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Toxicity evaluation by using intact sediments and sediment extracts
Marine Pollution Bulletin 50 (2005) 660–667 www.elsevier.com/locate/marpolbul
Toxicity evaluation by using intact sediments and sediment extracts Ann-Kristin Eriksson Wiklund *, Brita Sundelin Dag Broman Department of Applied Environmental Science, Stockholm University, SE-10691 Stockholm, Sweden
Abstract The toxicity of intact sediments and sediment extracts, from both an uncontaminated site and a site contaminated by pulp-mill effluents, was tested in a five months study. The deposit-feeding amphipod Monoporeia affinis was exposed in soft-bottom flowthrough water microcosms. To examine potential toxicity a set of reproduction endpoints was used including fecundity and different embryo aberrations such as malformed eggs. Among extracts, the aliphatic/monoaromatic and diaromatic fractions along with the total extract were shown to cause the highest toxicity measured as malformed eggs, while the polyaromatic fraction caused toxicity at background levels. A comparison between sediment extracts and pulp mill contaminated intact sediment, however, showed no toxicity of the intact sediment. Thus, the extraction procedure seems to increase bioavailability and subsequently toxicity as compared to the intact sediments in situ. In toxicity testing using fractionated extracts of sediments in a toxicity identification evaluation (TIE) procedures, caution should therefore be taken when assessing bioavailable contaminants in contaminated areas. This should be taken in account both in determining remediation priorities as well as in ecological risk assessments. 2005 Elsevier Ltd. All rights reserved. Keywords: Toxicity; Sediment extracts; Intact sediments; TIE; ERA
1. Introduction The pulp and paper industry was one of the main sources of organic contaminants in the Bothnian Sea in the 20th century (Jonsson, 2003). The most obvious effect found in early field studies was an increase in organic loading of the receiving waters resulting in high oxygen demand in both water column and sediments (Rosenberg, 1976). Reduced discharges of oxygen consumptive substances have led to recovered health status in both fish and sediment-living bottomfauna. Discriminating between effects of various bleaching processes has been the major issue in both field studies and laboratory experiments during the last decades. Severely deformed skeletons in fish was one of the first reported serious toxic effects of bleached kraft mill effluents *
Corresponding author. E-mail address: [email protected] (A.-K. Eriksson Wiklund). 0025-326X/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2005.02.030
(BKME) (Bengtsson, 1988), but in the last few years the focus has changed to reproduction disorders due to endocrine disrupting chemicals. The recent papers on reproduction effects of pulp mill effluent have mainly been concerned with fish (McMaster et al., 1996; Munkittrick et al., 2003; Jenkins et al., 2003; Sepu´lveda et al., 2003, 2004), while there are few studies on invertebrates, e.g. Sibley et al. (1997). Among those are laboratory experiments with pulp and paper mill effluents and sediments as well as field studies in recipients having demonstrated toxic effects on amphipod reproduction (Sundelin, 1988, 1989; Sundelin and Eriksson, 1998). When examining sediment toxicity, benthic species in whole sediment exposures are considered to be the best indicators (Burton et al., 1992) due to their direct contact with sediment and interstitial water. Among benthic species, amphipods are considered as particularly sensitive since they are the first to disappear from contaminated sediments (Swartz et al., 1982). The usefulness of amphipods is well established and they are often used
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in toxicity assessments in fresh, estuarine and marine waters (Chapman et al., 1998). The high lipid content of the amphipod Monoporeia affinis makes this species important in the transfer of organic chemicals from sediments upwards in the food chain as well as in the transfer of organic chemicals from parent to offspring (Eriksson Wiklund et al., 2003). Thus, we consider M. affinis as a relevant test species in bioassays for sediment toxicity in the Baltic. Toxicity identification and evaluation (TIE) procedures were developed by the US-EPA (Mount and Andersson-Carnahan, 1988, 1989; Norberg-King et al., 1992; Mount and Norberg-King, 1993). These procedures were originally designed for aqueous samples but methods for sediment testing are continuously being developed (Burgess et al., 2000). This strategy has grown increasingly popular since the 1990s, especially when pinpointing substances in multiple endocrine disrupting mixtures (Burnison et al., 2003). The TIE procedures are mainly used in connection with effluent discharge regulation, but are also used in environmental risk assessment (ERA) as well as in remediation work. From the TIE procedures, several bioassay/effect directed analyses (BDA/EDA) concepts have being developed. When examining sediment toxicity by these methods, sediment elutriates or sediment extracts in aqueous test procedures are normally used (e.g. Brack et al., 1999; Burgess et al., 2000; Phillips et al., 2003). 