURE 4

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Percentage of brown bullhead with melanistic lesions in Lake Memphremagog (South Bay and Scott's Cove) collected during 2014–2020. Numbers within bars are the total number of fish collected in each length category.

In 2023, as in previous years, only BBH 200 mm and greater were observed with melanistic lesions. A total of 78 BBH < 200 mm were captured and examined at six sites, including SC (n = 50), SML (n = 6), JR (n = 8), HC (n = 7), SB (n = 1) and FB (n = 6). Combining all sites, a total of 427 BBH 200 mm and greater were examined, and 26.5% had melanistic lesions. The length (mean ± standard error) of clinically normal BBH (241.5 ± 1.8) was significantly (p < 0.001) less than that of those with melanistic lesions (267.3 ± 3.2). The prevalence of melanistic lesions in BBH 200 mm and greater at the seven sites sampled in Lake Memphremagog varied from 18% at JR to 42% at HC (Figure 5).

FIGURE 5.

FIGURE 5

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Percentage of brown bullhead (200 mm and greater total length) with melanistic lesions at sites within Lake Memphremagog, 2023. Numbers within the bars indicate the total number captured at each site.

The 20 BBH (10 normal and 10 lesioned) collected for necropsies at each site were aged, sexed and condition factors computed. Of the 140 fish necropsied, 67 (48%) were female, and 73 (52%) were male. Clinically normal BBH ranged from 3 to 18 years (4.9 ± 0.3; mean ± standard error) but were significantly younger (p < 0.001) than those with melanistic lesions, which ranged from 4 to 8 years (5.3 ± 0.1). Those with lesions were also significantly (p = 0.01) longer (275 ± 4) than clinically normal BBH (262 ± 4). Individual sites varied in terms of morphometric differences between normal and lesioned fish and were not always significantly different (Table 1).

TABLE 1.

Morphometric and age comparisons of brown bullhead with and without black lesions from selected waterbodies in the northeastern United States and Quebec, Canada.

Site Type (sample size) Age (years) Length (mm) Weight (g) Condition factor (Ktl)
South Main Lake (SML) Normal (10) 5.1 ± 0.7a 282.5 ± 12.6 343.0 ± 52.7 1.5 ± 0.1
Lesion (10) 5.5 ± 0.3b 261.5 ± 11.8 295.0 ± 35.1 1.8 ± 0.4
Scott's Cove (SC) Normal (10) 5.6 ± 0.6 237.6 ± 7.4 201.1 ± 20.3 1.5 ± 0.2
Lesion (10) 5.1 ± 0.2 228.9 ± 3.2 159.8 ± 8.5 1.3 ± 0.1
North Main Lake (HC) Normal (10) 4.2 ± 0.2a 273.1 ± 6.1a 312.3 ± 27.7a 1.5 ± 0.1
Lesion (10) 5.1 ± 0.2b 303.9 ± 6.0b 417.5 ± 26.1b 1.5 ± 0.1
South Bay (JR) Normal (10) 4.1 ± 0.4a 239.4 ± 9.5a 200.6 ± 23.9 1.4 ± 0.1
Lesion (10) 5.2 ± 0.2b 263.7 ± 4.2b 254.1 ± 17.0 1.4 ± 0.1
Sargent's Bay (SB) Normal (10) 4.8 ± 0.4a 253.6 ± 7.4a 235.6 ± 23.4a 1.5 ± 0.2
Lesion (10) 5.5 ± 0.3b 288.3 ± 3.8b 341.8 ± 13.7b 1.4 ± 0.1
Fitch's Bay (FB) Normal (10) 4.5 ± 0.3a 259.9 ± 4.7 230.2 ± 16.1 1.3 ± 0.1
Lesion (10) 4.9 ± 0.1b 272.5 ± 2.8 269.2 ± 7.7 1.3 ± 0.1
Magog Area (MA) Normal (10) 5.8 ± 1.4 289.3 ± 11.3 341.5 ± 34.8 1.4 ± 0.1
Lesion (10) 6.1 ± 0.4 306.2 ± 11.4 336.9 ± 25.3 1.2 ± 0.1
Hermon Pond Normal (5) 6.6 ± 1.3 274.0 ± 4.3 272.2 ± 15.6 1.3 ± 0.1
Lesion (5) 5.5 ± 0.6 276.0 ± 7.3 259.9 ± 28.0 1.2 ± 0.0
Village Pond Normal (9) 4.1 ± 0.3 182.0 ± 6.1 77.9 ± 6.6 1.3 ± 0.0a
Lesion (16) 4.7 ± 0.2 194.4 ± 3.6 88.6 ± 5.3 1.2 ± 0.0b
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Note: Data presented as mean ± standard error of the mean. Values followed by a different letter indicate a significant difference (p < 0.05) between clinically normal and lesioned individuals at each site.

