Ecotoxicology (2014) 23:939–945 DOI 10.1007/s10646-014-1237-3
Humic substances of varying types increase survivorship of the freshwater shrimp Caridina sp. D to acid mine drainage Aleicia Holland • Leo J. Duivenvoorden Susan H. W. Kinnear
•
Accepted: 31 March 2014 / Published online: 9 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Differences relating to the ability of various types of humic substances (HS) to influence toxicity of pollutants have been reported in the literature, but there still remains a gap in understanding whether various HS will have the same influence on the toxicity of acid mine drainage (AMD). This study investigated differences in the ability of Aldrich humic acid (AHA), Suwannee River humic acid and Suwannee River fulvic acid to decrease toxicity of AMD to the freshwater shrimp (Caridina sp. D). Toxicity tests were conducted over 96 h and used Mount Morgan open pit water as source of AMD and Dee River water as control/diluents. Concentrations of 0–4 % AMD at 0 mg/L HS, 10 mg/L AHA, 10 mg/L Suwannee River humic acid and 10 mg/L Suwannee River fulvic acid were used. Significantly higher survival of shrimp was recorded in the HS treatments compared with the treatment containing no HS. No significant differences were found among HS type. HS considerably increased LC50 values irrespective of type, from 1.29 (0 mg/L HS) to 2.12 % (AHA); 2.19 (Suwannee River humic acid) and 2.22 % (Suwannee River fulvic acid). These results support previous work that HS decrease the toxicity of AMD to freshwater organisms, but with the novel finding that this ability occurs irrespective of HS type. These results increase the stock of knowledge regarding HS and may contribute to a possible remediation option for AMD environments.
A. Holland (&) L. J. Duivenvoorden S. H. W. Kinnear School of Medical and Applied Sciences, Centre for Environmental Management, Central Queensland University, Rockhampton, Queensland 4702, Australia e-mail: [email protected]
Keywords Dissolved organic carbon Metals DOC Toxicity Mount Morgan Mixture
Introduction Humic substances (HS) are found throughout terrestrial and aquatic ecosystems and are formed via the decomposition of organic matter. HS are generally divided into three main fractions, dependent on their solubility and adsorption properties: humic acids, fulvic acids and humin (Tipping 2002), with the first two commonly associated with aquatic environments. Dissolved HS can comprise up to 80 % of the dissolved organic carbon (DOC) in some aquatic ecosystems (Steinberg et al. 2008). Their unique chemical and biological properties allow them to interact with pollutants and organisms. Their high surface area, different functional groups (carboxyl, hydroxyl, carbonyl, amides, and others) and the presence of high-affinity binding sites allow HS to bind to pollutants, thus reducing their toxicity (Steinberg 2003). HS can decrease toxicity of metals (Schwartz et al. 2004; Trenfield et al. 2012; McGeer et al. 2002) and some metal mixtures (Kamunde and MacPhail 2011; Richards et al. 1999; Hutchinson and Sprague 1987). Decreased effects of environmental stressors such as pH and salinity have also been reported due to HS (Holland et al. 2014; Holland et al. 2013b; Suhett et al. 2011). The acidification and pollution of freshwaters with metals via acidic mine drainage (AMD) is a global problem. Waterways contaminated with AMD are often depauperate in aquatic organisms, especially sensitive taxa such as fish, crustaceans and some invertebrates. Recently it has been shown that a commercially available HS (Aldrich Humic acid (AHA)) can lessen the effects of AMD on freshwater shrimp (Holland et al. 2013a).
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However, results obtained via the use of commercial products such as AHA may not mimic natural HS, as differences in the ability of different HS (commercial; natural; terrestrial; aquatic; autochothonous; allochthonous; humic and fulvic) to decrease toxicity of pollutants have been reported (Wood et al. 2011). Therefore, we investigated the possible differences in the ability of different types of HS—AHA, Suwannee River fulvic acid (SRFA), and Suwannee River humic acid (SRHA) to decrease toxicity of AMD. AHA is a commercially available HS from SigmaAldrich, believed to be derived from terrestrial sources, whereas the SRHA and SRFA are isolates from the Suwannee River available from the International Humic Substances Society. We aimed to validate previous findings of Holland et al. (2013a), that HS decrease the toxicity of AMD.
Materials and methods Our experiments used AMD-contaminated water from the open cut pit at the now disused Mount Morgan Mine in Central Queensland (Australia) during January 2013. Diluent and control water consisted of Dee River water collected upstream of the influence of the mine. The Dee River flows adjacent to Mount Morgan Mine, and sites downstream of this point are highly contaminated by AMD. Shrimp (Caridina sp. D) were collected using a 250-lm dip net from the Dee River at sites upstream of the mine. On arrival in the laboratory, shrimp were acclimated for 48 h within a controlled climate room, at 25 °C and 16:8-h light:dark photoperiod, before starting experiments. The 96-hour static toxicity tests were conducted in 400 mL plastic containers, each containing 200 mL of test solution and three shrimps. Plastics used during each experimental trial were purchased new, acid washed and double rinsed using Dee River water before use. Every 24 h, the shrimp were checked for mortality, and any dead individuals were removed. Shrimp mortality was defined by shrimp displaying no movement and offering no reaction to gentle prodding. Shrimp were not fed during the trial as per the recommendations in the ‘Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians’ (ASTM 2007). Contents of the containers were aerated continuously throughout the trials. To limit contamination and the escape of shrimp, lids were placed on top of the test chambers. Test waters (treatments and controls) were prepared by diluting the AMD with filtered (0.7-lm, GF/F) Dee River water (collected upstream of the mine) containing 0.75 g/l MOPS (3-N morpholinopropanesulfonic acid) buffer and followed the procedure outlined in Holland et al. (2013a).
