Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Review of fluoride toxicity to aquatic organisms and its toxicity contribution in Volvo wastewater STEVEN FLEISS Degree project for Master of Science in Ecotoxicology 30 ECTS Department of Plant and Environmental Sciences University of Gothenburg June 2011 y Summary The literature on fluoride water chemistry and toxicity to aquatic life has been reviewed. Altogether 388 values on the toxicity of fluoride for a range of species (algae, invertebrates, fish) were compiled and used for estimating species sensitivity distributions (SSDs). This have been combined with laboratory tests on the copepod Nitocra spinipes using both fluoride and Volvo paint process wastewater at relevant salinity conditions, in order to define the potential impacts of fluoride to the local environment. A concentration of 1.16 mg F/L has been preliminary identified as the Predicted No Effect Concentration (PNEC), based on the compiled EC/LC50 values. The effect of salinity on toxicity and a ratio of LC50/NOEC derived from the Nitocra trials were used to derive a safety factor. The literature identified that juveniles and small individuals are most susceptible to fluoride. An increase in temperature as well as a decrease in water hardness increases the acute toxicity of fluoride. Because of the influence of ambient water characteristics a study on the chronic toxicity of the wastewater is proposed. This would allow the validation of the 1.16 mg F/L value as a water quality target under local conditions (water temperature, pH, salinity and hardness). Furthermore, more toxicity tests with the wastewater should be conducted to investigate the variability over time and to determine what is causing the acute toxicity besides fluoride. A TIE (Toxicity Identification Evaluation) approach is recommended for these studies. Table of Contents Summary.................................................................................................................................. 1 Lists of Figures and Tables.................................................................................................... 3 Acronyms and Abbreviations............................................................................................... 3 Background...........................................................................................................................5 Physical and Chemical Properties of Fluoride (F-) and Fluoride Salts ........................... 6 Toxicological Effects of Fluoride...................................................................................6 General Mechanisms of Toxicity .......................................................................................... 7 Methods and Approaches................................................................................................8 Literature compilation ........................................................................................................... 8 Species Sensitivity Distribution Curves and the 95th Percentile ...................................... 9 Laboratory Methods............................................................................................................. 10 Statistical Analysis of Exposure Trials............................................................................... 12 Literature Review of Ecotoxicological Effects ....................................................... 12 Algal Toxicity ........................................................................................................................ 13 Invertebrate toxicity ............................................................................................................. 13 Fish Toxicity .......................................................................................................................... 15 Sub-lethal Effects .................................................................................................................. 17 Summary of ecotoxicological effects.................................................................................. 18 Critical Factors that influence the Aquatic Toxicity of Fluoride..................... 19 Temperature .......................................................................................................................... 19 Water Hardness .................................................................................................................... 20 pH ........................................................................................................................................... 20 Salinity.................................................................................................................................... 20 Survey of Water Quality Criteria ............................................................................................. 21 Results of Laboratory Exposure Trials ..................................................................... 22 Comparative Toxicity of Effluent Solution and the Equivalent Fluoride Concentration........................................................................................................................ 22 LC50 of the Effluent Solution ............................................................................................... 25 LC50 of the Fluoride Solution............................................................................................... 26 Statistically Observed No Effect Concentrations ............................................................. 26 Influence of Salinity on Fluoride Toxicity......................................................................... 27 Influence of pH, Temperature and Water Hardness....................................................... 28 Summary and derivation of a preliminary PNEC for Fluoride ....................... 29 PNEC derivation................................................................................................................... 30 References: ......................................................................................................................... 33 2 Lists of Figures and Tables Figure 1. Invertebrate Acute LC50 SSD Figure 2. Invertebrate Chronic LC50 SSD Figure 3. Fish Acute LC50 SSD Figure 4. Fish Chronic LC50 SSD Figure 5. Fish Median Acute LC50 SSD Figure 6. Invertebrate EC50 SSD Figure 7. Nitocra 100% Effluent Comparisons Figure 8. Nitocra 50% Effluent Comparisons Figure 9. Nitocra 25% Effluent Comparisons Figure 10. Nitocra Mortality Distributions 12 13 14 14 15 16 22 22 23 23 Figure 11. Salinity Effects on Fluoride Figure 12. Salinity Effects on Effluent 25 26 Table 1. Literature search profile Table 2. Fluoride compounds and complexes of environmental concern Table 3. 95th Percentile and Sub-lethal Effects Summary Table 4. Water Quality Criteria for Protection of Aquatic Life Table 5. LC50 of N. spinipes exposed to Effluent Table 6. LC50 of N. spinipes exposed to Fluoride solution Table 7. No Observed Effect Concentrations from tests with N. spinipes in effluent and fluoride. 6 8 17 20 24 24 25 Appendix 1. Combined database (separate Excel File) Appendix 2. Nitocra database (separate Excel File) Acronyms and Abbreviations ATP EC50 LC50 Adenosine triphosphate Median effective concentration, concentration that causes 50% effect Median lethal concentration, concentration that causes 50% mortality 3 LOEC NOEC SSD PNEC ppt Lowest observed effect concentration No observed effect concentration Species Sensitivity Distribution Predicted No Effect Concentration Parts per thousand 4 Background Fluoride has been identified as a potential toxicant in the paint process at the Volvo factory at Torslanda. The University of Gothenburg has been commissioned by Volvo to review the literature with regard to the ecotoxicological hazard of fluoride for the aquatic environment, and with the aid of laboratory trials, validate under relevant environmental conditions a concentration at which no effect is predicted. The scope of the study was to review impacts of fluoride on different aquatic organisms in different aquatic environments (freshwater, estuary and marine) and to determine the impact it may have to the local environment. The influence of important water quality parameters (pH, temperature, hardness) on the toxicity of fluoride was analysed and water quality criteria for fluoride was reviewed. A major aim was to identify the most sensitive group of species and the critical confounding factors, and use this to calculate a predicted no effect concentration (PNEC) relevant to the receiving waters. This report hence collates information relating to the responses of different aquatic organisms to fluoride. The information is gathered from peerreviewed articles, US Environmental Protection Agency ECOTOX database as well as from reports from government agencies and international bodies. Ecotoxicological information that was gathered from the literature is tabulated in a separate Excel file to provide a comprehensive reference. Figures 1-6 in this report draw on that information and provide details on the distribution of toxicity values for selected groups of organisms, endpoints and exposure durations. Nitocra spinipes, a harpactoid copepod, was identified as an ideal test invertebrate species to use in the exposure trials, due to local occurrence and its tolerance to a wide range of salinities. Its historical use in ecotoxicological studies also allowed for standard procedures to be used. The exposure trials will be used to gauge the influence of fluoride within the effluent. This will be achieved by conducting comparisons between effluent solutions at differing dilutions and prepared solutions containing the equivalent fluoride concentrations. 