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Compatibility of hydroxypropyl-β-cyclodextrin with algal toxicity bioassays
Environmental Pollution 157 (2009) 135–140
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Compatibility of hydroxypropyl-b-cyclodextrin with algal toxicity bioassays Patricia Bi Fai, Alastair Grant, Brian J. Reid* School of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
Compatibility of the biomimetic HPCD extraction method with algal cell growth inhibition bioassays to assess toxicity of reference toxicants and environmental relevant herbicides.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2008 Received in revised form 11 July 2008 Accepted 18 July 2008
Numerous reports have indicated that hydrophobic organic compound bioaccessibility in sediment and soil can be determined by extraction using aqueous hydroxypropyl-b-cyclodextrin (HPCD) solutions. This study establishes the compatibility of HPCD with Selenastrum capricornutum and assesses whether its presence influences the toxicity of reference toxicants. Algal growth inhibition (72 h) showed no significant (P > 0.05) difference at HPCD concentrations up to and including 20 mM. HPCD presence did not influence the toxicity of the inorganic reference toxicant (ZnSO4), with IC50 values of 0.82 mM and 0.85 mM, in the presence and absence of HPCD (20 mM), respectively. However, HPCD presence (20 mM) reduced the toxicity of 2,4-dichlorophenol and the herbicides diuron and isoproturon. These reductions were attributed to inclusion complex formation between the toxicants and the HPCD cavity. Liberation of complexed toxicants, by sample manipulation prior to toxicity assessment, is proposed to provide a sensitive, high throughput, bioassay that reflects compound bioaccessibility. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Selenastrum capricornutum Cyclodextrin Toxicity Inhibition Herbicide Dosing Realistic exposure scenario
1. Introduction Sediments, soils and other particulate matter are well-known sinks for moderately to strongly hydrophobic organic compounds (HOCs) (Brack, 2003). Many compounds such as polycyclic aromatic hydrocarbons (PAHs), monoaromatic hydrocarbons, herbicides and other pesticides fall within this broad grouping. These compounds are often cause for concern on account of their carcinogenicity, teratogenicity, mutagencity and adverse ecological effects (Bostro¨m et al., 2002). When present in sediment and soil exposure to HOCs is typically through both the aqueous and solid phase. Thus, toxicity may be manifested as a result of exposure via pore water, ingested sediment, and direct contact with sediment particles. These pathways are important for soil dwelling organisms, benthic organisms as well as for algae, daphnids and fish (in case of re-suspension of the particles) (Brack et al., 1999). As such, ecotoxicity assessment of pore water (or aqueous elutriates obtained from sediment/soil) neglects two of these three obvious possible pathways of exposure and is therefore likely to underestimate toxicity. Sediment toxicity assessment is therefore necessary. However, prior to screening with most algal or microbial bioassays, HOCs associated with sediment/soil generally require extraction into a solution phase. For meaningful toxicity assessment to be made, it
* Corresponding author. Tel.: þ44 1603 592357; fax: þ44 1603 591327. E-mail address: [email protected] (B.J. Reid). 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.07.015
is imperative that extraction methods provide (i) an extract that is compatible with the bioassay and (ii) an extract that meaningfully reflects the manner in which the chemical residues are partitioned within sediment or soil. Where hydrophobic compounds are being screened it is widely recognised that extraction using water as a solvent is limited by compound aqueous solubility. Thus, while an aqueous extract is compatible with many bioassays, it would only contain a fraction of the hydrophobic compound that is bioaccessible. As a consequence, when these extracts are screened for toxicity, the risk associated with the sediment/soil associated residues is underestimated. In contrast to such ‘mild’ extraction methods harsh organic solvent extractions are often used to strip hydrophobic chemicals from sediment/soil with a view to evaluating these total residues and their toxicity. Significantly, such extracts are inherently incompatible with biosensors on account of the toxicity associated with the extraction solvent itself. As a consequence dilution (to circa 1%) in water or growth media is required prior to toxicity assessment. As the toxicity threshold of a compound is approached (or surpassed) this dilution results in loss of sensitivity (or a ‘false negative result’ being reported). More importantly, it is now widely recognised that harsh solvent techniques overestimate the bioaccessible fraction of HOCs in sediment/soil and as a consequence overestimate the risk associated with these residues (Alexander, 2000). Over the past decade, less exhaustive techniques have been investigated with a view to establishing extraction methodologies that reflect the bioaccessible fraction of organic contaminants
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present in soils/sediments (White et al., 1997; Tang and Alexander, 1999; Liste and Alexander, 2002; Reid et al., 2000a,b; Stokes et al., 2005). While favourable correlations have been identified between extractability using ‘mild’ solvents and organic compounds bioaccessibility to a range of receptors (Tang and Alexander, 1999; Liste and Alexander, 2002), many of these techniques have failed to provide consistent and representative results when applied to different sediment/soils and/or compounds (White et al., 1997; Johnson and Weber, 2001). An alternative approach makes use of aqueous solutions of hydroxypropyl-b-cyclodextrin (HPCD) to extract non-polar organic contaminants via a shake extraction (Reid et al., 2000a; Cuypers et al., 2002; Stokes et al., 2005; Hickman and Reid, 2005; Allan et al., 2006; Sabate et al., 2006). Cyclodextrins are a group of torus- or bucket-shaped cyclic macromolecules composed of a-1,4-linked glucose units. They are highly soluble in water due to their array of hydroxyl functional groups on the exterior of the torus. However, the interior exhibits a hydrophobic character and can therefore host one or more hydrophobic molecules; thus forming a host–guest inclusion complex (Mahedero et al., 2002; Dupuy et al., 2005). Aqueous solutions of cyclodextrin have been used to dissolve a range of low solubility contaminants to greater than their aqueous solubility limits (Reid et al., 2000a,b; Dupuy et al., 2005; Hickman and Reid, 2005; Stokes et al., 2005; Allan et al., 2006). Significantly, strong relationships between HPCD extractability and microbial bioaccessibility have been reported for a wide range of hydrophobic organic compounds (Reid et al., 2000a,b; Mahedero et al., 2002; Dupuy et al., 2005; Stokes et al., 2005). In this research the compatibility of aqueous HPCD solutions with Selenastrum capricornutum, now Pseudokirchneriella subcapitata, Pseudokirchneriella subspicata or Raphidocelis subcapitata (Bengtson Nash et al., 2005; Ma et al., 2006; Pavlic´ et al., 2006), was investigated in algal growth inhibition assays (up to 72 h) in order to determine the optimum HPCD concentration in extracts for algal bioassay testing. Having established optimal HPCD concentrations (with respect to their toxicity being not significantly different to the media only control), algal growth inhibition assays were undertaken to investigate the effect of HPCD presence on the toxicities of inorganic and organic reference toxicants, namely, ZnSO4 and 2,4dichlorophenol (DCP), respectively. Subsequently, dose–response relationships were established for two herbicides, namely, diuron and isoproturon. These compounds represent a large and important class of herbicides widely used for pre- and post-emergent control of weeds (Mahedero et al., 2002; Dupuy et al., 2005).
