A c c e p t e d P r e p r i nt
Aquatic Toxicology
BICARBONATE TOXICITY TO CERIODAPHNIA DUBIA AND THE FRESHWATER SHRIMP PARATYA AUSTRALIENSIS AND ITS INFLUENCE ON ZINC TOXICITY
CAROLINA LOPEZ VERA, ROSS V. HYNE, RON PATRA, SUNDERAM RAMASAMY, FLEUR PABLO, MORENO JULLI, AND BEN J.
KEFFORD
Environ Toxicol Chem., Accepted Article • DOI: 10.1002/etc.2545
Accepted Article
"Accepted Articles" are peer-reviewed, accepted manuscripts that have not been edited, formatted, or in any way altered by the authors since acceptance. They are citable by the Digital Object Identifier (DOI). After the manuscript is edited and formatted, it will be removed from the “Accepted Articles” Web site and published as an Early View article. Note that editing may introduce changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. SETAC cannot be held responsible for errors or consequences arising from the use of information contained in these manuscripts.
A c c e p t e d P r e p r i nt
Aquatic Toxicology
Environmental Toxicology and Chemistry DOI 10.1002/etc.2545
BICARBONATE TOXICITY TO CERIODAPHNIA DUBIA AND THE FRESHWATER SHRIMP PARATYA AUSTRALIENSIS AND ITS INFLUENCE ON ZINC TOXICITY
CAROLINA LOPEZ VERA,† ROSS V. HYNE,*‡ RON PATRA,‡ SUNDERAM RAMASAMY,‡ FLEUR PABLO,‡ MORENO JULLI,‡ AND BEN J. KEFFORD†
† Centre for Environmental Sustainability, University of Technology Sydney, Broadway, New South Wales, AUSTRALIA
‡ Centre for Ecotoxicology, Office of Environment and Heritage, Lidcombe, New South Wales, AUSTRALIA
Running title: Toxicity of bicarbonate and its influence on zinc toxicity
*Address correspondence to [email protected].
© 2014 SETAC
Submitted 20 September 2013; Returned for Revisions 28 January 2014; Accepted 30 January 2014
A c c e p t e d P r e p r i nt
Abstract: Bicarbonate is often a major ionic constituent associated with produced waters from methane gas extraction and coal mining but few studies have determined its specific toxicity. Currently the environmental risk of bicarbonate anion in water discharges is assessed based on the toxicity of sodium chloride or artificial sea water and is regulated via electrical conductivity. Increased NaHCO3 added to Ceriodaphnia dubia in synthetic or natural water gave similar 48-h EC10 values of 1750 ± 125 (mean ± SE) and 1670 ± 180 mg NaHCO3/L, respectively. Bicarbonate was toxic to C. dubia in both waters with conductivities above 1900 µS/cm. In contrast, when conductivity was elevated with NaCl, toxicity to C. dubia was only observed above 2800 µS/cm. Bicarbonate also impaired C. dubia reproduction with an EC10 of 340 mg NaHCO3/L. Major ion composition also influenced Zn bioavailability, a common co-occurring metal contaminant in coal mine waters with sub-lethal concentrations of NaHCO3 and elevated pH increasing Zn toxicity. Higher pH was the dominant parameter determining a 10-fold increase in the 48-h EC50 for Zn toxicity to C. dubia at pH 8.6 of 34 µg Zn/L (95% CL = 32–37) compared to the Zn toxicity at approximately circumneutral pH. Exposure of the freshwater shrimp Paratya australiensis (Atyidae) in natural water to increasing bicarbonate gave a mean 10-d LC10 of 850 ± 115 mg NaHCO3/L, associated with a mean conductivity EC10 of 1145 µS/cm, which is considerably lower than toxicity of NaCl and artificial sea water to this species reported elsewhere. Since toxicity was influenced by salt composition, specific ions should be regulated rather than conductivity alone in mine waste water discharges.
