review of fluoride toxicity to aquatic organisms and its toxicity … · 2011-11-30 · information...
TRANSCRIPT
STEVEN FLEISS
Degree project for Master of Science in Ecotoxicology 30 ECTS Department of Plant and Environmental Sciences University of Gothenburg June 2011
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Review of fluoride toxicity to aquatic organisms and its toxicity contribution in Volvo wastewater
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.
2
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
3
Lists of Figures and Tables
Figure 1. Invertebrate Acute LC50 SSD 12
Figure 2. Invertebrate Chronic LC50 SSD 13
Figure 3. Fish Acute LC50 SSD 14
Figure 4. Fish Chronic LC50 SSD 14
Figure 5. Fish Median Acute LC50 SSD 15
Figure 6. Invertebrate EC50 SSD 16
Figure 7. Nitocra 100% Effluent Comparisons 22
Figure 8. Nitocra 50% Effluent Comparisons 22
Figure 9. Nitocra 25% Effluent Comparisons 23
Figure 10. Nitocra Mortality Distributions 23
Figure 11. Salinity Effects on Fluoride 25
Figure 12. Salinity Effects on Effluent 26
Table 1. Literature search profile 6
Table 2. Fluoride compounds and complexes of environmental concern 8
Table 3. 95th Percentile and Sub-lethal Effects Summary 17
Table 4. Water Quality Criteria for Protection of Aquatic Life 20
Table 5. LC50 of N. spinipes exposed to Effluent 24
Table 6. LC50 of N. spinipes exposed to Fluoride solution 24
Table 7. No Observed Effect Concentrations from tests with N. spinipes in 25
effluent and fluoride.
Appendix 1. Combined database (separate Excel File)
Appendix 2. Nitocra database (separate Excel File)
Acronyms and Abbreviations
ATP Adenosine triphosphate
EC50 Median effective concentration, concentration that causes 50%
effect
LC50 Median lethal concentration, concentration that causes 50%
mortality
4
LOEC Lowest observed effect concentration
NOEC No observed effect concentration
SSD Species Sensitivity Distribution
PNEC Predicted No Effect Concentration
ppt Parts per thousand
5
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 peer-
reviewed 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.
6
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
Compound
CAS
Number
Fluoride
%
Molecular
Mass
Solubility
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
Very soluble
Sodium
Silicofluoride
(F6Na2Si)
16893-85-9 60.6 188.1
0.64-0.76g/100ml
@ 20-25oc
Aluminium
Fluoirde (AlF3) 7784-18-1 67.8 83.97
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.
7
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).
8
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
9
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
10
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
11
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
12
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.
13
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.
1 10 100 1000 100000
10
20
30
40
50
60
Caddisfly
Other Invertebrates
95th Percentile
N. sinipes
Fluoride Concentration (mg/L)
Sp
ec
ies
Ra
nk
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.
14
10 100 10000
2
4
6
8
10
12
Caddisfly
Mollusc
95th Percentile
Fluoride Concentration (mg/L)
Sp
ec
ies
Ra
nk
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.
15
1 10 100 10000
10
20
30
40
Oncorhynchus mykiss
Other Fish
95th Percentile
Fluoride Concentration (mg/L)
Sp
ec
ies
Ra
nk
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.
1 10 100 10000
10
20
30Oncorhynchus mykiss
Other Fish
95th Percentile
Fluoride Concentration (mg/L)
Sp
ec
ies
Ra
nk
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
16
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.
100 10000
2
4
6
8
Oncorhynchus mykiss
Pimephales promelas
Salmo trutta
Channa punctata
Gasterosteus aculeatus
Cyprinodon variegatus
Gambusia affinis
Fluoride Concentration (mg/L)
Sp
ec
ies
Ra
nk
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.
17
10 100 10000
10
20
30
40
50
Caddisfly
Other Invertebrates
95th Percentile
Fluoride Concentration (mg/L)
Sp
ec
ies
Ra
nk
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
18
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.
19
Table 3. Summary of SSDs (lower 95th Percentiles) and Sub-lethal Effects of
Fluoride
Species Group Exposure Endpoint Concentration
Chronic 95th % LC50 12.34 mg/L
Acute 95th % LC50 26.08 mg/L
Invertebrate
Acute 95th % EC50 19.2 mg/L
Chronic 95th % LC50 2.62 mg/L Fish
Acute 95th % LC50 15.98 mg/L
Salmon species Chronic Significant
disruption of
migration
0.5 mg/L
Algae Chronic* Lowest EC50 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).
20
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
21
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
2.0
10
Threshold levels for marine and
estuarine waters for maintenance of
aquatic ecosystems.
6-month median marine and estuarine
Single sample limit, marine and
estuarine.
22
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
23
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.
Effluen
t 100
%
Fluori
de
contr
ol0
20
40
60
80
100
Exposure medium
% m
ort
alit
y
Figure 7. Comparison of mortality distribution of N. snipines at 100%
effluent, 27 mg/L fluoride and control at 96 hours exposure.
24
Effluen
t 50%
Fluorid
e
contr
ol0
20
40
60
80
100
Exposure medium
% m
ort
alit
y
Figure 8. Comparison of mortality distribution of N. spinipes at 50%
effluent, 13.5mg/L fluoride and control at 96 hours exposure.
Effluen
t 25%
Fluorid
e
contr
ol
0
20
40
60
80
100
Exposure medium
% m
ort
ali
ty
Figure 9. Comparison of mortality distribution of N. spinipes at 25%
effluent, 6.75 mg/L fluoride and control at 96 hours exposure.
25
0.1 1 10 100 10000
20
40
60
80
100
Fluoride
Effluent
Concentration (mg F/L)
% M
ort
alit
y
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
Confidence
95% Upper
Confidence
% Trim
8ppt Salinity 25.38 22.67 28.41 44.61
All Salinities 22.71 16.14 31.94 39.28
26
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
Confidence
95% Upper
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
27
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.
1 8 170
10
20
30
Salinity ppt
% m
ort
ality
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.
28
0 10 20 300
20
40
60
80
1008ppt
17ppt
Fluoride Concentration (mg/l)
% m
ort
alit
y
Effluent
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
29
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.
30
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
31
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
32
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.
33
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