Application of a quantitative histological health index for Antarctic rock cod (Trematomus bernacchii) from Davis Station, East Antarctica

Highlights

• Histological alteration in Antarctic rock cod exposed to Davis Station wastewater.

• Severity of organ pathologies directly related to proximity to outfall.

• Histological health index indicates poor fish health within 4 km of outfall.

• Gill most severely affected organ followed by liver.

• Higher level of wastewater treatment required to minimise impact on marine biota.

Abstract

A quantitative Histological Health Index (HHI) was applied to Antarctic rock cod (Trematomus bernacchii) using gill, liver, spleen, kidney and gonad to assess the impact of wastewater effluent from Davis Station, East Antarctica. A total of 120 fish were collected from 6 sites in the Prydz Bay region of East Antarctica at varying distances from the wastewater outfall. The HHI revealed a greater severity of alteration in fish at the wastewater outfall, which decreased stepwise with distance. Gill and liver displayed the greatest severity of alteration in fish occurring in close proximity to the wastewater outfall, showing severe and pronounced alteration respectively. Findings of the HHI add to a growing weight of evidence indicating that the current level of wastewater treatment at Davis Station is insufficient to prevent impact to the surrounding environment. The HHI for T. bernacchii developed in this study is recommended as a useful risk assessment tool for assessing in situ, sub-lethal impacts from station-derived contamination in coastal regions throughout Antarctica.

Introduction

Histological biomarkers represent a powerful tool to assess the sub-lethal effects of toxicants to organisms in situ. Cellular alteration signifies the cumulative adverse physiological and biochemical consequences of long term exposure to contaminants (Hinton and Lauren, 1990). Pathological change in cell structure can impair organ function by disrupting key biological processes including detoxification, osmoregulation and reproduction (Couch and Fournie, 1993, Au, 2004). Alterations illustrate a definitive endpoint of historical exposure, intermediate between initial biochemical changes and reproductive capability and growth (Hinton and Lauren, 1990, Stentiford et al., 2003, Salamat et al., 2013).

Organs of particular importance for histological analysis in fish are the gills, liver, kidney, spleen and gonad (Hinton and Lauren, 1990, Teh et al., 1997, Pawert et al., 1998, Stentiford et al., 2003, Osman et al., 2010). Gill and liver in particular, represent histological biomarker organs of contaminant exposure (e.g. Hinton and Lauren, 1990, Heath, 1995, Pawert et al., 1998, Wood, 2001, Stentiford et al., 2003, van Dyk et al., 2009, Van Dyk et al., 2012, Osman et al., 2010, Sonne et al., 2014). Cellular alteration of any of these organs could significantly reduce overall fish health (Schlacher et al., 2007).

Prevalence of histological alterations provides a percentage measure of individuals affected (Bucher and Hofer, 1993, Nowak, 1996, Olojo et al., 2005). Further quantification of alteration in terms of number, size and/or area, allows specific comparison between individuals. However, quantification only allows judgements to be made based on individual organs and not overall severity in terms of a fish's health. Health indices apply scores representing multiple organ condition, which leads to an indication of overall fish health. Bernet et al. (1999) developed a histopathological fish health index which represents a culmination of these approaches. It includes a severity measure indicating the importance of alteration (in terms of reversibility and recovery) within an individual organ, and incorporates a number of organs to assess the overall condition or health of a fish. The cumulative histological fish health index value represents the degree of alteration of tissue and organ damage: the higher the fish health index the greater the degree and severity of damage to multiple organs. Zimmerli et al. (2007) has advanced the concept further and developed a scoring scheme for the organ index: an organ index value of 10 indicating normal/healthy structure, through to values exceeding 40, indicating severe modification of structure.

The fish index assessment is valuable in that it maximises the quantity of information obtained from each individual fish. Collection of fish in Antarctica is costly and logistically difficult. Constraints including unpredictable weather and sea ice extent generally allow only a very short window of opportunity for sampling near-shore marine ecosystems during the summer season. Permitting restrictions further limit the number of fish available for scientific purposes, reinforcing the necessity of gaining as much information as possible from each individual collected.

Current toxicological impact assessment in Antarctica has largely focused on macro-invertebrate community composition and toxicity tests (Stark et al., 2011, Marcus-Zamora et al., 2015), and as a consequence, incorporation of sub-lethal impacts on higher order predators is needed. Little is known in relation to the biology of the common Antarctic rock cod (Trematomus bernacchii), including life history, and potential pathology. Rock cod are considered key indicator species of station-derived contamination impact in Antarctica, and represent an ideal biomonitor in environmental impact assessments (Miller et al., 1998, Jiménez et al., 1999, Di Bello et al., 2007). Individuals are long lived, benthic, opportunistic feeders with a restricted home range of approximately 500 m, and consequently subject to exposure at, or within close proximity to the site of collection (Kawaguchi et al., 1989, Evans et al., 2000, Herbert et al., 2003). Two studies have considered histological alteration in T. bernacchii as potential biomarkers (Evans et al., 2000, Corbett et al., 2014). The gill and liver tissue of T. bernacchii showed greater prevalence of pathologies in individuals within close proximity to the U.S McMurdo Station (Evans et al., 2000). Similarly, gill and liver tissue of T. bernacchii exhibited histological alterations directly related to wastewater contamination from Australia's Davis Station (Corbett et al., 2014).