Keywords: Bicarbonate, Zinc, Alkalinity, Cladoceran, Major ions, Salinity
A c c e p t e d P r e p r i nt
INTRODUCTION
Coal bed methane extraction has increased world-wide over the past two decades since beginning in the
United States in the 1970s. The waters produced from the extraction process of the underlying coal beds as well as in discharge waters from coal mining are high in salinity. High levels of salinity are known to affect biota [1–3], especially freshwater organisms and ecosystems. Bicarbonate is one of the major ionic constituents associated with discharge waters from coal resource extraction along with metals and hydrocarbons naturally found within the coal bed [4,5]. Studies examining the toxicity of major ions to Ceriodaphnia dubia and Pimephales promales [6,7] suggested NaHCO3 as one of the salts most likely to be contributing to the toxicity of alkaline produced waters; however little is known about the toxicity of bicarbonate to aquatic life and its ecological effects. The ecological safety of disposal of saline coal mine waters cannot yet be determined. Existing salinity
sensitivity data are likely of limited relevance for predicting effects of discharge of most saline effluents from resource extraction because greater than 90% of toxicological data for salinity sensitivity of freshwater biota uses artificial sea water or sodium chloride (NaCl) as the salt source. Sea water is approximately 85% NaCl and in Australia most agricultural related salinity has ionic proportions similar to those of sea water [8]. Nonetheless, saline effluents from hydrocarbon extractions are not similar to sea water and are very variable. They often have higher proportions of bicarbonate (HCO3¯), sulphate (SO42¯) and boron in addition to metal and organic contaminants [4,5,9]. The proportions of ions in saline waters have major effects on its toxicity [6] and it has been recently
suggested that ionic proportions of HCO3¯ SO42¯, Ca2+ and Mg2+ [9] or HCO3¯ alone [10] may be more important in determining ecological effects than the total salinity. Yet the water quality guidelines in Australia, Canada or the United States do not give values to protect aquatic life for anions such as HCO3¯ and SO42¯. Following the United States Environmental Protection Agency (USEPA) procedure for calculating water quality criteria, Farag and Harper [10] proposed a criterion maximum concentration or an acute guideline value of 317 mg/L HCO3– and a criterion continuous concentration or chronic guideline value of 290 mg/L HCO3– for the protection of aquatic life.
A c c e p t e d P r e p r i nt
In addition, other studies have shown that bicarbonate alkalinity can modulate the toxicity of a number of metal ions such as Cu2+ and Pb2+, which are common contaminants in coal mine discharge waters, to C. dubia [11,12]. Currently discharge waters from coal mines in Australia are commonly only regulated by physico-chemical
parameters such as water salinity limits [http://www.epa.nsw.gov.au/prpoeo/index.htm]. There is a need to develop guidelines to improve the management of the major anions such as bicarbonate which can be a major contributor to the toxicity of these waters. Freshwater crustaceans are sensitive species to changes in water quality parameters such as alkalinity and hardness that can influence the toxicity of chloride, SO42¯ as well as metal ions [13–16]. This current study determined bicarbonate toxicity to two freshwater crustacean species in synthetic or natural water. The relationship of increasing bicarbonate or alkaline pH to the acute toxicity of zinc, a common contaminant in coal mine discharge water, to the cladoceran Ceriodaphnia dubia in synthetic water was also examined. MATERIALS AND METHODS Cladoceran culture methods The test organisms were < 24-h old neonates of the cladoceran, Ceriodaphnia dubia sensu stricto. This