3.3. Prevalence of Melanistic Lesions at Other Sites

A total of 62 BBHs ranging in length from 203 to 322 mm were captured and examined from Hermon Pond, Maine and melanistic lesions were present in 29%. Five clinically normal and five lesioned BBH were necropsied; three of the five BBH with melanistic lesions were male, and eight were female. For the 10 fish necropsied, there was no significant difference in length, weight, age or condition factor between normal and lesioned individuals (Table 1).

A total of 73 BBH were collected and examined at Village Pond, New Hampshire and ranged in length from 105 to 210 mm. Melanistic lesions were present in 22% of BBH > 158 mm. At this site, fish were not sexed, nor were gonads preserved for histology. Of the 25 necropsied, there was no significant difference in length, weight or age; however, the clinically normal fish had a greater mean condition factor (Table 1).

3.4. Histopathology

The skin of normal BBH is composed of epidermis and dermis with underlying hypodermis and subcutaneous layers. The epidermis contains columnar epithelial cells within the basal layers, which transition to squamous in the outer layer; large club (alarm cells) and smaller mucous (goblet cells). Occasionally, melanocytes were observed in the epidermis, as were taste buds and pit organs. The basement membrane of the epidermis separates it from the dermis, which is composed of a compact layer of fibrous connective tissue. A layer of melanocytes lies along the basement membrane (Figure 6A).

FIGURE 6.

FIGURE 6

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Microscopic appearance of skin and subcutaneous tissues of a visibly normal brown bullhead. (A) The epidermis of normal skin contains the basal layer of columnar epithelial cells (a), which become squamous (b) at the surface. Large club cells (c) and mucous cells (black arrow) are present, and the dermal layer of melanocytes is intact (white arrow). (B) Skin from a visually normal fish with a proliferation of normal (a) and vacuolated club cells (b). In the dense fibrous tissue of the dermis, areas of inflammation (c), sometimes with pigmented cells (arrow), are present. (C) Section from a visibly normal fish with areas of sloughing of epidermal and club cells (a) and necrotic cells (b). In some areas, the dermal melanocyte layer is disrupted (arrow). Inflammation in the underlying adipose layer (c), abnormal musculature (d) and the presence of encysted trematodes (e) are evident. (D) Higher magnification of (C). (E) In some fish, ceroid/lipofuscin pigment (a) was evident around the fat cells (b). The underlying muscle fibres (c) were abnormal, but there was less inflammation.

Pathological changes were noted in the skin and subcutaneous tissues of clinically normal BBH. These included the proliferation of club cells, some of which exhibited vacuolisation of the cytoplasm, foci of inflammation and pigment‐containing cells in the subcutaneous tissue (Figure 6B). In more advanced lesions, there was a loss of the epidermal layers with necrosis and sloughing of cells, many of them club cells. Encysted trematodes were also commonly observed in subcutaneous and underlying muscle tissues (Figure 6C). In a few samples, inflammation and necrosis of underlying muscle (Figure 6D) as well as accumulations of ceroid/lipofuscin around adipose cells in subcutaneous layers (Figure 6E) were observed.

Many of the melanistic lesions were non‐raised or slightly raised, and there were a variety of associated microscopic changes within these lesions. In some skin samples from areas with small, non‐raised melanistic lesions, there was a proliferation of club cells in the epidermis, as well as a proliferation of melanocytes in the dermis and subcutaneous tissue. Occasional melanin‐containing cells were present in the epidermis (Figure 7A). In other non‐raised to slightly raised skin lesions, smaller round cells containing yellowish‐brown and black pigments were observed in the epidermis. These are hypothesised to be melanomacrophages, which were often observed surrounding club cells, some of which were undergoing necrosis. The normal dermal melanocyte and subdermal layers were present (Figure 7B). In other fields, there was a loss of club cells, the epidermis appeared disorganised, and pseudocysts filled with melanomacrophages were present. The underlying dermis and connective tissue contained inflammatory cells and pigmented cells. The normal dermal layer of melanocytes was no longer present (Figure 7C). In some fish, the area around the degenerating muscle fascicles was replete with melanomacrophages containing yellowish‐brown ceroid/lipofuscin (Figure 7D). As the lesions progressed from non‐raised to slightly raised lesions, there was increased inflammation and melanistic pigmentation around adipose cells and degenerating muscle fascicles (Figure 7E).