1.1. Aim and strategy Several field studies performed 5–10 years before this study have demonstrated toxicity in the pulp mill area in terms of a changed structure of the meiofauna community and an increased level of malformed embryos of M. affinis (Sundelin and Eriksson, 1996, 1998). Large industrial efforts in altering and improving processes have since then been performed, i.e. changing the bleaching process from chlorine to elemental chlorine free (ECF) bleaching as well as substantially reducing effluents. In this study the sediment-dwelling amphipod M. affinis was exposed to five experimental treatments. Three treatments were based on samples from a pulp mill recipient using natural intact sediments, unfractionated and fractionated sediment extracts of varying aromaticity, while two treatments were based on samples from a reference site using natural intact and unfractionated sediment extracts. To simulate the mother system in situ and accordingly achieve ecologically relevant experimental conditions, we used the strategy of mixing the sediment extracts into a sediment matrix. The aims were to identify and compare the toxicity of the different sediment extracts and to examine whether the toxicity is found in the fractions or remains in the unfractioned extract (total extract). Furthermore, we wanted to examine the possibility of assessing bioavailable and toxic com-
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pounds and making field predictions by comparing the toxicity of untreated intact sediment with unfractionated sediment extract.
2. Material and methods 2.1. Test species M. affinis is found both in the reference area and in the vicinity of the contaminated area, thus fulfilling the requirement of ecological significance addressed by Batley et al. (2002) and underlined by Chapman (2002). M. affinis has been the dominating soft bottom species of the Baltic, further strengthening its relevance as a test species. It is a deposit feeder using the surface sediment as its main food source (Byre´n et al., 2002). The spring phytoplankton bloom is the most important food contribution but the amphipods continue to accumulate lipids throughout the summer months. The reproduction cycle starts in the late summer/early autumn with gonad maturation (oogenesis and spermatogenesis) triggered by the reduction of light (Segerstra˚le, 1970, 1971). Mating occurs in November, followed by three months of embryo development. The method used in this paper is a method for biomonitoring recommended by ICES and applied for effect monitoring of contaminated sediments in the Swedish ¨ stersjo¨, 1994–2003; Marine National Programme (O Bottniska viken, 1994–2000). The method includes different reproduction variables, e.g. the embryonic development in M. affinis. The variable Ômalformed embryosÕ has proven to be a powerful contaminant monitor in both laboratory experiments and field studies, while the variable Ôfemales carrying dead broodsÕ is a monitor of hypoxia tested in both laboratory experiments and field studies (Eriksson Wiklund and Sundelin, 2001, 2004). 2.2. Microcosm system The microcosms used were two-litre Erlenmeyer flasks containing approximately 4 cm of sediment (Sundelin, 1983). The microcosms were connected to a continuous water flow of 2.2 l h1. The incoming water was collected at 37 m depth and followed the natural temperature regime of 2–8 C during the experiment. Light was adjusted to natural day length. 2.3. Sampling Reference sediment, to be mixed with extracts or covered with intact pulp mill sediments, was collected with a modified Ockelman dredge, mesh size 450 lm, (Blomqvist and Lundgren, 1996) on a reference site within the Swedish National Monitoring Programme at position 5847 0 4700 N, 1743 0 9300 E. Intact contaminated