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A. Holland et al. Table 1 Chemical features of HS used during trials. Values are % content HS type
Carbon
Hydrogen
Nitrogen
AHA
40.15
3.6
0.92
SRFA
52.34
4.36
0.67
SRHA
52.63
4.28
1.17
MOPS was added to the experimental waters to stabilize pH drift as pilot trials showed dramatic increases in pH, greater than 1 pH unit, in water without MOPS over the first 24 h. MOPS was chosen as it has been previously recommended for use in ecotoxicology trials with metals as it has been shown to be non-toxic and does not bind metals (Kandegedara and Rorabacher 1999; De Schamphelaere et al. 2004) and has been successfully applied in other studies using AMD (Holland et al. 2013a). Test solutions consisted of 0, 1, 2, 3, or 4 % AMD in the presence of either no HS, 10 mg/L Aldrich Sigma Humic Acid, 10 mg/L Suwannee River fulvic acid (Standard II) or 10 mg/L Suwannee River humic acid (Standard II). The chemical features of each type of HS are provided in Table 1. Each treatment was replicated three times with 3 shrimp in each chamber, n = 9 each treatment each trial. Trials were repeated three times, back to back over time to validate results. During the experiment water quality parameters such as temperature, pH (TPS 80A), conductivity (TPS LC84), oxygen (TPS WP-82Y), and ammonia levels (Aquarium Pharmaceuticals Inc., freshwater total ammonia test kit) were measured every 24 h. Temperature loggers (TG-4100 Tiny Tag) recorded temperature readings every hour. Samples were collected for all trials and filtered using Sartorius 0.45-lm cellulose filters for measurements of dissolved fractions of metals. These samples were analysed using inductively coupled plasma mass spectroscopy (ICPS). The detection limits of the ICPS were as follows: Al 0.01 mg/L, Cd 0.0001 mg/L, Co 0.001 mg/L, Cu 0.001 mg/L, Mn 0.001 mg/L, Ni 0.001 mg/L and Zn 0.001 mg/L. Dee River water was analysed for DOC via filtration by Sartorius 0.45-lm cellulose acetate filters, as provided by ALS analytical services, and analysed using a Sievers 5310 CTOC Analyser 900 series. Cation and anion analyses were also conducted on river water using the following methods: Flame atomic absorption spectrometry was used to measure Ca, Mg, Na, and K, titration for Alkalinity, automated silver potentiometric method for Cl-, and the turbidimetric method for SO42-. (APHA 1998). Mortality results from all three trials were pooled and analysed using non-linear (three-parameter sigmoidal) regressions to determine LC50 values and their corresponding 95 % confidence limits (SigmaPlot 11.0).
Humic substances of varying types increase survivorship of the freshwater Table 2 Ranges of physicochemical variables recorded every 24 h during the 96 h trial period across all three trials, n = 135 % AMD
pH
Conductivity (lS/cm)
0
7.29–7.51
1
6.74–6.92
2 3
6.23–6.47 5.49–5.66
4
4.47–4.61
Oxygen (% sat)
Ammonia (ppm)
903–955
82–96
\0.25
1011–1094
81–95
\0.25
1127–1222 1298–1369
80–96 83–96
\0.25 \0.25
1399–1472
82–95
\0.25
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Multiple line and scatter plots of pooled survivorship data were generated to visualise differences in the proportion of organisms surviving between HS treatments. The use of parametric statistics is not recommended for survivorship data, for such data are not normally distributed and fail the homogeneity test. Consequently, the Kruskal–Wallis Test was applied to pooled 2 % AMD survivorship data after 96 h, with a significance level of p \ 0.05. This yielded clear differences between treatments with HS and those without (IBM SPSS Statistics 20).
Table 3 Composition and water quality of Dee River water (control, diluent water) and the 100 % Mount Morgan Open pit (AMD raw water source) Cond (lS/cm)
pH
DOC
Hardness (CaCO3)
Alkalinity (CaCO3)
Ca2?
Mg2?
Na?
K?
Cl-
SO4
Control/diluent waters
615–630
7.7
5–8
167–176
210–221
32.2–33.6
21.2–22.4
63.2–65.6
1.9–2.8
72–77
\0.01
100 % AMD
11,920
2.9
N/A
10,270
N/A
500
2200
574
8.1
200
&11,000*
Values shown for control/diluents waters is the range obtained for all four treatment waters (0 HS, AHA, SRFA and SRHA). Units are mg/L unless otherwise indicated * An approximate value for SO4 is provided as it exceeded the analytical range of the test
Table 4 Dissolved metal concentrations (mg/L) recorded for each AMD treatment % AMD
Treatment
0
Control
1
\0.0001
\0.001
0.008
Manganese 0.01
Nickel \0.001
Zinc
pH
0.019
7.4 6.8
0.06
0.0023
0.040
0.079
1.74
0.012
0.044
0.0016
0.036
0.042
1.84
0.010
0.060
SRFA
0.11
0.0013
0.030
0.105
1.49
0.008
0.098
SRHA
0.10
0.0015
0.031
0.095
1.57
0.008
0.052
12.10
0.0026
0.038
0.811
1.570
0.010
0.504
0 HS
0.03
0.0050
0.081
0.105
3.22
0.023
0.351
AHA
\0.01
0.0040
0.074
0.094
3.05
0.020
0.290
SRFA
\0.01
0.0043
0.078
0.077
3.27
0.021
0.269
SRHA
\0.01
0.0042
0.076
0.086
2.20
0.020
0.282
24.20
0.0052
0.076
1.622
3.140
0.019
1.008
0 HS
0.05
0.0082
0.126
0.313
4.86
0.036
1.340
AHA
\0.01
0.0073
0.118
0.498
4.65
0.032
0.976
SRFA SRHA
\0.01 \0.01
0.0075 0.0077
0.120 0.122
0.519 0.427
4.74 4.83
0.033 0.045
1.019 1.050
36.30
0.0079
0.115
2.433
4.71
0.029
1.512
2.37
0.0100
0.165
2.960
6.38
0.046
2.070
0 HS AHA
2.34
0.0109
0.168
2.880
6.93
0.047
2.060
SRFA
2.24
0.0111
0.165
2.830
6.71
0.046
2.040
SRHA Expected 100
Copper
0.07
Expected 4
Cobalt
0 HS
Expected 3
0.04
Cadmium
AHA
Expected 2
Aluminium
AMD source
2.73
0.0110
0.166
2.880
6.84
0.046
2.060
48.40
0.0105
0.153
3.244
6.280
0.038
2.016
1210.00
0.2620
3.820
81.100
157.000
0.962
50.400
6.3
5.5
4.5
2.9
