Environmental Toxicology and Chemistry, Vol. 33, No. 3, pp. 567–572, 2014 # 2013 SETAC Printed in the USA
INFLUENCE OF ALDRICH HUMIC ACID AND METAL PRECIPITATES ON SURVIVORSHIP OF MAYFLIES (ATALOPHLEBIA SPP.) TO ACID MINE DRAINAGE ALEICIA HOLLAND,* LEO J. DUIVENVOORDEN, and SUSAN H.W. KINNEAR School of Medical and Applied Sciences, Central Queensland University, Rockhampton, Queensland, Australia (Submitted 30 June 2013; Returned for Revision 17 August 2013; Accepted 9 November 2013) Abstract: Humic substances (HS) have been shown to decrease the toxicity of environmental stressors, but knowledge of their ability to influence the toxicity of multiple stressors such as metal mixtures and low pH associated with acid mine drainage (AMD) is still limited. The present study investigated the ability of HS to decrease toxicity of AMD to mayflies (Atalophlebia spp.). The AMD was collected from the Mount Morgan (Mount Morgan, Queensland, Australia) open pit. Mayflies were exposed to concentrations of AMD at 0%, 1%, 2%, 3%, and 4% in the presence of 0 mg/L, 10 mg/L, and 20 mg/L Aldrich humic acid (AHA). A U-shaped response was noted in all AHA treatments, with higher rates of mortality recorded in the 2% and 3% dilutions compared with 4%. This result was linked with increased precipitates in the lower concentrations. A follow-up trial showed significantly higher concentrations of precipitates in the 2% and 3% AMD dilutions in the 0 mg/L AHA treatment and higher precipitates in the 2% AMD, 10 mg/L and 20 mg/L AHA, treatments. Humic substances were shown to significantly increase survival of mayflies exposed to AMD by up to 50% in the 20 mg/L AHA treatment. Humic substances may have led to increased survival after AMD exposure through its ability to influence animal physiology and complex heavy metals. These results are valuable in understanding the ability of HS to influence the toxicity of multiple stressors. Environ Toxicol Chem 2014;33:567–572. # 2013 SETAC Keywords: Dissolved organic carbon
Mount Morgan
Acid mine drainage
Metal toxicity
Atalophlebia
negatively charged carboxylic acid groups in HS allow these compounds to bind to metals, thus reducing their toxicity [9]. A number of authors have also proposed a possible role of HS in decreasing the toxicity of low pH to aquatic organisms [10–12]. However, little is yet known about the ability of HS to decrease the toxicity of multiple stressors such as metal mixtures and low pH associated with AMD. The present study aimed to investigate the ability of HS to decrease the toxicity of AMD to mayflies and, in doing so, increase knowledge of the ability of HS to influence the toxicity of multiple environmental stressors. The present study was designed to inform the development of a new, sustainable treatment option for waterways severely affected by AMD.
INTRODUCTION
Acid mine drainage (AMD) occurs when sulfide minerals, particularly pyritic rocks, are exposed to atmospheric oxygen. This often occurs through mining activities and is a global problem [1]. Bacterially mediated oxidation of the exposed rocks results in the rapid formation of a highly acidic and metal rich leachate, known as AMD, that can severely impact both surface and ground water quality [2]. The formation of AMD is difficult to stop once it has begun and can produce highly contaminated leachates for decades or even centuries post mining, polluting surrounding waters. The effect of AMD on receiving waters is dependent on their buffering capacity and available dilution [2]. Impacts of AMD include a reduction in pH, elevation in metal concentrations, the formation of ochre, and increased sulfate concentration [3]. Adverse effects on biota may occur as a direct consequence of low pH or increased concentrations of dissolved heavy metals, as well as metal precipitates, cloaking the stream bed [4]. Acid mine drainage poses a serious threat to the biota of freshwater systems with detrimental effects on the community structure of these waterways. Acid mine drainage inhibits the growth and function of organisms across a broad range of trophic levels, including algae, aquatic invertebrates, and fish [4]. Humic substances (HS) are formed by the decomposition of organic matter and are found almost everywhere in nature. They have unique chemical and biological properties because of their high surface area and different functional groups (carboxyl, hydroxyl, carbonyl, amides, and others) [5]. It is widely known that HS influence the toxicity of heavy metals [6–8]. The presence of high-affinity binding sites and also large numbers of
MATERIALS AND METHODS
Mount Morgan mine is located approximately 40 km southsouthwest of Rockhampton, Queensland (Australia). Mount Morgan mine sits alongside the Dee River, Central Queensland. This river is severely impacted by AMD exiting the mine site, with estimates in 2007 suggesting that over 90 000 ML of AMD containing high levels of aluminium (Al), iron (Fe), copper (Cu), and zinc (Zn) have already been generated [13]. No fish or submerged or free floating macrophytes are present for at least 20 km downstream from the mine, with only tolerant invertebrate and algal species present [14]. Mount Morgan open pit was originally created while the ore was mined, but since the closure of the mine in 1990, the pit has been filling with highly contaminated AMD water. In 2011, AMD collected from the open pit was characterized by a pH of 2.7 and dissolved metal concentrations of 1780 mg/L Al, 101 mg/L Cu, 173 mg/L manganese (Mn), 51.8 mg/L Zn, and 51.8 mg/L Fe [15]. In January 2013, the pit reached capacity and the first uncontrolled release of pit water was recorded. Water contaminated with AMD was collected from the Mount Morgan mine open cut pit during January 2013 before the uncontrolled release, for use in
* Address correspondence to [emailprotected]. Published online 19 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2459 567
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Environ Toxicol Chem 33, 2014
the experimental trials. Uncontaminated Dee River water (upstream of the influence of the mine) was collected for use as diluent and for use in the control treatment. A map showing the location of collection sites is provided in Figure 1. Mayflies (Atalophlebia spp.) were collected from an uncontaminated section along the Dee River, upstream of the mine, via washing of rocks into a collection bucket. Mayflies could be observed on the underside of rocks, allowing for easy collection. Mayflies were then transported back to the laboratory in aerated buckets and placed in acclimation tanks within a controlled climate room, at 25 8C and a 16-h:8-h light:dark photoperiod, for 48 h before the experimental trials began. Mayflies were identified by using Dean’s guide [16], and identification was confirmed by P. Suter. Mayflies were chosen as the test organism because of their ease of collection and maintenance in the laboratory, and the existence of previous toxicity data for Leptophlebiidae in general to metals and AMD. Previous work has investigated the toxicity of cadmium to Atalophlebia australia [17], and the toxicity of Cu, Zn, and AMD has been investigated in other Leptophlebiidae taxa [18,19]. Toxicity tests were conducted over 96 h in 400-mL plastic containers, each containing 200 mL of test solution and 2 mayflies. Each treatment in each trial was replicated 3 times. Treatments consisted of 0% (control), 1%, 2%, 3%, and 4% AMD, with and without the presence of 10 mg/L and 20 mg/L AHA (Sigma Aldrich Humic Acid). The chemical features of the AHA used in the present study were: residue on ignition 29.9%, carbon content 40.15%, hydrogen content 3.60%, and nitrogen content 0.92%. Test waters (treatments and controls) were prepared using the method outlined in Holland et al. [15].