5 Physical and Chemical Properties of Fluoride (F-) and Fluoride Salts The following review focuses on the toxicity of the Fluoride ion (F-) (CAS No. 16984488) and fluoride salts that form in industrial processes and in the environment. Table 1 provides a brief description of some of the main fluoride complexes that have ecotoxicological data present in the Aquire database. The physical and chemical information was retrieved from the Hazardous Substance Data Bank (National Library of Medicine, 2010). Table 1. Fluoride compounds and complexes of environmental concern. Fluoride CAS Fluoride Molecular Solubility Compound Number % Mass Sodium Fluoride (NaF) 7681-49-4) 45.2 41.99 4.0-4.3g/100ml @ 15-25oc Calcium Fluoride (CaF2) 7789-75-5 48.7 78.1 0.0015g/100ml @ 18oc Hydrogen Fluoride (HF) 7664-39-3 95 20 Sodium Silicofluoride (F6Na2Si) 16893-85-9 60.6 188.1 Aluminium Fluoirde (AlF3) 7784-18-1 67.8 83.97 Very soluble 0.64-0.76g/100ml @ 20-25oc 0.559g/100ml @ 25oc Toxicological Effects of Fluoride Fluoride is present in the environment as the stable form of the super reactive element fluorine. Fluorine is the 17th most abundant element in the earth's crust, with fluoride detectable in almost all minerals. The main minerals are fluorspar-CaF2, Cryolite-Na3AlF6 and fluorapatite-Ca10F2(PO4)6. Fluoride naturally enters the aquatic system through weathering of alkalic and silicic igneous and sedimentary rocks, primarily shales, as well as from emissions from volcanic activity. Fluoride is typically found in freshwater at concentrations less than 1.0 mg/L, however, natural concentrations may exceed even 50.0mg/L (McNeely et al., 1979). An understanding of local natural fluoride levels is important in assessing the toxicological effects, because local populations may already be adapted to fluoride exposure. 6 To judge on the potential environmental impacts of fluoride, it is important to first review the current knowledge on its impact on the homeostasis within organisms. The benefits of fluoride are seen mostly in the hardening of teeth and the protection from caries (Barbier et al., 2010). However, current evidence is inconclusive whether fluoride is essential for any other biological function (Government of British Columbia, 1990). The most common ailment associated with an excess of fluoride is fluorosis. This condition relates to the retention of excess fluoride within the body and its deleterious integration into biochemical pathways, often as a substitute for calcium (Barbier et al., 2010). General Mechanisms of Toxicity A review by Barbier et al. (2010) has outlined a number of cellular processes in which fluoride can have a deleterious effects. Identified effects include disruption of enzyme activity (mostly inhibition), inhibition of protein secretion and synthesis, generation of reactive oxygen species (ROS), and alteration of gene expression. Fluoride disrupts enzyme activity by binding to functional amino acid groups that surround the active centre of an enzyme. This includes the inhibition of enzymes of the glycolytic pathway and the Krebs cycle (Barbier et al., 2010). Studies by Mendoza-Shulz et al. (2009) indicate that fluoride at micromolar concentrations can act as an anabolic agent and promote cell proliferation, whereas at millimolar concentrations it acts as an enzyme inhibitor on e.g. phosphatases, which play an important role in the ATP (cellular energy) production cycle and cellular respiration. Interruption of the signaling pathways involved in cell proliferation and apoptosis has also been attributed to fluoride, caused by the inhibition of protein synthesis and secretion (Barbier et al., 2010). Fluoride has also been associated with oxidative stress. Oxidative stress can lead to the degradation of cellular membranes and reduce mitochondrial fitness. The increase of oxidative stress leads to an increase in the expression of genes responsible for stress response (Barbier et al., 2010). 7 Methods and Approaches Literature compilation The approach for the literature search, including database and keywords used, is shown in Table 2. The literature search process involved the primary step of searching the US Environmental Protection Agencies ECOTOX database (Aquire, www.epa.gov/ecotox). Using the keyword ‘fluoride’, a query was undertaken of the aquatic data. This provided a list of fluoride containing compounds and their ecotoxicological effects. Only the information for inorganic fluoride compounds was extracted and tabulated. A search was also undertaken in Scopus journal database (www.scopus.com, Elsevier Publishers). Using key words ‘fluoride’, ‘environment’, ‘toxicity’ and ‘aquatic’, journals were investigated for relevant publications and the retrieved information was tabulated. A further search in those articles’ reference lists and citation lists was made to include any additional connections in the review. Finally, a search of Google and Google scholar using the key words ‘fluoride’ and ‘environmental’ was made, with a search emphasis on guidelines and criteria used by different government jurisdictions. Table 2. Literature search profile Search Location Keywords Google Fluoride + aquatic US EPA ECOTOX (Aquire) Fluoride Scopus Fluoride + aquatic + toxicity Biogeochemical + fluoride Fluoride + marine Culture + studies + fluoride + pollution Fluoride + effluent + marine Fluoride + chemical + biological + marine NaF + toxicity Camargo + fluoride Fluoride + environmental + toxicity + 2001-2010 Hazardous Substance Data Bank Sodium fluoride 8 Once all the data has been collected and cross-referenced to ensure that there is no duplication, the data were tabulated and species sensitivity distribution (SSD) curves of the reported LC50 and EC50 values were produced. The SSD information allows the calculation of the 95th percentile, which is used to determine a concentration that would protect 95% of species from the endpoint used in a particular curve. The 95th Percentile was calculated using the percentile function in excel and choosing the value that equated to being the 5th percentage point along the curve. A 95th percentile was calculated for both acute (< 4 days) and chronic (> 4 days) exposure for fish and invertebrate LC50 data points, as well as for the invertebrate EC50 values. To obtain a Predicted No Effect Concentration (PNEC), a safety factor (assessment factor), in this case 10, was applied to the lowest or most appropriate 95th percentile, in order to produce a water quality target which is believed to illicit no effect on the aquatic species in the environment. Species Sensitivity Distribution Curves and the 95th Percentile A SSD curve is the visualisation of ecotoxicological data derived from test on a specific taxon, a selected species assemblage, or a natural community (Posthuma et al., 2002). The SSD is used to estimate the distribution of toxicity endpoints, using the toxicological information available. SSD’s are most sensitive when using a range of species and NOECs as the toxicological endpoint. The information presented in this paper is organised into separate effect endpoints, in our case either EC50 or LC50 with the corresponding observed concentrations for those species tested. NOECs have not been used as there are not sufficient data points to provide a robust estimate. Newman et