The Assessment of Impacts from Mining Wastes on Water Quality and Aquatic Ecosystems Using Freshwater Macroinvertebrate Communities and Novel Bio-Assay Tests

7th Australian Stream Management Conference - Full Paper

The assessment of impacts from mining wastes on water quality and aquatic ecosystems using freshwater macroinvertebrate communities and novel bio-assay tests. 1 2 3 4 Rebecca Sullivan , Dr Ian Wright , Dr Adrian Renshaw , and Mitchell Wilks

1. School of Science and Health, University of Western Sydney, Locked Bag 1797, South PenrithDC, 1797. [email protected] 2. School of Science and Health, University of Western Sydney, Locked Bag 1797, South PenrithDC, 1797. [email protected] 3. School of Science and Health, University of Western Sydney, Locked Bag 1797, South PenrithDC, 1797. [email protected] 4. School of Science and Health, University of Western Sydney, Locked Bag 1797, South PenrithDC, 1797. [email protected]

Key Points • The water quality and macroinvertebrate assemblages from two sites in the Lithgow area were strongly modified by two different types of mining (mixed metal and coal). • The extent and severity of these modifications differs from site to site. • Both sites have demonstrated a continual decline of macroinvertebrate abundance, family richness and modifications of water quality above ANZECC ecosystem protection guidelines. • Novel international bio-assays (not previously used in Australian waters) using New Zealand mudsnail (P.antipodarum) were most successful when tested in the contaminated waters of Daylight creek (mixed metal waste) and moderately successful with downstream waters of Wollangambe River (coal mine waste).

Abstract The water quality and macroinvertebrate assemblages from two sites in the Lithgow area were strongly modified by two different types of mining (mixed metals and coal).The extent and severity of these modifications differ from site to site. The abandoned Sunny Corner mixed metal mine site impacted on Daylight creek significantly more than Clarence colliery on Wollangambe River. Both sites demonstrated a decline of macroinvertebrate abundance, family richness and degradation of water quality in comparison to ANZECC ecosystem protection guidelines. A novel bio-assay (not previously used in Australian) using the introduced New Zealand Mudsnail (P.antipodarum) provided a rapid indication of the mining wastewaters impact on macroinvertebrate communities.

Keywords Wastewater, Mining, Lithgow, Daylight Creek, Wollangambe River, New Zealand Mudsnail, macroinvertebrates, water quality

Introduction Coal and metaliferous mining generates large volumes of contaminated wastewater (Johnson 2003). Wright et. al., (2011) found that often in NSW this wastewater is disposed of directly into adjacent rivers and streams. Waste water from coal mining is often chemically characterized by elevated levels of salts (salinity), low pH (acid mine drainage) and elevated metal loads (Wright et al., 2011). Additionally due to acid mine drainage (a phenomenon often associated with mine waste) acidic wastewater reacts with the surrounding environment making soil metals bioavailable to freshwater ecosystem (Wright et al., 2011). Management of mining wastewater is a global concern. Metal pollution is persistent in the environment, can be present at levels toxic to survival of organisms and bio-accumulates throughout the food web. Chapmen (1983) found evidence to suggest that contaminated mining wastewater can impact the environment up to 20km (dependent on flow) downstream of the discharge. This is of particularly concern in high conservation geographical regions such as those in this study (Chapmen et al., 1983).

Active mine sites such as Clarence Colliery, Lithgow NSW, are regulated by the NSW Environmental Protection Agency using Environmental Protection License 726 (Centennial Coal 2012). This license regulates the disposal of mine waste water including permitted volumes and pollutant concentrations to manage the impact from mining on the environment (Davies and Wright 2014). Sunny Corner’s historic mining area is an abandoned mine site (“a mine where no mining lease or title presently exists”: McKay et. al. 2012). Management of these areas falls on local, state, territory governments and sometimes land owners and/or industry on a basis of proven “significant liability” (Minerals Council Australia 2011).

