June 19th 2014
Aquatic ecotoxicity of surfactants:
identifiying the unknown hazards of widely used compounds.
R.B. Mulders 12 EC
10167501 Bachelor thesis
Supervisor: Jort Hammer Examiner: Michiel Kraak IBED
Abstract
Surfactants are widely used and produced compounds. All Surfactants share a similar basic design, consisting of a hydrophobic tail and a hydrophilic head, giving them their surface active properties. Although surfactants are applied and produced in high volumes, little is known about the aquatic ecotoxicity of these compounds, primarily because this is hard to test. The most commonly used technique for estimating the hazard of an organic compound by calculating the octanol-water partitioning coefficient, but because of the surface active properties of surfactants they tend to form a third layer at the interface between water and octanol. Hence a better method to estimate the hydrophobicity of surfactants and link this to yet missing toxicity data is needed. This project focused on the latter by providing these missing toxicity data. Since preliminary research indicated that polar head group type and hydrophobic carbon chain length may influence toxicity, the aim of this research was to determine the relation between the molecular structure and the aquatic ecotoxicity of surfactants. To this purpose Daphnia magna acute immobilization tests were executed with various surfactants. In addition a new method for measuring the bioavailability of surfactants was tested, using a solid phase micro extraction (SPME) fiber to resemble tissue uptake. Thirteen out of the sixteen surfactants selected turned out to be insoluble below their lethal dose.
concentration response relationships were obtained for C11COOH, C8FCOOH and C11SO4
from which EC50 values were derived respectively. Since C11COOH was more than 18
times more toxic than C11SO4 the hydrophilic head group did influence toxicity. Based on
comparisons with other studies, carbon chain length is likely to influence toxicity as well. The fibers were tested with both C11SO4 and C8FCOOH and for C8FCOOH a fiber
concentration response relationship could be obtained. In conclusion, the molecular structure of a surfactant does influence toxicity and the SPME fiber has proven to be a viable method to eventually substitute Daphnids in aquatic ecotoxicity research. Introduction
Surfactants are commonly applied in a wide variety of domestical and industrial products including paints, textiles, cleaning detergents, personal care products, pharmaceuticals, pesticides and polymers (Hodges, et al., 2006). They are also used in industrial
processes for mining, oil recovery and in the pulp and paper industry (Ying, 2006). The wide variety of applications for surfactants is based on their surface active properties. These properties give surfactants the ability to act as water repellent, or to enclose fat particles in micelles enabling them to dissolve in water. Surfactants share a similar basic design (figure 1), consisting of a
hydrophobic tail and a hydrophilic head (Ying, 2006). The hydrophobic tail consists of a non-polar carbon chain saturated with hydrogen or fluor atoms. The hydrophilic head is polar and can form hydrogen bridges with water (Ying, 2006). In the case of anionic surfactants, which are the most widely used
surfactants, the head is negatively charged (Sandbacka, et al., 2000). The annual global production of synthetic
surfactants was 7.2 million tons in the year 1993, of which 55% was anionic (Di Corcia, 1998).
Although surfactants are ubiquitously applied and produced in high volumes, little is known about the aquatic ecotoxicity of these substances. The most commonly used technique for estimating the fate of a substance is by determining the octanol-water
partitioning coefficient (Kow) (Konnecker, et al., 2011). The Kow value is used as a measure for the hydrophobicity of a substance and therefore gives an indication of the partitioning of the substance into an organism (Ding & Peijnenburg, 2013). Hence the Kow value
Figure 1. Basic surfactant structure.
C H3 C H2 C H2 C H2 C H2 C H2 C H2 C H2 C H2 C H2 C H2 C H2 CH3
hydrophobic
“alkyl chain”
hydrophilic
“head
group”
serves as a proxy for the potential toxicity of a compound. Although this method generates reliable results for the majority of organic compounds, it is not applicable for surfactants, since due to their surface active properties
surfactants tend to form a third layer at the interface between water and octanol and a correct Kow can therefore not be determined (Konnecker, et al., 2011). To identify the unknown environmental hazards of these ubiquitously applied compounds, there is thus an urgent need for developing a better method to
estimate the hydrophobicity and
bioavailability of surfactants and to link these to yet missing toxicity data. To asses bioavailability a new method has been developed, offering a reliable and easier way to asses surfactant
concentrations in water. In this method a polyacrylate coated fiber is used to simulate the uptake of surfactants from the water by organisms. The fiber only absorbs the free available concentration in the water without depleting the system, indicating the bioavailability of the surfactant (Leslie, et al., 2002). During this research this method was tested in order to find a direct link between toxicity and bioavailability. In determining the toxicity of surfactants there is need for generalization, since the group of surfactants consists of numerous different molecules. Yet, Kow values can not be calculated for
surfactants and therefore it is important to investigate the influence of other molecular descriptors of surfactants on their aquatic ecotoxicity.
