Comparison of heavy metals, parasites and ...

Environmental Research 165 (2018) 306–316

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Comparison of heavy metals, parasites and histopathology in sculpins (Myoxocephalus spp.) from two sites at a lead-zinc mine in North East Greenland

T

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Rasmus Dyrmose Nørregaarda,b, , Mai Dangd, Lis Bacha, Ole Geertz-Hansenb, Kim Gustavsona, Peter Aastrupa, Pall S. Leifssonc, Jens Søndergaarda, Barbara Nowaka,d, Christian Sonnea a

Aarhus University, Faculty of Science and Technology, Department of Bioscience, Arctic Research Centre, Frederiksborgvej 399, 4000 Roskilde, Denmark Greenland Institute of Natural Resources, Department of Environment and Mineral Resources, Nuuk, Greenland University of Copenhagen, Faculty of Health, Department of Pathobiology, Frederiksberg, Denmark d Institute of Marine and Antarctic Studies, University of Tasmania, Launceston, Tasmania 7250, Australia b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Arctic Pollution Heavy metal Monitoring Fish Effects

The former lead-zinc mine at Mestersvig, Greenland, continues to contaminate the surrounding environment despite its operations ceasing over 50 years ago. Elevated concentrations of heavy metals are found in water, sediment and biota in the terrestrial, freshwater and marine environments. To shed light on the present contamination and its potential effects on local fish we investigated gill and liver histology of sculpins (Myoxocephalus spp.) around the former mining area. Two species of sculpins were caught; shorthorn sculpins (M. scorpius; n = 16) and fourhorn sculpins (M. quadricornis; n = 17) at a contaminated site, Nyhavn, and shorthorn sculpins (M. scorpius; n = 25) at the reference site. In a previous study we found histopathological changes in the tissues of the sculpins, and we suspected this to be related to elevated heavy metal tissue concentrations. Concentrations of Fe, Hg, Mn, Pb, Se and Zn were significantly higher in sculpins at Nyhavn compared to the reference site. Reference NOED and LOEC thresholds for biochemistry, tissue lesions, growth, survival and reproduction for hepatic Hg, As, Cd and Pb from the ERED database were exceeded in both sculpin species. Histopathological investigations of the sculpins gills revealed significant increases in the prevalence of hyperplastic epithelium, inflammation, intensity of neutral and total mucus cells and chloride cells along with an increased infection of colonial Peritricha. At the contaminated Nyhavn site, fourhorn sculpins had a significantly higher prevalence of chondroplastic tissue and intensity of neutral, mixed and total mucus cells in the gills compared to the shorthorn sculpins. The data indicate that both sculpin species could be useful indicator species for environmental monitoring of metal pollution in Arctic areas. However, confounding effects of gender and species should be investigated further. Effects on other biomarkers as well as baseline measurements should be included in future environmental monitoring efforts around mining activities in Greenland.

1. Introduction There is a rising international interest in the mineral rich underground of Greenland which has increased the exploration activities considerably during the last decade. The exploitation of minerals inevitably leads to the risk of dispersal of elements, including environmental contamination of heavy metals, to the environment. Greenland has a long history of mining and before proper environmental management was implemented, mining operations caused significant contamination of the local environment (Johansen et al., 2008, 2010; Schiedek et al., 2009). The Mestersvig lead-zinc mine in the high artic

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part of East Greenland operated from 1956 to 1963 and resulted in a significant long-term contamination on the surrounding terrestrial area, rivers and coastal waters. Elevated concentrations of especially lead (Pb) and zinc (Zn) in seawater sediments and surrounding biota is found (Johansen et al., 2008). In this area as well as at other mining sites in Greenland, sculpins have been used as a key monitoring species to assess the pollution by mining activities as it is common and relatively sedentary (Sonne et al., 2014). Typically, metal concentrations in sculpin liver and muscle have been used as a proxy for metal exposure of coastal waters, but such analyses lack information on the effects of exposure.

Corresponding author at: Aarhus University, Faculty of Science and Technology, Department of Bioscience, Arctic Research Centre, Frederiksborgvej 399, 4000 Roskilde, Denmark. E-mail address: [email protected] (R.D. Nørregaard).

https://doi.org/10.1016/j.envres.2018.04.016 Received 2 January 2018; Received in revised form 13 April 2018; Accepted 18 April 2018 0013-9351/ © 2018 Elsevier Inc. All rights reserved.


R.D. Nørregaard et al.

concentrate was loaded and shipped by ship. To facilitate transport of concentrate from the mine, a road was constructed between the mine and Nyhavn where the concentrate was stored and loaded onto barges. Concentrate spillage from the loading operation including 4-tons bags and whole barge loads of about 30 t of concentrate is the likely cause of the lead and zinc pollution of sediment and the seawater around the harbour (Johansen et al., 2008). In 1979, Pb and Zn concentrations were measured in seawater up to 0.51 µg/L and 0.58 µg/L, respectively (Johansen et al., 2008). In 1986, a detailed study of the marine sediment contamination was conducted and Pb concentrations over 10 mg/ g were found within 300 m of the quay (Johansen et al., 2008).