species is taxonomically similar to C. dubia Richard, 1894 the type species (D. Berner, pers comm.). The cultures have been maintained in the Centre for Ecotoxicology (NSW) laboratories based on the procedures previously described [11] using dechlorinated Sydney tap water. Residual chlorine was measured and removed by the addition of sodium thiosulphate. This thiosulphate-treated Sydney tap water was supplemented with 5% Perrier mineral water and filtered seawater to a conductivity of 500 µS/cm at 25C 1. This water was used as the cladoceran culture and test water (alkalinity 50–69 mg CaCO3/L and hardness 80–100 mg CaCO3/L). Ten neonates from each cladoceran culture were assessed weekly by determining the number of young produced in three broods compared against a threshold level (mean of 16 young per cladoceran) to ensure that the cladoceran cultures were in a healthy state. The cladocerans were fed with two species of green algae, Ankistrodesmus sp. and Pseudokirchneriella subcapitata (50,000 cells/mL of each species) as well as Yeast, Cerophyl® and Trout Chow (YCT) food supplement (0.1 mL YCT/animal) [17]. The YCT supplement was prepared based on the USEPA protocol [17] except that a dried powdered blend of
barley and wheat grass (Lifestream International Ltd) was used instead of Cerophyl and a 3:2 mixture of fish food
A c c e p t e d P r e p r i nt
flakes (Sera Vipan) and Sera micron (Sera) used instead of Trout Chow. Synthetic water
The control water for toxicity testing was USEPA synthetic soft water (hardness 44 mg CaCO3/L; alkalinity
30 mg CaCO3/L; conductivity 160 to 180 µS/cm) [17] made from the following analytical reagent grade chemicals (BDH-Merck); sodium bicarbonate (NaHCO3), calcium sulfate, magnesium sulfate and potassium chloride added to purified Milli-Q water (Millipore Australia Pty Ltd). For toxicity tests involving varying concentrations of NaHCO3, the USEPA synthetic soft water was modified by the addition of 5 mM of the buffer 3morpholinopropanesulfonic acid (MOPS) together with 4 mM of MOPS sodium salt (Sigma-Aldrich) to pH 7.1. To examine the effect of high pH alone with no added NaHCO3 on Zn toxicity, 4 mM Piperazine-N,N'-bis (4butanesulfonic acid) (PIPBS) (GFS Chemicals) was added to the modified USEPA soft water and the pH adjusted to 8.1 or 8.6 with NaOH. These buffers were selected on the basis to reduce the rise in pH associated with the added NaHCO3 to increase the alkalinity or to determine Zn toxicity at higher pH to C. dubia with no added NaHCO3 present.
Cladoceran acute toxicity test The cladoceran acute toxicity test method used was based on the USEPA acute toxicity test protocol [17].
Neonates (
Aquatic Toxicology
BICARBONATE TOXICITY TO CERIODAPHNIA DUBIA AND THE FRESHWATER SHRIMP PARATYA AUSTRALIENSIS AND ITS INFLUENCE ON ZINC TOXICITY
CAROLINA LOPEZ VERA, ROSS V. HYNE, RON PATRA, SUNDERAM RAMASAMY, FLEUR PABLO, MORENO JULLI, AND BEN J.
KEFFORD
Environ Toxicol Chem., Accepted Article • DOI: 10.1002/etc.2545
Accepted Article
"Accepted Articles" are peer-reviewed, accepted manuscripts that have not been edited, formatted, or in any way altered by the authors since acceptance. They are citable by the Digital Object Identifier (DOI). After the manuscript is edited and formatted, it will be removed from the “Accepted Articles” Web site and published as an Early View article. Note that editing may introduce changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. SETAC cannot be held responsible for errors or consequences arising from the use of information contained in these manuscripts.
A c c e p t e d P r e p r i nt
Aquatic Toxicology
Environmental Toxicology and Chemistry DOI 10.1002/etc.2545
BICARBONATE TOXICITY TO CERIODAPHNIA DUBIA AND THE FRESHWATER SHRIMP PARATYA AUSTRALIENSIS AND ITS INFLUENCE ON ZINC TOXICITY
CAROLINA LOPEZ VERA,† ROSS V. HYNE,*‡ RON PATRA,‡ SUNDERAM RAMASAMY,‡ FLEUR PABLO,‡ MORENO JULLI,‡ AND BEN J. KEFFORD†
† Centre for Environmental Sustainability, University of Technology Sydney, Broadway, New South Wales, AUSTRALIA
‡ Centre for Ecotoxicology, Office of Environment and Heritage, Lidcombe, New South Wales, AUSTRALIA
Running title: Toxicity of bicarbonate and its influence on zinc toxicity
*Address correspondence to [email protected].