maintained by transferring 1 mL of stock culture weekly to 50 mL of new growth medium and 3–4 days old cells used for all tests. The cell doubling time for this alga is approximately 10 h (Gorski et al., 2006). The Spectra MaxÒ M2 microplate reader from Molecular Devices Corporation (CA, USA) was used to obtain absorbance readings at 430 nm (A430) for the growth inhibition assay. All reported absorbance values have been corrected for background absorbance by microplate and algal medium. In order to establish bioassay compatibility with HPCD a stock solution of HPCD was prepared and serially diluted in MILLI Q water to obtain concentrations from 10 to 50 mM. These solutions were then used in the assessment of inhibition of growth (as described above) after exposure times of 24 h, 48 h and 72 h. Having established maximum HPCD concentrations that resulted in no inhibition in algal growth with respect to the media only controls, dose–response relationships in the presence (20 mM) and absence (media only) of HPCD were established for two reference toxicants, namely, an inorganic species (ZnSO47H2O; 0.03–17.4 mM (Zn)) and an organic compound (2,4-DCP; 0.005–10 mM). Finally, the dose–response relationships in the presence (20 mM) and absence (media only) of HPCD were established for two priority herbicides: diuron (0.01–0.4 mM) and isoproturon (0.02–1665 mM). For tests with all toxicants in the absence of HPCD, a stock toxicant concentration was prepared and serially diluted to obtain desired concentrations in the ranges given above. These were then introduced into microplates (a separate microplate for each toxicant) and test controls (algal growth medium) included. In order to obtain equivalent concentrations of toxicants in HPCD the toxicants were prepared to twice the desired concentrations and 40 mM HPCD prepared separately. These were then mixed in a 1:1 ratio before introducing the toxicants in the microplates. Test controls as well as solvent controls containing algal growth medium and HPCD (final concentration of 20 mM) were included. The rest of the procedure followed standard protocol (Environment Canada, 1992). Each experimental microplate had five replicates per test concentration (four replicates used as solvent controls were included).
2.3. Statistical analysis Growth inhibition after 72 h was obtained by subtracting the initial absorbance readings from the corresponding final value. One-way analysis of variance (ANOVA) was used to determine the maximum HPCD concentration that did not cause significant growth inhibition of S. capricornutum. Concentrations causing 50% growth inhibition (IC50) were calculated for each toxicant using the Excel macro REGTOX (http://eric.vindimian.9online.fr/en_index.html). The model used in this macro is written in the form proposed by Duggleby (1981) in which two parameters: Hill number (H) and EC50, are characteristics of the probability function written as follows: f ðxÞ ¼
xnH xnH
þ ECnH 50
where x is the dose and f(x) is a probability function of the dose varying from 0 to 1 with the dose. When x ¼ EC50, f(x) is 0.5. Confidence intervals were estimated using a non-parametric bootstrapping method. Two-way ANOVA was used to determine which variables (solvent type or
0.12 24 h 48 h 72 h
2. Materials and methods
0.10
An axenic culture of the S. capricornutum strain CCAP 278/4, equivalent to the American Type Culture Collection strain ATCC 22662 (Environment Canada, 1992) was obtained from the Scottish Association for Marine Science (SAMS) Research Services, UK. S. capricornutum was cultured in an incubator under continuous fluorescent lighting (quantal flux of 96 mmol photons/m2/s), at 24 C and 100 rpm. The growth medium, 1 AAP (Algal Assay Procedure) medium was prepared according to Blaise et al. (2000) and contained the following chemicals: CaCl2$2H2O, CoCl2$6H2O, CuCl2$2H2O, MgCl2$6H2O, FeCl3$6H2O, H3BO3, K2HPO4, MgSO4, MnCl2$4H2O, Na2EDTA$2H2O, Na2MoO4$2H2O, NaHCO3, NaNO3 and ZnCl2. Diuron and 2,4-DCP were PESTANALÒ analytical standards obtained from Sigma–Aldrich (Dorset, UK). Isoproturon (ALPHA IPU 500) was obtained as a suspension concentrate formulation 500 g/L (46.3% w/w) from Makhteshim Agan (Berkshire, UK). All other chemicals were reagent grade and obtained either from Sigma–Aldrich or Fisher Scientific (Leicestershire, UK).
Growth (A430) a.u.
2.1. S. capricornutum and chemicals
0.08
∗
0.06
∗ ∗
0.04
0.02
A microplate-based algal growth inhibition (specifically, inhibition of cell reproduction) assay was carried out according to the standard protocol set by Environment Canada (1992) with little modification in the algal inoculum preparation as in Fai et al. (2007). A continuous supply of ‘‘healthy’’ cells for tests was
∗
30
40
∗
0.00 0
2.2. Algal growth inhibition bioassays
∗
10
20
50
[Hydroxypropyl-β-Cyclodextrin] mM Fig. 1. Growth inhibition of S. capricornutum exposed to increasing concentrations of hydroxypropyl-b-cyclodextrin following 24 h (white), 48 h (black) and 72 h (hatched). An asterisk above a bar indicates significant difference with respect to control at 0.05 level; n ¼ 5; error bars ¼ 1 standard deviation; a.u. ¼ arbitrary unit.
Growth inhibition (%)
P.B. Fai et al. / Environmental Pollution 157 (2009) 135–140
100
A
B
137
C
50
0
Growth medium HPCD -50 10-8
10-7
10-6
10-5
ZnSO4.7H2O (M)
10-4
Growth medium HPCD 10-8
10-7
10-6
10-5
Growth medium HPCD
10-4
10-8
10-7
ZnSO4.7H2O (M)
10-6
10-5
10-4
ZnSO4.7H2O (M)
Fig. 2. Dose–response for S. capricornutum exposed to the reference toxicant ZnSO4$7H2O in media (open) and 20 mM hydroxypropyl-b-cyclodextrin (closed) at 24 h (A), 48 h (B) and 72 h (C). Error bars ¼ 1 standard deviation.
toxicant concentrations) explained observed inhibition in growth with respect to toxicant and solvent influence. Statistical analyses were done using Microsoft Excel and SPSS 12.0.1 for Windows.