FIGURE 7.

FIGURE 7

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Microscopic appearance of non‐raised or slightly raised melanistic lesions. (A) Melanistic spot with a proliferation of club cells (a) in the epidermis and melanocytes in the dermis and subdermal tissue (b). A few melanocytes (arrows) in the epidermis. (B) Non‐raised melanistic lesion in which club cells, some of which are vacuolated (a), are surrounded by pigmented cells. Foci of pigmented cells (b) appear to be replacing necrotic club and epithelial cells. The dermal melanocyte layer (arrows) is intact. (C) Section of a slightly raised melanistic lesion in which the epidermis contains very few club cells (a) near the surface and pseudocysts of melanomacrophages (b). There are some melanocytes above the basement membrane (arrow) and inflammation and pigmented cells in the dense connective tissue (bracket). (D) In some fish around the subcutaneous muscle fibres (a), there was an accumulation of ceroid/lipofuscin (b). (E) In more advanced lesions, the inflammation (arrows) of underlying adipose tissue (a) and focal areas of degenerating muscle bundles (b) included melanomacrophages and melanocytes.

The raised, more advanced neoplastic lesions also varied in microscopic appearance. In some samples, the epidermis was thickened with large pseudocysts containing the less pigmented presumptive melanomacrophages, while there was a proliferation of melanocytes in the dermis and subcutaneous tissues (Figure 8A). The cell populations in the two areas had very different morphology and staining characteristics. The neoplastic melanocytes, primarily in the dermis and subdermal areas, with fewer in the epidermis, were pleomorphic and varied in pigment content. Some cells within this area were fusiform with little to no pigment (Figure 8B). Most of the pigmented cells (putative melanomacrophages) in the epidermis were smaller, round cells with an eccentric nucleus and yellowish‐brownish to black pigment. Free black granules, believed to be melanosomes, were present in the epidermal tissue (Figure 8C). In other raised lesions, the epidermis was thinner, the large pseudocysts were not observed, but there were pigmented cells present. The underlying dermis, subcutaneous and muscle layers were disrupted and filled with pigmented cells and inflammation (Figure 8D). The epidermis in these more advanced lesions contained pigmented cells, often in vacuolated areas and inflammatory cells. The epidermal epithelial cells were often surrounded by small intercellular spaces formed by projections of the cell membranes (Figure 8E).

FIGURE 8.

FIGURE 8

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Microscopic appearance of advanced, raised melanistic lesions. (A) Section with underlying proliferation of neoplastic melanocytes (a) and epidermis (b) with a proliferation of melanomacrophages. (B) Melanocytes (a) in the dermal and subcutaneous layers varied in morphology, size and pigment content. Some cells were elongated (arrows). (C) Melanomacrophages (arrows) in the epidermis were small, round cells with an eccentric nucleus and contained varying amounts of pigment. Free melanosomes were also present (red arrows). (D) Raised lesion composed primarily of proliferating melanocytes (a) in the dermis and underlying tissues. The epidermis (b) contains foci of pigmented cells (arrow). (E) Higher magnification of epidermis (a), which is vacuolated, contains pigmented cells (b) and inflammatory cells (white arrows). Small vacuoles are formed around the epithelial cells by projections of the cell membrane (black arrows).

Microscopic lesions, not associated with metastases, were present in other organs of both clinically normal and lesioned BBH. The gills contained cellular changes, including hyperplasia leading to fusion of the secondary lamellae (Figure 9A), cell necrosis and inflammation (Figure 9B) and the accumulation of ceroid/lipofuscin‐containing cells, as well as occasional melanin‐containing cells (Figure 9C). Large and numerous macrophage aggregates containing primarily ceroid/lipofuscin pigments were observed in the spleen (Figure 9D) and the anterior and posterior kidneys. A low incidence of liver neoplasia (6% in Lake Memphremagog and 10% in Hermon Pond) was also observed.

FIGURE 9.