Different polar head groups, or
differences in hydrophobic carbon chain length may influence the toxicity of organic compounds. Preliminary research has shown a relationship between
surfactant carbon chain length and toxicity (Barmentlo, et al., 2014). Due to longer carbon chain length of the
compound the hydrophobicity increases. This will cause the surfactant to
accumulate more into body tissue, and increases toxicity. However, an increase in hydrophobicity will also make the surfactant less bioavailable because of a decrease in solubility. Another aspect is the type of atoms connected to the carbon chain. Some surfactants are saturated with Fluor instead of hydrogen. The strong bond between fluor and carbon atoms makes the surfactant molecule more resilient against
degradation by acid, heat and microbes, making it more persistent (Ding & Peijnenburg, 2013). The properties of the fluor atoms also reduce both inter- and intramolecular interaction, making the fluor saturated tail very water and oil repelling (Ding & Peijnenburg, 2013). Because of this decrease in inter- and intramolecular interactions it is expected that the role of the head becomes more prominent, making it less likely to accumulate into body tissue, and reduce toxicity.
In table 1 a summary of EC50 values of
previous research is given, indicating that there is a difference in EC50 value
between different kinds of polar head groups, while also an increase in aquatic toxicity with increasing tail length is shown.
Given the above, the aim of this research is to determine the influence of
hydrophobic chain length and polar head group type on the aquatic ecotoxicity of surfactants.
Materials and Methods
The four types of surfactants tested are 1)Alkyl Carboxylates, 2)Perfluoro Alkyl Carboxylates, 3)Alkyl sulfates and 4)Alkyl sulfonates. These surfactant groups have a different head groups, while Perfluoro Alkyl Carboxylates have a carbon chain saturated with fluor atoms. An overview of the test compounds is given in figure 2.
To assess the acute aquatic toxicity of the selected compounds acute 48h toxicity tests with Daphnia magna were performed according to OECD guideline 202: Daphnia magna acute
immobilization test (48h) (OECD, 2004). The daphnids were obtained from Ecofide (Weesp) and the surfactants were
ordered from Sigma-Aldrich
(Zwijndrecht, the Netherlands). The toxicity tests were performed with Daphnia neonates (aged less than 48 hours). Four different surfactant concentrations were tested. Daphnid survival was determined after 24 hours and finally after 48 hours. For some compounds a maximum solubility test was performed, during such a test a significant amount of compound was added to Daphnia magna buffer and stirred for 24 hours. Thereafter it was left in darkness for 24 hours to reach an equilibrium. The top layer of the fluid was used for a Daphnia magna immobility test. A polyacrylate SPME fiber was used to determine the free available concentration of surfactants in the test medium. The fibers were
thermally conditioned at 120 degrees, has a length of 4cm and a coating
thickness of 35mm (Leslie, et al., 2002). The fibers were put into tubes containing the same medium as used in the
Daphnia magna tests.The fibers were shaken at 150rpm for 3 weeks to reach equilibrium. Sodium azide was added to prevent microorganisms from growing, the concentration sodium azide added to test solution was 50mg/L. After three weeks samples were taken and the surfactant concentration the fiber was analyzed. The analysis of the
concentration of surfactant that partitioned to the fiber was performed using a Shimadzu LCMS. To calculate and plot the EC50-values a non-linear
regression model was used (Haanstra, et al., 1985). Calculations and plots were performed using Microsoft Excel. Results
Alkyl carboxylates
The alkyl carboxylate with the shortest carbon chain length, C11, was the only
one for which a dose response
relationship was obtained, all daphnids in
Figure 2. Test compounds. colums 1-5: (1)Scientific Name, (2)chemical structure, (3)basic structure
the control group survived. The three other chain lengths, C13 till C15, were
insoluble, resulting in random or complete Daphnia magna survival. In figure 3 the relation between the
nominal and the actual concentration of C11COOH in the Daphnia test medium is
visualized. As seen in the graph, the actual C11COOH concentration was 24%
of the nominal concentration.