3. Results and discussion 3.1. HPCD toxicity Dose–responses were apparent following 24 h of incubation but were more resolved following 48 h and 72 h (Fig. 1). In the media only controls and at all HPCD concentrations it was noted that while considerable growth inhibition occurred between 24 h and 48 h, growth inhibition was much greater between 48 h and 72 h. HPCD concentrations of 10 mM and 20 mM did not significantly (P > 0.05) inhibit S. capricornutum growth at any of the exposure times relative to the control (Fig. 1). However, HPCD concentrations 30 mM consistently resulted in significant (P < 0.05) inhibition in algal growth at 48 h and 72 h with respect to the media only control. Toxicity, with respect to cyclodextrins, has been proposed on account of inclusion complexation of membrane components, particularly cholesterol and phospholipids resulting in destruction of the cell membrane leading to cytotoxicity (LeroyLechat et al., 1994). By the same mechanism, cyclodextrins give rise to perturbation in the surface tension of lipid monolayers and destruction of liposomes (Irie et al., 1992). In addition to these mechanisms, it was noted (observational/not measured) that aqueous solutions of HPCD became increasingly more viscous with increasing concentration. Increased viscosity would also have impaired gas diffusion (specifically CO2) and as a consequence may have limited photosynthesis. A HPCD concentration of 20 mM was selected for all further experiments using reference toxicants and herbicides. 3.2. Dose–response: reference toxicants Dose–responses were apparent for ZnSO4 following 24 h although there were sometimes large confidence limits associated with measurements at 24 h (attributed to the small extent of algal growth) (Fig. 2). The dose–response did not change substantially from 24 to 72 h (Fig. 2). Indeed, IC50 values were almost identical at 48 h and 72 h. The effect of ZnSO4 on S. capricornutum growth inhibition was observed within the same concentration range, irrespective of the presence or absence of HPCD (Fig. 2), with IC50 values at 48 h and 72 h for ZnSO4 in growth media or HPCD being virtually identical. Two-way ANOVA indicted ZnSO4 concentration alone explained 97% of its effect on growth inhibition; the effect of solvent (as well as solvent–concentration interaction) on growth inhibition was not significant at the 0.05 level. The aqueous 72 h IC50 value derived in this work (Table 1) was consistent with Zn
IC50 values previously reported in the literature (0.76–1.5 mM; Vasseur et al., 1988). Regarding 2,4-DCP, dose–responses were again distinguishable, albeit with larger confidence limits, following 24 h. The 72 h IC50 growth inhibition value derived here for 2,4-DCP in media only (11.7 mM) was consistent with 2,4-DCP aqueous IC50 values previously reported (86 mM; Shigeoka et al., 1988). As was the case for ZnSO4 exposure times of 48 h and 72 h made very little difference to the determined IC50 value. In contrast, however, to the inorganic reference toxicant (ZnSO4), 2,4-DCP toxicity was markedly influenced by the presence of HPCD (20 mM). Onset of inhibition to algal growth in the presence and absence of HPCD was observed at 2,4DCP concentrations around 104 mM and 105 mM, respectively; with 100% algal growth inhibition being observed at 2,4-DCP concentrations around 103 mM and 104 mM, respectively (Fig. 3). Thus, 2,4-DCP toxicity was decreased by an order of magnitude when HPCD was present. IC50 values in the absence/presence of HPCD were observed to differ by a factor of 41 (Table 1). The observed decrease in toxicity was expected given the affinity between 2,4-DCP molecules and the HPCD cavities. As a consequence of 2,4-DCP/HPCD inclusion complex formation the genuine solution phase 2,4-DCP concentration would have been significantly lower; it follows that algal cells were in effect being exposed to lower concentrations and toxicity decreased correspondingly (Fig. 3). Zinc would not have been subject to complexation/association with HPCD and as a consequence toxicity was not influenced (Fig. 2).
3.3. Dose–response: herbicides Inhibition of S. capricornutum growth in response to increasing concentrations of diuron (Fig. 4) and isoproturon (Fig. 5) was observed to be proportional to herbicide concentration both in the presence and absence of HPCD. However, as was the case with 2,4DCP (Fig. 3) there was a marked decrease in herbicide toxicity in the presence of HPCD. Indeed, no inhibition in algal growth was observed for these toxicants in the presence of HPCD at
Table 1 Selenastrum capricornutum IC50 (72 h) growth inhibition values and associated 95% confidence intervals obtained for all toxicants Toxicant
72 h IC50 growth inhibition (95% confidence limits) mM Media (A)
HPCD (B)
ZnSO4 2,4-Dichlorophenol Diuron Isoproturon
0.85 11.7 0.03 0.03
0.82 475 0.4 10.8
(0.78–0.94) (10.7–20.6) (0.03–0.04) (0.02–0.05)
(0.76–0.9) (415–533) (0.3–0.5) (6.4–20.1)
Proportion (B/A)
0.96 41 13 360
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Growth inhibition (%)
138
150
Growth medium HPCD
A
B
100
100
100
C
50
50
50
0 0
Growth medium HPCD
0 10-6
10-5
10-4
10-3
10-2
10-1
10-6
2,4-Dichlorophenol (M)
10-5
10-4
10-3
10-2
10-1
Growth medium HPCD
-50 10-6
10-5
2,4-Dichlorophenol (M)
10-4
10-3
10-2
10-1
2,4-Dichlorophenol (M)
Fig. 3. Dose–response for S. capricornutum exposed to the reference toxicant 2,4-dichlorophenol in media (open) and 20 mM hydroxypropyl-b-cyclodextrin (closed) at 24 h (A), 48 h (B) and 72 h (C). Error bars ¼ 1 standard deviation.
concentrations that resulted in 100% algal growth inhibition in media only samples. Toxicity was abated to a larger extent in the case of isoproturon (an ‘up-concentration’ shift in algal growth inhibition of between two and three orders of magnitude; Fig. 5) compared to diuron (an ‘up-concentration’ shift in algal growth inhibition of about one order of magnitude; Fig. 4). Correspondingly, the 72 h IC50s for isoproturon and diuron in HPCD were, respectively, 360 and 13 times greater than their respective IC50s in growth media (Table 1). The aqueous IC50 values derived in this work (Table 1) were consistent with diuron and isoproturon IC50 values previously reported in the literature: diuron (0.03 mM; El Jay et al., 1997; isoproturon: 0.24 mM; Pavlic´ et al., 2006). In keeping with the observations for 2,4-DCP, toxicity decreased for both of the phenylurea herbicides (diuron and isoproturon) tested when present with HPCD (20 mM). Previous studies have demonstrated that b-cyclodextrin and HPCD form relatively stable inclusion complexes with phenylurea herbicides (Ishiwata and Kamiya, 1999; Mahedero et al., 2002; Dupuy et al., 2005). Thus, the reduction in toxicity, as observed in the present study, strongly suggests that the inclusion complexes decreased the opportunity for compound–algae interaction of the organic compounds tested. The inclusion behaviour of b-cyclodextrin and herbicides have been reported to depend on the size fit between ‘‘guest’’ and host resulting in an inclusion ratio of 1:1 for fenuron, monuron and diuron, and an inclusion ratio of 2:1 for isoproturon (Dupuy et al., 2005). Thus, isoproturon would have been more effectively precluded from the free aqueous phase on account of this 2:1 complexation compared to the situation for diuron where a less shielded 1:1 complex would have been formed.
environmental contaminants (Environment Canada, 1992; Blaise et al., 2000; USEPA, 2002). Results presented here have indicted that concentrations of HPCD 20 mM resulted in no significant growth inhibition of this micro algal species with respect to media only controls (Fig. 1). Significantly, aqueous HPCD extraction has been shown to closely mimic the mass transfer mechanisms that govern the bioavailability of HOCs in sediment/soil (Reid et al., 2000a,b; Cuypers et al., 2002; Hickman and Reid, 2005; Stokes et al., 2005; Allan et al., 2006; Sabate et al., 2006). Thus, it is arguably representative to use aqueous HPCD solutions to extract HOCs from sediment/soil with a view to using these extracts in toxicity assessment; as such an approach will facilitate the toxicity assessment of the entire bioaccessible pool, rather than the fraction present in the interstitial/pore-water. Clearly, loss of sensitivity by the sensor when exposed to organic toxicants in the presence of HPCD (Figs. 3–5) has implications for the application of S. capricornutum in the screening of HPCD aqueous extracts derived from sediments and soils. However, it should be borne in mind (given the hydrophobic properties of many pesticides) that soil/sediment concentration will be much greater than those in the aqueous pore water phase (on account of partitioning in favour of the mineral and organic components present). The option remains to use an aqueous cyclodextrin extraction to liberate the putative bioaccessible fraction of toxicants with a view to their quantification by a chemical means and to complement these measurements with toxicity screening on a second extract derived by conventional solvent extraction methods. However, we and others suggest that when screening an extract it is, arguably, better to screen one that is representative of the entire bioaccessible fraction, albeit with reduced sensor sensitivity, than to screen an extract that does not reflect the entire bioaccessible pool with higher sensor sensitivity. Although not investigated in this study, liquid–liquid–liquid exchange could be used to abate the issues of toxicity suppression in the presence of
3.4. Towards more realistic sediment/soil toxicity assessment S. capricornutum is one of the major standard test organisms used in toxicity tests internationally to assess the risk of
Growth inhibition (%)
100 100
A
B
50
100
C
50 50
0 Growth medium HPCD 10-8
10-7
10-6
10-5
Diuron (M)
10-4
10-3
Growth medium HPCD
0 10-8
10-7
10-6
10-5
Diuron (M)
10-4
10-3
Growth medium HPCD
0 10-8
10-7
10-6
10-5
10-4
10-3
Diuron (M)
Fig. 4. Dose–response for S. capricornutum exposed to the herbicide diuron in media (open) and 20 mM hydroxypropyl-b-cyclodextrin (closed) at 24 h (A), 48 h (B) and 72 h (C). Error bars ¼ 1 standard deviation.