FIGURE 9

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Cellular changes in tissues of brown bullhead, indicative of oxidative damage. (A) Areas of epithelial proliferation and fusion of secondary lamellae (a) in the gill. (B) Higher magnification of the area within the box illustrates vacuolated areas with cellular debris (a), hyperplasia and disruption of the secondary lamellae (b) and inflammation (white arrows). (C) Within the proliferating epithelial cells between the secondary lamella were cells containing yellowish‐brown ceroid/lipofuscin pigment (a). These cells were also present in the tissue of the primary filament (white arrows), as were melanin‐containing cells (black arrows). D. Splenic tissue had abundant, large macrophage aggregates (a).

3.5. Contaminant Analyses

3.5.1. Elemental Concentrations

Skin tissue was analysed for 23 elements (Table S1). Seven elements (aluminium, beryllium, cadmium, thallium, bismuth, thorium and uranium) were not detected in any samples. At Lake Memphremagog, vanadium, molybdenum, silver, tin and antimony were infrequently detected and were at levels slightly above the level of quantification. Chromium was detected in four samples from skin with melanistic lesions, with concentrations ranging from 0.1 to 0.84 μg/g, but was not detected in normal skin. Nickel and lead were detected at low incidence and low concentrations in both normal and melanistic skin samples. Manganese (Mn), cobalt (Co), copper (Cu), zinc (Zn), selenium (Se) and barium (Ba) were detected in all samples, while arsenic (As) and iron (Fe) were detected above the LOQ in most samples (88% for As; 98% for Fe). For these eight elements, we compared concentrations in clinically normal and melanistic skin. For sample non‐detects, we substituted the LOQ as the concentration for statistical comparisons. All eight elements were significantly different between clinically normal BBH and those with melanistic lesions and were higher in skin from melanistic lesions, except Ba, which was higher in skin from clinically normal BBH (Table 2).

TABLE 2.

Comparison of concentrations of elements (μg per gram dry weight) that were significantly different in brown bullhead with and without melanistic skin lesions from Lake Memphremagog, Vermont, USA and Quebec, Canada.

Elements Melanistic lesions (n = 17) Normal skin (n = 17) Significance
Iron (Fe) 105.0 ± 17 34.0 ± 5.8 p < 0.001
Manganese (Mn) 5.2 ± 1.1 2.0 ± 1.8 p < 0.001
Cobalt (Co) 0.11 ± 0.01 0.07 ± 0.01 p = 0.002
Copper (Cu) 3.1 ± 0.2 2.5 ± 0.3 p = 0.037
Zinc (Zn) 613.4 ± 88.4 72.2 ± 8.4 p < 0.001
Arsenic (As) 2.6 ± 0.6 0.3 ± 0.1 p < 0.001
Selenium (Se) 3.6 ± 0.5 1.1 ± 0.1 p < 0.001
Barium (Ba) 1.2 ± 0.2 3.1 ± 0.4 p < 0.001
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At Hermon Pond, Zn, As, Se and Ba were significantly different, higher in the skin of fish with melanistic lesions relative to clinically normal skin. At both Lake Memphremagog and Hermon Pond, As, Se and Zn had the greatest differences in skin samples between normal and lesioned BBH (Figure 10).

FIGURE 10.

FIGURE 10

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Metal concentrations (bar is mean with standard error bar) in melanistic lesions (black bars) and normal skin (grey bars) of the brown bullhead Ameiurus nebulosus collected in Lake Memphremagog, Vermont, USA and Quebec, Canada (top panels) and Hermon Pond, Maine (bottom panels). (A) Arsenic. (B) Selenium. (C) Zinc.

4. Discussion

The melanistic lesions observed in BBH collected at the three study water bodies ranged from small non‐raised black spots to larger non‐raised or slightly raised melanistic areas to large raised lesions. Many affected fish exhibited both non‐raised and raised lesions. Microscopic skin pathology indicated cell damage, preneoplastic proliferative lesions and neoplasia. Our findings suggest chronic exposure to initiators and promoters of oxidative damage, inflammation, cell necrosis and proliferation of multiple cell types may ultimately lead to carcinogenesis. Early pathological changes included proliferation of club cells in the epidermis, inflammation and foci of ceroid/lipofuscin accumulation (hallmarks of oxidative damage) in the dermis and subcutaneous tissues. Additionally, other tissues, such as the gill, anterior kidney and spleen, showed evidence of oxidative damage as indicated by the accumulation of ceroid/lipofuscin pigments and macrophage aggregates.