Values for dissolved metals are the average of 3 readings, one for each trial (n = 3). Expected values calculated based on the measured concentrations recorded in the 100 % AMD
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Fig. 1 % Survival of shrimp exposed to AMD (Mount Morgan open pit water) and different HS where: a 0 HS, b Sigma Aldrich humic acid (AHA), c Suwannee River fulvic acid (SRFA), d Suwannee River humic acid (SRHA). Data points are the mean of pooled data from all three trials ± SE
Results Water quality The pH decreased with increases in AMD while conductivity increased, in all trials (Table 2). Oxygen levels were consistently above 80 % saturation and ammonia was always \0.25 mg/L (Table 2). Ionic composition of the control/diluent waters and AMD are provided in Table 3. Concentrations of dissolved metals increased with increases in AMD (Table 4). Toxicity Shrimp survival was 100 % in control treatments for AHA, SRFA and SRHA during all three trials, but one shrimp did not survive in the control treatments for 0 HS during trial 1 (Fig. 1). A significant difference in survival at 2 % AMD was found between HS treatments (v2 = 12.7, p = 0.005), with follow up analyses showing significantly higher survival in all treatments with HS irrespective of type (AHA: v2 = 8.8, p = 0.003; SRFA: v2 = 6.6, p = 0.01; SRHA: v2 = 9.1, p = 0.003) (Fig. 1). Survival did not differ significantly between the AHA, SRFA and SRHA treatments (v2 = 0.2, p = 0.913) (Fig. 1), indicating that all types of HS behaved similarly in increasing survival to AMD. Nonlinear regressions revealed that Mount Morgan open pit
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Fig. 2 Concentration response plot for Mount Morgan open pit water (AMD) and Dee River water, with 0 HS, Sigma Aldrich Humic acid (AHA), Suwannee River fulvic acid (SRFA), and Suwannee River humic acid (SRHA). Curves represent the non-linear regression (three-parameter sigmoidal) of pooled data from the three trials at 96 h
AMD is considerably more toxic without the addition of HS, with LC50 values of 1.29 % (95 percentiles: 1.09–1.53 %) 0HS; 2.12 % (95 percentiles: 1.90–2.67 %) AHA; 2.22 % (95 percentiles: 2.01–2.50 %) SRFA; and 2.19 % (95 percentiles: 2.04–2.70 %) SRHA, recorded for each treatment (Fig. 2). All LC50 values recorded for the different HS types were within the 95 percentiles of the
Humic substances of varying types increase survivorship of the freshwater
others, indicating that there is probably no difference in survival among types of HS.
Discussion Metal concentrations and pH varied with increases in AMD during this study, with increases in dissolved metals and decreases in pH recorded. Concentrations of Al and Cu in all AMD treatments (0–4 %) were much lower than expected based on calculations using the measured concentrations in the 100 % AMD (Table 4). This is likely due to the effect of pH on metal speciation and precipitation, with Al generally in its precipitate form at pH values greater than 5 (Gensemer and Playle 1999). White precipitates were present in all AMD treatments. Therefore, the low measured concentrations of dissolved Al are likely due to the precipitation of Al in treatment water. The formation of Al precipitates also reduces the amount of dissolved Cu in solution due to co-precipitation (Lee et al. 2002). As the pH decreases it is expected that differences between measured and expected metal concentrations will decrease. This can be observed for Cu in the 4 % AMD. Zn concentrations were also shown to be lower in the 0–3 % AMD treatments with concentrations in the 4 % similar between the measured and expected values. This is also likely to be due to the co-precipitation of Zn on to the Al precipitates (Lee et al. 2002). Concentrations of the other metals did not vary greatly from the expected concentrations indicating correct dilution had occurred (Table 4). Adding as little as 2 % AMD to the treatment with no HS resulted in a mortality rate of over 80 %, and as little as 1.3 % AMD caused 50 % mortality in the exposed shrimp populations. The addition of HS increased the survivorship of shrimp exposed to AMD irrespective of HS type, with the commercial AHA corresponding with LC50 values similar to those of SRFA and SRHA. We also found no significant difference in the impacts of the Suwannee humic and fulvic acids. The lack of difference between the commercial AHA and the SRHA and SRFA may be attributed to all three of these HS being optically dark products (Schwartz et al. 2004). Variation in the ability of different HS to decrease toxicity of metals has been linked with HS optical properties, with optically dark, allochthonous HS shown to decrease toxicity of metals better than optically light, autochthonous HS (Schwartz et al. 2004; Al-Reasi et al. 2011; Wood et al. 2011). Previously, this feature has been suggested to be due to a greater proportion of HA in the optically dark HS (Schwartz et al. 2004; Wood et al. 2011; Ryan et al. 2004), however, our results show that SRFA can decrease toxicity of AMD similarly to SRHA. Decreased toxicity of metals in the presence of HS has often been attributed to complexation, which decreases