P.B. Fai et al. / Environmental Pollution 157 (2009) 135–140
139
Growth inhibition (%)
150
A
100
B
100
C
100 50 50
50 0 0
0 Growth medium HPCD
-50 10-8
10-7
10-6
10-5
10-4
Isoproturon (M)
10-3
10-2
Growth medium HPCD
-50 10-8
10-7
10-6
10-5
10-4
Isoproturon (M)
10-3
10-2
Growth medium HPCD 10-8
10-7
10-6
10-5
10-4
10-3
10-2
Isoproturon (M)
Fig. 5. Dose–response for S. capricornutum exposed to the herbicide isoproturon in media (open) and 20 mM hydroxypropyl-b-cyclodextrin (closed) at 24 h (A), 48 h (B) and 72 h (C). Error bars ¼ 1 standard deviation.
HPCD while maintaining the value of the HPCD extraction as a biomimetic for bioaccessibility assessment (Reid et al., 2000a,b; Cuypers et al., 2002; Mahedero et al., 2002; Dupuy et al., 2005; Hickman and Reid, 2005; Stokes et al., 2005; Allan et al., 2006). Thus, a HPCD extract could be exchanged into a strongly solvating, and water immiscible, solvent (such as dichloromethane (DCM)), the DCM evaporation to dryness and toxicants subsequent redissolving in a low toxicity solvent such as dimethyl sulfoxide (Okumura et al., 2001) prior to dilution in media and toxicity screening. Of course this sort of sample manipulation is not without its disadvantages, for example, solvent consumption as well as loss of toxicants during transfer and drying, and possibility of low solubility compounds not re-dissolving in the final solvent. Alternatively, incorporation of a step in sample preparation to release the organic toxicants from the cyclodextrin cavities may abate the reduction in algal sensitivity. Based on these initial results further studies are warranted to investigate applicability to a wider range of pesticides and other HOCs (e.g. PAHs and priority monoaromatic compounds such as benzene, toluene, ethylbenzene and xylene) and the possibility of enhancing algal sensitivity by further sample manipulation as described above. Acknowledgements The authors would like to thank the LINK Bioremediation program and the Natural Environment Research Council (NERC) for contributing funding towards this work (grant nos. NER/T/S/2003/ 00107 and NE/C513185) and the UK Commonwealth Scholarship Commission. References Alexander, M., 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environmental Science and Technology 34 (20), 4259–4265. Allan, I.J., Semple, K.T., Hare, R., Reid, B.J., 2006. Prediction of mono- and polycyclic aromatic hydrocarbon degradation in spiked soils using cyclodextrin extraction. Environmental Pollution 144 (2), 562–571. Blaise, C., Forget, G., Trottier, S., 2000. Toxicity screening of aqueous samples using a cost-effective 72-h exposure Selenastrum capricornutum assay. Environmental Toxicology 15 (4), 352–359. Bengtson Nash, S.M., Quayle, P.A., Schreiber, U., Muller, J.F., 2005. The selection of a model microalgal species as biomaterial for a novel aquatic phytotoxicity assay. Aquatic Toxicology 72 (4), 315–326. Bostro¨m, C.-E., Gerde, P., Hanberg, A., Jernstro¨m, B., Johansson, C., Kyrklund, T., Rannug, A., To¨rnqvist, M., Victorin, K., Westerholm, R., 2002. Cancer risk assessment, indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air. Environmental Health Perspectives 110 (Suppl. 3), 451–488. Brack, W., Altenburger, R., Ensenbach, U., Moder, M., Segner, H., Schuurmann, G., 1999. Bioassay-directed identification of organic toxicants in river sediment in the industrial region of Bitterfeld (Germany) – a contribution to hazard assessment. Archives of Environmental Contamination and Toxicology 37 (2), 164–174.
Brack, W., 2003. Effect-directed analysis: a promising tool for the identification of organic toxicants in complex mixtures? Analytical and Bioanalytical Chemistry 377 (3), 397–407. Cuypers, C., Pancras, T., Grotenhuis, T., Rulkens, W., 2002. The estimation of PAH bioavailability in contaminated sediments using hydroxypropyl-[beta]-cyclodextrin and Triton X-100 extraction techniques. Chemosphere 46 (8), 1235–1245. Duggleby, R.G., 1981. A nonlinear regression program for small computers. Analytical Biochemistry 110 (1), 9–18. Dupuy, N., Barbry, D., Bria, M., Marquis, S., Vrielynck, L., Kister, J., 2005. 1H NMR study of inclusion compounds of phenylurea derivatives in [beta]-cyclodextrin. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 61 (6), 1051–1057. El Jay, A., Ducruet, J.-M., Duval, J.-C., Pelletier, J.P., 1997. A high-sensitive chlorophyll fluorescence assay for monitoring herbicide inhibition of photosystem II in the chlorophyte Selenastrum capricornutum: comparison with effect on cell growth. Archives of Hydrobiology 140 (2), 273–286. Environment Canada, 1992. Biological Test Method: Growth Inhibition Test Using the Freshwater Alga Selenastrum capricornutum. EPS 1/RM/25. Environment Canada, Ottawa, Ontario, 41 pp. Fai, P.B., Grant, A., Reid, B., 2007. Chlorophyll a fluorescence as a biomarker for rapid toxicity assessment. Environmental Toxicology and Chemistry 26 (7), 1520–1531. Gorski, P.R., Armstrong, D.E., Hurley, J.P., Shafer, M.M., 2006. Speciation of aqueous methylmercury influences uptake by a freshwater alga (Selenastrum capricornutum). Environmental Toxicology and Chemistry 25 (2), 534–540. Hickman, Z.A., Reid, B.J., 2005. Towards a more appropriate water based extraction for the assessment of organic contaminant availability. Environmental Pollution 138 (2), 299–306. Irie, T., Fukunaga, K., Pitha, J., 1992. Hydroxypropylcyclodextrins in parenteral use. I: lipid dissolution and effects on lipid transfers in vitro. Journal of Pharmaceutical Sciences 81 (6), 521–523. Ishiwata, S., Kamiya, M., 1999. Effects of humic acids on the inclusion complexation of cyclodextrins with organophosphorus pesticides. Chemosphere 38 (10), 2219–2226. Johnson, M.D., Weber, W.J., 2001. Rapid prediction of long-term rates of contaminant desorption from soils and sediments. Environmental Science and Technology 35 (2), 427–433. Leroy-Lechat, F., Wouessidjewe, D., Andreux, J.-P., Puisieux, F., Duchene, D., 1994. Evaluation of the cytotoxicity of cyclodextrins and hydroxypropylated derivatives. International Journal of Pharmaceutics 101 (1–2), 97–103. Liste, H.-H., Alexander, M., 2002. Butanol extraction to predict bioavailability of PAHs in soil. Chemosphere 46 (7), 1011–1017. Ma, J.Y., Wang, S.F., Wang, P.W., Ma, L.J., Chen, X.L., Xu, R.F., 2006. Toxicity assessment of 40 herbicides to the green alga Raphidocelis subcapitata. Ecotoxicology and Environmental Safety 63 (3), 456–462. ˜ a, A.M.D.L., Bautista, A., Aaron, J.J., 2002. An investigation of Mahedero, M.C., Pen inclusion complexes of cyclodextrins with phenylurea herbicides by photochemically-induced fluorescence. Analytical Applications. Journal of Inclusion Phenomena and Macrocyclic Chemistry 42 (1), 61–70. Okumura, Y., Koyama, J., Takaku, H., Satoh, H., 2001. Influence of organic solvents on the growth of marine microalgae. Archives of Environmental Contamination and Toxicology 41 (2), p123–128. Pavlic´, Zˇ., Stjepanovic´, B., Horvatic´, J., Persˇic´, V., Puntaric´, D., Culig, J., 2006. Comparative sensitivity of green algae to herbicides using erlenmeyer flask and microplate growth-inhibition assays. Bulletin of Environmental Contamination and Toxicology 76, 883–890. Reid, B.J., Stokes, J.D., Jones, K.C., Semple, K.T., 2000a. Nonexhaustive cyclodextrinbased extraction technique for the evaluation of PAH bioavailability. Environmental Science and Technology 34 (15), 3174–3179. Reid, B.J., Jones, K.C., Semple, K.T., 2000b. Bioavailability of persistent organic pollutants in soils and sediments – a perspective on mechanisms, consequences and assessment. Environmental Pollution 108 (1), 103–112. Sabate, J., Vinas, M., Solanas, A.M., 2006. Bioavailability assessment and environmental fate of polycyclic aromatic hydrocarbons in biostimulated creosotecontaminated soil. Chemosphere 63 (10), 1648–1659. Shigeoka, T., Sato, Y., Takeda, Y., Yoshida, K., Yamauchi, F., 1988. Acute toxicity of chlorophenols to green algae, Selenastrum capricornutum and Chlorella vulgaris,