Adverse effects of exposure to heavy metals include effects on the nerve, reproductive and immune system, liver and kidney functions and hormonal balance in fish (Authman et al., 2015; Adams and Sonne, 2013; Depew et al., 2012; Crump and Trudeau, 2009; Scheuhammer et al., 2007; Oost et al., 2003; Matta et al., 2001). The main target for Pb and Zn waterborne exposure are the gills, where acute concentrations induce gill lesions eventually leading to hypoxia and suffocation (Authman et al., 2015). Specifically, Pb is known to cause adverse effects on reproduction, nerve and immune system including lesions, deformities and oxidative stress (Delistraty and Stone, 2007; Olojo et al., 2005; Shah and Altindag, 2005; Ercal et al., 2001; Katti and Sathyanesan, 1983). High Zn exposure has been shown to cause numerous different structural changes in gills, liver, kidneys and skeletal muscle changes in respiratory and cardiac systems as well as inhibition of growth and spawning (Authman et al., 2015; Dyk et al., 2007; Cariño and Cruz, 1990; Mallat, 1985; Nemcsók et al., 1981). Based on this, we decided to initiate studies of fish health around the Mestersvig lead-zinc mine in East Greenland. In Dang et al. (2017), a number of different histopathological lesions and parasites were identified in sculpins at Nyhavn. To further expand on the observations described in Dang et al. (2017), we wanted to compare histopathological data and an extended number of metals from the polluted site with a reference site. To that effect, a subset of the elemental and parasitic data from the polluted site Nyhavn published in Dang et al. (2017) were reused in the present investigation. We wanted to use histopathological data as effect markers for the heavy metal residues in liver and muscle to evaluate the effects of the Mestersvig lead-zinc mine. As such, the specific aims of the study were; i) to compare inter-site concentrations of heavy metals in sculpin from two sites around Mestersvig and ii) to evaluate a relationship between the prevalence of histopathological lesions and heavy metal concentrations in liver and muscle.

2.2. Sculpin sampling Sculpins were caught in August 2014 at Nyhavn and a reference site (Fig. 1) with gill nets deployed in the afternoon and sampled in the following morning. The area is known to host both shorthorn sculpins (Myoxocephalus scorpius) and fourhorn sculpins (Myoxocephalus quadricornis). All fish were identified to species. At Nyhavn a total of 17 fourhorn sculpins and 16 shorthorn sculpins were euthanized and sampled while at the reference site samples from only shorthorn sculpins (n = 25) were collected. The sculpins caught at Nyhavn and described in Dang et al. (2017) were reused in this study. Total length (TL, mm), total weight (g) and sex were recorded for each specimen before dissection. A sterile stainless-steel scalpel was used to dissect the fish and the sampling environment was kept clean and wiped off between fish to avoid contamination by the surrounding surfaces. For histological assessments, samples were carefully collected from the posterior lobe of the liver and the second right gill arch. These were stored in 4% buffered formalin until processing. Samples of the posterior lobe of the liver and of the right posterior muscle were collected for element analysis and individually placed in sterile polyethylene zip-lock bags and kept frozen (−20 °C) until element analysis. Otoliths were collected and the age of the fish determined using the “break and burn method” described in Brogan and Anderl (2012). A total of 25 and 33 sculpins were caught at the reference site and Nyhavn respectively.

2. Materials and methods 2.1. Study site The Mestersvig lead-zinc mine is located approximately 10 km inland from the coast (Fig. 1) at 72°11'N; 28°8'W. In 1979 it became clear that the mining operation at Mestersvig had caused widespread pollution (Johansen et al., 2008). The contamination of the coastal water is located around the harbour, Nyhavn (Fig. 1) where lead and zinc

2.3. Element analysis Liver and muscle samples were analysed for 13 elements relevant for the mineralogy of the area and thus local environmental pollution. Subsamples (1 g wet weight) were microwave digested (Anton Paar

Fig. 1. Mestersvig with the lead-zinc mine (Blyminen), the airfield, Nyhavn, Noret and the reference site as well as the main road connecting the mine with the airfield and Nyhavn (red dotted line). Sculpin sampling sites are marked with “X”. The reference station is approximately 8 km from Nyhavn. 307



1 cm−2 of surface area. Intensity of parasite in the gills was calculated as the number of infected filaments/total filaments. For more information on the histological methods, diagnoses and quantification in fish studies see Leonardi et al. (2009), Adams et al. (2010), Adams and Sonne (2013), Sonne et al. (2014) and Dang et al. (2017). Histological samples were not collected from every fish, and as such liver histology was evaluated in 25 and 32 of the sculpins caught at the reference site and Nyhavn respectively, while gill histology was determined in 22 and 26 sculpins respectively.

Multiwave 3000) in Teflon bombs in 4 ml/4 ml Merck Suprapure HNO3/milliQ water. Digestion solutions were diluted with milliQ water and analysed for chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), cadmium (Cd), mercury (Hg) and lead (Pb). All metal analyses were performed using an Agilent 7500ce ICP-MS at the accredited trace element laboratory at Department of Bioscience, Roskilde, Denmark. Analytical quality was assured by analysing blanks, duplicates and the certified reference materials DOLT-5, DORM-4 and TORT-2 (sediment, fish liver, fish protein and lobster, respectively) from the National Research Council Canada (www.nrc-cnrc.gc.com) along with the samples. The trace element laboratory is accredited for analyses of Cr, Ni, Cu, Zn, As, Se, Cd and Pb in biota with < 20% precision (2 standard deviations, SD) and participates twice a year in the international Quasimeme intercalibration program for laboratory performance (www.quasimeme.org). The recovery of elements in the certified reference materials in the present study was between 82% and 120% and the detection limit (determined as 3 SD of the concentrations measured in the blank samples) for each element is shown in Table 2. Element concentrations were determined in all 25 and 33 of the sculpins caught at the reference site and Nyhavn respectively. It should be noted that the Nyhavn part of the element data presented here, was also presented in Dang et al. (2017).

2.5. Statistics To test for differences in the biometrical data (length, weight and liver weight) at each site, two-way ANOVAs were applied. Since shorthorn sculpins were the only species caught at the reference site, any site differences were analysed using only shorthorn sculpins. Any species differences between fourhorn and shorthorn sculpins were analysed using only Nyhavn specimens. The scaled mass index (SMI) model was used to calculate condition indices and a linear model with age and gender as main effects were applied to test for differences between the sites (Peig and Green, 2009). Element concentrations in different organs were log transformed and differences between sites, species and tissues were analysed with linear models (LMs). Site or species differences in prevalence of lesions and parasite infection intensity were tested with binomial general linear models and linear models respectively. To test for correlations and site or species differences between chloride and mucus cell intensities a first order nested general linear mixed-effects model with (fishID: filament) as the random effect. Poisson general linear models (GLMs) were applied to correlate total lesions counts and element concentrations in liver or muscle. Statistical analyses were preformed using Rstudio (Rstudio version 0.99.491, 2016) and the level of significance was set to p < 0.05.