© 2014 SETAC
Submitted 20 September 2013; Returned for Revisions 28 January 2014; Accepted 30 January 2014
A c c e p t e d P r e p r i nt
Abstract: Bicarbonate is often a major ionic constituent associated with produced waters from methane gas extraction and coal mining but few studies have determined its specific toxicity. Currently the environmental risk of bicarbonate anion in water discharges is assessed based on the toxicity of sodium chloride or artificial sea water and is regulated via electrical conductivity. Increased NaHCO3 added to Ceriodaphnia dubia in synthetic or natural water gave similar 48-h EC10 values of 1750 ± 125 (mean ± SE) and 1670 ± 180 mg NaHCO3/L, respectively. Bicarbonate was toxic to C. dubia in both waters with conductivities above 1900 µS/cm. In contrast, when conductivity was elevated with NaCl, toxicity to C. dubia was only observed above 2800 µS/cm. Bicarbonate also impaired C. dubia reproduction with an EC10 of 340 mg NaHCO3/L. Major ion composition also influenced Zn bioavailability, a common co-occurring metal contaminant in coal mine waters with sub-lethal concentrations of NaHCO3 and elevated pH increasing Zn toxicity. Higher pH was the dominant parameter determining a 10-fold increase in the 48-h EC50 for Zn toxicity to C. dubia at pH 8.6 of 34 µg Zn/L (95% CL = 32–37) compared to the Zn toxicity at approximately circumneutral pH. Exposure of the freshwater shrimp Paratya australiensis (Atyidae) in natural water to increasing bicarbonate gave a mean 10-d LC10 of 850 ± 115 mg NaHCO3/L, associated with a mean conductivity EC10 of 1145 µS/cm, which is considerably lower than toxicity of NaCl and artificial sea water to this species reported elsewhere. Since toxicity was influenced by salt composition, specific ions should be regulated rather than conductivity alone in mine waste water discharges.
Keywords: Bicarbonate, Zinc, Alkalinity, Cladoceran, Major ions, Salinity
A c c e p t e d P r e p r i nt
INTRODUCTION
Coal bed methane extraction has increased world-wide over the past two decades since beginning in the
United States in the 1970s. The waters produced from the extraction process of the underlying coal beds as well as in discharge waters from coal mining are high in salinity. High levels of salinity are known to affect biota [1–3], especially freshwater organisms and ecosystems. Bicarbonate is one of the major ionic constituents associated with discharge waters from coal resource extraction along with metals and hydrocarbons naturally found within the coal bed [4,5]. Studies examining the toxicity of major ions to Ceriodaphnia dubia and Pimephales promales [6,7] suggested NaHCO3 as one of the salts most likely to be contributing to the toxicity of alkaline produced waters; however little is known about the toxicity of bicarbonate to aquatic life and its ecological effects. The ecological safety of disposal of saline coal mine waters cannot yet be determined. Existing salinity
sensitivity data are likely of limited relevance for predicting effects of discharge of most saline effluents from resource extraction because greater than 90% of toxicological data for salinity sensitivity of freshwater biota uses artificial sea water or sodium chloride (NaCl) as the salt source. Sea water is approximately 85% NaCl and in Australia most agricultural related salinity has ionic proportions similar to those of sea water [8]. Nonetheless, saline effluents from hydrocarbon extractions are not similar to sea water and are very variable. They often have higher proportions of bicarbonate (HCO3¯), sulphate (SO42¯) and boron in addition to metal and organic contaminants [4,5,9]. The proportions of ions in saline waters have major effects on its toxicity [6] and it has been recently
suggested that ionic proportions of HCO3¯ SO42¯, Ca2+ and Mg2+ [9] or HCO3¯ alone [10] may be more important in determining ecological effects than the total salinity. Yet the water quality guidelines in Australia, Canada or the United States do not give values to protect aquatic life for anions such as HCO3¯ and SO42¯. Following the United States Environmental Protection Agency (USEPA) procedure for calculating water quality criteria, Farag and Harper [10] proposed a criterion maximum concentration or an acute guideline value of 317 mg/L HCO3– and a criterion continuous concentration or chronic guideline value of 290 mg/L HCO3– for the protection of aquatic life.