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and quantitative structure activity relationships. Environmental Toxicology and Chemistry 7 (10), 847–854. Stokes, J.D., Wilkinson, A., Reid, B.J., Jones, K.C., Semple, K.T., 2005. Prediction of polycyclic aromatic hydrocarbon biodegradation in contaminated soils using an aqueous hydroxypropyl-beta-cyclodextrin extraction technique. Environmental Toxicology and Chemistry 24 (6), 1325–1330. Tang, J., Alexander, M.,1999. Mild extractability and bioavailability of polycyclic aromatic hydrocarbons in soil. Environmental Toxicology and Chemistry 18 (12), 2711–2714.
USEPA, 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. EPA-821-R-02-013. U.S. Environment Protection Agency, Washington, DC, 350 p. Vasseur, P., Pandard, P., Burnel, D., 1988. Influence of some experimental factors on metal toxicity to Selenastrum capricornutum. Toxicity Assessment 3 (3), 331–343. White, J.C., Kelsey, J.W., Hatzinger, P.B., Alexander, M., 1997. Factors affecting sequestration and bioavailability of phenanthrene in soils. Environmental Toxicology and Chemistry 16 (10), 2040–2045.
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Compatibility of hydroxypropyl-b-cyclodextrin with algal toxicity bioassays Patricia Bi Fai, Alastair Grant, Brian J. Reid* School of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
Compatibility of the biomimetic HPCD extraction method with algal cell growth inhibition bioassays to assess toxicity of reference toxicants and environmental relevant herbicides.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2008 Received in revised form 11 July 2008 Accepted 18 July 2008
Numerous reports have indicated that hydrophobic organic compound bioaccessibility in sediment and soil can be determined by extraction using aqueous hydroxypropyl-b-cyclodextrin (HPCD) solutions. This study establishes the compatibility of HPCD with Selenastrum capricornutum and assesses whether its presence influences the toxicity of reference toxicants. Algal growth inhibition (72 h) showed no significant (P > 0.05) difference at HPCD concentrations up to and including 20 mM. HPCD presence did not influence the toxicity of the inorganic reference toxicant (ZnSO4), with IC50 values of 0.82 mM and 0.85 mM, in the presence and absence of HPCD (20 mM), respectively. However, HPCD presence (20 mM) reduced the toxicity of 2,4-dichlorophenol and the herbicides diuron and isoproturon. These reductions were attributed to inclusion complex formation between the toxicants and the HPCD cavity. Liberation of complexed toxicants, by sample manipulation prior to toxicity assessment, is proposed to provide a sensitive, high throughput, bioassay that reflects compound bioaccessibility. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Selenastrum capricornutum Cyclodextrin Toxicity Inhibition Herbicide Dosing Realistic exposure scenario
1. Introduction Sediments, soils and other particulate matter are well-known sinks for moderately to strongly hydrophobic organic compounds (HOCs) (Brack, 2003). Many compounds such as polycyclic aromatic hydrocarbons (PAHs), monoaromatic hydrocarbons, herbicides and other pesticides fall within this broad grouping. These compounds are often cause for concern on account of their carcinogenicity, teratogenicity, mutagencity and adverse ecological effects (Bostro¨m et al., 2002). When present in sediment and soil exposure to HOCs is typically through both the aqueous and solid phase. Thus, toxicity may be manifested as a result of exposure via pore water, ingested sediment, and direct contact with sediment particles. These pathways are important for soil dwelling organisms, benthic organisms as well as for algae, daphnids and fish (in case of re-suspension of the particles) (Brack et al., 1999). As such, ecotoxicity assessment of pore water (or aqueous elutriates obtained from sediment/soil) neglects two of these three obvious possible pathways of exposure and is therefore likely to underestimate toxicity. Sediment toxicity assessment is therefore necessary. However, prior to screening with most algal or microbial bioassays, HOCs associated with sediment/soil generally require extraction into a solution phase. For meaningful toxicity assessment to be made, it
* Corresponding author. Tel.: þ44 1603 592357; fax: þ44 1603 591327. E-mail address: [email protected] (B.J. Reid). 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.07.015
is imperative that extraction methods provide (i) an extract that is compatible with the bioassay and (ii) an extract that meaningfully reflects the manner in which the chemical residues are partitioned within sediment or soil. Where hydrophobic compounds are being screened it is widely recognised that extraction using water as a solvent is limited by compound aqueous solubility. Thus, while an aqueous extract is compatible with many bioassays, it would only contain a fraction of the hydrophobic compound that is bioaccessible. As a consequence, when these extracts are screened for toxicity, the risk associated with the sediment/soil associated residues is underestimated. In contrast to such ‘mild’ extraction methods harsh organic solvent extractions are often used to strip hydrophobic chemicals from sediment/soil with a view to evaluating these total residues and their toxicity. Significantly, such extracts are inherently incompatible with biosensors on account of the toxicity associated with the extraction solvent itself. As a consequence dilution (to circa 1%) in water or growth media is required prior to toxicity assessment. As the toxicity threshold of a compound is approached (or surpassed) this dilution results in loss of sensitivity (or a ‘false negative result’ being reported). More importantly, it is now widely recognised that harsh solvent techniques overestimate the bioaccessible fraction of HOCs in sediment/soil and as a consequence overestimate the risk associated with these residues (Alexander, 2000). Over the past decade, less exhaustive techniques have been investigated with a view to establishing extraction methodologies that reflect the bioaccessible fraction of organic contaminants