2.4. Histology Liver and gill samples were trimmed, processed using standard protocols for histology, embedded in paraffin, sectioned at about 4 µm, and stained with haematoxylin and eosin (HE) and Alcian Blue/ Periodic Acid Schiff (AB/PAS). The prevalence of lesions in the gills (congestions, telangiectasis, partial epithelium lifting, complete epithelium lifting, necrosis, epithelial hyperplasia, partial fusion, complete fusion, mononuclear cell infiltrations and chondroplasia) and the liver (haemorrhage, cytoplasmic alterations, nuclear alterations, necrosis, megalocytic hepatosis, bile duct proliferation, mononuclear cell infiltrations and granuloma) were evaluated using low- and high-power light microscope fields (50–400). Completely lifted epithelium and fused lamellae were categorized as such only when 100% of the lamellae were affected. Telangiectasis is used to describe dilations in the lamellae capillaries given the blood spaces is defined and separated by pillar cells, not the three-part wall structure of normal blood vessels, where such a dilation would be classified as an aneurism. All these findings were categorised as “present” or “absent”. Mucus cells were quantified using Alcian Blue/Periodic Acid Schiff (AB/PAS) staining (pH 1 and 2.5, Jones and Reid, 1978) and chloride cells were quantified using Na+/K ATPase antibody (Nowak et al., 2013) stained slides. The filaments for quantification of these cells were selected so that they represented a regular distribution across the gill arch. The number of mucous and chloride cells was counted in 10 inter-lamellar units in the middle of the selected filaments. Parasites were identified as far as possible (group or family or genus) on the basis of histology (Bruno et al., 2006). Parasite burden was determined based on prevalence (percentage of infected fish) and intensity (number of parasite per infected fish) (Bush et al., 1997). Individual intensity of a particular parasite in the liver was calculated as the number of those parasites in

3. Results 3.1. Sculpin biometrics The biometrical data are shown in Table 1. There were no gender specific differences at either site for weight, length and liver weight. At Nyhavn, the fourhorn sculpins weighted significantly less than the shorthorn sculpins (two-way ANOVA, p < 0.01). Mean age across both sites was approximately 5 years and there were no significant differences between site, species and gender. To check for the effect of site on condition index, the scaled mass index (SMI) model was applied to the biometry (see Table 1 for mean SMI scores). The condition indices calculated with the SMI model showed no significant differences between gender, species or site. The log relationship between length and weight for all sculpins caught at Mestersvig is shown in Fig. 2. 3.2. Heavy metal tissue concentrations by species and gender Mean heavy metal concentrations for the 12 elements in muscle and liver tissue at Nyhavn and at the reference site are shown in Table 2. While there was no significant difference in tissue concentrations

Table 1 Sculpins biometrics from the two sites sampled in August 2014. All data is given as mean ± SD. Site

Species

Gender

n

Age (y)

Length (cm)

Weight (g)

Liver weight (g)

SMI score

Reference

Shorthorn

Nyhavn

Fourhorn

Female Male Female Male Female Male

17 8 8 9 10 6

4.9 4.6 4.9 5.6 4.7 4.8

22.2 21.8 22.3 22.3 23.5 20.8

131 ± 30 117 ± 19 99 ± 38 88 ± 22 147 ± 42 108 ± 27

4.93 3.84 3.84 3.00 4.07 3.25

147 ± 81 114 ± 51 115 ± 87 94 ± 51 196 ± 111 91 ± 52

Shorthorn

± ± ± ± ± ±

0.8 0.9 1.7 2.2 0.8 1.0

308

± ± ± ± ± ±

2.0 1.7 2.2 1.5 2.1 1.4

± ± ± ± ± ±

3.67 0.99 2.63 1.00 1.43 1.05



all fish showed partly fused lamellae (Fig. 3C), while completely fused lamellae was found in 25% of the male fourhorn sculpins from Nyhavn. Mononuclear infiltrates were most prevalent in sculpins caught at Nyhavn, where 50% of both the fourhorn genders and respectively 100% and 50% of female and male shorthorn sculpins showed mononuclear cellular infiltrations. The prevalence of mononuclear infiltrates where significantly higher in the shorthorn sculpins at Nyhavn compared to the reference site (GLM, p < 0.001). Chondroplasia (Fig. 3E), hyperplasia of cartilaginous cells, was observed almost exclusively in fourhorn sculpins at Nyhavn, where 67% of the female and 100% of the male specimens displayed the lesion, leading to a statistically significant prevalence of the lesion between shorthorn and fourhorn sculpins at Nyhavn (GLM, p = 0.005). These lesions were often associated with a cyst of unknown origin (Fig. 3F) with a dark basophilic border surrounding a more eosinophilic centre. The number of neutral mucus cells (GL mixed-model, p = 0.014), total number of mucus cells (GL mixed-model, p < 0.001) and number of chloride cells (GL mixedmodel, p < 0.001) were significantly higher in the Nyhavn shorthorn sculpins compared to the references specimens (Table 4, Fig. 3D). While no significant species differences were found between fourhorn and shorthorn sculpins for chloride cells; neutral (GL mixed-model, p < 0.001), mixed (GL mixed-model, p < 0.001) and total numbers of mucus cells (GL mixed-model, p < 0.001) were at Nyhavn observed in significantly higher numbers in the fourhorn sculpins. Several parasites were identified in the sculpins; solitary Peritricha, Trichodinids, solitary Ciliates, Trematoda and Trypanosoma, but the only parasite that consistently appeared across multiple specimens was colonial Peritricha (Figs. 3D, E). The intensity of colonial Peritricha was species, site and gender specific (Table 6); at Nyhavn the male shorthorn sculpins showed a higher intensity than female (LM, p = 0.048) but the shorthorn sculpins generally showed a lower intensity than the fourhorn sculpins (LM, p < 0.001). Finally, the female shorthorn sculpins at Nyhavn had a significantly lower intensity of colonial peritricha than the shorthorn sculpins at the reference site (LM, p = 0.019).