Historically, wastewater in Antarctica has been discharged directly into the sea (Bargagli, 2008, Tin et al., 2009). A 2005 survey of research station discharge revealed 37% of permanently manned stations and 69% of ‘summer only’ stations lacked wastewater treatment (Gröndahl et al., 2009). In addition, existing treatment facilities risk inefficiency or even failure during peak occupancy periods (Gröndahl et al., 2009, Stark et al., 2015). Wastewater treatment system operation and design must be robust and flexible enough to cope with conditions unique to the Antarctic environment: extreme cold and dry climatic conditions, high energy requirement for heating, pumps and motors, considerable daily and seasonal variation in volume and timing of discharge (Connor, 2008, Smith and Riddle, 2009, Stark et al., 2015). Faecal material accounts for a large portion of discharged effluent due to lack of available water for dilution (Stark et al., 2015).

As a signatory to the Protocol on Environmental Protection to the Antarctic Treaty (The Madrid Protocol), Australia and other treaty nations are required to reduce waste as far as practical (Annex III, article 1), to assess the potential of impacts prior to any activity beginning and ‘monitor key environmental indicators to assess impact’ after commencement of an activity (Annex 1, Article 5). Wastewater may be macerated and discharged directly into the sea at stations with a population of 30 people or more, taking into account the ‘assimilative capacity of the receiving marine environment’ (Annex III, Article 5).

At present, Davis Station wastewater is only macerated before discharge at the shoreline. The wastewater concentration is higher than standard municipal wastewater concentration, and is highly variable in terms of composition, volume and timing of release (Stark et al., 2015). The plume can extend up to 1.5 km south, 1.25 km north and 100 m west of the outfall (Stark et al., 2011), whereas sewage nitrogen assimilation has been detected in T. bernacchii up to and beyond 4 km downstream of the discharge point in a southerly direction (Corbett et al., 2015-a).

A recent pilot study detected an increase in prevalence and severity of histological alteration in gill and liver tissue of T. bernacchii close to the Davis Station wastewater outfall (Corbett et al., 2014). Even though this study provided direct evidence of an effect of exposure on resident fish, it did not attempt to identify overall fish health, and any future evaluation based on these preliminary findings could be biased due to the ‘simple’ singular organ scoring system adopted. Application of a Histological Health Index (HHI) which integrates multiple alterations evident in key organs into a single index value, as well as overall health by combining key organ indices, enables direct comparison between organs, and between fish across spatial and temporal scales (Bernet et al., 1999). Comparison is not based on the specific organ function, but on the alteration type itself, and on the effect the alteration has on organ function (Bernet et al., 1999). HHI classifies individual alterations into one of five reaction patterns or categories: circulatory disturbance, regressive changes, progressive changes, inflammatory response and tumours (Bernet et al., 1999). Each alteration is set a predetermined importance factor from 1 to 3 based on pathological importance, ranging from minimal importance, where the lesion is easily reversible if exposure to the causative stressor ceases, through to marked pathological importance, where the lesion is irreversible and leads to partial or total loss of organ function. Overall fish health can then be determined by collating all organ indices together to calculate (based on a score value) which combines both severity (based on semi-quantitative values) and importance of alteration per individual, allowing further comparison between individuals.

Effectively, the HHI assessment tool identifies individual organ condition as well as overall fish health to be quantified. HHIs should include all important biomarker organs likely to be impacted by toxicant exposure, including gill, liver, kidney, spleen and gonad (Hinton and Lauren, 1990, Teh et al., 1997, Pawert et al., 1998, Stentiford et al., 2003, Osman et al., 2009). To remove subjectivity relating to the semi-quantitative category used by Bernet et al., for this HHI application each alteration was recorded quantitatively (i.e. numerical enumeration to provide a defined quantitative range for each specific severity category). The fully quantitative approach defines exactly what constitutes a mild, moderate or severe alteration, which allows fish health indices for specific species to be compared directly across spatial and temporal scales, and studies, providing greater consistency and objective analyses between examiners. A fully quantitative HHI was applied specifically to assess the level of effect and impact on health of T. bernacchii exposed to wastewater discharge from Davis Station.