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present in soils/sediments (White et al., 1997; Tang and Alexander, 1999; Liste and Alexander, 2002; Reid et al., 2000a,b; Stokes et al., 2005). While favourable correlations have been identified between extractability using ‘mild’ solvents and organic compounds bioaccessibility to a range of receptors (Tang and Alexander, 1999; Liste and Alexander, 2002), many of these techniques have failed to provide consistent and representative results when applied to different sediment/soils and/or compounds (White et al., 1997; Johnson and Weber, 2001). An alternative approach makes use of aqueous solutions of hydroxypropyl-b-cyclodextrin (HPCD) to extract non-polar organic contaminants via a shake extraction (Reid et al., 2000a; Cuypers et al., 2002; Stokes et al., 2005; Hickman and Reid, 2005; Allan et al., 2006; Sabate et al., 2006). Cyclodextrins are a group of torus- or bucket-shaped cyclic macromolecules composed of a-1,4-linked glucose units. They are highly soluble in water due to their array of hydroxyl functional groups on the exterior of the torus. However, the interior exhibits a hydrophobic character and can therefore host one or more hydrophobic molecules; thus forming a host–guest inclusion complex (Mahedero et al., 2002; Dupuy et al., 2005). Aqueous solutions of cyclodextrin have been used to dissolve a range of low solubility contaminants to greater than their aqueous solubility limits (Reid et al., 2000a,b; Dupuy et al., 2005; Hickman and Reid, 2005; Stokes et al., 2005; Allan et al., 2006). Significantly, strong relationships between HPCD extractability and microbial bioaccessibility have been reported for a wide range of hydrophobic organic compounds (Reid et al., 2000a,b; Mahedero et al., 2002; Dupuy et al., 2005; Stokes et al., 2005). In this research the compatibility of aqueous HPCD solutions with Selenastrum capricornutum, now Pseudokirchneriella subcapitata, Pseudokirchneriella subspicata or Raphidocelis subcapitata (Bengtson Nash et al., 2005; Ma et al., 2006; Pavlic´ et al., 2006), was investigated in algal growth inhibition assays (up to 72 h) in order to determine the optimum HPCD concentration in extracts for algal bioassay testing. Having established optimal HPCD concentrations (with respect to their toxicity being not significantly different to the media only control), algal growth inhibition assays were undertaken to investigate the effect of HPCD presence on the toxicities of inorganic and organic reference toxicants, namely, ZnSO4 and 2,4dichlorophenol (DCP), respectively. Subsequently, dose–response relationships were established for two herbicides, namely, diuron and isoproturon. These compounds represent a large and important class of herbicides widely used for pre- and post-emergent control of weeds (Mahedero et al., 2002; Dupuy et al., 2005).
maintained by transferring 1 mL of stock culture weekly to 50 mL of new growth medium and 3–4 days old cells used for all tests. The cell doubling time for this alga is approximately 10 h (Gorski et al., 2006). The Spectra MaxÒ M2 microplate reader from Molecular Devices Corporation (CA, USA) was used to obtain absorbance readings at 430 nm (A430) for the growth inhibition assay. All reported absorbance values have been corrected for background absorbance by microplate and algal medium. In order to establish bioassay compatibility with HPCD a stock solution of HPCD was prepared and serially diluted in MILLI Q water to obtain concentrations from 10 to 50 mM. These solutions were then used in the assessment of inhibition of growth (as described above) after exposure times of 24 h, 48 h and 72 h. Having established maximum HPCD concentrations that resulted in no inhibition in algal growth with respect to the media only controls, dose–response relationships in the presence (20 mM) and absence (media only) of HPCD were established for two reference toxicants, namely, an inorganic species (ZnSO47H2O; 0.03–17.4 mM (Zn)) and an organic compound (2,4-DCP; 0.005–10 mM). Finally, the dose–response relationships in the presence (20 mM) and absence (media only) of HPCD were established for two priority herbicides: diuron (0.01–0.4 mM) and isoproturon (0.02–1665 mM). For tests with all toxicants in the absence of HPCD, a stock toxicant concentration was prepared and serially diluted to obtain desired concentrations in the ranges given above. These were then introduced into microplates (a separate microplate for each toxicant) and test controls (algal growth medium) included. In order to obtain equivalent concentrations of toxicants in HPCD the toxicants were prepared to twice the desired concentrations and 40 mM HPCD prepared separately. These were then mixed in a 1:1 ratio before introducing the toxicants in the microplates. Test controls as well as solvent controls containing algal growth medium and HPCD (final concentration of 20 mM) were included. The rest of the procedure followed standard protocol (Environment Canada, 1992). Each experimental microplate had five replicates per test concentration (four replicates used as solvent controls were included).
2.3. Statistical analysis Growth inhibition after 72 h was obtained by subtracting the initial absorbance readings from the corresponding final value. One-way analysis of variance (ANOVA) was used to determine the maximum HPCD concentration that did not cause significant growth inhibition of S. capricornutum. Concentrations causing 50% growth inhibition (IC50) were calculated for each toxicant using the Excel macro REGTOX (http://eric.vindimian.9online.fr/en_index.html). The model used in this macro is written in the form proposed by Duggleby (1981) in which two parameters: Hill number (H) and EC50, are characteristics of the probability function written as follows: f ðxÞ ¼
xnH xnH
þ ECnH 50
where x is the dose and f(x) is a probability function of the dose varying from 0 to 1 with the dose. When x ¼ EC50, f(x) is 0.5. Confidence intervals were estimated using a non-parametric bootstrapping method. Two-way ANOVA was used to determine which variables (solvent type or
0.12 24 h 48 h 72 h
2. Materials and methods
0.10
An axenic culture of the S. capricornutum strain CCAP 278/4, equivalent to the American Type Culture Collection strain ATCC 22662 (Environment Canada, 1992) was obtained from the Scottish Association for Marine Science (SAMS) Research Services, UK. S. capricornutum was cultured in an incubator under continuous fluorescent lighting (quantal flux of 96 mmol photons/m2/s), at 24 C and 100 rpm. The growth medium, 1 AAP (Algal Assay Procedure) medium was prepared according to Blaise et al. (2000) and contained the following chemicals: CaCl2$2H2O, CoCl2$6H2O, CuCl2$2H2O, MgCl2$6H2O, FeCl3$6H2O, H3BO3, K2HPO4, MgSO4, MnCl2$4H2O, Na2EDTA$2H2O, Na2MoO4$2H2O, NaHCO3, NaNO3 and ZnCl2. Diuron and 2,4-DCP were PESTANALÒ analytical standards obtained from Sigma–Aldrich (Dorset, UK). Isoproturon (ALPHA IPU 500) was obtained as a suspension concentrate formulation 500 g/L (46.3% w/w) from Makhteshim Agan (Berkshire, UK). All other chemicals were reagent grade and obtained either from Sigma–Aldrich or Fisher Scientific (Leicestershire, UK).