A c c e p t e d P r e p r i nt
In addition, other studies have shown that bicarbonate alkalinity can modulate the toxicity of a number of metal ions such as Cu2+ and Pb2+, which are common contaminants in coal mine discharge waters, to C. dubia [11,12]. Currently discharge waters from coal mines in Australia are commonly only regulated by physico-chemical
parameters such as water salinity limits [http://www.epa.nsw.gov.au/prpoeo/index.htm]. There is a need to develop guidelines to improve the management of the major anions such as bicarbonate which can be a major contributor to the toxicity of these waters. Freshwater crustaceans are sensitive species to changes in water quality parameters such as alkalinity and hardness that can influence the toxicity of chloride, SO42¯ as well as metal ions [13–16]. This current study determined bicarbonate toxicity to two freshwater crustacean species in synthetic or natural water. The relationship of increasing bicarbonate or alkaline pH to the acute toxicity of zinc, a common contaminant in coal mine discharge water, to the cladoceran Ceriodaphnia dubia in synthetic water was also examined. MATERIALS AND METHODS Cladoceran culture methods The test organisms were < 24-h old neonates of the cladoceran, Ceriodaphnia dubia sensu stricto. This
species is taxonomically similar to C. dubia Richard, 1894 the type species (D. Berner, pers comm.). The cultures have been maintained in the Centre for Ecotoxicology (NSW) laboratories based on the procedures previously described [11] using dechlorinated Sydney tap water. Residual chlorine was measured and removed by the addition of sodium thiosulphate. This thiosulphate-treated Sydney tap water was supplemented with 5% Perrier mineral water and filtered seawater to a conductivity of 500 µS/cm at 25C 1. This water was used as the cladoceran culture and test water (alkalinity 50–69 mg CaCO3/L and hardness 80–100 mg CaCO3/L). Ten neonates from each cladoceran culture were assessed weekly by determining the number of young produced in three broods compared against a threshold level (mean of 16 young per cladoceran) to ensure that the cladoceran cultures were in a healthy state. The cladocerans were fed with two species of green algae, Ankistrodesmus sp. and Pseudokirchneriella subcapitata (50,000 cells/mL of each species) as well as Yeast, Cerophyl® and Trout Chow (YCT) food supplement (0.1 mL YCT/animal) [17]. The YCT supplement was prepared based on the USEPA protocol [17] except that a dried powdered blend of
barley and wheat grass (Lifestream International Ltd) was used instead of Cerophyl and a 3:2 mixture of fish food
A c c e p t e d P r e p r i nt
flakes (Sera Vipan) and Sera micron (Sera) used instead of Trout Chow. Synthetic water
The control water for toxicity testing was USEPA synthetic soft water (hardness 44 mg CaCO3/L; alkalinity
30 mg CaCO3/L; conductivity 160 to 180 µS/cm) [17] made from the following analytical reagent grade chemicals (BDH-Merck); sodium bicarbonate (NaHCO3), calcium sulfate, magnesium sulfate and potassium chloride added to purified Milli-Q water (Millipore Australia Pty Ltd). For toxicity tests involving varying concentrations of NaHCO3, the USEPA synthetic soft water was modified by the addition of 5 mM of the buffer 3morpholinopropanesulfonic acid (MOPS) together with 4 mM of MOPS sodium salt (Sigma-Aldrich) to pH 7.1. To examine the effect of high pH alone with no added NaHCO3 on Zn toxicity, 4 mM Piperazine-N,N'-bis (4butanesulfonic acid) (PIPBS) (GFS Chemicals) was added to the modified USEPA soft water and the pH adjusted to 8.1 or 8.6 with NaOH. These buffers were selected on the basis to reduce the rise in pH associated with the added NaHCO3 to increase the alkalinity or to determine Zn toxicity at higher pH to C. dubia with no added NaHCO3 present.
Cladoceran acute toxicity test The cladoceran acute toxicity test method used was based on the USEPA acute toxicity test protocol [17].
Neonates (