Fig. 2. Weight length relationship for all sculpins caught at Mestersvig in 2014 represented on log scales. Dotted line is linear correlation and the grey area corresponds to the 95% confidence intervals.

between male and female shorthorn sculpins at either site, the male fourhorn sculpins at Nyhavn had significantly higher liver concentrations of Co and Se than females (LM, p < 0.05). At Nyhavn, significantly higher liver concentrations of Cu, Zn, Hg and Pb along with muscle concentrations of Cu, Zn, Hg and Pb were found in fourhorn compared to shorthorn sculpins (LM, p < 0.05). In shorthorn sculpins, concentrations of liver Fe and Mn along with muscle Se were significantly higher (LM, p < 0.05).

3.5. Liver histopathology 3.3. Heavy metals tissue concentrations between sites Liver samples were fully evaluated in 57 sculpins for histopathological lesions (Table 5) and parasites (Table 6). Megalocytic hepatosis (Fig. 4A) was defined as the hepatocytes 3 times larger than the average hepatocyte and it was the most common lesion observed (> 33%). This lesion was sometimes associated with cytoplasmic or nuclear alterations, but mostly associated with enlarged nucleoli. At Nyhavn, the prevalence of megalocytic hepatosis was significantly increased in female fourhorn sculpins compared to male fourhorns (GLM, p = 0.025). Nuclear alterations included hepatocytic nuclei exhibiting pleomorphism, pyknosis or other alterations of general shape or structure. At the reference site the prevalence of nuclear alterations for the female was 82% and for male shorthorn sculpins it was 75%, while at Nyhavn the lesions were only found in 56% of females and 50% of the male shorthorn sculpins. The difference between the two sites was statistically significant (GLM, p = 0.002). Mononuclear infiltrates (Fig. 4C) were found in high numbers across both sites and species (> 38%). The highest prevalence was found in male fourhorn sculpins at Nyhavn where 89% of the individuals exhibited the lesion. Prevalence among the female fourhorn sculpins at Nyhavn was 38%, which was significantly lower than in the male fourhorn sculpins (GLM, p = 0.008). Granulomas (Fig. 4B) were present in up to 18% of the fish, and were observed both with and without an inflammatory response in the surrounding tissues. Bile duct proliferation, i.e. an abnormally high number of bile ducts in an area (Fig. 4D), was also generally low across both the sites and species (0–17%). Haemorrhages, cytoplasmic alterations, necrosis (Fig. 4E), and interstitial fibrosis (Fig. 4F) were all observed in generally higher numbers, and no statistically significant patterns could be determined between sites, gender or species. Two

Significantly higher concentrations of Fe, Hg, Mn, Pb, Se and Zn were found in shorthorn sculpins at Nyhavn compared to reference site shorthorn sculpins (Table 2, LM, p < 0.05). For Fe, the highest concentrations were found in muscle while Zn, Mn and Hg were found in higher concentrations in liver tissue. Pb and Se concentrations were higher in both liver and muscle in the shorthorn sculpins at Nyhavn than at the reference site. Of the 12 elements analysed, As, Co, Cu, Fe, Mn, Se, Cd and Zn were present in significantly higher concentrations in the liver than muscle of shorthorn sculpins at both Nyhavn and at the reference site (LM, p < 0.05). In contrast, Hg and Pb concentrations were significantly higher in the muscle than in liver at both sites (LM, p < 0.05). 3.4. Gill histopathology A total of 48 gill samples from the two sites were examined for histological lesions (Tables 3, 4) and parasites (Table 6). The most common lesions observed were apical or basal congestion which occurred in 75–100% of the specimens. Epithelial lifting (Fig. 3B) was found in 33–100% of the fish, while telangiectasis (Fig. 3A) was also found in relatively high prevalence (17–75%). These lesions were present across both sites, species and genders. Only fish from Nyhavn showed necrosis in the gills, but the prevalence was not statistically significant. Occurrence of hyperplastic epithelium of the secondary lamella was significantly higher in the specimens from Nyhavn (GLM, p < 0.001), where more than 50% of both fourhorn and shorthorn sculpins of both genders displayed the lesion. Between 0% and 38% of 309

Fourhorn

Nyhavn

310

Nyhavn

D.L. Reference

Station

Shorthorn

D.L. Reference

Male

Female

Male

Female

Male

Female

Gender

Zn 0.074 21.32 ± 3.10 1.94 ± 0.64 19.57 ± 2.92 2.03 ± 0.35 31.44 ± 2.70 4.96 ± 1.49 29.45 ± 5.34 4.74 ± 1.17 24.04 ± 2.98 2.23 ± 0.96 24.36 ± 5.85 2.03 ± 0.35

As 0.010 6.37 ± 0.85 ± 8.25 ± 0.76 ± 2.79 ± 1.07 ± 3.62 ± 0.91 ± 4.27 ± 0.86 ± 4.15 ± 0.68 ± 3.50 0.53 5.40 0.35 1.26 0.28 1.16 0.34 1.86 0.58 2.18 0.39

Se 0.072 1.16 ± 0.13 ± 1.31 ± 0.13 ± 1.43 ± 0.15 ± 2.26 ± 0.14 ± 1.47 ± 0.18 ± 1.75 ± 0.15 ±

Metal concentrations in sculpin tissues (µg/g ww)