Growth (A430) a.u.
2.1. S. capricornutum and chemicals
0.08
∗
0.06
∗ ∗
0.04
0.02
A microplate-based algal growth inhibition (specifically, inhibition of cell reproduction) assay was carried out according to the standard protocol set by Environment Canada (1992) with little modification in the algal inoculum preparation as in Fai et al. (2007). A continuous supply of ‘‘healthy’’ cells for tests was
∗
30
40
∗
0.00 0
2.2. Algal growth inhibition bioassays
∗
10
20
50
[Hydroxypropyl-β-Cyclodextrin] mM Fig. 1. Growth inhibition of S. capricornutum exposed to increasing concentrations of hydroxypropyl-b-cyclodextrin following 24 h (white), 48 h (black) and 72 h (hatched). An asterisk above a bar indicates significant difference with respect to control at 0.05 level; n ¼ 5; error bars ¼ 1 standard deviation; a.u. ¼ arbitrary unit.
Growth inhibition (%)
P.B. Fai et al. / Environmental Pollution 157 (2009) 135–140
100
A
B
137
C
50
0
Growth medium HPCD -50 10-8
10-7
10-6
10-5
ZnSO4.7H2O (M)
10-4
Growth medium HPCD 10-8
10-7
10-6
10-5
Growth medium HPCD
10-4
10-8
10-7
ZnSO4.7H2O (M)
10-6
10-5
10-4
ZnSO4.7H2O (M)
Fig. 2. Dose–response for S. capricornutum exposed to the reference toxicant ZnSO4$7H2O in media (open) and 20 mM hydroxypropyl-b-cyclodextrin (closed) at 24 h (A), 48 h (B) and 72 h (C). Error bars ¼ 1 standard deviation.
toxicant concentrations) explained observed inhibition in growth with respect to toxicant and solvent influence. Statistical analyses were done using Microsoft Excel and SPSS 12.0.1 for Windows.
3. Results and discussion 3.1. HPCD toxicity Dose–responses were apparent following 24 h of incubation but were more resolved following 48 h and 72 h (Fig. 1). In the media only controls and at all HPCD concentrations it was noted that while considerable growth inhibition occurred between 24 h and 48 h, growth inhibition was much greater between 48 h and 72 h. HPCD concentrations of 10 mM and 20 mM did not significantly (P > 0.05) inhibit S. capricornutum growth at any of the exposure times relative to the control (Fig. 1). However, HPCD concentrations 30 mM consistently resulted in significant (P < 0.05) inhibition in algal growth at 48 h and 72 h with respect to the media only control. Toxicity, with respect to cyclodextrins, has been proposed on account of inclusion complexation of membrane components, particularly cholesterol and phospholipids resulting in destruction of the cell membrane leading to cytotoxicity (LeroyLechat et al., 1994). By the same mechanism, cyclodextrins give rise to perturbation in the surface tension of lipid monolayers and destruction of liposomes (Irie et al., 1992). In addition to these mechanisms, it was noted (observational/not measured) that aqueous solutions of HPCD became increasingly more viscous with increasing concentration. Increased viscosity would also have impaired gas diffusion (specifically CO2) and as a consequence may have limited photosynthesis. A HPCD concentration of 20 mM was selected for all further experiments using reference toxicants and herbicides. 3.2. Dose–response: reference toxicants Dose–responses were apparent for ZnSO4 following 24 h although there were sometimes large confidence limits associated with measurements at 24 h (attributed to the small extent of algal growth) (Fig. 2). The dose–response did not change substantially from 24 to 72 h (Fig. 2). Indeed, IC50 values were almost identical at 48 h and 72 h. The effect of ZnSO4 on S. capricornutum growth inhibition was observed within the same concentration range, irrespective of the presence or absence of HPCD (Fig. 2), with IC50 values at 48 h and 72 h for ZnSO4 in growth media or HPCD being virtually identical. Two-way ANOVA indicted ZnSO4 concentration alone explained 97% of its effect on growth inhibition; the effect of solvent (as well as solvent–concentration interaction) on growth inhibition was not significant at the 0.05 level. The aqueous 72 h IC50 value derived in this work (Table 1) was consistent with Zn
IC50 values previously reported in the literature (0.76–1.5 mM; Vasseur et al., 1988). Regarding 2,4-DCP, dose–responses were again distinguishable, albeit with larger confidence limits, following 24 h. The 72 h IC50 growth inhibition value derived here for 2,4-DCP in media only (11.7 mM) was consistent with 2,4-DCP aqueous IC50 values previously reported (86 mM; Shigeoka et al., 1988). As was the case for ZnSO4 exposure times of 48 h and 72 h made very little difference to the determined IC50 value. In contrast, however, to the inorganic reference toxicant (ZnSO4), 2,4-DCP toxicity was markedly influenced by the presence of HPCD (20 mM). Onset of inhibition to algal growth in the presence and absence of HPCD was observed at 2,4DCP concentrations around 104 mM and 105 mM, respectively; with 100% algal growth inhibition being observed at 2,4-DCP concentrations around 103 mM and 104 mM, respectively (Fig. 3). Thus, 2,4-DCP toxicity was decreased by an order of magnitude when HPCD was present. IC50 values in the absence/presence of HPCD were observed to differ by a factor of 41 (Table 1). The observed decrease in toxicity was expected given the affinity between 2,4-DCP molecules and the HPCD cavities. As a consequence of 2,4-DCP/HPCD inclusion complex formation the genuine solution phase 2,4-DCP concentration would have been significantly lower; it follows that algal cells were in effect being exposed to lower concentrations and toxicity decreased correspondingly (Fig. 3). Zinc would not have been subject to complexation/association with HPCD and as a consequence toxicity was not influenced (Fig. 2).
3.3. Dose–response: herbicides Inhibition of S. capricornutum growth in response to increasing concentrations of diuron (Fig. 4) and isoproturon (Fig. 5) was observed to be proportional to herbicide concentration both in the presence and absence of HPCD. However, as was the case with 2,4DCP (Fig. 3) there was a marked decrease in herbicide toxicity in the presence of HPCD. Indeed, no inhibition in algal growth was observed for these toxicants in the presence of HPCD at
Table 1 Selenastrum capricornutum IC50 (72 h) growth inhibition values and associated 95% confidence intervals obtained for all toxicants Toxicant
72 h IC50 growth inhibition (95% confidence limits) mM Media (A)
HPCD (B)
ZnSO4 2,4-Dichlorophenol Diuron Isoproturon
0.85 11.7 0.03 0.03
0.82 475 0.4 10.8
(0.78–0.94) (10.7–20.6) (0.03–0.04) (0.02–0.05)
(0.76–0.9) (415–533) (0.3–0.5) (6.4–20.1)
Proportion (B/A)
0.96 41 13 360
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Growth inhibition (%)
138
150
Growth medium HPCD
A
B
100
100
100
C
50
50
50
0 0
Growth medium HPCD
0 10-6
10-5
10-4
10-3
10-2
10-1
10-6
2,4-Dichlorophenol (M)
10-5
10-4
10-3
10-2
10-1
Growth medium HPCD
-50 10-6
10-5
2,4-Dichlorophenol (M)
10-4
10-3
10-2
10-1
2,4-Dichlorophenol (M)
Fig. 3. Dose–response for S. capricornutum exposed to the reference toxicant 2,4-dichlorophenol in media (open) and 20 mM hydroxypropyl-b-cyclodextrin (closed) at 24 h (A), 48 h (B) and 72 h (C). Error bars ¼ 1 standard deviation.