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bioavailability and affects metal speciation. In all HS treatments, HS precipitates formed in treatments containing AMD, indicating metal complexation. Slight differences in average metal concentrations were shown between treatments for some metals, however, this did not follow a constant pattern with HS treatments sometimes recording lower concentrations and sometimes recording higher. This may be due to the heterogeneous nature of AMD leading to slight differences in metal concentrations within treatments and not due to the complexation with HS. The lack of obvious effect of HS on metal concentrations does not suggest a lack of complexation: rather the complexed metal/HS particles may still be small enough to pass through the 0.45 lm filter and be measured as dissolved metals. The complexation of metals with HS may have accounted for the increased survivorship of shrimp reported for all HS treatments in the 1 and 2 % AMD treatment. The decreased effect of HS at the higher AMD treatments is likely due to HS being unable to bind enough metals to reduce toxicity within these treatments, as metals concentrations in the 3 and 4 % AMD increased dramatically; for example: there is approximately five times more Cu in the 3 % AMD treatments compared with the 2 % AMD. The increased concentrations of metals along with a decrease in pH proved too toxic for freshwater shrimp even in the presence of HS. pH has also been shown to affect HS solubilisation and its ability to bind metals, with decreases in pH linked with decreased solubilisation and metal binding (Liu and Gonzalez 2000). It is unclear the effect pH had on solubilisation and metal binding within the current study as HS-metal precipitates were formed in all AMD treatments. The decreased effect at 3 and 4 % AMD may also be linked with the pH effects on metal binding at the lower pH in the 3 % AMD treatment (pH 5.5), however, as mentioned above, it may also be due to the increase in toxicants: lower pH and higher metal level in this AMD treatment. Humic acids have a greater affinity than fulvic acid for complexing metals, with greater amounts of metals often reported to be chelated by humic rather than fulvic acids (Lombartini et al. 1994; Tan 2003). This difference has been suggested to be due to difference in size (HA larger), complexity (HA more complex) and the presence of COOH groups (higher in FA) and phenolic-OH groups (higher in HA). The greater amounts of phenolic-OH groups in HA allow it to perform acidic reactions plus an interactive effect (complexation), whereas the higher amounts of COOH in FA allow for predominately acidic reactions to occur (Tan 2003). In the current study, we found no clear difference in the ability of HA (Aldrich or Suwannee) to complex and remove metals, compared with SRFA. This may be due to the effects of acidity on HA decreasing the amount of binding sites available for dissolved metals. As acidity increases, HA may become well protonated. As this
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occurs, the overall morphology of the molecule changes such that it impacts on the number and availability of binding sites, leading to a decrease in their ability to bind metal ions. This is why HA precipitates at pH 2 or below. Also the presence of high amounts of H? ions increases competition for binding sites and may make it harder for metal ions to bind to the HA molecule. Another possible explanation for the increased survivorship of shrimp to AMD in HS treatments is that HS may have affected shrimp at the physiological level. Previous work has shown that HS can bind to and be taken up by cell membranes, influencing their permeability, activating enzymes and defence proteins, and limiting ion loss (Campbell et al. 1997; Bedulina et al. 2010; Steinberg et al. 2007; Wood et al. 2003). However the methods applied in this study were not precise enough to closely examine the effects of HS on the physiological functions. Acknowledgments This study was supported by the Women’s Equal Opportunity Postgraduate Research Award, the Centre for Environmental Management, and Central Queensland University. The authors would like to acknowledge Dr Satish Choy for confirmation of shrimp identification, Heather Smyth for help with water chemistry, and Associate Professor Steve Mckillup for help with statistical design. Thanks to the two reviewers whose contributions significantly improved the manuscript. Conflict of interest of interest.
The authors declare that they have no conflict
References Al-Reasi HA, Wood CM, Smith DS (2011) Physicochemical and spectroscopic properties of natural organic matter (NOM) from various sources and implications for ameliorative effects on metal toxicity to aquatic biota. Aquat Toxicol 103(3–4): 179–190. doi:10.1016/j.aquatox.2011.02.015 APHA (1998) Standard methods for the examination of water and wastewater, 20th edn. APHA, Washington ASTM (2007) Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians. ASTM International, West Conshohocken Bedulina DS, Timofeyev MA, Zimmer M, Zwirnmann E, Menzel R, Steinberg CEW (2010) Different natural organic matter isolates cause similar stress response in the freshwater amphipod, Gammarus pulex. Environ Sci Pollut Res 17:261–269 Campbell PGC, Twiss MR, Wilkinson KJ (1997) Accumulation of natural organic matter on the surfaces of living cells: implications for the interaction of toxic solutes with aquatic biota. Can J Fish Aquat Sci 54(11):2543–2554 De Schamphelaere KAC, Heijerick DG, Janssen CR (2004) Comparison of the effect of different pH buffering techniques on the toxicity of copper and zinc to Daphnia magna and Pseudokirchneriella subcapitata. Ecotoxicology 13(7): 697–705 Gensemer RW, Playle RC (1999) The bioavailability and toxicity of aluminium in aquatic environments. Crit Rev Environ Sci Technol 29(4):315–450 Holland A, Duivenvoorden LJ, Kinnear SHW (2013a) Humic substances increase survival of freshwater shrimp Caridina sp.