Potamopyrgus antipodarum the invasive New Zealand Mudsnail has previously been used in the northern hemisphere to assess the effects of salinity and mining metal pollution (Duff et. al 2003; Gust et al., 2010, Gust et. al. 2011).This species is highly invasive, tolerant of abiotic and polluted conditions and is similar in characteristics (sessile animals, that can

Sullivan, R., Wright, I., Renshaw, A. & Wilks, M. (2014). The assessment of impacts from mining wastes on water quality and aquatic ecosystems using freshwater macroinvertebrate communities and novel bio-assay tests, in Vietz, G; Rutherfurd, I.D, and Hughes, R. (editors), Proceedings of the 7th Australian Stream Management Conference. Townsville, Queensland, Pages 269-276. 269 7ASM Full Paper

Sullivan et.al. - Water quality and macroinvertebrate impacts of mining in LithgowCity bioaccumulate toxins in their tissues) to bivalve molluscs such as oysters and mussels which are already proven to be valuable indicators (Alonso 2008, Gust 2010, Gust 2011). Potamopyrgus antipodarum were chosen to be used as biomonitoring species as they are common in urban streams in the Hawkesbury-Nepean catchment and the Sydney basin. These bio-assay tests are novel because they have not been used to test in Australian freshwater conditions.

This study aimed to assess, using a multidisciplinary approach, the impact of mining waste discharge on freshwater ecosystems in the Marrangaroo district, Lithgow, NSW Australia. It also aimed to compare two different mining types (coal and mixed metal) in the same district area. Another aim was to assess if a novel bio-assay method currently in use in the Northern hemisphere can demonstrate the effects of these impacts on macroinvertebrates. This research is significant because its compares coal mine and metal mine waste impacts in a relatively undisturbed area. It will also fill knowledge gaps about the conditions of the area and highlight the existing need of proper management for conservation of the ecosystems.

Methods

Site Description Field-work was carried out on September 2013 and January 2014 in the district of rural-north Marrangaroo, a part of Lithgow City LGA (approximately 112-200km North-West of Sydney Metropolitan Area and 20km west of Lithgow). Three sample sites were studied across two waterways (Wollangambe River and Daylight Creek) and two different catchments (Upper Wollangambe River Catchment and Turon River Catchment). Two sampling sites were waterways receiving mining effluent (WD, SC Figure: 1). A third site was a reference site (WU) upstream of mining activity on Wollangambe River. A reference site for Daylight creek was omitted due to the headwaters of this creek receiving mining effluent.

Figure1. A: Geographical location of sample sites in NSW. B: Wollangambe River Downstream of the mine site; C: Daylight Creek; D: Reference Site upstream of the mine Site Wollangambe River.

All three sites were flowing and were typical of dry weather flow conditions. The exception is Wollangambe River downstream site (WD) as flow is increased significantly by treated effluent discharge averaging 457ML per a month (Centennial Coal 2012;2013). This treated effluent is from Centennial Coals and SK Energy Australia PTY Ltd.’s, active Clarence Colliery (Centennial Coal 2012; 2013). Daylight Creek receives effluent from a number of highly eroded old workings from the non-active Sunny Corner mining area. These contribute to the loading of a large variety of trace metals as acid drainage leaches into Daylight Creek (Haynes, Chaudhury, Buckney, & Khan, 2003) causing pollution for 20km downstream (Chapmen et al., 1983). Previous studies (Haynes, Chaudhury, Buckney, & Khan, 2003; Chapmen et al., 1983) have described Daylight Creek as an extremely polluted creek where green algae flourishes and fauna of the creek is largely non-existent. Both sites are surrounded by state forest and the upper Wollangambe River is located just outside the boundaries of The Blue Mountains National Park.