concentrations that resulted in 100% algal growth inhibition in media only samples. Toxicity was abated to a larger extent in the case of isoproturon (an ‘up-concentration’ shift in algal growth inhibition of between two and three orders of magnitude; Fig. 5) compared to diuron (an ‘up-concentration’ shift in algal growth inhibition of about one order of magnitude; Fig. 4). Correspondingly, the 72 h IC50s for isoproturon and diuron in HPCD were, respectively, 360 and 13 times greater than their respective IC50s in growth media (Table 1). The aqueous IC50 values derived in this work (Table 1) were consistent with diuron and isoproturon IC50 values previously reported in the literature: diuron (0.03 mM; El Jay et al., 1997; isoproturon: 0.24 mM; Pavlic´ et al., 2006). In keeping with the observations for 2,4-DCP, toxicity decreased for both of the phenylurea herbicides (diuron and isoproturon) tested when present with HPCD (20 mM). Previous studies have demonstrated that b-cyclodextrin and HPCD form relatively stable inclusion complexes with phenylurea herbicides (Ishiwata and Kamiya, 1999; Mahedero et al., 2002; Dupuy et al., 2005). Thus, the reduction in toxicity, as observed in the present study, strongly suggests that the inclusion complexes decreased the opportunity for compound–algae interaction of the organic compounds tested. The inclusion behaviour of b-cyclodextrin and herbicides have been reported to depend on the size fit between ‘‘guest’’ and host resulting in an inclusion ratio of 1:1 for fenuron, monuron and diuron, and an inclusion ratio of 2:1 for isoproturon (Dupuy et al., 2005). Thus, isoproturon would have been more effectively precluded from the free aqueous phase on account of this 2:1 complexation compared to the situation for diuron where a less shielded 1:1 complex would have been formed.
environmental contaminants (Environment Canada, 1992; Blaise et al., 2000; USEPA, 2002). Results presented here have indicted that concentrations of HPCD 20 mM resulted in no significant growth inhibition of this micro algal species with respect to media only controls (Fig. 1). Significantly, aqueous HPCD extraction has been shown to closely mimic the mass transfer mechanisms that govern the bioavailability of HOCs in sediment/soil (Reid et al., 2000a,b; Cuypers et al., 2002; Hickman and Reid, 2005; Stokes et al., 2005; Allan et al., 2006; Sabate et al., 2006). Thus, it is arguably representative to use aqueous HPCD solutions to extract HOCs from sediment/soil with a view to using these extracts in toxicity assessment; as such an approach will facilitate the toxicity assessment of the entire bioaccessible pool, rather than the fraction present in the interstitial/pore-water. Clearly, loss of sensitivity by the sensor when exposed to organic toxicants in the presence of HPCD (Figs. 3–5) has implications for the application of S. capricornutum in the screening of HPCD aqueous extracts derived from sediments and soils. However, it should be borne in mind (given the hydrophobic properties of many pesticides) that soil/sediment concentration will be much greater than those in the aqueous pore water phase (on account of partitioning in favour of the mineral and organic components present). The option remains to use an aqueous cyclodextrin extraction to liberate the putative bioaccessible fraction of toxicants with a view to their quantification by a chemical means and to complement these measurements with toxicity screening on a second extract derived by conventional solvent extraction methods. However, we and others suggest that when screening an extract it is, arguably, better to screen one that is representative of the entire bioaccessible fraction, albeit with reduced sensor sensitivity, than to screen an extract that does not reflect the entire bioaccessible pool with higher sensor sensitivity. Although not investigated in this study, liquid–liquid–liquid exchange could be used to abate the issues of toxicity suppression in the presence of
3.4. Towards more realistic sediment/soil toxicity assessment S. capricornutum is one of the major standard test organisms used in toxicity tests internationally to assess the risk of
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Fig. 4. Dose–response for S. capricornutum exposed to the herbicide diuron in media (open) and 20 mM hydroxypropyl-b-cyclodextrin (closed) at 24 h (A), 48 h (B) and 72 h (C). Error bars ¼ 1 standard deviation.
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Fig. 5. Dose–response for S. capricornutum exposed to the herbicide isoproturon in media (open) and 20 mM hydroxypropyl-b-cyclodextrin (closed) at 24 h (A), 48 h (B) and 72 h (C). Error bars ¼ 1 standard deviation.
HPCD while maintaining the value of the HPCD extraction as a biomimetic for bioaccessibility assessment (Reid et al., 2000a,b; Cuypers et al., 2002; Mahedero et al., 2002; Dupuy et al., 2005; Hickman and Reid, 2005; Stokes et al., 2005; Allan et al., 2006). Thus, a HPCD extract could be exchanged into a strongly solvating, and water immiscible, solvent (such as dichloromethane (DCM)), the DCM evaporation to dryness and toxicants subsequent redissolving in a low toxicity solvent such as dimethyl sulfoxide (Okumura et al., 2001) prior to dilution in media and toxicity screening. Of course this sort of sample manipulation is not without its disadvantages, for example, solvent consumption as well as loss of toxicants during transfer and drying, and possibility of low solubility compounds not re-dissolving in the final solvent. Alternatively, incorporation of a step in sample preparation to release the organic toxicants from the cyclodextrin cavities may abate the reduction in algal sensitivity. Based on these initial results further studies are warranted to investigate applicability to a wider range of pesticides and other HOCs (e.g. PAHs and priority monoaromatic compounds such as benzene, toluene, ethylbenzene and xylene) and the possibility of enhancing algal sensitivity by further sample manipulation as described above. Acknowledgements The authors would like to thank the LINK Bioremediation program and the Natural Environment Research Council (NERC) for contributing funding towards this work (grant nos. NER/T/S/2003/ 00107 and NE/C513185) and the UK Commonwealth Scholarship Commission. References Alexander, M., 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environmental Science and Technology 34 (20), 4259–4265. Allan, I.J., Semple, K.T., Hare, R., Reid, B.J., 2006. Prediction of mono- and polycyclic aromatic hydrocarbon degradation in spiked soils using cyclodextrin extraction. Environmental Pollution 144 (2), 562–571. Blaise, C., Forget, G., Trottier, S., 2000. Toxicity screening of aqueous samples using a cost-effective 72-h exposure Selenastrum capricornutum assay. Environmental Toxicology 15 (4), 352–359. Bengtson Nash, S.M., Quayle, P.A., Schreiber, U., Muller, J.F., 2005. The selection of a model microalgal species as biomaterial for a novel aquatic phytotoxicity assay. Aquatic Toxicology 72 (4), 315–326. Bostro¨m, C.-E., Gerde, P., Hanberg, A., Jernstro¨m, B., Johansson, C., Kyrklund, T., Rannug, A., To¨rnqvist, M., Victorin, K., Westerholm, R., 2002. Cancer risk assessment, indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air. Environmental Health Perspectives 110 (Suppl. 3), 451–488. Brack, W., Altenburger, R., Ensenbach, U., Moder, M., Segner, H., Schuurmann, G., 1999. Bioassay-directed identification of organic toxicants in river sediment in the industrial region of Bitterfeld (Germany) – a contribution to hazard assessment. Archives of Environmental Contamination and Toxicology 37 (2), 164–174.
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