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D to acid mine drainage. Arch Environ Contam Toxicol 64:263–272. doi:10.1007/s00244-012-9823-y Holland A, Duivenvoorden LJ, Kinnear SHW (2013b) Humic substances increase the survivorship rates of freshwater shrimp exposed to acidified waters of varying hardness. Ann Environ Sci 7:49–58 Holland A, Duivenvoorden L, Kinnear SW (2014) The double-edged sword of humic substances: contrasting their effect on respiratory stress in eastern rainbow fish exposed to low pH. Environ Sci Pollut Res 21:1701–1707. doi:10.1007/s11356-013-2031-0 Hutchinson NJ, Sprague JB (1987) Reduced lethality of Al, Zn and Cu mixtures to American flagfish by complexation with humic substances in acidified soft waters. Environ Toxicol Chem 6(10):755–765 Kamunde C, MacPhail R (2011) Effect of humic acid during concurrent chronic waterborne exposure of rainbow trout (Oncorhynchus mykiss) to copper, cadmium and zinc. Ecotoxicol Environ Saf 74(3):259–269 Kandegedara A, Rorabacher DB (1999) Noncomplexing tertiary amines as ‘‘better’’ buffers covering the range of pH 3–11. Temperature dependence of their acid dissociation constants. Anal Chem 71(15):3140–3144 Lee G, Bigham JM, Faure G (2002) Removal of trace metals be coprecipitation with Fe, Al and Mn from natural waters contaminated with acid mine drainage in the Ducktown Mining District, Tennessee. Appl Geochem 17:569–581 Liu A, Gonzalez RD (2000) Modeling adsorption of copper (II), cadmium (II) and lead (II) on purified humic acid. Langmuir 16:3902–3909 Lombartini JC, Tan KH, Pape C (1994) The nature of humic acidapatite interaction products and their availability to plant growth. Commun Soil Sci Plant Anal 25:2355–2369 McGeer JC, Szebedinszky C, McDonald DG, Wood CM (2002) The role of dissolved organic carbon in moderating the bioavailability and toxicity of Cu to rainbow trout during chronic waterborne exposure. Comp Biochem Physiol C Toxicol Pharmacol 133(1–2):147–160 Richards JG, Burnison BK, Playle RC (1999) Natural and commercial dissolved organic matter protects against the physiological effects of a combined cadmium and copper exposure on rainbow trout (Oncorhynchus mykiss). Can J Fish Aquat Sci 56:407–418 Ryan AC, Van Genderen EJ, Tomasso JR, Klaine SJ (2004) Influence of natural organic matter source on copper toxicity to larval fathead minnows (Pimephales promelas): implications for the biotic ligand model. Environ Toxicol Chem 23(6):1567–1574. doi:10.1897/02-476 Schwartz ML, Curtis PJ, Playle RC (2004) Influence of natural organic matter source on acute copper, lead, and cadmium toxicity to rainbow trout (Oncorhynchus mykiss). Environ Toxicol Chem 23(12):2889–2899 Steinberg CEW (2003) Ecology of humic substances in freshwaters. Springer-Verlag Belin Heidelberg, New York Steinberg CEW, Saul N, Pietsch K, Meinelt T, Rienau S, Menzel R (2007) Dissolved humic substance facilitate fish life in extreme aquatic environments and have the potential to extend the lifespan of Caenorhabditis elegans. Ann Environ Sci 1:81–90 Steinberg CEW, Meinelt T, Timofeyev MA, Bittner M, Menzel R (2008) Humic substances—Part 2: interactions with organisms. Environ Sci Pollut Res 15(2):128–135 Suhett AL, Steinberg CEW, Santangelo JM, Bozelli RL, Farjalla VF (2011) Natural dissolved humic substances increase the lifespan and promote transgenerational resistance to salt stress in the cladoceran Moina macrocopa. Environ Sci Pollut Res. doi:10. 1007/s11356-011-0455-y Tan K (2003) Humic matter in soil and the environment: principles and controversies. CRC Press, New York
Humic substances of varying types increase survivorship of the freshwater Tipping E (2002) Cation binding by humic substances. Cambridge University Press, Cambridge Trenfield MA, Markich SJ, Ng JC, Noller B, van Dam RA (2012) Dissolved organic carbon reduces the toxicity of aluminum to three tropical freshwater organisms. Environ Toxicol Chem 31(2):427–436. doi:10.1002/etc.1704 Wood CM, Matsuo AYO, Wilson RW, Gonzalez RJ, Patrick ML, Playle RC, Val AL (2003) Protection by natural blackwater
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Humic substances of varying types increase survivorship of the freshwater shrimp Caridina sp. D to acid mine drainage Aleicia Holland • Leo J. Duivenvoorden Susan H. W. Kinnear
•
Accepted: 31 March 2014 / Published online: 9 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Differences relating to the ability of various types of humic substances (HS) to influence toxicity of pollutants have been reported in the literature, but there still remains a gap in understanding whether various HS will have the same influence on the toxicity of acid mine drainage (AMD). This study investigated differences in the ability of Aldrich humic acid (AHA), Suwannee River humic acid and Suwannee River fulvic acid to decrease toxicity of AMD to the freshwater shrimp (Caridina sp. D). Toxicity tests were conducted over 96 h and used Mount Morgan open pit water as source of AMD and Dee River water as control/diluents. Concentrations of 0–4 % AMD at 0 mg/L HS, 10 mg/L AHA, 10 mg/L Suwannee River humic acid and 10 mg/L Suwannee River fulvic acid were used. Significantly higher survival of shrimp was recorded in the HS treatments compared with the treatment containing no HS. No significant differences were found among HS type. HS considerably increased LC50 values irrespective of type, from 1.29 (0 mg/L HS) to 2.12 % (AHA); 2.19 (Suwannee River humic acid) and 2.22 % (Suwannee River fulvic acid). These results support previous work that HS decrease the toxicity of AMD to freshwater organisms, but with the novel finding that this ability occurs irrespective of HS type. These results increase the stock of knowledge regarding HS and may contribute to a possible remediation option for AMD environments.
A. Holland (&) L. J. Duivenvoorden S. H. W. Kinnear School of Medical and Applied Sciences, Centre for Environmental Management, Central Queensland University, Rockhampton, Queensland 4702, Australia e-mail: [email protected]
Keywords Dissolved organic carbon Metals DOC Toxicity Mount Morgan Mixture
Introduction Humic substances (HS) are found throughout terrestrial and aquatic ecosystems and are formed via the decomposition of organic matter. HS are generally divided into three main fractions, dependent on their solubility and adsorption properties: humic acids, fulvic acids and humin (Tipping 2002), with the first two commonly associated with aquatic environments. Dissolved HS can comprise up to 80 % of the dissolved organic carbon (DOC) in some aquatic ecosystems (Steinberg et al. 2008). Their unique chemical and biological properties allow them to interact with pollutants and organisms. Their high surface area, different functional groups (carboxyl, hydroxyl, carbonyl, amides, and others) and the presence of high-affinity binding sites allow HS to bind to pollutants, thus reducing their toxicity (Steinberg 2003). HS can decrease toxicity of metals (Schwartz et al. 2004; Trenfield et al. 2012; McGeer et al. 2002) and some metal mixtures (Kamunde and MacPhail 2011; Richards et al. 1999; Hutchinson and Sprague 1987). Decreased effects of environmental stressors such as pH and salinity have also been reported due to HS (Holland et al. 2014; Holland et al. 2013b; Suhett et al. 2011). The acidification and pollution of freshwaters with metals via acidic mine drainage (AMD) is a global problem. Waterways contaminated with AMD are often depauperate in aquatic organisms, especially sensitive taxa such as fish, crustaceans and some invertebrates. Recently it has been shown that a commercially available HS (Aldrich Humic acid (AHA)) can lessen the effects of AMD on freshwater shrimp (Holland et al. 2013a).