al. (2000) recommends 15-50 data points, with more numbers and greater variance in species providing a more rigorous SSD. The effect concentrations are organized from lowest to highest and each is assigned a rank from 1 to n. The distribution curve is then constructed by plotting the concentration against the rank. The distribution of species used in the ecotoxicological studies is not even, for example Oncorhynchus mykiss dominates the fish studies. As multiple data for a single species can distort a curve, those species that dominate the input values have been highlighted. The 95th percentile is used to determine what data value would encompass 95% of the data range. It is used in environmental management as a tool to determine at what concentration of a toxicant 95% of species that are present 9 in the ecosystem are protected. The 95th percentile determined for the data sets only represents the endpoints and species used in each SSD. It must be noted that the 95th percentile of LC50 data only protects 95% of species from exposure to concentrations that are lethal to 50% of individuals. Posthuma et al. (2002) highlight a number of issues that need to be kept in mind when SSDs are constructed, the most important being an awareness of the origin of the data. Many of the concentrations generated have come from laboratory experiments, where the response of a species may be different to that of one exposed to variable field conditions. To deal with this uncertainty, a safety factor is often applied, with the level determined by its applicability (may be too conservative and propose a concentration below natural background levels) and the size of the data set. The REACH guidance document Guidance on Information Requirements and Chemical Safety Assessment (European Chemicals Agency, 2008) recommends using an assessment factor of between 5 and 1 (depending on input data) for SSD’s conducted for freshwater environments and a factor of 10 for marine environments. These safety factors are based on the input data being NOEC or EC10 values. The SSD in this review are conducted using LC50 and EC50 data, instead of NOEC values, therefore a safety factor of 10 needs to be applied, as LC50 and EC50 values do not offer protection to species. Laboratory Methods The laboratory methods for culturing Nitocra spinipes follow those described by Dave et al. (1993). Cultures of Nitocra spinipes started with the addition of 10-15 egg-carrying females to a container with 100 ml Nitocra standard culture medium (8ppt saline natural seawater solution). After 2 weeks, juveniles (those without egg sacs) from these cultures were used for the toxicity tests. The toxicity tests were conducted in 4 x 6 cell plates that hold approximately 4 ml of medium in each cell. Only 2.5 ml of test medium were used for the toxicity tests. The toxicity testing for both experiments was made with 5 juveniles which were placed in each cell with test medium and exposed for 96 hours. Observations and survival rates were examined after 96 hours. Each test concentration was replicated n times, depending on the aim of the exposure trial. Each exposure plate also had at least 2 cells dedicated to a control 10 solution of Nitocra standard culture medium. The Nitocra were not fed for the duration of the trial. To ensure that the exposure times were similar and to minimize the transfer time into the test cells among treatments, individuals were removed from the culture medium and placed into a smaller vessel up to 6 hrs before the exposure was started. The medium in the smaller vessel was of the same salinity as the test medium. Preparation of Effluent Water Samples of effluent discharge were collected from the manufacturing plant, frozen, and then delivered to University of Gothenburg. Chemical analysis was undertaken on an additional sample by Göteborgs Kemanalys AB to determine the fluoride concentration. The effluent was prepared for exposures by defrosting a sample and warming it to 22 oC. The salinity of the effluent was determined by a hand salinity refractometer. The sample was then split into three, and NaCl was added to obtain solutions with salinities of 17 ppt, 8 ppt and 1 ppt. The pH of these solutions was then adjusted to between pH 7-8. The desired effluent concentration for the trial was obtained by mixing the stock solution with the standard Nitocra culture medium or the salt adjusted Nitocra culture solution. The exposure in 1ppt salinity used MilliQ salt-adjusted water. Preparation of Fluoride Solution A stock fluoride solution was made by adding NaF to MilliQ water to a concentration of 4.77 g NaF/l or 2.16 g F-/l. Depending on the exposure trial undertaken, no more than an hour before the trial was to begin, 15 ml solutions were made using the fluoride stock solution and the Nitocra culture medium (8 ppt salinity) or a salt adjusted Nitocra culture medium (17 ppt). Those trials testing the effects at 1 ppt salinity used the fluoride stock solution and salt-adjusted MilliQ water. Exposure trials The trials were organised so as to determine the following: 1. LC50 and NOEC of the effluent solution 2. The effect of salinity on fluoride 3. LC50 and NOEC of the fluoride solution 11 4. Whether the effluent solution and a solution with equivalent fluoride exhibited the same mortality rates. LC50 and NOEC were determined by producing a dilution series. The salinity effects were recorded by producing a limited dilution series of fluoride solution (typically 3 concentrations), with each concentration tested at a salinity of 1 ppt, 8 ppt and 17 ppt. The difference between mortality in effluent and the equivalent fluoride solution was determined at 100% effluent and 27 mg F/l fluoride solution. An exposure trial with 12 replicates of the effluent, fluoride solution and control was conducted. For lower concentrations, comparisons were made by collating data from the other exposure trials. Statistical Analysis of Exposure Trials LC50 concentrations for the N. spinipes were calculated using the Trimmed Spearman-Karber method, using software available from the US EPA (http://www.epa.gov/eerd/stat2.htm). The Weibull model was used to compare the slopes of the dose response curves of fluoride and effluent solutions. Comparisons between and within the effluent solutions and fluoride solutions were undertaken using a one-way ANOVA, while comparisons between increasing salinity and increasing effluent/fluoride were done using a two-way ANOVA. Comparisons between concentrations were analysed using Tukey’s post test. The NOEC was calculated using the Dunnett’s multiple comparison test. All statistics and plotting other than the LC50 was done on the program GraphPad Prism version 5. Literature Review of Ecotoxicological Effects This section documents the findings of the literature search. This review provides a table of the relevant ecotoxicological studies that have been undertaken for fluoride (Appendix 1). An illustration of the species sensitivity distribution for invertebrates and fish is provided in figures 1-6. Additional critical points of discussion from the literature have also been included where appropriate. 12 Algal Toxicity Algal data has not been provided below as a SSD, as the results from the literature do not provide a uniform response to the presence of fluoride. Camargo (2003) highlights this in the review of fluoride toxicity to aquatic organisms. Studies have shown that some species of algae will respond with growth inhibition (e.g. Amphidinium carteri (Antia and Klut, 1981), some show growth enhancement (e.g. Chaetoceros gracilis) (Antia. and Klut, 1981) and others remain unaffected (e.g. Nannochloris oculata) (Oliveira et al., 1978). Therefore, to provide only those data points that indicate a negative effect would not be representative of this organism group. However, the lowest EC50 value was determined at 82 mg/L for Skeletonema costatum after chronic exposure. The lowest observed effect concentration is 2 mg/L, which did inhibit growth of the Chlorella pyrenoidosa by 37% in a freshwater environment (Groth, 1975). Invertebrate toxicity The species sensitivity distribution for invertebrate toxicity (LC50s) for acute exposure (4 days or less) and chronic exposure (more than 4 days) is shown in Figures 1 and 2. 60 Species Rank 50 40 Caddisfly Other Invertebrates 95th Percentile N. sinipes 30 20 10 0 1 10 100 1000 10000 Fluoride Concentration (mg/L) Figure 1: LC50 Invertebrate sensitivity to fluoride at acute exposure duration (4 days or less). SSD is separated into values from caddisfly studies (all species) and all other invertebrates. The 95th percentile was calculated on the basis of all data. The LC50 of N. spinipes from the exposure trials undertaken for this report are included for reference. 