The melanistic lesions were first reported in Lake Memphremagog in 2012 (Blazer, Shaw, et al. 2020). They had not been documented in earlier game fish surveys in which BBH were captured; however, BBH were not the target species in these surveys. Specific monitoring of BBH did not occur until after the lesions were reported by the public in 2012 (personal communication J. Kratzer, VFWD). Tropical storm Irene caused widespread flooding and runoff in the area in 2011, and there is public concern that the storm introduced carcinogens into the Memphremagog drainage. Monitoring to date has largely focused on grabbing water samples and whole‐body or fillet samples. There are a few studies in other fish species that include contaminant analyses of the skin. Those that have been conducted show skin can accumulate specific contaminants, including metals (Uysal 2011), at higher concentrations than other tissues. Studies have assessed the effect of removing skin from fillets for human consumption on chemical concentrations. For example, the concentration of lipophilic contaminants (polychlorinated biphenyls, DDT, DDE, DDD, mirex, α‐chlordane, hexachlorobenzene and octachlorostyrene) in salmonid samples was significantly reduced (median 17%–37%) after skin removal (Zhang et al. 2013). Consequently, focus on accumulation in specific tissues showing visible or microscopic changes may be necessary to understand the adverse effects observed.

The elevated concentrations of As in the skin of fish with melanistic lesions compared with visibly normal fish indicate that uptake and exposure to As may be an important risk factor. Sources of As include industry and agriculture, as it is used to produce paints, insecticides, other pesticides and wood preservatives and is also found in soil amendments, chicken litter and sludges from water treatment plants (Missimer et al. 2018). Natural As sources include bedrock weathering, which results in elevated concentrations in soils, sediment and groundwater. In New Hampshire and Maine, nearly 30% of wells in bedrock aquifers have elevated As levels (Ayotte et al. 2003), exceeding 10 μg/L, the US Environmental Protection Agency maximum contaminant level. Wells in north central and eastern Vermont, including the Lake Memphremagog drainage, also have elevated As concentrations (Corenthal 2010). If the As source is primarily natural (and not a recent contaminant), it is likely that other risk factors are necessary for the progression of the lesions to melanoma.

Arsenic is a Class 1 carcinogen that tends to accumulate in the skin, and chronic human exposure is associated with skin lesions, including hyperpigmentation, hyperkeratosis and an increased risk of skin cancers, including melanoma (Yu et al. 2006; Karagas et al. 2015; Wang et al. 2025). Oxidative damage is considered a major factor in As carcinogenesis; hence, the signs of oxidative damage in skin and other organs of the sampled BBH support a potential role for As in the development of these lesions. Arsenic has been shown to cause genetic damage in addition to cellular damage. Genetic analyses of the lesioned skin from this study found many single‐nucleotide variants of both nuclear and mitochondrial DNA (Curd et al., unpublished manuscript). In humans, chromosomal abnormalities and altered growth factors have been identified as possible modes of action for As, which has been shown to be a co‐carcinogen or promoter of carcinogenesis, particularly as a co‐mutagen with UVR. For instance, As has a synergistic effect, leading to a more than twofold enrichment of the mutational burden of UVR alone (Speer et al. 2023). However, mitochondrial damage and mutations are also common with As exposure alone (Lee and Yu 2016). Heteroplastic mitochondrial DNA deletions occur and, with continued exposure, result in varying deletion patterns (Partridge et al. 2007). These As‐related mutations or silencing of pro‐apoptotic genes are associated with the genomic instability observed by allowing the survival of damaged cells (Bhattacharjee et al. 2013).

Elevated concentrations of Zn in skin from lesioned BBH may be an additional risk factor. Unlike As, Zn is an essential mineral that, at high concentrations, may be toxic. Disruption of physiological and biochemical mechanisms causing ion regulatory effects and oxidative damage (Loro et al. 2012) as well as immunomodulation (Dunier 1996) are reported in some Zn‐exposed fishes. In mice, high dietary Zn promotes melanoma tumour progression (Narayan et al. 2024). Consequently, Zn may act synergistically with As and other stressors in lesion progression of BBH.