Shorthorn

Species

Station

0.28 0.03 0.42 0.02 0.25 0.03 0.60 0.03 0.30 0.07 0.44 0.03

Liver Muscle Liver Muscle Liver Muscle Liver Muscle Liver Muscle Liver Muscle

Organ

Ag 0.003 0.03 ± < D.L 0.06 ± < D.L 0.02 ± < D.L 0.03 ± < D.L 0.03 ± < D.L 0.03 ± < D.L 0.02

0.03

0.02

0.01

0.05

0.03

17 17 8 8 8 8 9 9 10 10 6 6

n

Cd 0.005 0.50 ± < D.L 0.48 ± < D.L 0.77 ± < D.L 0.63 ± < D.L 0.46 ± < D.L 0.74 ± < D.L 0.43

0.17

0.24

0.46

0.37

0.31

Fe 0.455 45.20 ± 22.21 1.82 ± 0.75 43.12 ± 21.14 1.66 ± 0.35 17.19 ± 4.89 4.15 ± 1.85 19.07 ± 6.34 4.96 ± 5.04 39.79 ± 13.42 5.04 ± 8.13 48.58 ± 23.93 5.57 ± 8.29

Hg 0.009 0.02 ± 0.01 0.014 ± 0.007 0.02 ± 0.01 0.012 ± 0.005 0.12 ± 0.03 0.039 ± 0.012 0.11 ± 0.05 0.043 ± 0.011 0.03 ± 0.01 0.016 ± 0.007 0.03 ± 0.01 0.015 ± 0.007

Ni 0.063 0.07 ± 0.04 < D.L 0.08 ± 0.06 < D.L < D.L < D.L < D.L < D.L < D.L < D.L 0.09 ± 0.05 < D.L

Pb 0.008 0.01 ± < D.L < D.L < D.L 1.22 ± 0.38 ± 1.37 ± 0.27 ± 0.09 ± < D.L 0.20 ± 0.18 ±

Metal concentrations in sculpin tissues (µg/g ww)

0.33 0.34

0.39 0.61 0.65 0.43 0.08

0.01

Cr 0.137 < D.L < D.L < D.L < D.L < D.L < D.L < D.L < D.L < D.L < D.L < D.L < D.L

Co 0.004 0.06 ± 0.03 0.004 ± 0.001 0.09 ± 0.07 0.004 ± 0.001 0.06 ± 0.03 0.004 ± 0.001 0.12 ± 0.03 0.005 ± 0.003 0.06 ± 0.02 0.005 ± 0.002 0.08 ± 0.02 0.005 ± 0.002

Table 2 Sculpins element tissue concentrations (µg/g ww) at Nyhavn and the reference site from sculpins caught in August 2014. All data is given as mean ± SD. D.L.: detection limit.

Mn 0.108 1.13 ± 0.37 ± 1.08 ± 0.50 ± 0.88 ± 0.43 ± 0.79 ± 0.46 ± 1.44 ± 0.42 ± 1.61 ± 0.58 ±

Cu 0.080 1.29 ± 0.09 ± 1.91 ± 0.09 ± 2.48 ± 0.16 ± 2.96 ± 0.19 ± 1.30 ± 0.13 ± 1.59 ± 0.11 ±

0.28 0.09 0.20 0.29 0.23 0.13 0.23 0.33 0.33 0.17 0.61 0.53

0.43 0.03 0.95 0.02 1.08 0.06 1.30 0.07 0.36 0.05 0.47 0.02





Table 3 Prevalence (%) of lesions found in the gills of shorthorn sculpins at the reference site (9) and shorthorn (12) and fourhorn (14) sculpins at Nyhavn. Analysed with binomial general linear model (p < 0.05). C = Congestions, T = Telangiectasis, ELP = Epithelium Lift, Partly, ELC = Epithelium Lift, Completely, N = Necrosis, EHP = Epithelium Hyperplasia, FP = Fusions of lamellae, Partly, FC = Fusions, Completely, MNCI = Mononuclear Cell Infiltrations, CP = Chondroplasia. Site

Species

Gender

n

Prevalence (%) of lesions

Reference

Shorthorn

Nyhavn

Fourhorn

Both Female Male Both Female Male Both Female Male

9 6 3 14 6 8 12 8 4

C 89 83 100 93 100 88 92 100 75

Shorthorn

a-b

= significant differences between sites.

c-d

T 33 17 67 64 50 75 67 63 75

ELP 100 100 100 100 100 100 83 88 75

ELC 33 33 33 71 83 63 42 38 50

N 0 0 0 21 17 25 25 13 50

EHP 11b 17 0 50 50 50 67a 63 75

FP 22 33 0 21 0 38 17 25 0

FC 0 0 0 14 0 25 0 0 0

MNCI 22b 33 0 50 50 50 83a 100 50

CP 0 0 0 86c 67 100 17d 25 0

= significant differences between species at Nyhavn.

Table 4 Mean intensity of acidic (MA), neutral (MNEU), mixed (MM), total (MT) mucus cells and chloride cells (CC) in shorthorn and fourhorn at Nyhavn and the reference site. Values are average number of cells per inter lamella unit (ILU) ∓ S.D with number of replicates in parentheses. Analysed with a first order nested general linear model (p < 0.05). Site

Species

Mean intensity (cells/ILU) of mucus and chloride cells in gills ∓ S.D

Reference Nyhavn

Shorthorn Fourhorn Shorthorn

MA 0 ∓ 0 (19) 0.01 ∓ 0.11 (11) 0 ∓ 0 (11)

a-b


c-d

MNEU 0.16 ∓ 0.43 (19)b 1.54 ∓ 1.45 (11)c 0.25 ∓ 0.53 (11)a,d

MM 0.002 ∓ 0.46 (19) 0.23 ∓ 0.48 (11)c 0.013 ∓ 0.11 (11)d

MT 0.13 ∓ 0.39 (19)b 1.77 ∓ 1.58 (11)c 0.27 ∓ 0.55 (11)a,d

CC 3.52 ∓ 1.63 (9)b 4.78 ∓ 2.16 (9)a

= significant differences between species at Nyhavn.