A clear dose response relationship was obtained for the effect of C11COOH on
Daphnia magna survival, visualized in figure 4, from which an EC50 value of
0.7mg/L was derived (CI 0,595-0.810mg/L).
Perfluoro Alkyl Carboxylates Only the perfluor with the shortest carbon chain length, C8FCOOH, produced
data for a dose response relationship. The three other chain lengths, C9 till C11,
were insoluble resulting in complete Daphnia magna survival. In figure 5 the relation between the nominal and the
actual concentration of C8FCOOH in the
Daphnia test medium is visualised. As seen in the graph, the actual C8FCOOH
concentration was 92% of the nominal concentration.
A clear dose response relationship was obtained for the effect of C8FCOOH on
Daphnia magna survival, visualized in figure 4, from which an EC50 value of
425mg/L was derived (CI 311-539mg/L).
In figure 7 the C8FCOOH concentration in
the fibers is plotted against the C8FCOOH
concentration in the test medium in which the fibers were shaken during three weeks. The fiber uptake is logarithmic. y = 0.2367x R² = 0.9127 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 5 10 A ct u a l co n ce n tra tio n (mg/ L) Nominal concentration (mg/L) actual vs nominal Lineair (actual vs nominal) 0 10 20 30 40 50 60 70 80 90 100 0.01 0.10 1.00 10.00 100.00 M o bil ity (% ) Concentration (mg/L) C11COOH Data C11COOH Model Fit C11COOH EC50 = 0.7mg/L y = 0.9194x R² = 0.997 0 500 1000 1500 2000 2500 3000 0 1000 2000 3000 A ct u a l co n ce n tra tio n (mg/ L) Nominal concentration (mg/L) Actual vs Nominal Lineair (Actual vs Nominal) 0 10 20 30 40 50 60 70 80 90 100 0.01 1 100 10000 M o bil ity (% ) Concentration C8FCOOH (mg/L) Data Model Fit EC 50 = 425 mg/L
Figure 6. The C8FCOOH dose response
relationship on Daphnia magna. The EC50 value is 425mg/L.
Figure 5. The actual C8FCOOH concentration (mg/L) plotted against the nominal
concentration (mg/L).
Figure 4. The C11COOH dose response
relationship on Daphnia magna. The EC50 value is 0.7mg/L.
Figure 3. The actual C11COOH concentration (mg/L) plotted against the nominal
In figure 8 the Daphnia magna survival
is plotted against the C8FCOOH
concentration in the fiber resulting in a clear dose response relationship from
which an EC50fiber value of 2558mg/L was
derived (CI 2418-2698mg/L).
Alkyl sulfates
The alkyl sulfate with the shortest carbon chain length, C11SO4, was the only one
which produced data for a dose response relationship. In figure 9 the relation between the nominal and the actual
concentration is shown. As seen in the
graph, the actual C11SO4 concentration was 74% of the nominal concentration.
A clear dose response relationship was obtained for the effect of C11SO4 on Daphnia magna survival, visualized in figure 4, from which an EC50 value of
13.0mg/L was derived (CI 12.7-13.3mg/L).
For the longer carbon chain lengths: C13,
C15 and C16, a maximum solubility test
was performed. The results of this test are shown in table 2.
Compound Immobility after 48h (%)
C13SO4 88
C14SO4 0
C15SO4 24
In figure 11 the C11SO4 concentration in
the fibers is plotted against the C11SO4
concentration in the test medium in which the fibers were shaken during R² = 0.6477 0 1000 2000 3000 4000 5000 0 100 200 300 400 Fi b e r co n cent ra ti on ( m g/ L) Water concentration (mg/L)
C8FCOOH fiber concentration vs water concentration Log. (C8FCOOH fiber concentration vs water concentration)
0 10 20 30 40 50 60 70 80 90 100 1 10 100 1000 10000 Im m ob ili ty ( % ) Fiberconcentration (mg/L) Data Model Fit EC50 = 2558mg/L y = 0.7762x R² = 0.9824 0 10 20 30 40 50 60 70 0 50 100 A ct u a l co n ce n tra tio n (mg/ L) Nominal concentration (mg/L) Actual conc vs nominal conc 0 10 20 30 40 50 60 70 80 90 100 0.01 0.1 1 10 100 M o bil ity (% ) Concentration (mg/L) C11SO4 Data C11SO4 Model Fit C11SO4 EC50 = 13.0mg/L
Table 2. Results of the maximum solubility
tests for C13SO4, C14SO4 and C15SO4.