Changes associated with three main cell types (club cells, melanocytes and melanomacrophages) were observed in the progression of the melanistic lesions. Early lesions in clinically normal skin involved the proliferation of club cells, a cell present in certain fish species. Epidermal club cells are conventionally known as alarm substance cells because these cells may release chemical alarm molecules to conspecifics when the skin is damaged. More recently, the role of club cells in protection against UVR and in immunity has been recognised (Chivers et al. 2007; Pandey et al. 2021). Club cells respond to numerous stressors, including metal exposure (Iger et al. 1994). Specifically, hyperplasia or proliferation of club cells has been reported in response to As exposures (Sankhla et al. 2024). The proteinaceous content of the club cells may also lead to increased accumulation of substances known to bind to proteins, such as per‐ and polyfluoroalkyl substances (PFAS) (Liu et al. 2019). Consequently, the proliferation and subsequent necrosis and depletion of these cells may be a response to chronic exposure to multiple environmental toxicants.

Numerous studies have shown that fish melanocytes respond to tissue damage from a variety of infectious and non‐infectious insults. For instance, certain trematode parasites initiate a melanocyte response that is involved in the encystment of these parasites, leading to what has been termed black spot disease (Charo‐Karisa et al. 2021). Melanistic areas or blotchy bass syndrome is caused by an Adomavirus, which induces a melanocytic response in the epidermis (Blazer, Young, et al. 2020; Iwanowicz et al. 2025). Inflammation in Zebrafish (Zebra Danio, Danio rerio ) has been shown to lead to the recruitment of melanocytes and melanoblasts to sites of tissue damage (Lévesque et al. 2013). Melanin is a complex polymer that can absorb and neutralise free radicals and other potentially toxic compounds. Numerous chemicals have been shown to bind to melanin, ultimately leading to chronic lesions. These include metals (including Cu, Mn and Zn), herbicides, dyes and polycyclic aromatic compounds; co‐exposures to metals and herbicides may increase binding affinity. This binding of toxic compounds may be intended to serve as a protective mechanism, but when exposure is chronic, it leads to adverse effects (reviewed by Larsson (1993)). The observation of BBH melanistic lesions only in adults suggests chronic exposure may be important.

Pigmented macrophage, or melanomacrophage, aggregates, as illustrated in Figure 9D, have been used for many years as bioindicators of exposure to environmental stressors (Wolke 1992; Agius and Roberts 2003) and have been shown to accumulate metals (Barst et al. 2015). Early studies suggested that the melanin granules within these cells result from phagocytosis of melanosomes released by damaged melanocytes. More recently, melanomacrophages are believed to be sites of primary melanogenesis, functioning to sequester products of cellular degradation, including ROS damage. Lipogenic pigments (ceroid and lipofuscin) are derived from cellular components through the process of oxidation (Agius and Roberts 2003). In studying melanised focal changes in the muscle of salmonids, amelanotic macrophage phenotypes were found to develop in situ into pigmented melanomacrophages as the condition progresses (Bjørgen and Koppang 2024; Bjørgen et al. 2024). Fish have two main types of macrophages. The M1 macrophages are primarily involved in acute inflammatory responses, while M2 macrophages are more associated with tissue repair (Forlenza et al. 2011). The presence of M2‐polarised melanomacrophages in the salmonid melanistic lesions suggested this polarisation plays a role in activating melanin synthesis within macrophages (Bjørgen and Koppang 2024). Moreover, these cells have been suggested to be involved in detoxification processes of environmental contaminants, in part due to the scavenger action of melanin (Fenoglio et al. 2005). Further research would be necessary to definitively identify the cell types and elucidate responses within the melanistic lesions.

A second hypothesis that has been proposed for the melanistic lesions is that of a new type of transmissible tumour. However, there is currently no evidence for actual transmission; rather, this is based on the number of genetic variants (mitochondrial and nuclear) in samples of skin from raised lesions versus the brain tissue (Curd et al., unpublished manuscript). Transmissible tumours are defined as those that have evolved the ability to infect another individual through direct transfer of a parasitic cancer clone (Ostrander et al. 2016). Certain factors have been identified as necessary for the successful horizontal transmission of cancer, including the shedding of a large number of ‘cancer’ cells that have the ability to survive outside the host, evade the new host's immune system and proliferate within the new host (Dujon et al. 2020). Several observations raise questions about the plausibility of this hypothesis for the melanistic lesions of BBH. First, the lesions are seen throughout a large lake, as well as in various other unconnected waterbodies in the Northeast. BBHs have been used as an indicator species due to their small home range and benthic behaviour, leading to exposure to both waterborne and sediment‐associated contaminants (Sakaris et al. 2005; Rafferty et al. 2009). While we are unaware of any movement studies of BBH in Lake Memphremagog, other studies have indicated limited long‐distance movement. In the Presque Isle Bay, Lake Erie AOC, a telemetry tagging study tracked movement associated with spawning. Site fidelity of adults was observed, suggesting they are a useful indicator species, as most adults are long‐term residents of a site (Millard et al. 2009). Similarly, an ultrasonic telemetry study verified the residency of adult BBH in the Anacostia River (Sakaris et al. 2005), raising questions of how a parasitic cancer cell of BBH moves throughout a large lake (50 km in length, 1780 km2 watershed), as well as to other distant water bodies in a few years. The ability of cells to survive in lake or pond water and invade and proliferate has not been demonstrated.