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However, results obtained via the use of commercial products such as AHA may not mimic natural HS, as differences in the ability of different HS (commercial; natural; terrestrial; aquatic; autochothonous; allochthonous; humic and fulvic) to decrease toxicity of pollutants have been reported (Wood et al. 2011). Therefore, we investigated the possible differences in the ability of different types of HS—AHA, Suwannee River fulvic acid (SRFA), and Suwannee River humic acid (SRHA) to decrease toxicity of AMD. AHA is a commercially available HS from SigmaAldrich, believed to be derived from terrestrial sources, whereas the SRHA and SRFA are isolates from the Suwannee River available from the International Humic Substances Society. We aimed to validate previous findings of Holland et al. (2013a), that HS decrease the toxicity of AMD.
Materials and methods Our experiments used AMD-contaminated water from the open cut pit at the now disused Mount Morgan Mine in Central Queensland (Australia) during January 2013. Diluent and control water consisted of Dee River water collected upstream of the influence of the mine. The Dee River flows adjacent to Mount Morgan Mine, and sites downstream of this point are highly contaminated by AMD. Shrimp (Caridina sp. D) were collected using a 250-lm dip net from the Dee River at sites upstream of the mine. On arrival in the laboratory, shrimp were acclimated for 48 h within a controlled climate room, at 25 °C and 16:8-h light:dark photoperiod, before starting experiments. The 96-hour static toxicity tests were conducted in 400 mL plastic containers, each containing 200 mL of test solution and three shrimps. Plastics used during each experimental trial were purchased new, acid washed and double rinsed using Dee River water before use. Every 24 h, the shrimp were checked for mortality, and any dead individuals were removed. Shrimp mortality was defined by shrimp displaying no movement and offering no reaction to gentle prodding. Shrimp were not fed during the trial as per the recommendations in the ‘Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians’ (ASTM 2007). Contents of the containers were aerated continuously throughout the trials. To limit contamination and the escape of shrimp, lids were placed on top of the test chambers. Test waters (treatments and controls) were prepared by diluting the AMD with filtered (0.7-lm, GF/F) Dee River water (collected upstream of the mine) containing 0.75 g/l MOPS (3-N morpholinopropanesulfonic acid) buffer and followed the procedure outlined in Holland et al. (2013a).
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A. Holland et al. Table 1 Chemical features of HS used during trials. Values are % content HS type
Carbon
Hydrogen
Nitrogen
AHA
40.15
3.6
0.92
SRFA
52.34
4.36
0.67
SRHA
52.63
4.28
1.17
MOPS was added to the experimental waters to stabilize pH drift as pilot trials showed dramatic increases in pH, greater than 1 pH unit, in water without MOPS over the first 24 h. MOPS was chosen as it has been previously recommended for use in ecotoxicology trials with metals as it has been shown to be non-toxic and does not bind metals (Kandegedara and Rorabacher 1999; De Schamphelaere et al. 2004) and has been successfully applied in other studies using AMD (Holland et al. 2013a). Test solutions consisted of 0, 1, 2, 3, or 4 % AMD in the presence of either no HS, 10 mg/L Aldrich Sigma Humic Acid, 10 mg/L Suwannee River fulvic acid (Standard II) or 10 mg/L Suwannee River humic acid (Standard II). The chemical features of each type of HS are provided in Table 1. Each treatment was replicated three times with 3 shrimp in each chamber, n = 9 each treatment each trial. Trials were repeated three times, back to back over time to validate results. During the experiment water quality parameters such as temperature, pH (TPS 80A), conductivity (TPS LC84), oxygen (TPS WP-82Y), and ammonia levels (Aquarium Pharmaceuticals Inc., freshwater total ammonia test kit) were measured every 24 h. Temperature loggers (TG-4100 Tiny Tag) recorded temperature readings every hour. Samples were collected for all trials and filtered using Sartorius 0.45-lm cellulose filters for measurements of dissolved fractions of metals. These samples were analysed using inductively coupled plasma mass spectroscopy (ICPS). The detection limits of the ICPS were as follows: Al 0.01 mg/L, Cd 0.0001 mg/L, Co 0.001 mg/L, Cu 0.001 mg/L, Mn 0.001 mg/L, Ni 0.001 mg/L and Zn 0.001 mg/L. Dee River water was analysed for DOC via filtration by Sartorius 0.45-lm cellulose acetate filters, as provided by ALS analytical services, and analysed using a Sievers 5310 CTOC Analyser 900 series. Cation and anion analyses were also conducted on river water using the following methods: Flame atomic absorption spectrometry was used to measure Ca, Mg, Na, and K, titration for Alkalinity, automated silver potentiometric method for Cl-, and the turbidimetric method for SO42-. (APHA 1998). Mortality results from all three trials were pooled and analysed using non-linear (three-parameter sigmoidal) regressions to determine LC50 values and their corresponding 95 % confidence limits (SigmaPlot 11.0).
Humic substances of varying types increase survivorship of the freshwater Table 2 Ranges of physicochemical variables recorded every 24 h during the 96 h trial period across all three trials, n = 135 % AMD
pH
Conductivity (lS/cm)
0
7.29–7.51
1
6.74–6.92
2 3
6.23–6.47 5.49–5.66
4
4.47–4.61
Oxygen (% sat)
Ammonia (ppm)
903–955
82–96
\0.25
1011–1094
81–95
\0.25
1127–1222 1298–1369
80–96 83–96
\0.25 \0.25
1399–1472
82–95
\0.25
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Multiple line and scatter plots of pooled survivorship data were generated to visualise differences in the proportion of organisms surviving between HS treatments. The use of parametric statistics is not recommended for survivorship data, for such data are not normally distributed and fail the homogeneity test. Consequently, the Kruskal–Wallis Test was applied to pooled 2 % AMD survivorship data after 96 h, with a significance level of p \ 0.05. This yielded clear differences between treatments with HS and those without (IBM SPSS Statistics 20).