Table 5 Prevalence (%) of lesions found in the liver of shorthorn sculpins at the reference site (25) and shorthorn (15) and fourhorn (17) sculpins at Nyhavn. Analysed with binomial general linear model (p < 0.05). HH = Haemorrhage, HPA = Cytoplasmic Alterations, HNA = Nuclear Alterations, N = Necrosis, MCH = Megalocytic Hepatosis, BDP = Bileduct Proliferation, MNCI = Mononuclear Cell Infiltrations, G = Granuloma, IF = Interstitial fibrosis. Site

Species

Gender

n

Prevalence (%) of lesions

Reference

Shorthorn

Nyhavn

Fourhorn

Both Female Male Both Female Male Both Female Male

25 17 8 17 8 9 15 9 6

HH 12 6 25 18 13 22 20 22 17

Shorthorn

a-b


e-f

HPA 40 35 50 24 25 22 40 44 33

HNA 80a 82 75 41 50 33 53b 56 50

N 28 29 25 35 25 44 47 44 50

MCH 68 71 63 47 63e 33f 53 56 50

BDP 12 12 13 6 0 11 7 0 17

MNCI 52 53 50 65 38f 89e 60 56 67

G 12 18 0 6 0 11 7 11 0

IF 44 47 38 53 50 56 40 44 33

= significant differences between genders.

Table 6 Parasites intensity in gills (% filaments) and liver (number/cm2 surface) in shorthorn and fourhorn sculpins at Nyhavn and the reference site. Values are average intensity ∓ S.D with replicates in parentheses. Analysed with linear model (p < 0.05). COLP = colonial Peritricha in gill filaments, APIC = Apicomplexa in liver, ACDS = acid fast spores in liver. Site

Species

Gender

Intensity of parasites in gills (% filaments) and liver (% surface) ∓ S.D

Reference

Shorthorn

Both

COLP 29.9 ∓ 28.1 (23)a

APIC 1.2 ∓ 2.4 (23)

Female Male Both Female Male Both Female Male

32.5 ∓ 28.9 (16) 22.3 ∓ 27.0 (7) 91.1 ∓ 15.4 (14)c 96.9 ∓ 6.3 (6) 87.9 ∓ 18.5 (8) 18.3 ∓ 25.1 (12)b,d 8.7 ∓ 15.2 (8)f 43.8 ∓ 31.8 (4)e

0.8 ∓ 1.6 (17) 2.1 ∓ 3.6 (8) 0. ∓ 0 (17) 0 ∓ 0 (8) 0 ∓ 0 (9) 0.9 ∓ 2.0 (15) 1.1 ∓ 2.4 (9) 0.4 ∓ 0.9 (6)

Nyhavn

Fourhorn

Shorthorn

a-b


c-d

= significant differences between species at Nyhavn. 311

e-f

= significant differences between genders.

ACDS 1.5 ∓ 3.73 (25) 1.9 ∓ 4.4 (17) 0.4 ∓ 0.8 (8) 0.2 ∓ 0.4 (17) 0 ∓ 0 (8)f 0.3 ∓ 0.5 (9)e 0.6 ∓ 1.9 (15) 0.1 ∓ 0.4 (9)f 1.5 ∓ 3.4 (6)e



Fig. 3. Histology observed in the sculpin gills. H&E staining. (A) Telangiectasia in secondary lamella. (B) Epithelial lifting in secondary lamella. (C) Complete fusions of secondary lamella. (D) Mucus cell hyperplasia (large arrow), colonial Peritricha (small arrows). (E) Chondroplasia (large arrow), Colonial peritricha (small arrows). (F) Basophilic cyst in the centre of a region with chondroplasia.

peritricha was positively correlated with hepatic concentrations of Zn, As, Cu and Co and muscle concentrations of As (GLM, p < 0.05). Intensity of chloride cells were positively correlated with hepatic Pb and Se concentrations and negatively correlated with liver concentrations of Cu, Cd and Ag, while the intensity of total mucus cells was positively correlated with hepatic Pb and Se and negatively correlated with hepatic Cu, Fe, Co and Ag concentrations (GLM, p < 0.05).

distinct parasites were found in the liver of the sculpins; Apicomplexa and acid fast spores. Both parasites were present in equal numbers across the two species at Nyhavn, between the two sites and the genders, with the exception of Nyhavn male sculpins of both species who had a significantly higher intensity of acid spores than the females (fourhorn: LM, p = 0.009, shorthorn: LM, p = 0.011). 3.6. Heavy metals and histopathology

4. Discussion Including only shorthorn sculpins, fish from Nyhavn had a significantly higher number of total gill lesions and the total number of gill lesions were positively correlated with hepatic concentrations of Hg, Zn, Pb and Mn (GLM, p < 0.05). Analysing the lesions and parasites in the shorthorn sculpins revealed several correlations to the heavy metal concentrations measured in the liver and muscle tissues. Prevalence of hyperplastic gill epithelium were positively correlated with hepatic Zn, Pb, Mn and Se concentrations as well as the concentration of Pb in muscle (GLM, p < 0.05). The prevalence of mononuclear infiltrations in the gills were positively correlated with hepatic concentrations of Pb and Mn and muscle Pb concentrations, while the prevalence of hepatic nuclear alterations was negatively correlated with hepatic concentrations of Zn, Se and Pb (GLM, p < 0.05). The intensity of colonial