Figure 10. The C11SO4 dose response relationship on Daphnia magna. The EC50 value is 13.0mg/L.
Figure 9. The actual C11SO4 concentration (mg/L) plotted against the nominal concentration (mg/L).
Figure 8. Dose response relation of Daphnia magna against the C8FCOOH fiber concentration. The dotted line marks the highest measured fiber concentration, the EC50-fiber is 2558mg/L.
Figure 7. The C8FCOOH fiber concentration plotted against the C8FCOOH water
three weeks. The fiber uptake is logarithmic.
Due to the bad logarithmic fit, no Dose
response curve of Daphnia magna against the C11SO4fiber concentration could be made.
Alkyl Sulfonates
All alkyl sulfonates were insoluble, for all
four of these compounds; C11SO3,
C13SO4, C14SO4 and C15SO4 the lowest
attempted concentration was 0.5mg/L. Summary
Table 3 gives an overview of the
successful tested compounds with their
nominal concentration loss and their EC50
values. Compound Concentration loss (%) EC50 and CI (mg/L) C11COOH 76 0.7 (0,595-0.810) C8FCOOH 8 425 (311-539) C11SO4 22 13.0 (12.7-13.3)
Conclusion and discussion
SPME Fibers
The purpose of the fibers was to create a proxy for the internal surfactant
concentration in a test organism, making the use of living daphnids unnecessary in the future. Because of the difference in test concentrations between the fibers and the Daphnia magna survival tests with C8FCOOH and C11SO4 the
logarithmic fit needed to be significant to calculate an EC50-fiber. Therefore only the
EC50-fiber of C8FCOOH was of use.
This EC50-fiber concentration could now be
used to bypass the use of Daphnids and only test with fibers for this particular compound. Because of the direct link between fiber concentration and daphnid response. Obviously more research need to be done so a more accurate standard could be made, but nevertheless this proves to be a promising method for biomimetic aquatic toxicity assessment. Polar head group
C11COOH and C11SO4 both have the
same carbon chain length, but different
polar head groups. Because the EC50
values differ at a factor of 18.5 one can conclude that the polar head group indeed influences the aquatic toxicity of surfactants.
Carbon chain length
For the assessment of carbon chain length on aquatic toxicity, the study performed by Barmentlo et al. in 2013 on Perfluoro Alkyl Carboxylates was used. Barmentlo et al. tested the aquatic
toxicity on Daphnia magna for C3FCOOH,
C5FCOOH and C7FCOOH. In figure 13 the
dose response curves of the data of Barmentlo et al. and from the present
C8FCOOH Daphnia magna test are
visualized. R² = 0.3442 0 1000 2000 3000 4000 5000 6000 7000 8000 0 200 400 600 800 1000 F ibe r co n ce n tra tio n (mg/ L) Water concentration (mg/L)
C11SO4 fiber concentration vs water concentration
Table 3. Summary of successful tested
compounds, their concentration loss and their EC50 values.
Figure 11. The C11SO4 fiber concentration plotted against the C11SO4water concentration.
As seen in figure 13 the presently
obtained C8FCOOH EC50 value is higher
than the EC50 value of C7FCOOH, which is
against expectations, even though it is still lower than the EC50 values for
C5FCOOH and C3FCOOH. An explanation
could be the temporal variation, another one could be the source of the daphnids. Barmentlo et al. obtained theirs from Aquasense, while for this study the daphnids were obtained from Ecofide. This could account for the observed sensitivity of the daphnids.
Maximum solubility
When the daphnids were added to the test solution during the maximum solubility tests part of the diluted surfactant concentration went solid and therefore formed a physical stress factor. This is probably the cause of the
mortalities during these tests, since the Daphnid survival was spread irregularly between the different replications. The toxicity of C13SO4 and the slight toxicity
of C15SO4 is therefore probably due to
the physical limitations the daphnids experienced being unable to move due
to the hindrance of the surfactants in solid form.