Sullivan et.al. - Water quality and macroinvertebrate impacts of mining in LithgowCity

Water Sample Collection and Analysis Physical sampling methods followed those of Wright & Burgin 2009 in a similar study conducted on the Grose River. Water samples were collected immediately prior to the collection of macroinvertebrates to minimise disturbance due to kick- sampling. At each site, water quality (electrical conductivity and pH) was monitored in situ at the center of the waterway using a portable calibrated field chemistry meter according to manufacturer’s instructions (TPS Aqua pH-Conductivity Meter). Replicated measurements (n = 10 at each site) of water quality samples were conducted on each sampling occasion. Upstream reference sampling was not possible at Daylight Creek due to mining activity (Sunny Corner Mining Area) occurring at the headwaters of the creek. An adjacent stream was proposed but isolation of the site, contamination from mining of nearby streams and hazardous conditions prevented sampling of a reference stream.

Macroinvertebrate Sampling Kick sampling method was used to collect macroinvertebrates using the same methods as Rosenberg & Resh, 1993 and Wright & Burgin 2009. The kick net had a frame of 30 x 30 cm and 250 μm size mesh. Sampling was achieved by disturbing the stream bottom for a period of 30 seconds over a 900 cm² area, immediately upstream of the net. The net contents, including stream detritus and macroinvertebrates, were immediately placed into a sealed and labeled storage container and preserved in 70% ethanol. Five replicate samples from each site were collected on each sampling occasion.

In the laboratory, the samples were sorted under a dissecting microscope (X 40) to extract the macroinvertebrates from stream detritus (e.g., leaves, sticks, rocks, gravel). Macroinvertebrate identification was determined using the identification keys recommended by Hawking (1994). All insect groups were identified to family as these data have been demonstrated to provide adequate taxonomic resolution for impact assessment (Wright et al., 1995). Two non-insect groups (Oligochaeta, Hydracarina) were not identified to the family level due to identification difficulties.

Data Analysis Multivariate analyses of macroinvertebrate community studies have been demonstrated to be a sound technique to evaluate the response of macroinvertebrates to water pollution (Wright & Burgin 2009). Non-metric multidimensional scaling (NMDS) was performed on the similarity matrix, computed with square-root transformed macroinvertebrate taxon abundance data, using the Bray-Curtis dissimilarity measure (Clarke, 1993; Warwick, 1993). Two-dimensional ordination plots represented the dissimilarity among samples. Macroinvertebrate data from a reference site was used to test differences by two-way analysis of similarity (ANOSIM: Clarke, 1993) between the reference site and sites downstream of the two waste discharges. These multivariate analyses were achieved using the software package PRIMER version 5 (Clarke, 1993).

A number of biotic indices have been developed to enable interpretation of stream macroinvertebrate results from biological assessment of waterways. Two popular and simple indices are ‘taxa richness’ and ‘total abundance’ (see Resh and Jackson, 1993). The EPT (Ephemeroptera, Plecoptera and Trichoptera) index is another commonly used biotic index, based on the proportion of macroinvertebrates in a sample belong to these three common macroinvertebrate orders that have a well- known sensitivity to degraded water quality (Lenat, 1988; Lenat and Penrose, 1996). It was reported to be one of the most sensitive biotic indices for assessing ecological damage from coal mine pollution from a non-operational coal mine in the nearby Grose Valley (Wright & Burgin 2009).

Laboratory Rapid 48hr bio-assay tests using the New Zealand Mudsnail (P.antipodarum) Adult P. antipodarum organisms were obtained from natural populations collected in an urban creek of the Sydney Basin a week before beginning of the experiments. Bio-assay toxicity tests using P.antipodarum were carried out in laboratory conditions following methods of Brown (1980) and Barbour et. al., (2012). The potentially toxic nature of the Daylight Creek and Wollangambe River was determined by addition of 5 specimens, each around 4.5mm in height, to water samples at room temperature. To mimic natural dilution further downstream and to assess toxic thresholds a series of dilutions (100%, 10%, 1%, 0.1%) were also assessed (Barbour et. al., 2012). These dilutions were made by mixing distilled water with effluent receiving water (WD, SC). Three controls were also carried out, natural creek water (stock water), distilled (for dilution treatments) and Upstream Wollangambe River (WU). Three replicates of 5 snails in each testing condition were carried out based on the number of snails available. In the interest of observing if there was any recovery of snails displaying toxic response to test waters snails were not removed after being recorded as immobilised or stressed. Following a method used by Brown (1980) after a suitable period animals were dislodged by stirring of the water with a paintbrush. Snails not suffering from toxic effect within a few seconds extended their foot, using it to flip themselves back over and attach to the test