4.1. Heavy metal concentrations Sculpins are used as a biomonitoring species to assess environmental pollution in Greenland; as it is a relatively sedentary species, common in coastal waters, and has been shown to accumulate metals from the environment (Bach and Asmund, 2013; Søndergaard et al., 2015). Effects of gender and organ on heavy metal uptake in sculpins has previously been studied (Kaarsholm and Verland, 2016; Sonne et al., 2014; Dietz et al., 2013). Sonne et al. (2014) showed that female sculpins had higher liver concentrations of Fe, Co, Ni, Cu and Se than males while Kaarsholm and Verland (2016) showed large differences in metal concentrations between organs (gills, liver and muscle) in 312



Fig. 4. Histology observed in the sculpin livers. H&E staining. (A) Megalocytic hepatosis. (B) Granuloma. (C) Focal mononuclear infiltrations. (D) Bileduct proliferation. (E) Necrotic tissue. (F) Interstitial fibrosis.

this is not the case for the site at Nyhavn located in the Kong Oscars Fjord system with a salinity of usually > 30 ppm (Overgaard et al., 1983). The biometric data on length, weight and age were normally distributed along with the calculated condition indexes, and the parameters were not deviate significantly between the two sculpin species. No specific literature exists on the diet for these two sculpin species in East Greenland. However, generally for the Arctic, reported food items for both species include benthic crustaceans, worms and fish (Atkinson and Percy, 1992; Houston, 1990; Morrow, 1980; Mukhomediyarov, 1967). Without knowledge of the above mentioned parameters for the East Greenland sculpins, it is hard to conclude whether the species-specific differences are due to differences in intrinsic factors or e.g. a lack of data points. Referencing the Environmental Residue-Effects Database (ERED; https://ered.el.erdc.dren.mil) and including sculpins specimens from the two sites, the lowest observed effect dose (LOED) for biochemistry, tissue lesions, growth, survival and reproduction in fathead minnows (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss) were exceed for hepatic Cd (0.42–1.8 μg/g ww) in 23 (56%) of the shorthorn sculpins and 13 (76%) of the fourhorn sculpins. Similarly, the hepatic Pb concentrations for no observed effect dose (NOED) for biochemistry (0.32 μg/g ww) were exceeded in 17 (100%) of the fourhorn sculpins,

sculpins caught outside Scoresbysund, East Greenland. Gender differences may be due to differences in growth, spawning, metabolism and diet (AMAP, 2005, 1998). In this study, we found gender differences in heavy metal concentrations in the fourhorn sculpins at Nyhavn, where liver Co and Se concentrations were significantly highest in the males. The lack of gender difference for shorthorn sculpins in this study may originate from a skewed dataset, e.g. the number of female shorthorn sculpins caught at Nyhavn is 17, compared to 8 males. The fourhorn and shorthorn sculpins at Nyhavn exhibited distinct differences in heavy metal concentrations. Liver and muscle concentrations of Cu, Zn, Hg and Pb are all higher in the fourhorn sculpins, while liver concentrations of Fe and Mn and muscle concentrations of Se were higher in shorthorn sculpins. Species-specific differences in uptake and accumulation are well known for fish (Dietz et al., 2013). Intrinsic factors known to affect the bioaccumulation of metals include spatial habitat, fish age and diet (Jezierska and Witeska, 2006). A previous study by Dang et al. (2017) on sculpins in Mestersvig suggests that habitat differences are the likely cause for differences in metal accumulation between the two species. Fourhorn sculpins have an increased tolerance for lower salinity and this may allow fourhorn sculpins to enter more polluted areas caused by e.g. the wash of tailings through the Tunnelelv (Dang et al., 2017). While such a scenario might be true for an area highly influenced by freshwater, e.g. Noret (Fig. 1), 313



bucket, time spent caught in the net, or time out of seawater when collecting the fish from the net caused the telangiectasis and epithelial lifting due to fixation delays. Statistically, the total number of gill lesions was higher at Nyhavn than at the reference site, however, no such difference was present for liver lesions. The significantly higher concentrations of Fe, Hg, Mn, Pb, Se and Zn found in liver and/or muscle in sculpins from Nyhavn could potentially be a cofactor in the development of the gill lesions found in the present study (Authman et al., 2015; Lujic et al., 2015; Sonne et al., 2014; Poleksic et al., 2010; Olojo et al., 2005; Jezierska and Witeska, 2004; Lemly, 2002; Dalzell and Macfarlane, 1999; Tao et al., 1999; Mallat, 1985). The Nyhavn specific increases were prevalence of hyperplastic gill epithelium, mononuclear infiltrates or inflammation in gills, intensity of neutral and total mucus cells and chloride cells along with an increased infection of colonial Peritricha. The prevalence of hyperplastic epithelium in gills was positively correlated with liver Zn, Pb, Se and Mn concentrations, and at least Zn and Pb have previously been linked to this specific response (Subashkumar and Selvanayagam, 2014; Olojo et al., 2005; Parashar and Banerjee, 1999). The prevalence of inflammation in the gills was positively correlated with Pb and Mn and both of these metals have previously been linked to immune responses in fish (Guernic et al., 2016; Morcillo et al., 2016; Morcillo et al., 2015; Nilantika et al., 2014). Colonial Peritricha do not cause severe infections in healthy fish (Lom and Dyková, 1992), and while, specifically, in the female shorthorn sculpins at the reference site a greater occurrence of colonial Peritricha was observed, a more general positive correlation with Zn, As, Cu and Co were found, suggesting that the fish with higher heavy metal concentration are more stressed. Similarly, due to the protective nature of mucus, an increased number of mucus cells suggested that the affected fish live in more adverse conditions (Dang et al., 2017; Paulino et al., 2012; Shephard, 1994), including exposure to metals such as As, Fe and Zn (Authman et al., 2015; Jezierska and Witeska, 2004; Mallat, 1985). The intensity of the total mucus cells was positively correlated with Pb and Se, and while correlation with the heavy metal concentrations was not shown for the neutral mucus cells, they did correlate positively with increasing intensity of the colonial peritricha infection. This implies that the increased mucus cell numbers occur as a consequence of not only increased heavy metal concentrations in the environment, but also to a more severely infected condition. Two distinct histopathologies were identified as species-specific, both occurring almost exclusively in fourhorn sculpins at Nyhavn; prevalence of chondroplasia and intensity of neutral, mixed and total mucus cells in gills. This is in agreement with our previous observations (Dang et al., 2017). Occurrences of gross cartilage deformities in fourhorn sculpins have been reported by Bengtsson et al. (1988) and Bengtsson and Larsson (1986) in fourhorn sculpins exposed to metals. For more in-depth information on species-specific histopathological lesions as well as parasite identification and categorization in fourhorn and shorthorn sculpins caught at Mestersvig the reader is referred to Dang et al. (2017). Other known causes for histological effects in fish are e.g. starvation, temperature, salinity, oxygen saturation, parasites, virus, bacteria and oxidative stress (Roberts, 2012; Ernst et al., 2006), so a definite causation between the metal concentrations in sculpin and histological effects presented here cannot be established. However, presented together with the fact that various reference LOED and NOEC thresholds for hepatic Hg, As, Cd and Pb were exceeded in a large percentage of the fish, we consider it likely that the sculpins caught at the Nyhavn were experiencing increased levels of stress.