Comparison with EC50 values from
literature
There is a remarkable difference
between the presently obtained EC50
values and those reported in literature.
C11COOH has a EC50 value of 0.7mg/L
while literature reported 12mg, which is
about 17 times higher. The EC50 of
C8FCOOH is 425mg/L while literature
mentions a value of 151mg, which is
almost 3 times lower. The EC50 value of
C11SO4 is 13mg, which again is much
lower than literature suggests, between 235mg and 400mg, so about 30 times. These discrepancies again underline the difficulties in research on surfactants and their effects on the environment. With this research a great step has been made to enhance our knowledge of
surfactants. In conclusion, the molecular
structure of a surfactant does influence toxicity and the SPME fiber has proven to be a viable method to eventually
substitute Daphnids in aquatic ecotoxicity research.
Figure 12. Dose response curves of both the data of Barmentlo et al.; C3FCOOH, C5FCOOH and C7FCOOH,
Chain
length 24-h EC50 24-h EC50
24-h 24-h
EC50 48-h EC50 48-h EC50 IC50 24-h EC50 EC50
Dapnia magna Dapnia magna Ceriodapnia Dapnia magna Daphnia magna Daphnia magna Daphnia magna Dapnia magna dubia
static system static system
flow-through static
system
test
(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) CxSO4 CxSO4 CxSO4 CxSO3 CxFCOOH CxFCOOH CxFCOOH CxCOOH
C3 6095 182 C5 891 C7 253 212 476.5 C8 4350 >900 3200 151 C9 2300 163 65 C10 800 470 133 C11 79 12 C12 80 25 5.55 220 8-47 C13 42 C14 37 1.58 60 C15 0.59 C16 0.15 C17 C18 >0.69 (no mortality) 4.2 C19 References
Barmentlo, S. H. et al., 2014. Acute and chronic toxicity of new perfluoroalkyl substances to Daphnia magna.. Di Corcia, A., 1998. Characterisation of surfactants and their biointermediates by liquid chromatography-mass spectrometry. Journal of Chromatography A. 794, pp. 165-185.
Ding, G. & Peijnenburg, W. J. G. M., 2013. Physicochemical Properties and Aquatic Toxicity of Poly- and Perfluorinated Compounds. Critical reviews in environmental science and technology, pp. 598-678. Haanstra, L., Doelman, P. & Oude Voshaar, J. H., 1985. The use of sigmoidal dose response curves in soil ecotoxilogical research. Plant and Soil 84, pp. 293-297.
HERA, 2013. Fatty Acid Salts (Soap) Environmental Risk Assessment, s.l.: s.n.
Hodges, G., Roberts, D. W., Marshall, S. J. & Dearden, J. C., 2006. The aquatic toxicity of anionic surfactants to Daphnia magna - A comparitive QSAR study of linear alkylbenzene sulphonates and ester sulphonates.
Chemosphere 63, pp. 1443-1450.
Konnecker, G. et al., 2011. Environmental properties and aquatic hazard assessment of anionic surfactants: Physico-chemical, environmental fate and ecotoxicity properties. Ecotoxicology and environmental safety 74, pp. 1445-1460.
Table 1. EC50 values from previous research (Barmentlo, et al., 2014), (Ding & Peijnenburg, 2013),
Leslie, H. A. et al., 2002. Biomimetic solid-phase microextraction to predict body residues. Environmental toxicology and chemistry 21, pp. 229-234.
OECD, 2004. Test No. 202: Daphnia sp. Acute Immobilisation Test. In: OECD Guidelines for the Testing of Chemicals. s.l.:OECD publishing, p. Section 2.
RIVM, 2012. Case studies on the Development and Application of in-Silico Techniques for Environmental hazard and Risk assessment , s.l.: s.n.
Sandbacka, M., Christianson, I. & Isomaa, B., 2000. The Acute Toxicity of Surfactants on Fish Cells, Daphnia magna and Fish. A Comparative Study. Toxicology in Vitro 14, pp. 61-68.
Ying, G.-G., 2006. Fate, behavior and effects of surfactants and their degradation products in the environment. Environment International 32, pp. 417-431.