13 12 Species Rank 10 8 Caddisfly Mollusc 6 95th Percentile 4 2 0 10 100 1000 Fluoride Concentration (mg/L) Figure 2. LC50 invertebrate sensitivity to fluoride at chronic exposure duration (> 4 days). Data points have been separated into caddisfly (different species) and molluscs. The 95th percentile has been determined on the basis of all data points. Figure 1 and 2 plot the range of 50% lethal concentration doses that have been described in the literature for invertebrates at different exposure durations. The lowest acute LC50 concentration is 10.5 mg/L for Mysidopsis bahia (Mysid shrimp) which was tested in seawater. The lowest chronic LC50 concentration is 11.5 mg/L for Hydropsyche bronta (caddisfly), which was tested in freshwater with a hardness of 40.2 CaCO3 mg/L at a temperature of 18oC. Figures 1 and 2 also indicate that when comparing the invertebrate 95th percentiles of acute (26.08 mg F/L) and chronic (12.34 mg F/L) exposure, an increase in exposure time reduces the LC50 concentration by half In addition to the LC50 information provided in figure 2, a mortality study by Sparks et al. (1983) highlighted the sensitivity of the fingernail clam (Musculium transversum). Their study showed that that this small (2-4mm) and quickly reproducing (maturity in 33 days) clam was also sensitive to fluoride. This study found that the clams exposed to 2.8 mg F/L over 8 week period, suffered 60% mortality compared to 25% mortality of the controls. 14 Species Rank 40 Oncorhynchus mykiss Other Fish 30 95th Percentile 20 10 0 1 10 100 1000 Fluoride Concentration (mg/L) Figure 3. LC50 fish sensitivity distribution for exposure less than 4 days (acute). Distribution separated into Oncorhynchus mykiss (rainbow trout) and other fish species. The 95th percentile is calculated using all data points. Species Rank 30 Oncorhynchus mykiss Other Fish 95th Percentile 20 10 0 1 10 100 1000 Fluoride Concentration (mg/L) Figure 4. LC50 fish sensitivity distribution for exposure more than 4 days (chronic). Distribution separated into Oncorhynchus mykiss (rainbow trout) and other fish species. The 95th percentile is calculated using all data points. Fish Toxicity The species sensitivity distribution for fish toxicity (LC50s) for acute exposure (4 days or less) and chronic exposure (more than 4 days) is shown in Figures 3 and 4. Figure 3 and 4 plot the range of LC50 values that have been described in the literature for fish at different exposure durations. Oncorhynchus mykiss shows 15 both the lowest acute LC50 concentration (7.0 mg/L) and chronic LC50 (2.3 mg/L). Both tests were conducted in soft water, with the acute test having a zero hardness value. Figures 3 and 4 also indicate that when comparing the Fish 95th percentiles of acute (15.98 mg F/L) and chronic (2.62 mg F/L) exposure, there is a 6 times reduction in LC50 concentration in response to the increase in exposure time. Species Rank 8 Oncorhynchus mykiss Pimephales promelas Salmo trutta Channa punctata Gasterosteus aculeatus Cyprinodon variegatus Gambusia affinis 6 4 2 0 100 1000 Fluoride Concentration (mg/L) Figure 5. Distribution of median LC50 concentrations of the fish species used in the SSDs on acute toxicity. Figure 5 shows the spread of LC50 values of each fish species used in the acute SSD. It should be noted that although Oncorhynchus mykiss did provide the most sensitive response at 7.0 mg/L, the median for all Oncorhynchus mykiss data points is 124.5 mg/L. 16 Species Rank 50 40 30 20 Caddisfly Other Invertebrates 10 95th Percentile 0 10 100 1000 Fluoride Concentration (mg/L) Figure 6. EC50 studies with invertebrate species (exposure < 4 days). Sub-lethal Effects Species sensitivity distribution for studies of sub-lethal responses of invertebrates (EC50) are shown in Fig. 6. Similar figures for fish were not available in the literature. The majority of EC50 values plotted in figure 6 come from studies that have examined the response of caddisfly larvae to various fluoride concentrations. The 95th percentile for the EC50s-SSD is 19.2 mg/L. All of the observations were made for exposures of 4 days or less. No chronic data for EC50’s were available. Other studies (Shi et al., 2009; Pillai and Mane, 1985; Pankhurst et al, 1980; Camargo, 2003; Damkaer and Day 1989) have observed sub-lethal effects, without quantifying an EC50 value. A brief description of their observations is hence provided below. A number of studies indicated that exposure of fluoride can reduce growth. Observations during a bioaccumulation study by Shi et al. (2009) with juvenile sturgeon fish indicated that at increasing concentrations (10, 25 and 60 mg F/l) there was a significant inhibition of growth over 90 days compared to the control, with decrease in growth following an increase in concentration. They also observed that fish exposed to concentrations over 25 mg F/l displayed alterations in their respiration and violent erratic movements. The study attributed the diminished growth to the impairment of physiological processes, such as enzyme inhibition (as discussed earlier), but also to 17 histopathological changes. These changes include the increase in mucous cells in the epithelium of the head region and the gills. They note that the behavioural changes observed are similar to those identified in other studies. These histopathological changes were noted in another fish species Labeo rohita, which was exposed to 15 mg/L NaF for a period of 120 days. Observations were made every 30 days and even after the first time point, it was noted that there was significant swelling at the tip of secondary gill lamellae and clubbing of lamellae, as well as pathological conditions that included mucoid metaplasia and lamellar hyperplasia (Bhatnagar et al., 2007). The same study also observed that the intestine exhibited flattening and fusion of villi and that the kidney showed renal architecture damage. Pillai and Mane (1985) demonstrated a delayed egg hatching of the freshwater fish species Catla catla. A 1hr delay occurred at 3.66 mg/L fluoride compared to the control, with concentrations of 7.34 incurring a 2hr delay in hatching. Pankhurst et al. (1980) tested the brine shrimp (Artemia salina) and found that at 5 mg/L the shrimp larvae demonstrated significant growth impairment. A study of toxicity to freshwater mussel juveniles by Keller and Augspurger (2005) observed growth inhibition in mussels juveniles. The experiment evaluated the LC50s of a selection of freshwater mussels and determined that there was no significant different between 96 hr and 216 hr (9 days) LC50 concentrations. There was however a significant difference in shell length growth between the exposure scenarios. The study indicated that sub-lethal responses to fluoride was identified at 31 mg F/l. Although not lethal, mussels that are smaller are more prone to predation and hence have a reduced reproduction success. Damkaer and Day (1989) have been cited in many reviews for their studies of the migration pattern of different fish species. It was found that a concentration of 0.5 mg F/L could disrupt the migration run of the salmon species chinook (O. tschawytscha), chum (O. keta) and coho (O. kisutch). It was noted that aluminum levels in the river may have been a confounding factor. Summary of ecotoxicological effects The table below is a summary of the calculated 95th percentiles for invertebrates and Fish, as well as the lowest sub lethal effect concentrations for fish and algae. 18 Table 3. Summary of SSDs (lower 95th Percentiles) and Sub-lethal Effects of Fluoride Species Group Exposure Endpoint Concentration Invertebrate Chronic 95th % LC50 12.34 mg/L Acute 95th 26.08 mg/L Acute 95th % EC50 Chronic 95th % LC50 2.62 mg/L Acute 95th % LC50 15.98 mg/L Salmon species Chronic Significant disruption of migration 0.5 mg/L Algae Chronic* Lowest EC50 Fish % LC50 19.2 mg/L 82 mg/L *Algal studies longer than 3 days are considered long-term Critical Factors that influence the Aquatic Toxicity of Fluoride In the aquatic environment, transport and transformation of inorganic fluorides are influenced by pH, hardness, and the presence of ion-exchange materials such as clays (Environment Canada, 1994). In freshwater environments, dissolved inorganic fluoride is maintained in solution under conditions of low pH and hardness. An increased hardness limits the equilibrium solubility of the fluoride ion as complexes with magnesium and calcium ions form precipitates. Below are some observations of the effect these water quality parameters can have on the toxicity of fluoride. Temperature The environment protection division of the British Columbian Government indicates that the uptake rate of fluoride doubles for every 10oC rise in temperature. A study by Angelovic et al. (1961) found that when exposed to the same concentration (25mg F/l), an increase in temperature from 7.2 to 23.9oC would decrease the time for lethal effects to be observed in rainbow trout. This effect was also demonstrated for Daphnia magna, with 48-hr LC50 dropping from 304 to 251 to 200 mg/L, with every 5 degree increase from 15oC (Fieser et al., 1986). 