Table 7 Mean Pb concentrations (µg/g dw) ± SD in sculpin liver and muscle sampled at the Mestersvig mine from 1985 to 2014. Pb concentrations from the years 1985 – 2001 are from Johansen et al. (2008). Site

Year

n

Pb concentrations (µg/g dw)

Nyhavn

1985a 1991a 1996a 2001a 2014 Shorthorn Fourhorn 1991a 1996a 2001a 2014b

3 11 13 10 33 16 17 10 4 10 25

Muscle < 0.15 0.39 0.06 0.02 0.87 ± 1.61 0.46 ± 1.02 1.27 ± 1.97 < 0.15 0.05 0.03 0.02 ± 0.01

Reference

a b

Liver 0.32 4.13 0.36 0.1 2.13 ± 0.39 ± 3.76 ± < 0.4 0.07 0.04 0.02 ±

2.05 0.59 1.50

0.04

Species unidentified. Only shorthorns caught at this site.

while LOEDs for growth and survival were exceed for hepatic As (11.6 μg/g ww) in 4 (10%) of the shorthorn sculpins. Lastly, for hepatic Hg the LOED for growth (0.01 μg/g ww) were exceed in 23 (56%) of the shorthorn sculpins and in 17 (100%) of the fourhorn sculpins, while the NOED for survival (0.1 μg/g ww) were exceed in 7 (17%) of the shorthorn sculpins and in 9 (53%) of the fourhorn sculpins. Pb concentrations in muscle and liver tissue from sculpins caught at Mestersvig in this and previous studies are shown in Table 7 (1985 – 2001). Historically, awareness of species differences has not been taken into account in environmental assessment and the two sculpin species have generally been treated as Myoxocephalus spp. The data show a major difference in heavy metal concentrations related to species, i.e. when both fourhorn and shorthorn are included in the 2014 data, the mean muscle and liver Pb concentrations are 0.87 and 2.13 µg/g dry weight (dw) respectively, and for shorthorn only the concentrations drop to 0.46 and 0.39 µg/g dw respectively. Importantly, Table 7 also illustrates temporal fluctuations in Pb tissue concentrations and based on the species-specific analyses reported in present paper, theses fluctuations might very well originate from different sampling ratios of fourhorn to shorthorn sculpins and/or different age distributions between years. Due to the species-specific differences in heavy metal concentrations and because fourhorn sculpins were not found at both sites, differences in heavy metal concentrations between sites were only explored for the shorthorn sculpins. The significant site differences included Fe in muscle, Mn, Zn and Hg in liver and Pb and Se in both liver and muscle. All these concentrations were higher in the Nyhavn sculpins. Sonne et al. (2014) reported a similar gradient in heavy metal concentrations in liver of sculpins sampled around the former lead-zinc mine Black Angel at Maarmorilik, West Greenland. The most pronounced gradients reported by Sonne et al. (2014) was Pb, As and Hg which decreased as a function of source distance. 4.2. Histology The prevalence of telangiectasis and epithelial lifting were very high in all of the sculpin gills examined in this study. Due to the high numbers across all the individuals, some of these lesions are likely to be the result of sampling artifacts and/or post mortem changes rather than true pathology. Both telangiectasis and epithelial lifting are a common artifact due to sampling and fixing procedures (Wolf et al., 2015). Telangiectasis has previously been reported as a post-mortem artefact due to euthanasia, while epithelial lifting is induced by poor fixation and water quality (Wolf et al., 2015). Fish used in the present study were dissected within one hour of landing. Though the fish were kept in seawater, it is still likely that conditions with a high number of fish per

5. Conclusion The lead-zinc mine at Mestersvig in East-Greenland ceased operation in 1963 and despite being closed for more than 50 years contamination of heavy metals are still found in the surrounding marine environment. Concentrations of heavy metals in liver and muscle, 314



lesion prevalence and intensity along with infection condition were higher in shorthorn sculpins caught at contaminated site (Nyhavn) than at the reference site. Reference NOEC and LOEC thresholds for biochemistry, tissue lesions, growth, survival and reproduction for Hg, As, Cd and Pb were exceeded in several of the fish. Species-specific analyses revealed increased heavy metal concentrations in fourhorn sculpins compared to shorthorn sculpins at Nyhavn as well as differences in lesion prevalence and positive correlations between gill lesions and heavy metal concentrations in both liver and muscle were established. This study indicates that both sculpin species can be a useful species for environmental monitoring of contamination and toxic effects of mining project in arctic areas, while confounding effects of gender, species and age should be investigated further.

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