Table 3 Composition and water quality of Dee River water (control, diluent water) and the 100 % Mount Morgan Open pit (AMD raw water source) Cond (lS/cm)
pH
DOC
Hardness (CaCO3)
Alkalinity (CaCO3)
Ca2?
Mg2?
Na?
K?
Cl-
SO4
Control/diluent waters
615–630
7.7
5–8
167–176
210–221
32.2–33.6
21.2–22.4
63.2–65.6
1.9–2.8
72–77
\0.01
100 % AMD
11,920
2.9
N/A
10,270
N/A
500
2200
574
8.1
200
&11,000*
Values shown for control/diluents waters is the range obtained for all four treatment waters (0 HS, AHA, SRFA and SRHA). Units are mg/L unless otherwise indicated * An approximate value for SO4 is provided as it exceeded the analytical range of the test
Table 4 Dissolved metal concentrations (mg/L) recorded for each AMD treatment % AMD
Treatment
0
Control
1
\0.0001
\0.001
0.008
Manganese 0.01
Nickel \0.001
Zinc
pH
0.019
7.4 6.8
0.06
0.0023
0.040
0.079
1.74
0.012
0.044
0.0016
0.036
0.042
1.84
0.010
0.060
SRFA
0.11
0.0013
0.030
0.105
1.49
0.008
0.098
SRHA
0.10
0.0015
0.031
0.095
1.57
0.008
0.052
12.10
0.0026
0.038
0.811
1.570
0.010
0.504
0 HS
0.03
0.0050
0.081
0.105
3.22
0.023
0.351
AHA
\0.01
0.0040
0.074
0.094
3.05
0.020
0.290
SRFA
\0.01
0.0043
0.078
0.077
3.27
0.021
0.269
SRHA
\0.01
0.0042
0.076
0.086
2.20
0.020
0.282
24.20
0.0052
0.076
1.622
3.140
0.019
1.008
0 HS
0.05
0.0082
0.126
0.313
4.86
0.036
1.340
AHA
\0.01
0.0073
0.118
0.498
4.65
0.032
0.976
SRFA SRHA
\0.01 \0.01
0.0075 0.0077
0.120 0.122
0.519 0.427
4.74 4.83
0.033 0.045
1.019 1.050
36.30
0.0079
0.115
2.433
4.71
0.029
1.512
2.37
0.0100
0.165
2.960
6.38
0.046
2.070
0 HS AHA
2.34
0.0109
0.168
2.880
6.93
0.047
2.060
SRFA
2.24
0.0111
0.165
2.830
6.71
0.046
2.040
SRHA Expected 100
Copper
0.07
Expected 4
Cobalt
0 HS
Expected 3
0.04
Cadmium
AHA
Expected 2
Aluminium
AMD source
2.73
0.0110
0.166
2.880
6.84
0.046
2.060
48.40
0.0105
0.153
3.244
6.280
0.038
2.016
1210.00
0.2620
3.820
81.100
157.000
0.962
50.400
6.3
5.5
4.5
2.9
Values for dissolved metals are the average of 3 readings, one for each trial (n = 3). Expected values calculated based on the measured concentrations recorded in the 100 % AMD
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A. Holland et al.
Fig. 1 % Survival of shrimp exposed to AMD (Mount Morgan open pit water) and different HS where: a 0 HS, b Sigma Aldrich humic acid (AHA), c Suwannee River fulvic acid (SRFA), d Suwannee River humic acid (SRHA). Data points are the mean of pooled data from all three trials ± SE
Results Water quality The pH decreased with increases in AMD while conductivity increased, in all trials (Table 2). Oxygen levels were consistently above 80 % saturation and ammonia was always \0.25 mg/L (Table 2). Ionic composition of the control/diluent waters and AMD are provided in Table 3. Concentrations of dissolved metals increased with increases in AMD (Table 4). Toxicity Shrimp survival was 100 % in control treatments for AHA, SRFA and SRHA during all three trials, but one shrimp did not survive in the control treatments for 0 HS during trial 1 (Fig. 1). A significant difference in survival at 2 % AMD was found between HS treatments (v2 = 12.7, p = 0.005), with follow up analyses showing significantly higher survival in all treatments with HS irrespective of type (AHA: v2 = 8.8, p = 0.003; SRFA: v2 = 6.6, p = 0.01; SRHA: v2 = 9.1, p = 0.003) (Fig. 1). Survival did not differ significantly between the AHA, SRFA and SRHA treatments (v2 = 0.2, p = 0.913) (Fig. 1), indicating that all types of HS behaved similarly in increasing survival to AMD. Nonlinear regressions revealed that Mount Morgan open pit
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Fig. 2 Concentration response plot for Mount Morgan open pit water (AMD) and Dee River water, with 0 HS, Sigma Aldrich Humic acid (AHA), Suwannee River fulvic acid (SRFA), and Suwannee River humic acid (SRHA). Curves represent the non-linear regression (three-parameter sigmoidal) of pooled data from the three trials at 96 h
AMD is considerably more toxic without the addition of HS, with LC50 values of 1.29 % (95 percentiles: 1.09–1.53 %) 0HS; 2.12 % (95 percentiles: 1.90–2.67 %) AHA; 2.22 % (95 percentiles: 2.01–2.50 %) SRFA; and 2.19 % (95 percentiles: 2.04–2.70 %) SRHA, recorded for each treatment (Fig. 2). All LC50 values recorded for the different HS types were within the 95 percentiles of the
Humic substances of varying types increase survivorship of the freshwater
others, indicating that there is probably no difference in survival among types of HS.