19 Water Hardness The ability of water hardness to offer protection has been discussed in a number of papers (Camargo, 2003; Giguere and Campbell, 2004). The British Columbian Environmental Protection Division suggests that much of the benefits observed from the use of experimental hard water is in fact due to the precipitation of CaF2, which in turn reduces the free fluoride concentration (Government of British Columbia, 1990). However, Giguere and Campbell (2004) compiled data from all available studies and determined that there was no relationship between fluoride toxicity and calculated dissolved calcium concentrations. A study of the LC50 for Oncorhynchus mykiss (rainbow trout) in increasing water hardness did indicate a relationship between dissolved calcium and free fluoride concentration (Pimentel and Bulkley, 1983). Fish exposed to fluoride in a water hardness of 17mg CaCO3/L had a LC50 of 51mg F/L, whereas fish exposed to in a water hardness of 49, 182 and 385 mg CaCO3/L had LC50s of 128, 140 and 193 mg F/L, respectively. Giguere and Campbell (2004) hypothesised that there are three mechanisms that could explain the trend above. Firstly, the test organism is benefiting from the presence of the hardness cations (Ca2+, Mg2+), either externally, at epithelial membranes, or internally. Secondly, complexation between the fluoride ions and the hardness cations, which reduces the free fluoride concentration. Thirdly, precipitation of Calcium Fluoride (CaF2) in the aquatic media, which also reduces the effective fluoride concentration. pH A study by Rai et al. (1997) using the algae C. vulgaris indicated that pH alters the toxicity of fluoride. They found that in general, the toxicity of fluoride towards the algae increased with a downshift in pH. They also studied the effect of AlCl3 and found that in combination with NaF, the toxicity had an additive effect at pH 6.8, but a synergistic effect at pH 6.0 and 4.5. These interactions should be the focus of further studies to determine the influence on other species. Salinity Studies of freshwater organisms have indicated that the lower the salinity the more sensitive the organism is to fluoride. Camargo (2003) found that the mortality of rainbow trout exposed to a maximum concentration of 25 mg F/l decreased with an increase in chloride ions (0 – 9 mg Cl/l). He speculated that 20 the increase in chloride ions may facilitate fluoride excretion from the organism. A similar response was observed in the net-spinning caddisfly (Camargo, 2003). Pankhurst et al. (1980) conducted an experiment on the effect of fluoride effluent on marine organisms. Their study indicated that the effluent was affecting the sessile organisms that encrust the substrate for up to 400m from the point of effluent release. Their measurement of effluent dispersal showed that mixing was rapid and near background levels were recorded at 5m from the outfall. The outfall is located in a high tidal area, but they do also indicate that the effluent rapidly reacts or precipitates on entry to the sea. Nevertheless, concentrations of 1.00 to 1.90 mg/L compared to 0.90 mg/L background, indicated an increase that still modified the encrusting community, which included anemones, ascidians and sponges. Survey of Water Quality Criteria Water quality criteria for fluoride in various countries and provinces are shown in Table 4. There are slightly different protection objectives, with Canada pursuing a conservative approach, with the aim of protecting all species, while Australia aims to maintain aquatic ecosystems, without specifying a percentage of species the quality objective aims to protect. Table 4. Water Quality Criteria for Protection of Aquatic Life Country/Province mg/L Conditions Canada 0.12 Interim British Columbia 0.2 0.3 1.5 waters <50mg/L CaCO3 waters >50mg/L CaCO3 estuarine or marine Great Britain 1.5 1.8 95th percentile, salmonid or cyprinid fish 98th percentile, salmonid or cyprinid fish Australia 1.5 Threshold levels for marine and estuarine waters for maintenance of aquatic ecosystems. 2.0 6-month median marine and estuarine 10 Single sample limit, marine and estuarine. 21 Values taken from Ambient Water Quality Criteria for Fluoride, British Columbia Environmental Protection Division. (Government of British Columbia, 1990) The Government of British Columbia’s Ambient Water Quality Criteria for Fluoride provided a rationale to their freshwater quality criteria. Their most sensitive LC50 species was rainbow or brown trout fingerlings, which had a LC50 of 4.8 +/- 2.5 mg F/L in water hardness of 44 mg/L CaCO3 (Angelovic et al., 1961). The freshwater environments typical of coastal British Columbian streams have a lower CaCO3 (10 mg/L) and therefore only 2.0 mg F/L would be required for a similar toxicity (Pimental and Bulkley, 1983). The water temperature in the Angelovic et al. (1961) experiment was 18.0oC, however the temperature likely to be encountered in British Columbian waters is 12oC. This would increase the LC50 value by a factor of 2 to 4.0 mg F/L. To determine a chronic exposure level, a factor of 0.05 was applied to the adjusted LC50 value (corresponding to an assessment factor of 20), giving the criteria value of 0.2 mg F/L. Application of a factor of 0.01 was also considered. However a criteria value of 0.04 mg F/L is unrealistic given it is lower than natural background levels (Government of British Columbia, 1990). Results of Laboratory Exposure Trials Results are presented from toxicity tests with effluent and corresponding test solutions containing only fluoride (NaF). A major aim was to determine whether fluoride can explain the toxicity of the complete wastewater, or whether additional (unknown) components also contribute. The identification of responsible toxicant(s) is/are important if reduction of effluent toxicity is to be accomplished. Comparative Toxicity of Effluent Solution and the Equivalent Fluoride Concentration Three different effluent concentrations (100%, 50% and 25%) were tested for acute toxicity to Nitocra as well as their equivalent fluoride concentrations (27mg F/L 13.5mg F/L and 6.75mg F/L) to determine if there were any statistically significant differences in their toxicity. Using a one way-ANOVA, it was demonstrated that at effluent concentrations of 100% and 50% there was a significant difference (P<0.05) between the means of the effluent solution, the equivalent fluoride solution and the control. At 25% effluent 22 concentration (6.75mg F/L), no significance was found between the different exposure media. Using the Tukey Multiple Comparison Test, at effluent concentrations of 100% and 50%, there was significant difference (P<0.05) between the effluent solution and both the fluoride solution and control. There was no significance between the fluoride solution and the control. It must be noted that the data obtained for the 100% effluent comparison was gathered from an exposure held concurrently with each exposure medium having twelve replicates. The data points gathered for the 50% and 25% comparisons are accumulated from different exposure trials. 100 % mortality 80 60 40 20 l tr o co n e or id Fl u Ef flu en t 10 0% 0 Exposure medium Figure 7. Comparison of mortality distribution of N. snipines at 100% effluent, 27 mg/L fluoride and control at 96 hours exposure. 23 100 % mortality 80 60 40 20 tr ol e rid co n Ef fl u en Fl uo t5 0% 0 Exposure medium Figure 8. Comparison of mortality distribution of N. spinipes at 50% effluent, 13.5mg/L fluoride and control at 96 hours exposure. 100 % mortality 80 60 40 20 ol nt r co Fl uo rid e Ef flu en t 25 % 0 Exposure medium Figure 9. Comparison of mortality distribution of N. spinipes at 25% effluent, 6.75 mg/L fluoride and control at 96 hours exposure. 