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sediments and sediments for extraction and fractionation were collected with a Kajak-corer close to the pulp mill at position 6137 0 N, 1708 0 E. Amphipods were collected at position 5850 0 5000 N, 1747 0 1600 E. To collect amphipods sediment was sieved through a, 1.0 mm mesh size net, while the reference sediment was sieved through a 0.5 mm mesh size net to avoid predators. 2.4. Chemical fractionation The wet sediment samples were placed in preextracted cellulose thimbles and Soxhlet extracted with toluene for 24 h. The apparatus was equipped with a Dean–Stark trap for collecting water. After toluene evaporation (<10 Torr; 35–40 C), the residue was cleaned on a silica column with n-hexane as a mobile phase. To fractionate the extracts we used a HPLCsystem (Zebu¨hr et al., 1989) consisting of a MERCKHitachi L-6200 pump, Rheodyne 7125 valve injector, Rheodynhe 7067005 automatic valve station and switching valves and MERCK-Hitachi 1-4200 UV–VIS detector. A semipreparative Bondpak amino column (300 · 7.8 mm, Waters) was used with n-hexane as the mobile phase. Three fraction were collected: aliphatics/monoaromatics, diaromatics and polyaromatics. At a rate of 3 ml min1, the aliphatics/monoaromatics were eluted first, followed by the diaromatics in a forward direction (Zebu¨hr et al., 1989). The column was then back flushed (5 ml min1) and the polyaromatics eluted as one narrow peak. The compounds were monitored at 254 nm. Low-boiling aliphatics/monoaromatics were probably lost during the evaporation steps. 2.5. Experimental design The amphipods were exposed to nine different series (five replicates of each), including an aliphatic/monoaromatic fraction, a diaromatic fraction, a polyaromatic fraction, a recombination of those three fractions, a total unfractionated extract, an intact contaminated sediment, a total reference unfractioned extract, an intact reference sediment, and a blank extract. Sediment extract originating from 204 g dry weight sediment (corresponding to the amount of added intact sediment, see below) was dissolved in 25 ml acetone before being slowly blended in 550 ml of continuously stirred reference sediment. The sediment-extract mixture was stirred for an additional hour and then allowed to settle for one week. To each microcosm, 100 ml sediment extract mixture or 100 ml intact sediment was added on top of 300 ml reference sediment and allowed to settle in the aquaria for two days before the water flow of 2.2 l h1 was started. To avoid effects of acetone the test organisms (50 preadults) were added after one more day. The experiment started in the middle of August and ended in the middle of January.
2.6. Biological analyses Adults and embryos of M. affinis were examined for fertilisation rate, fecundity, egg developmental stage and different aberrations from normal development such as malformed embryos, dead and undifferentiated eggs and females carrying a dead brood according to Sundelin and Eriksson (1998). Variables studied in females were fertilisation success, fecundity (eggs/female), and dead brood. Embryos were examined for the developmental stage, percentage of malformed embryos, unfertilised/ undeveloped (henceforth called undifferentiated) eggs and dead eggs and embryos (for further information, see Sundelin and Eriksson, 1998). 2.7. Statistical evaluation The statistical evaluations were performed by logit analyses (embryo variables and dead brood) (Aldrich and Nelson, 1984; Demaris, 1992). Analysis of variance was used to test the non-dichotomous data, egg development and fecundity. Significance of a < 0.05 was applied.
3. Results Different types of malformations were identified, several of them caused by malfunction of the inner vitellin membrane, resulting in leakage of stored lipids into the space between egg and vitellin membranes. Other less frequently occurring malformations were enlarged embryos, shortened extremities appendages, deformed midguts and eyespots. In the statistical analysis the different types of embryo malformations have been merged into one category. There was no difference in toxicity, measured as percentage of malformed embryos, between the extracted reference sediment and the intact reference sediment or the blank extract. In all further comparisons the extracted reference sediment will be used as reference. Large differences in toxicity were found between experimental sediment series, the aliphatic/monoaromatic fraction, diaromatic fraction and the total unfractionated extract from pulp mill recipient exerting the largest toxicity with approximately 11%, 14% and 12% malformed embryos respectively. All extracts from the contaminated site exerted toxicity as compared to the sediment extract from the reference site (Fig. 1a, Table 1). The contaminated unfractionated total extract was more toxic than the polyaromatic fraction but did not differ from the aliphatic/monoaromatic fraction and the diaromatic fraction. The toxicity of the recombined extract was significantly lower than that of the single fractions. The polyaromatic fraction exerted lower toxicity than both the aliphatic/monoaromatic fraction and the diaromatic fraction, which did not differ in
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Fig. 1. Results from 5 months of exposure of the amphipod Monoporeia affinis to series of fractionated and unfractionated sediment extracts as well as intact sediments. The bars represent mean values of five replicates + standard error. (a) malformed embryos, (b) undifferentiated embryos, (c) dead eggs.