A. Holland et al.
Organisms were checked every 24 h for mortality, and dead individuals were removed daily to avoid any adverse influence on the remaining test organisms. The criteria for determining mortality were no movement and no reaction to gentle prodding. Mayflies were not provided with food during the 48-h acclimation period; however, algae and detritus may have been present in the holding tank. During the 96-h trial period, organisms were not fed, as recommended by ASTM International [20], to eliminate the possibility that heavy metals may bind to food items. Test solutions were maintained using the static technique. Air was supplied to the containers continuously from an electric aerator. Lids were placed on top of the test chambers to prevent contamination and the escape of test subjects. All trials, when possible, followed the recommendations made in ASTM International’s guidelines for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians [20]. During the experiments, temperature, pH (TPS 80A; TPS Pty Ltd, Australia), conductivity (TPS LC84; TPS Pty Ltd., Australia), oxygen (TPS WP-82Y; TPS Pty Ltd, Australia), and total ammonia levels (Aquarium Pharmaceuticals freshwater total ammonia test kit; Rocky Pet World) were measured every 24 h in each container unless 100% mortality was recorded, at which point readings were no longer taken. All measuring devices were purchased from Labtek. Temperature loggers (TG4100 Tiny Tag; Gemini Data Loggers) were used to record temperature readings every hour. Disturbance to test organisms was minimal during water quality analyses. For trials 2 and 3, metal samples were collected, filtered with Sartorius 0.45-mm cellulose acetate filters, and placed in bottles containing nitric
Figure 1. Map of Mount Morgan, Central Queensland, Australia, showing the location of the collection site for acid mine drainage (AMD), control/diluents water, and mayflies.
Mayfly survival: Influence of HS, precipitates, and AMD
Environ Toxicol Chem 33, 2014
acid provided by Analytical Laboratory Services for determination of dissolved fractions. Metals were analyzed using inductively coupled plasma mass spectroscopy. Metal samples were collected after 24 h. Previous studies have shown no difference in metal concentrations over time [15]. A follow-up trial was conducted to calculate the quantity of precipitates forming in the different AMD concentrations (1%, 2%, 3%, and 4%) at 0 mg/L, 10 mg/L, and 20 mg/L AHA. Solutions were prepared following the procedure outlined above and left for 24 h. Three replicates were used for each treatment. Glass-fiber filters (GF/F) were dried in an oven at 70 8C for 24 h and then placed in desiccator for 2 h to cool, before the starting weight was recorded. Solutions were then filtered through 1 filter per treatment to collect the precipitates from the 200 mL of solution. Filters were then placed back in the oven and dried for another 24 h, placed in a desiccator for 2 h, and then weighed. The starting weight of the filter was subtracted from the end weight to determine the amount of precipitates collected. Statistical analysis
Multiple line and scatter plots were prepared for each trial to show the differences in the proportion of organisms surviving between treatments (Sigmaplot 11.0). One-way analysis of variance (ANOVA) was used to determine any significant differences in metal and precipitate concentrations between HS treatments (IBM SPSS Statistics 20). When ANOVAs were applied, the usual testing for normal distribution and hom*ogeneity of variances were performed. A Kruskal–Wallis test was applied to pooled 4% AMD survivorship data after 96 h with a significance level of p < 0.05. Survivorship data are not normally distributed and fail the hom*ogeneity test; therefore, the use of parametric statistics is not recommended. Nonlinear regressions (3-parameter sigmoidal) were conducted on pooled survival data (except for 4% AMD) to determine the median lethal dose (LC50) values (Sigmaplot 11.0). RESULTS
Water quality
The ionic composition and water quality of the raw treatment water (100% AMD) and the control/dilution water (Dee River water) are provided in Table 1. Key water quality parameters recorded during the 96-h trial periods are provided in Table 2. The pH remained relatively stable ( 0.4) in all treatments and in all trials (Table 2). Oxygen levels were above 85%; temperatures were 24 1 8C, and ammonia was