24 100 % Mortality 80 60 Fluoride Effluent 40 20 0 0.1 1 10 100 1000 Concentration (mg F/L) Figure 10. Comparison of mortality distribution of N. spinipes between effluent and fluoride concentrations at 96 hours exposure. Figure 10 depicts the slope of the mortality rates of Nitocra for the two different exposure media using a Weibull concentration response analysis plot. Using a Deming (model II) linear regression, there was no significant difference (P=0.2388) between the slopes. However, the toxicity of the effluent was higher than for corresponding fluoride solutions. This suggests a higher bioavailability of fluoride in the effluent or additional toxic components in the effluent. LC50 of the Effluent Solution An assessment of the LC50 concentration of the effluent was undertaken using the results collated from both exposures undertaken in all salinities as well as in salinity of 8ppt. The LC50 values were calculated using the Trimmed Spearman-Karber method. The Probit method and the Moving Average method were deemed unsuitable for use due to the low number of data available that caused more than 50% mortality. Table 5. LC50 of N. spinipes exposed to Effluent Medium LC50 95% Lower 95% Upper % Trim Confidence Confidence 8ppt Salinity 25.38 22.67 28.41 44.61 All Salinities 22.71 16.14 31.94 39.28 25 The results in Table 5 indicate no major effect of salinity on the toxicity of the effluent. LC50 of the Fluoride Solution As per the effluent solution assessment, an LC50 was calculated for exposures undertaken only in 8ppt salinity as well as data using all salinity. To maintain consistency, the LC50 for the fluoride solutions were also determined using the Trimmed Spearman-Karber method. Table 6. LC50 of N. spinipes exposed to Fluoride solution Medium LC50 95% Lower 95% Upper Confidence Confidence % Trim 8ppt Salinity 278.29 248.17 312.06 0.0 All Salinities 259.28 234.04 286.02 0.0 The results in Table 6 indicate no major effect of salinity on the toxicity of the fluoride. Statistically Observed No Effect Concentrations Data were analysed only from those exposures undertaken at salinities of 8 ppt. Using an ANOVA, it was determined that there was a significant difference in mortality between increasing concentrations in both the effluent solution and the fluoride solution. A Dunnett’s Multiple Comparison Test was used to determine at which concentration there was a significant difference from the controls. The highest non significant and lowest significant concentrations are tabled below. Table 7. No Observed Effect Concentrations from tests with N. spinipes in effluent and fluoride. Exposure Medium No Observed Effect Concentration Lowest Observed Effect Concentration Effluent Solution 6.75mg F/L 13.5mg F/L Fluoride Solution 216mg F/L 337mg F/L 26 Influence of Salinity on Fluoride Toxicity When comparing the toxicity of fluoride in 1 ppt, 8 ppt and 17 ppt salinities, a highly significant interaction (P= 0.0046) was found between the change in salinity and the mortality of Nitocra at increasing fluoride concentrations. However, when a comparison was made for exposures only in 8 ppt and 17 ppt salinity, no significant (P= 0.3483) interaction between salinity and fluoride was found. % mortality 30 20 10 0 1 8 17 Salinity ppt Figure 11. Comparison of mean mortality distribution of N. spinipes at different salinities at 96 hours exposure. When comparing the mean of mortality (when pooling all comparable fluoride concentration exposures) for 1 ppt (14%), 8 ppt (9.3%) and 17 ppt (10.1%), the mortality rate in 1ppt salinity was 50% higher than in 8ppt and 17ppt salinity. 27 Effluent 100 8ppt 17ppt % mortality 80 60 40 20 0 0 10 20 30 Fluoride Concentration (mg/l) Figure 12. Comparison of mortality distribution of N. spinipes at different salinities over increasing effluent concentrations at 96 hours exposure. When comparing the toxicity of the effluent solution in 8 ppt and 17 ppt salinities, no significant interaction (P= 0.2682) between salinity and effluent solution contributing to the mortality of Nitocra was found. The increase in effluent solution concentration was found to be the only significant (P= 0.0004) contributor to Nitocra mortality. Influence of pH, Temperature and Water Hardness The effect that pH, temperature and water hardness have on the toxicity of fluoride was not determined in the Nitocra exposure trials. An effort was made to maintain these factors at a constant rate to enable more reliable comparison between the effluent and fluoride solutions. Although during the Nitocra exposures, the effect of pH was not tested, an adjustment of the pH to a range between 7 and 8 was undertaken. It was noted that in pilot trials, where the effluent solution was not lowered from pH10, there was near 100% mortality within the first 24 hours, for concentrations as low as 25% effluent solution (6.75 mgF/l). These mortality rates were not observed once the pH had been lowered in the effluent solution. Water hardness was not measured, but it was observed that precipitates did form in pilot fluoride stock solutions of 27 mg/L at 8 ppt and 17 ppt, and 28 noticeably during the trials within the test cells of fluoride solution at concentrations above 252 mg/L. Summary and derivation of a preliminary PNEC for fluoride The Nitocra exposure trials indicate that there is a difference in toxicity between the effluent solution and a solution of equivalent fluoride. The LC50 of the effluent of 22.71 mg F/L was 11 times less than that of the fluoride LC50 of 259.28mg F/L. The acute NOEC for the effluent solution of 6.75mg F/L was 32 times less than for the fluoride solution 216 mg F/L. The exposure trials also indicated that there is roughly a 50% increase in mortality for fluoride exposures in freshwater (1 ppt) than estuarine (8 ppt, 17 ppt) waters. The lowest LC50 values retrieved in the literature review were in a range of 2.3-7.3 mg F/L, recorded for the rainbow trout Oncorhynchus mykiss. This study is the same as used for British Columbia’s rationale for their freshwater criteria. The 95th percentile is slightly higher (16 mg F/L). Invertebrates show a slightly lower sensitivity after short-term exposure to fluoride (95th percentile is 26 mg/L). A similar relation emerges for the data on chronic exposure. Using the 95th percentile of the LC50 values for chronic exposure yields a value of 2.62 mg F/L for fish and 12.34 mg F/L for invertebrates. Data on sub-lethal effects (EC50) are comparatively sparse. Even for acute exposures, the 95th percentile could only be calculated for invertebrates (19.2 mg F/L). For neither invertebrates nor fish were sufficient data found on sub-lethal effects after long-term exposure. However, assuming that a prolongation of the exposure lowers the invertebrate EC50’s similar as the LC50’s (which were lowered by a factor of 2.1, from 26 mg F/L to 12.34 mg F/L), a 95th percentile of roughly 8 mg F/L would be estimated for chronic exposure of invertebrates. It should, however, be noted that only a limited amount of data was available for the calculation of the SSD for chronic LC50’s. The factor of 2.1 can hence only be regarded as a rough approximation. 29 The lowest EC concentration for an algal species was 2 mg/L, which inhibited growth by 37%. The lowest EC50 for an algal species was 82 mg/L. Both values indicate a generally lower sensitivity of this group of organisms. Additional data were found in literature that should be considered for an assessment of the toxicity of fluoride. Evidence in Labeo rohita suggests that there are non-lethal impacts at concentrations of 15 mg F/L when exposed for 30 days, involving changes in gill filaments, intestinal villi and renal cell architecture. At concentrations of 0.5 mg/L, which is clearly lower than the lowest LC50, the disruption of salmonid migration was observed. Pankhurst et al. (1980) indicate the possibility that similarly low fluoride concentrations (around 1 mg/L above background levels) might disrupt recolonisation by juvenile marine encrusting species. It may be worthwhile to investigate whether any sponge or anemone species inhabit the Volvo discharge area. PNEC derivation The 2.62 mg F/L value (95th percentile, long-term (> 4 days) LC50, fish) could be adopted as the starting point for developing a quality criterion for fluoride. This value is similar to the acute NOEC for Nitocra, but this value already considers the differences in species sensitivity (within fish, but also in comparison to algae and invertebrates) and is for chronic exposures. Extrapolations are needed from lethality to sub-lethal endpoints, from high effects (50%) to low (no) effect and from laboratory to field situations (i.e. considering ecological effects). The REACH guidance starts with a default assessment factor of 1 000 for assessments in the limnic aquatic environment, if the so-called “base set” of ecotoxicological data (one EC/LC50 of each, algae, daphnids and fish) is at hand. This factor covers “intra- and inter-laboratory variation of toxicity data; intra- and inter-species variations (biological variance); short-term to long-term toxicity extrapolation; laboratory data to field impact extrapolation.” (REACH guidance document, chapter R.10). In the following it is assumed that the overall factor of 1 000 can be broken down into 4 equally sized portions of 5.6, i.e. 1) a factor of 5.6 for the laboratory variations in the data, 2) a factor of 5.6 for the species variations, 3) a factor of 5.6 for the short-term to long-term extrapolation. This factor actually contains two elements: (3a) the extrapolation across the time 30 scale (i.e. the effect of prolonged exposures) and (3b) the extrapolation from mortality to sub-lethal endpoints. 