toxicity. Unlike the extracted pulp mill sediment, the intact sediment from the contaminated site did not exert any significant toxicity (4% malformed embryos) compared to the reference sediment extract. The toxicity of the intact reference sediment and the blank extract did not differ significantly from that of the extracted reference sediment. Undifferentiated eggs were significantly more abundant in the intact reference sediment and the recombined extract of pulp mill sediment compared to the extracted reference sediment (Table 1, Fig. 1b). Other sediments did not differ from the extracted reference sediment. A lower frequency of dead eggs was found in the recombined fractionated extracts, unfractionated extract and intact contaminated sediment compared to the extracted reference sediment (Table 1, Fig. 1c). In the diaromatic and polyaromatic fractions and the intact reference
sediment, however, a higher frequency of dead eggs was found.
4. Discussion 4.1. Toxicity The major part of the toxicity in this pulp mill recipient study is found in the aliphatic/monoaromatic fraction and the diaromatic fraction of the sediment. In other parts of the Baltic Sea the major part of the toxicity has been found in the polyaromatic fraction (Sundberg et al., 2005). The total extract, on the other hand, does not show any additional toxicity. Knowledge about specific compounds in the aliphatic/monoaromatic fraction is limited, but a relatively low persistence of these
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Table 1 Statistical parameters from the logit analyses Dependent variable
Treatment
Regression SE coefficient
p
Malformed
Intact ref sediment Aliphatic extract Diaromatic extract Polyaromatic extract Recombined extract Total extract Intact recipient Blank extract
0.41 1.35 1.73 0.69 0.85 1.40 0.21 0.29
0.21 0.20 0.16 0.17 0.17 0.15 0.21 0.19
ns <0.001 <0.001 <0.001 <0.001 <0.001 ns ns
Dead
Intact ref sediment Aliphatic extract Diaromatic extract Polyaromatic extract Recombined extract Total extract Intact recipient Blank extract
0.93 0.97 0.96 0.67 0.52 0.88 0.91 0.92
0.22 0.56 0.22 0.21 0.29 0.31 0.38 0.21
<0.001 <0.001 0.01 <0.001 ns 0.05 <0.001 <0.001
Undifferentiated
Intact ref sediment Aliphatic extract Diaromatic extract Polyaromatic extract Recombined extract Total extract Intact recipient Blank extract
1.14 0.41 0.26 0.06 0.92 0.10 0.14 0.15
0.17 0.34 0.24 0.20 0.16 0.18 0.21 0.21
<0.001 ns ns ns <0.001 ns ns ns
compounds has been recorded in an earlier study (Zebu¨hr pers. comm.). In the diaromatic fraction compounds like PCBs, PCDDs and PCDFs will be retained (Zebu¨hr et al., 1989). Results from an earlier survey showed increased sediment concentrations of PCDD/F in the vicinity of the pulp mill (Na¨f et al., 1992). PAHs, expected to be retained in the polyaromatic fraction, are not related to pulp mill effluents. This is reflected in the low toxicity found in this fraction. There are several possible explanations of the difference in toxicity between fractions. One possible explanation is a higher toxicity or higher concentrations of toxic compounds in the low aromatic fractions. A lower binding capacity to the sediment, making these substances more bioavailable, is another possible explanation. Complex contaminant mixtures may not exert an additive effect but act both synergistically and/or antagonistically (Altenburger et al., 2003). In this experiment two of the fractions showed a higher toxicity than the recombination of fractions, indicating antagonistic behaviour. Nirmala et al. (1999) observed antagonistic effects on embryological survival in Japanese medaka, while Nasci et al. (2000) observed antagonistic effects in clams exposed to natural sediments including complex contaminant mixtures. Bosveld and Vandenberg (1994) reported that several PCDD/Fs in combination with PCBs might cause both synergistic and antagonistic effects. There is a large difference in toxicity between the recombined and the unfractionated total extract. The