Microscopic pathology supports the progression of cellular and mutational (genetic) changes. Melanoma is one of the most highly mutated tumours of humans (Davis et al. 2018). For instance, in one study, the frequency of somatic mutations showed a variation from 0.1 to 100 per megabase in melanoma cells (Alexandrov et al. 2013). Interestingly, normal human skin has been shown to have two to six mutations per megabase, similar to some other tumour types (Martincorena et al. 2015). While UVR exposure is considered a major driver in melanoma in mammalian species, it accounts for only 40%–50% of the attributable risk for cutaneous melanoma (Meyskens and Yang 2010). Oxidative stress plays a role in the initiation and progression of melanoma (reviewed by Venza et al. 2015), and there is increasing evidence for the role of metals in melanoma development (Meyskens and Yang 2010; Narayan et al. 2024). Additionally, transgenic Zebra Danio models have been used for many years to study melanoma progression and potential treatments. While transgenic zebrafish tumours can be initiated with two genetic mutations, it takes 4–6 months for the tumours to develop, and by that time thousands more mutations have developed (Kansler et al. 2017). Consequently, it will be important to identify genetic mutations (rate and types) in various stages of the BBH melanistic lesions.

It seems likely that the skin of fish, including BBH, which are constantly exposed to environmental contaminants both through water and sediment, might have an even greater mutation rate. The difference in single nucleotide variants among tissues has been demonstrated, varies greatly across cell types and depends on the source of the mutations (Gomez‐Ramos et al. 2014; Yu et al. 2024). Consequently, comparing genetic variants in the brain versus advanced lesions in the skin may not be appropriate. Extracts from raised lesions, as depicted in Figure 8A,D, would contain DNA from the affected epithelial cells and the smaller, rounded melanomacrophages in the epidermis, as well as the proliferating and neoplastic dermal melanocytes, raising the question as to which of these cells is the hypothetical clonal, transmissible cancer cell. As stated by Bjørgen and Koppang (2024), ‘fish are not swimming humans’ and may have evolved their own solutions to both biological and chemical challenges, which we have yet to understand. Consequently, it will be necessary to compare DNA variants and transcriptomics in skin from clinically normal fish, normal‐appearing skin from affected individuals and early to advanced lesions to understand the factors associated with these lesions and the responses BBH have acquired to respond to these stressors. Identifying potential initiators and promoters of these pathological responses could inform better management of fish, human and ecosystem health.

The potential synergistic effects of As with other environmental factors, such as algal toxins, particularly cyanotoxins, viruses and as yet unidentified contaminants on pathological and genetic variants need to be considered. Climate change and eutrophication from an overabundance of nutrients like phosphorus have led to optimal conditions for cyanobacterial blooms and production of their multiple toxins, many of which are not well‐studied (reviewed by Falfushynska et al. (2023)). Lake Memphremagog suffers from eutrophication and harmful algal blooms that may be severe (Foulon et al. 2020); eutrophication in the other ponds containing BBH with melanistic lesions suggests that algal toxins could also occur in these waterbodies (Bunyon et al. 2023). Cyanotoxins have been linked to both the initiation and promotion of cancer, causing oxidative stress, DNA damage and disruption of cellular signalling pathways (Rajput et al. 2024). Certain cyanotoxins have been shown to cause dermal toxicity in humans; however, a direct correlation between cyanobacterial exposure and melanoma was not found; yet, based on epidemiological data, certain cyanotoxins may be an important risk factor for melanoma (Backović et al. 2020).

Viruses are another potential risk factor to consider. Tissue DNA and RNA sequences were screened for microbial organisms, including viruses. While viruses were detected, none were found to be specifically associated with the tumour tissue analysed (Curd et al., unpublished manuscript). Further analyses may be necessary to determine if any viruses could be initiators of early lesions.