4) a factor of 5.6 for the laboratory to field impact extrapolation. The calculation of 95th percentiles inherently considers possible variations in the ecotoxicological data (i.e. factors 1 and 2). However, factors 3 and 4 need to be accounted for. Using the factors above, this yields a total assessment factor of 31 which should then be applied to the initial value of 2.62 mg F/L, resulting in a preliminary PNEC of 0.08 mg F/L. This value is actually quite consistent with the water quality criterion of 0.04 mg F/L that was initially determined by the Government of British Columbia (1990). It might also be equally overprotective, as the value does not consider (a) local conditions, especially water hardness and (b) the presence of natural background levels of fluoride, which renders local populations more tolerant than the laboratory populations that were used for determining the values that made up the SSD. Additionally, the initial value of 2.62 mg F/L is based on long-term mortality data, i.e. it already includes part of the short-term to long-term extrapolation (Factor 3a is accounted for). Therefore, using the information gained from the literature review and from the Nitocra exposure trials, the following factors should be applied to this value: • multiply by a factor of 1.5 to account for an increase in salinity • divide by a factor of 3.36 to account for the change in LC50 to NOEC as observed in the Nitocra exposure These factors take into account some of the local factors, such as salinity, but have not included the benefits of lower temperatures. Hence a total assessment factor of 2.25 seems justifiable, leading to a PNEC of 1.16 mg F/L for the estuarine aquatic environment. This value is quite consistent with the water quality targets for estuarine environments outlined in table 4. The laboratory exposure trial indicates that there are other factors contributing to the toxicity of the process water other than fluoride. A TIE (Toxicity Identification Evaluation) approach is recommended for further studies to determine what other constituents of the wastewater are contributing to the acute toxicity beside fluoride 31 The literature indicates that in freshwater environments, Oncorhynchus mykiss (rainbow trout) and Musculium transversum (fingernail clam) seem to be the most sensitive fish and invertebrate species respectively. It is also evident that juvenile life stages of nearly all species are the individuals that are most susceptible to an elevation of fluoride. The juvenile Nitocra indicated a much higher tolerance (10 times) to acute exposure to fluoride than the lowest 95th percentile most sensitive invertebrate species. This study constitutes a first step in identifying risk to those organisms inhabiting the vicinity of any fluoride discharge. It is recommended that any future species sensitivity studies have some focus on the response of invertebrates to chronic exposure, with sub-lethal responses a key endpoint. The most sensitive species identified in the literature, Oncorhynchus mykiss, needs to be represented in any future study, with the current PNEC determined largely from the response of the juvenile stage of this fish. It is recommended that a long-term exposure study is undertaken using juvenile Oncorhynchus mykiss or a local salmonid, with water quality parameters specific to the local area considered to determine whether a maximum concentration of 1.16 mg F/L can be used as an ecological quality target concentration for the total fluoride in the waterway after dilution from the discharge point. 32 References Angelovic, J.W., Sigler, W.F., Neuhold, J.M. (1961) Temperature and fluorosis in rainbow trout. J. Water Pollut. Control Fed. (April), 371–381. Antia, N.J., Klut, M.E. (1981) Fluoride addition effects on euryhaline phytoplankter growth in nutrient-enriched seawater at an estuarine level of salinity. Botanica Marina 24, 147–152. Bhatnagar, C.A., Bhatnagar, M.B., Regarc, B.C. (2007) Fluoride induced histopathological changes in gill, kidney, and intestine of freshwater teleost, Labeo rohita. Fluoride. 40 (1):55-61 Barbier, O., Arreola-Mendoza, L., Del Razo, L.M. (2010) Molecular mechanisms of fluoride toxicity. Chemico-Biological Interactions 188:319-333 Bringman G., and Kuhn, R. (1959) Comparative Water-Toxicological Investigations on Bacteria, Algae, and Daphnia. Gesund.-Ing. 80(4):115-120 Bringman G., and Kuhn, R. (1959) Water Toxicological Studies with Protozoa as Test Organisms. TR-80-0058, Literature Research Company :13 p. (GER) (ENG TRANSL) Gesund.Ing.80:239-242 Camargo, J.A. (1991a) Ecotoxicological analysis of the influence of an industrial effluent on fish populations in a regulated stream. Aquacult. Fish. Manag. 22, 509–518. Camargo, J.A.(1991b) Ecotoxicological study of the influence of an industrial effluent on a net-spinning caddisfly assemblage in a regulated river. Water Air Soil Pollut. 60, 263–277. Camargo, J.A. (2003) Fluoride toxicity to aquatic organisms: a review. Chemosphere 50: 251-264 Camargo, J.A., Tarazona, J.V. (1990) Acute toxicity to freshwater macroinvertebrates of fluoride ion (F_) in soft water. Bull. Environ. Contam. Toxicol. 45, 883–887. Camargo, J.A., Tarazona, J.V. (1991) Short-term toxicity of fluoride ion (F_) in soft water to rainbow trout and brown trout. Chemosphere 22, 605–611. Camargo, J.A., Ward, J.V., Martin, K.L. (1992a) The relative sensitivity of competing hydropsychid species to fluoride toxicity in the Cache la Poudre River (Colorado). Arch. Environ. Contam. Toxicol. 22, 107–113. Camargo, J.A., Garc_ıa de Jal_on, D., Mu~noz, M.J., Tarazona, J.V. (1992b) Sublethal effects of sodium fluoride (NaF) on net-spinning caddisflies (Trichoptera). Aquatic Insects 14, 23–30. 33 Camargo, J.A., La Point, T.W. (1995) Fluoride toxicity to aquatic life: a proposal of safe concentrations for five species of Palearctic freshwater invertebrates. Arch. Environ. Contam. Toxicol. 29, 159–163. Canadian Council of Ministers of the Environment (2002) Canadian Water Quality Guidelines for the Protection of Aquatic Life :Inorganic Fluoride. In: Canadian environmental quality guidelines, 1999, Canadian Council of Ministers of the Environment, Winnipeg. Cardwell, R.D., C.E. Woelke, C.E., Carr, M.I, and Sanborn, E.W. (1979) Toxic Substance and Water Quality Effects on Larval Marine Organisms. Tech.Rep.No.45, State of Washington, Dep.of Fish, Olympia, WA :71 p. Connell, A.D., and Airey, D.D. (1979) The Chronic Effects of Fluoride on the Estuarine Amphipods Grandidierella lutosa and G. lignorum. Water Res.16(8):1313-1317 (1982) / S.Afr.J.Sci. 75(12):566 (ABS) Damkaer, D.M., Dey, D.B. (1989) Evidence for fluoride effects on salmon passage at John Day dam, Columbia river, 1982– 1986. N. Am. J. Fish. Manag. 9, 154–162. Dave, G. (1984) Effects of fluoride on growth, reproduction and survival in Daphnia magna. Comp. Biochem. Physiol. 78C, 425–431. Dave, G., Björnestad, E., Efraimsen, H. and Tarkpea, M. (1993) Precision of the Nitocra spinipes Acute Toxicity Test and the Effect of Salinity on Toxicity of the Reference Toxicant Potassium Bichromate. Environ. Toxicol. and Water Qual.: An International Journal. 