Sullivan et.al. - Water quality and macroinvertebrate impacts of mining in LithgowCity container within 1 minute (class: ‘normal survival’). Snails suffering mild to moderate toxic effect were able to regain their stance, however, were unable to maintain their grip when tilted (approximately 50°) and lightly shaken (class: ‘stressed’). Brown (1980) recognized death after no movement was detected for 10 minutes. However, confirming death is difficult due to the closure of the gastropod’s operculum. As a result in this experiment ‘immobilisation’ will be measured as an endpoint. Immobilisation was defined as a toxic response where a snail has lost the ability to function normally. Loss of normal function means the snail can no longer extend its foot outside of the shell nor regain stance. Rapid bioassays were recorded every 30 minutes for the first 4 hours, then at 12 hour intervals till the conclusion of the experiment (48 hours).

Results/ Discussion Physiochemical and Water Chemistry Results

Figure 2 (left) Mean (plus/minus standard error of mean) pH values recorded for this study and compared to ANZECC default values. Reference results (WU) are green and the yellow (WD) is Wollangambe downstream of the coal mine and red (SC) is Daylight creek downstream of the Sunny corner mining area. (Right) Mean (plus/minus standard error of mean) EC/salinity recorded for this study and compared to ANZECC default values

During the past year there have been several incidents (Clarence’s regulatory data) where concentrations of metals and or amount of wastewater discharged to Wollangambe River have exceeded that set out in the guidelines of the EPL 726 for Clarence Colliery (Centennial Coal, 2012). The impact of mine discharges on the water quality/chemistry of these local freshwater sites (Figure 2 (left and right)) was clearly apparent. The pH varied significantly between different types of mining (P<0.05) and between the different sites (P<0.05). Mean pH was consistently acidic, below ANZECC ecosystem protection guidelines at the reference site (mean 5.4) and the Sunny Corner mine (mean 3.6). The pH of the Wollangambe River below the coal mine was more alkaline in comparison (mean 6.9) (Figure 2 (left). Salinity also varied highly significantly between the waterways affected by the two different types of mining wastes. It was lowest at reference (WU) site (mean 30 μS/cm), higher at the coal mine site (WD) (mean 383 μS/cm) and by far highest at the mixed metal (Sunny Corner) mine (SC) (mean 529 μS/cm) (Figure 2 (right)).

Belmer et al (2014) (separate paper) reports that metals zinc and nickel at the reference site (WU) had mean concentrations of <10μg/L (+/- SE zinc: 2 μg/L, nickel: 0 μg/L ). Mean levels of zinc was much higher at sites receiving mine effluent (WD and SC). Wollangambe River downstream of the coal mine had concentrations of zinc and nickel 10 times higher than the upstream reference site (zinc 101-136μg/L (+/- SE ~25 μg/L) and nickel 83-132 μg/L (+/- SE ~20 μg/L); Belmer et al. 2014). Copper and lead concentrations at the reference site (WU) and below the coal mine (WD) were both below detection levels (mean <1 μg/L; unpublished data). Daylight Creek downstream of the abandoned Sunny Corner mining area reported the highest concentrations of metals many times above the reference and ANZECC (2000) guidelines. Mean concentrations recorded form this site (SC) were Nickel (42 μg/L), Zinc (32600 μg/L), copper (1420 μg/L) and lead (1370 μg/L) (unpublished data). Given that Wollangambe River water hardness was classified as ‘very hard’ (with mean hardness levels of 1c. 200 mg/L CaCO3; ANZECC, 2000; Belmer et al. 2014)), the recommended hardness-modified trigger value (HMTV) for protecting 99% of species for total zinc levels was 12.5 μg/L. Nickel concentrations measured in the Wollangambe River (WD) below the coal mine were always above the HMTV of 41.6 μg/L (Belmer et al., 2014).