PFAS concentrations were measured in the plasma of BBH from five of the stations (Blazer et al. 2026). Concentrations were low, and affected individuals did not have higher concentrations than clinically normal individuals. However, measuring PFAS in actual skin samples, as was done with the metals, may be important as PFAS preferentially bind to proteins (Liu et al. 2019) and hence could accumulate within the club cells, eventually leading to oxidative damage and necrosis. Concentrations of PFAS were greater in skin‐on fillets than skin‐off in channel catfish Ictalurus punctatus and other fish species (Figueroa‐Muñoz et al. 2025). Skin was also one of the main tissue targets for PFAS in rainbow trout Oncorhynchus mykiss (Goeritz et al. 2013).

In conclusion, there is still much to be learned about the melanistic lesions of fishes, potential synergistic effects of biological and chemical risk factors and the roles of melanocytes and classical immune cells such as macrophages in the development and progression of the observed lesions. The results presented here suggest As and Zn may play a role in the development of melanistic lesions. Melanocytes and melanomacrophages respond to oxidative damage, but it is not yet known if chronic exposure to As and bioaccumulation eventually leads to the neoplastic lesions or whether additional risk factors such as Zn, cyanotoxins, UVB or other contaminants act as promoters of carcinogenesis. Ongoing studies are focused on additional chemical testing of skin samples and transcriptome analyses to identify important pathways involved in the initiation and progression of these lesions. Utilising laser microdissection of histological sections to identify genetic variants and differential gene expression within the multiple cell types (club cells, melanocytes and melanomacrophages) could aid in the accurate determination of contributing factors. Addressing the hypothesis of a transmissible tumour will include attempts to determine the survivability of melanistic cells in lake and pond water and the ability of affected BBH to transmit the cancer to visibly normal individuals from the same water body, as well as to individuals from unaffected sites through cohabitation.

Author Contributions

Matthew Pehrson: investigation, writing – review and editing, resources. Matthew Bodnar: investigation, methodology, writing – review and editing. Thomas Jones: conceptualization, investigation, writing – review and editing, resources. Cheyenne R. Smith: visualization, writing – review and editing, investigation. Peter Emerson: conceptualization, investigation, funding acquisition, writing – review and editing, resources. Mark J. Henderson: writing – review and editing, project administration. Danielle Cleveland: investigation, writing – review and editing, validation, methodology, formal analysis. Patricia M. Mazik: writing – review and editing, funding acquisition, project administration. David R. Russell: investigation, writing – review and editing, resources. Vicki S. Blazer: conceptualization, investigation, funding acquisition, writing – original draft, methodology.

Funding

Funding was provided by the Eastern Ecological Science Center, US Geological Survey Ecosystem Mission Area's Cooperative Fish and Wildlife Research Unit and Environmental Health Programs and Vermont Dingle‐Johnson via the Sport Fish Restoration Act and Wildlife Service.

Ethics Statement

The protocol and procedures were reviewed and approved by the US Geological Survey Eastern Ecological Science Center Institutional Animal Care and Use Committee.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Elements analysed in skin tissue of brown bullhead and estimated detection and quantification limits for analyses.

JFD-49-e70207-s001.docx (16KB, docx)

Acknowledgements

We thank the numerous fish biologists and technicians in Vermont, New Hampshire, Maine and Quebec, Canada, who assisted in the Brown Bullhead collections. We are especially indebted to Jean‐Sebastien Messier with the Ministry of the Environment, Climate Change Action, Wildlife and Parks, for assistance with site location and sampling in Quebec, David Largy from the Memphremagog Regional County Municipality, city of Magog and Robert Benoit for providing shelter for field sampling; Lindsey Hartzell and Lydia Reed for assistance with fish necropsies; Adam Sperry and Sean Walsh for histopathology slide preparations and Vanessa Melton for assistance with microwave digestion of skin samples for elemental analyses. We also thank Stephanie Gordon for assistance with maps. Any use of trade, firm or product names is for descriptive purposes only and does not imply endorsement by the US government.

Data Availability Statement

The data that support the findings of this study are openly available in ScienceBase at https://doi.org/10.5066/P1RTUEKV.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1: Elements analysed in skin tissue of brown bullhead and estimated detection and quantification limits for analyses.

JFD-49-e70207-s001.docx (16KB, docx)

Data Availability Statement

The data that support the findings of this study are openly available in ScienceBase at https://doi.org/10.5066/P1RTUEKV.

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