8, 271-277. Ellis, M.M. (1937) Detection and Measurement of Stream Pollution. In: Bull.Bur.Fish.No.22, U.S.Dep.Commerce, Washington, DC :365-437 European Chemicals Agency (2008) Guidance on information requirements and chemical safety assessment: Chapter R.10: Characterisation of dose [concentration]-response for environment. Guidance for the implementation of REACH. European Commission. Fieser, A.H., Sykora, J.L., Kostalos, M.S., Wu, Y.C., Weyel, D.W. (1986) Effect of fluorides on survival and reproduction of Daphnia magna. J. Water Pollut. Control Fed. 58, 82–86. Giguere, A., Campbell, P.G.C. (2004) Fluoride toxicity towards freshwater organisms and hardness effects - Review and reanalysis of existing data [Toxicité de l'ion fluorure envers les organismes d'eau douce et effets de la dureté - Revue et nouvelle analyse de données existantes]. Revue des Sciences de l’Eau. Volume 17 (3):373-393 Government of British Columbia, Ministry of Environment: Environmental Protection Division (1990) Ambient Water Quality Criteria for Fluoride: Overview report. Government of British Columbia, Victoria, British Columbia, Canada V8W 9V1 Groth, E. (1975) An Evaluation of the Potential for Ecological Damage by Chronic Low-Level Environmental Pollution by Fluoride. Fluoride 8(4): 224-240 34 Harry, H.W., and Aldrich, D.W. (1963) The Distress Syndrome in Taphius glabratus (Say) as a Reaction to Toxic Concentrations of Inorganic Ions. Malacologia 1(2): 283-289 Hazardous Substances Data Bank (HSDB). National Library of Medicine, National Toxicology Information Program, Bethesda, MD. http://toxnet.nlm.nih.gov/cgi-bin/sis/search. Heitmuller, P.T., Hollister, T.A., Parrish, P.R. (1981) Acute toxicity of 54 industrial chemicals to sheepshead minnows (Cyprinodon variegatus). Bull. Environ. Contam. Toxicol. 27, 596–604. Hekman, W.E., Budd, K., Palmer, G.R., MacArthur, J.D. (1984) Responses of certain freshwater planktonic algae to fluoride. J. Phycol. 20, 243–249. Hemens, J., Warwick, R.J. (1972) The effects of fluoride on estuarine organisms. Water Res. 6, 1301–1308. Hemens, J., Warwick, R.J., Oliff, W.D. (1975) Effect of extended exposure to low fluoride concentration on estuarine fish and crustacea. Prog. Water Technol. 7, 579–585. Herbert, D. W.M., and Shurben, D.S. (1964) The Toxicity of Fluoride to Rainbow Trout. Water Waster Treat. Journ. 10:141-142 Holland, G.A., Lasater, J.E., Neumann, E.D., and Eldridge, W.E. (1960) Toxic Effects of Organic and Inorganic Pollutants on Young Salmon and Trout. Res.Bull.No.5, State of Washington Dept.Fish., Seattle, WA :263 p. Hickey, C.W. (1989) Sensitivity of Four New Zealand Cladoceran Species and Daphnia magna to Aquatic Toxicants. N.Z.J.Mar.Freshwater Res. 23(1):131-137 Joy, C.M., Balakrishnan, K.P. (1990) Effect of fluoride on axenic cultures of diatoms. Water Air Soil Pollut. 49, 241–249. Keller, A.E. (2000) As Per Aquire Database: Personal Communication to U.S. EPA: Water Quality and Toxicity Data for Unpublished Unionid Mussel Tests. Memo to R.Pepin and C.Roberts,U.S.EPA Region 5,Chicago, IL :14 p. (Author Communication Used). Keller, A.E., Augspurger, T. (2005) Toxicity of Fluoride to the Endangered Unionid Mussel, Alasmidonta raveneliana, and Surrogate Species. Bull. Environ. Contam. Toxicol. 74:242-249 Klein, L. (1958) Aspects of River Pollution. Butterworth Scientific Publications, London. Academic Press, Inc. N.Y. Kobayashi, N. (1971) Fertilized Sea Urchin Eggs as an Indicatory Material for Marine Pollution Bioassay, Preliminary Experiments. Mar. Bio. Lab 18(6): 379-406 Kuhn, R., Pattard, M., Pernak, K.-D., Winter, A. (1989) Results of the harmful effects of water pollutants to Daphnia magna in the 21 day reproduction test. Water Res. 23, 501–510. 35 LeBlanc, G.A. (1980) Acute toxicity of priority pollutants to water flea (Daphnia magna). Bull. Environ. Contam. Toxicol. 24, 684–691. LeBlanc, G.A. (1984) Interspecies relationships in acute toxicity of chemicals to aquatic organisms. Environ. Toxicol. Chem. 3, 47–60. McClurg, T.P. (1984) Effects of fluoride, cadmium and mercury on the estuarine prawn Penaeus indicus. Water SA 10, 40–45. McNeeley, R. N., Neimanis, V. P., and Dwyer, L. (1979) Water Quality Source Book. A Guide to Water Quality Parameters. Inland Waters Directorate, Water Quality Branch, Environment Canada. MacPhee, C., and Ruelle, R. (1969) Lethal Effects of 1888 Chemicals upon Four Species of Fish from Western North America. Bull. No. 3, Forest, Wildl. And Range. Stn, Univ. of Idaho, Moscow, ID:112p. Mendoza-Schulz, A., Solano-Agama, C., Arreola-Mendoza, L., Reyes-M.rquez, B., Barbier, O., Del Razo, L.M., Mendoza-Garrido, M.E. (2009) The effects of fluoride on cell migration, cell proliferation, and cell metabolism in GH4C1 pituitary tumour cells, Toxicol. Lett. 190: 179–186. Moore, D.J. (1971) The uptake and concentration of fluoride by the blue crab, Callinectes sapidus. Chesapeake Sci. 12, 1–13. Nell, J.A., Livanos, G. (1988) Effects of fluoride concentration in seawater on growth and fluoride accumulation by Sydney rock oyster (Saccostrea commercialis) and flat oyster (Ostrea angasi) spat. Water Res. 22, 749–753. Newman, M.C., Ownby, D.R., Mezin, L.C., Powell, D.C., Christensen, T.R., Lerberg, S.B., and Anderson, B.A. (2000) Applying species sensitivity distributions in ecological risk assessment: assumptions of distribution type and sufficient number of species. Environ. Toxicol. Chem. 19, 508-515 Neuhold, J.M., Sigler, W.F. (1960) Effects of sodium fluoride on carp and rainbow trout. Trans. Am. Fish. Soc. 89, 358–370. Neuhold, J.M., Sigler, W.F. (1961) Chlorides affect the toxicity of fluoride in rainbow trout. Science 135, 732–733. Oliveira, L., Antia, N.J., Bisalputra, T. (1978) Culture studies on the effects from fluoride pollution on the growth of marine phytoplankters. J. Fish Res. Board Can. 35, 1500–1504. Oota, Y. (1971) Disappearance of Rhythmicity in Growth Response to Dark- and Light-Breaks in Lemna gibba G3 due to Iron Deficiency. Plant Cell Physiol. 12:255-266 36 Pankhurst, N.W., Boyden, C.R. and Wilson, J.B. (1980) The effect of a fluoride effluent on marine organisms. Environmental Pollution (series A) 23:299-312. Pillai, K.S., Mane, U.H. (1984) The effect of fluoride on fertilized eggs of a freshwater fish, Catla catla (Hamilton). Toxicol. Lett. 22, 139–144. Pillai, K.S., Mane, U.H. (1985) Effect of fluoride effluent on fry of Catla catla (Hamilton). Fluoride 18, 104–110. Pimentel, R., Bulkley, R.V. (1983) Influence of water hardness on fluoride toxicity to rainbow trout. Environ. Toxicol. Chem. 2, 381–386. Portman, J.E., and Wilson, K.W. (1971) The Toxicity of 140 Substances to the Brown Shrimp and Other Marine Animals. Shellfish Information Leaflet No.22 (2nd Ed.), Ministry of Agric.Fish.Food, Fish.Lab.Burnham-on-Crouch, Essex, and Fish Exp.Station Conway, North Wales :12 p. Posthuma, L., Suter II, G.W., and Traas, T.P. (2002) Species Sensitivity Distributions in Ecotoxicology .Chapter 1. Lewis Publishers, Florida, USA. Rai, L.C., Husaini, Y., Mallick, N. (1998) pH-altered interaction of aluminium and fluoride on nutrient uptake, photosynthesis and other variables of Chlorella vulgaris. Aquatic Toxicol. 42, 67–84. Saxena, R.,Gupta, R., Tripathi, M., and Gopal, K. (2001) Fluoride Induced Haematological Alterations in the Freshwater Fish Channa punctatus. J.Ecophysiol.Occup.Health 1(1/2):139-146 Shi, X., Zhuang, P., Zhang, L., Feng, G., Chen, L., Liu, J., Qu, L., Wang, R. (2009) The Bioaccumulation of fluoride ion (F-) in siberian sturgeon (Acipenser baerii) under laboratory conditions. Chemosphere 75: 376-380 Smith, A.O., Woodson, B.R., (1965) The effects of fluoride on the growth of Chlorella pyrenoidosa. Virginia J. Sci. 16, 1–8. Smith, L.R., Holsen, T.M., Ibay, N.C., Block, R.M., Leon, A.B. (1985) Studies on the acute toxicity of fluoride ion to stickleback, fathead minnow and rainbow trout. Chemosphere 14, 1383–1389. Sparks, R.E., Sandusky, M.J., Paparo, A.A. (1983) Identification of the water quality factors which prevent fingernail clams from recolonizing the Illinois River phase III. Water Resource Centre, University of Illinois at Urbana-Champaign, Urbana, IL. United States Environmental Protection Agency (http://www.epa.gov/eerd/stat2.htm) Vallin, S. (1968) Giftverkan av Fluor pa Fisk (The Toxicity of Fluoride to Fish). Vatten 24(1): 51-52 37 Wallen, I.E., Greer, W.C., and Lasater, R. (1957). Toxicity to Gambusia affinis of Certain Pure Chemicals in Turbid Waters. Sewage Ind.Wastes 29(6):695-711 Woodwiss, F.S., and Fretewell, G. (1974) The Toxicity of Sewerage Effluents, Industrial Discharge and Some Chemical Substances to Brown Trout (Salmo trutta), in the Trent River Authority Area. Wat. Poll. Control (G.B.) 73: 396 Wright, D.A. (1977) Toxicity of fluoride to brown trout fry (Salmo trutta). Environ. Pollut. 12, 57–62. 38