Discussion Metal concentrations and pH varied with increases in AMD during this study, with increases in dissolved metals and decreases in pH recorded. Concentrations of Al and Cu in all AMD treatments (0–4 %) were much lower than expected based on calculations using the measured concentrations in the 100 % AMD (Table 4). This is likely due to the effect of pH on metal speciation and precipitation, with Al generally in its precipitate form at pH values greater than 5 (Gensemer and Playle 1999). White precipitates were present in all AMD treatments. Therefore, the low measured concentrations of dissolved Al are likely due to the precipitation of Al in treatment water. The formation of Al precipitates also reduces the amount of dissolved Cu in solution due to co-precipitation (Lee et al. 2002). As the pH decreases it is expected that differences between measured and expected metal concentrations will decrease. This can be observed for Cu in the 4 % AMD. Zn concentrations were also shown to be lower in the 0–3 % AMD treatments with concentrations in the 4 % similar between the measured and expected values. This is also likely to be due to the co-precipitation of Zn on to the Al precipitates (Lee et al. 2002). Concentrations of the other metals did not vary greatly from the expected concentrations indicating correct dilution had occurred (Table 4). Adding as little as 2 % AMD to the treatment with no HS resulted in a mortality rate of over 80 %, and as little as 1.3 % AMD caused 50 % mortality in the exposed shrimp populations. The addition of HS increased the survivorship of shrimp exposed to AMD irrespective of HS type, with the commercial AHA corresponding with LC50 values similar to those of SRFA and SRHA. We also found no significant difference in the impacts of the Suwannee humic and fulvic acids. The lack of difference between the commercial AHA and the SRHA and SRFA may be attributed to all three of these HS being optically dark products (Schwartz et al. 2004). Variation in the ability of different HS to decrease toxicity of metals has been linked with HS optical properties, with optically dark, allochthonous HS shown to decrease toxicity of metals better than optically light, autochthonous HS (Schwartz et al. 2004; Al-Reasi et al. 2011; Wood et al. 2011). Previously, this feature has been suggested to be due to a greater proportion of HA in the optically dark HS (Schwartz et al. 2004; Wood et al. 2011; Ryan et al. 2004), however, our results show that SRFA can decrease toxicity of AMD similarly to SRHA. Decreased toxicity of metals in the presence of HS has often been attributed to complexation, which decreases
943
bioavailability and affects metal speciation. In all HS treatments, HS precipitates formed in treatments containing AMD, indicating metal complexation. Slight differences in average metal concentrations were shown between treatments for some metals, however, this did not follow a constant pattern with HS treatments sometimes recording lower concentrations and sometimes recording higher. This may be due to the heterogeneous nature of AMD leading to slight differences in metal concentrations within treatments and not due to the complexation with HS. The lack of obvious effect of HS on metal concentrations does not suggest a lack of complexation: rather the complexed metal/HS particles may still be small enough to pass through the 0.45 lm filter and be measured as dissolved metals. The complexation of metals with HS may have accounted for the increased survivorship of shrimp reported for all HS treatments in the 1 and 2 % AMD treatment. The decreased effect of HS at the higher AMD treatments is likely due to HS being unable to bind enough metals to reduce toxicity within these treatments, as metals concentrations in the 3 and 4 % AMD increased dramatically; for example: there is approximately five times more Cu in the 3 % AMD treatments compared with the 2 % AMD. The increased concentrations of metals along with a decrease in pH proved too toxic for freshwater shrimp even in the presence of HS. pH has also been shown to affect HS solubilisation and its ability to bind metals, with decreases in pH linked with decreased solubilisation and metal binding (Liu and Gonzalez 2000). It is unclear the effect pH had on solubilisation and metal binding within the current study as HS-metal precipitates were formed in all AMD treatments. The decreased effect at 3 and 4 % AMD may also be linked with the pH effects on metal binding at the lower pH in the 3 % AMD treatment (pH 5.5), however, as mentioned above, it may also be due to the increase in toxicants: lower pH and higher metal level in this AMD treatment. Humic acids have a greater affinity than fulvic acid for complexing metals, with greater amounts of metals often reported to be chelated by humic rather than fulvic acids (Lombartini et al. 1994; Tan 2003). This difference has been suggested to be due to difference in size (HA larger), complexity (HA more complex) and the presence of COOH groups (higher in FA) and phenolic-OH groups (higher in HA). The greater amounts of phenolic-OH groups in HA allow it to perform acidic reactions plus an interactive effect (complexation), whereas the higher amounts of COOH in FA allow for predominately acidic reactions to occur (Tan 2003). In the current study, we found no clear difference in the ability of HA (Aldrich or Suwannee) to complex and remove metals, compared with SRFA. This may be due to the effects of acidity on HA decreasing the amount of binding sites available for dissolved metals. As acidity increases, HA may become well protonated. As this
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occurs, the overall morphology of the molecule changes such that it impacts on the number and availability of binding sites, leading to a decrease in their ability to bind metal ions. This is why HA precipitates at pH 2 or below. Also the presence of high amounts of H? ions increases competition for binding sites and may make it harder for metal ions to bind to the HA molecule. Another possible explanation for the increased survivorship of shrimp to AMD in HS treatments is that HS may have affected shrimp at the physiological level. Previous work has shown that HS can bind to and be taken up by cell membranes, influencing their permeability, activating enzymes and defence proteins, and limiting ion loss (Campbell et al. 1997; Bedulina et al. 2010; Steinberg et al. 2007; Wood et al. 2003). However the methods applied in this study were not precise enough to closely examine the effects of HS on the physiological functions. Acknowledgments This study was supported by the Women’s Equal Opportunity Postgraduate Research Award, the Centre for Environmental Management, and Central Queensland University. The authors would like to acknowledge Dr Satish Choy for confirmation of shrimp identification, Heather Smyth for help with water chemistry, and Associate Professor Steve Mckillup for help with statistical design. Thanks to the two reviewers whose contributions significantly improved the manuscript. Conflict of interest of interest.
The authors declare that they have no conflict
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