Sullivan et.al. - Water quality and macroinvertebrate impacts of mining in LithgowCity

The water quality at both the mine impacted sites failed several ANZECC (2000) WQ guidelines for ecosystem protection including the metals detailed above. One of these was pH, however (WD) fell within an acceptable level and the reference site (WU) failed to fall within the recommended default guideline range of 6.5 to 9 for upland streams in SE Australia (Figure 2 (left)). Salinity below the coal mine (WD) (383 μS/cm) was above the upper default guideline range (30-350 μS/cm) and the mixed metal mine was well above the default range (SC) (529 μS/cm) (Figure 2 (right)). The reference sites were at or below the lower guideline threshold.

Macroinvertebrate Results A total of 846 macroinvertebrates from 25 taxa (mostly families) were collected for this study. Based on the collection using identical methods and sampling effort 85.3% of all invertebrates were collected from the reference site above the mine coal mine (WU). A total of 122 invertebrates (14.4% of the total) were collected downstream of the coal mine (WD) and 2 invertebrates (0.24%) were collected in Daylight Creek below Sunny Corner Mining area (Figure 3 (left). Mean family richness was highest at the reference site upstream of the coal mine discharge (13 families). Mean family richness was much lower below the coal mine (mean 3.4 families) and was much lower again below Sunny Corner mining area (mean 0.4 families) (figure 3 (middle)). The mean proportion of invertebrates in the sensitive EPT orders differed significantly at the three sampling sites (P<0.01). It was 33.4% at the reference site (WU). In comparison, the EPT proportion was 8.8% below the coal mine and no EPT animals were collected below Sunny Corner.

The macroinvertebrate community structure differed significantly according to sampling location (Global R=0.67, p<1.0%). The nMDS plot (Fig. 3 right) shows that 5 macroinvertebrate samples from the reference site (green triangles ‘WU’) are positioned well separated from the samples collected downstream of the mines. The relative distance of these samples is due to ecological distance due different numbers and types of macroinvertebrate groups. The 5 samples below Sunny Corner (light blue squares ‘SC) are ecologically further away from the reference site and the 5 samples below the coal mine (dark blue triangles ‘WD’) are marginally closer to the reference samples (Figure 3 (right)). ANOSIM results confirm the statistically differences, with the pairwise contrast between reference samples and downstream coal mine (WD) having an R statistic of 0.964 (and the significance level is very high). The pairwise difference between Sunny Corner and reference samples was 1.0 (and very high significance) indicating that the community was essentially completely different

Figure 3 (left) Total number of macroinvertebrates samples at each site. (Middle) Mean number of families sampled at each site ((plus/minus standard error of mean). (Right) NMDS ordination of macroinvertebrate data collected for this study. Stress = 0.05. Each symbol represents a macroinvertebrate sample. ‘Reference’ samples (upstream of the coal mine) are upward green triangles. Samples collected below the coal mine are downward dark blue triangles and samples collected downstream of Sunny Corner are light blue squares. Snail bioassay results The only site that recorded 100% normal survival of all snails was the urban creek, from where the samples were collected (Figure. 4). The other two control treatments (water from Wollangambe River ‘upstream’ of the mine or distilled water had a large proportion of snails immobilized or showing signs of stress (Figure 4). The snails in 100% and 10% of Sunny Corner water all were immobilized after the bioassay. Even the 1% dilution of Sunny Corner water resulted in all snails being either immobilized or showing signs of stress (Figure 3 right). The snail results from the Wollangambe River, downstream of the coal mine, showed a less toxic effect than for the Sunny Corner samples. For both the 100% and 10% dilution (of river water collected below the coal mine) the close to half of the snails were showing signs of stress, at the end of the bioassay. A small number approximately 1 snail in each of these samples (Distilled, Upstream and WD (100%, 10%) became immobilized. The

Sullivan et.al. - Water quality and macroinvertebrate impacts of mining in LithgowCity majority of the snails in the 1 % and 0.1% dilution survived and none were immobilized at the end of the experiment. All snail bioassay results varied highly significantly (P<0.05), with normal survival, signs of stress and immobilization results having a treatment by time interactions. Snail health was monitored during the experiment. The snail health scores fell rapidly for SC 100% and SC 10% (within 3-5mins). In comparison all dilutions fell more gradually for SC 1% and SC 0.1% and for all dilutions for Wollangambe River (100% to 0.1%).

Figure 4 Percentage (plus/minus standard error of mean) response of snails at the conclusion of the bioassay experiments for different treatments. Normal survival represents the proportion of snails that exhibited ‘normal’ movement and activity. Stressed represents the proportion of snails showing abnormal movement. Immobilized represents snails that showed no movement at all Wollangambe downstream experiments conveyed varied success when compared to the control results. There are numerous possible reasons as to why this occurred. Recovery of snail condition was observed in distilled, WU and WD 100%-0.1%. This is similar to results in a publication by Leclair et al., (2011). Leclair (2011) suggests that acclimatizing P.antipodarum, to conditions for 48 hours prior to laboratory bioassay experiments doubles survival rate. Therefore increasing the duration of this test to include a period of acclimatization (not exceeding 216 hrs for the total experiment) in further studies could provide a more conclusive result. Natural mortality, induced handling effects and/or other factors might have contributed to the small amount of immobilization (~1 snail per a sample) in the WU and distilled tests (Leclair et al., 2011). Selection of dilution water continues to be a problematic issue for P.antipodarum tests. These snails are tolerant of a large range of conditions (preferring polluted). Though preliminary tests in the laboratory by the lead authors have demonstrated that this snail cannot survive in 100% tap water and tank rain water. However over a period of a few months these snails survived in distilled water significantly better than any other dilution water. The snail immobilization from exposure to wastewaters is similar to that observed in Chapmen et. al., (1983) and Gust et.al., (2010, 2011).

A correlation between loss of sensitive macroinvertebrate groups, modifications of water quality and the immobilization of this snail suggests that mining discharge is having a negative impact on macroinvertebrate communities and assemblages in this local area.

Conclusion These results indicate that the discharge from Clarence Colliery and Sunny Corner Mining area is causing modifications, suggestive of pollution impact to water quality and benthic macroinvertebrate community assemblages. Results show levels above the ANZECC guidelines. All test sites downstream of the mine when compared to the upstream reference sites showed a decline of macroinvertebrate biodiversity and abundance. As expected, unmanaged and unregulated effluent impacting Daylight Creek resulted in much more degraded water quality conditions, loss of biodiversity, abundance and sudden immobilisation of snails. Whereas current metal levels permitted by Clarence Collieries EPL 726 and management seems to reduce the impact to a lesser degree it was clear that the discharge limits are not sensitive enough to protect macroinvertebrates and to manage adverse impacts on water quality. P.antipodarum behavioral assays proved to be a potentially useful presumptive rapid assessment tool for identification of severely mine impacted freshwater ecosystems. However further testing including development of a standardized method and understanding of the limitation in Australian waters needs to be undertaken. With further development this invasive species has potential as a holistic indicator of wastewater effluent for a large range of habitats and geographical locations.

Sullivan et.al. - Water quality and macroinvertebrate impacts of mining in LithgowCity

Acknowledgments Authors would like to thank Sue Cusbert, Michael Franklin and Maree Gorham (UWS technical staff) for providing sampling equipment and helpful advice. This study was undertaken by a UWS honours student.

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