unfractionated extract contains polar substances extracted by toluene that are omitted in the fractions and in the recombination of the fractions by a cleanup process on silica-column. The unfractionised extract might contain substances like phenols and chloroguaiacols. Bleached kraft mill effluents affect the reproductive physiology of fish by reducing concentrations of plasma sex steroids, gonadosomatic index and expression of secondary sex characteristics (Sepu´lveda et al., 2003). The mechanisms behind the effects in invertebrates are unknown. Both laboratory experiments and field studies have shown malformed embryos to be a powerful tool for monitoring effects of contaminants (Sundelin, 1983, 1988; Sundelin and Eriksson, 1998), a conclusion that was further confirmed in this study. Effects of pulp mill effluents on fish fecundity and embryo development have been reported (Sepu´lveda et al., 2003). Our experiment showed no similar effects on amphipod fecundity. Since the contaminant exposure in the fish experiments occurred after vitellogenesis, when lipophilic chemicals are transferred into oocytes, the authors hypothesised a sub-optimal timing as a possible cause of the lack of effects in their experiments (Sepu´lveda et al., 2003). The timing could not, however, cause the lack of effects on fecundity and embryo development in our experiments covering a complete reproductive cycle including lipid transfer into in the oocytes. In field studies a close relationship between food availability and lipid content and fecundity of amphipods has been shown (Sundelin et al., 2003). Possible toxic effects of contaminants on fecundity might thus be confounded by availability of food resources. Fecundity thus for this species must be considered as a non-optimal field monitor of toxicity (Eriksson Wiklund and Sundelin, 2004). 4.2. Comparison with in situ conditions The great difference in toxicity between the intact sediments and the fractionated sediments indicates increased bioavailability due to the extraction procedure. The strategy of identifying the toxicity in different fractions of extracted sediment is particularly valuable in sediments or wastewater contaminated by various unknown chemicals, where it may facilitate the search for responsible toxicants. This strategy has also been successful in examining the outcome of remediation work (Eriksson et al., 1996). Observing toxicity in one specific fractionated extract, however, does not make it possible to predict that this fraction is the most bioavailable in situ. Results must be validated by field measurements or bioassays using intact sediments (Anderson et al., 2001). Bioavailability and subsequent toxicity may differ between experiments based on intact sediments and
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sediments spiked with contaminants. Contaminants bound into natural intact sediments are generally more strongly sorbed to the sediment and thus more slowly desorbed than contaminants in unaged laboratory spiked sediment. Also, greater toxicity is generally shown in laboratory tests than in in situ processes (Sasson-Brickson and Burton, 1991). Several authors (e.g. Simpson et al., 2000) therefore call for caution when extrapolating results from laboratory experiments to natural conditions. Several authors have drawn attention to certain weaknesses inherent in the TIE procedures, i.e. increased bioavailability caused by chemical manipulations and difficulties in extrapolating laboratory results to field conditions (Desbrow et al., 1998; Ho et al., 2002). Also, the identification of a compound in a fraction (the confirmation step in the TIE) does not prove that this compound per se caused the effect (Brack, 2003). In traditional toxicity testing a common strategy is to spike sediments with contaminants, but in a procedure aiming at identifying potent chemicals in contaminated sediments, the fractionated extracts are generally tested in aqueous solutions or pore water. This methodology might be inappropriate for sediment living organisms, which are generally exposed to two contaminant sources, the solid phase that often constitutes their food and the interstitial and overlying water (Chapman et al., 2002). As far as we know this is the first study where sediment extracts are mixed into the sediments and compared to intact natural sediment. This process closely mimics natural conditions, thus providing high ecological relevance. The results clearly demonstrate that caution is needed before extrapolating results from TIE/ BRD/EDA procedures to natural conditions. Since these procedures are developed as tools in risk assessment, an overestimated risk based on a delusive degree of bioavailability might lead to unnecessary remediation work and restoration activities. Acknowledgments We would like to acknowledge the following persons for skilful performances: Carina Na¨f participated in the early phase of this project and is also a co-author of an earlier paper, Eva Ha˚kansson assisted during the amphipod exposure and Kerstin Grunder extracted the sediments and made the fractionations. References Aldrich, J.H., Nelson, F.D., 1984. Linear Probability, Logit and Probit Models. Series Quantitative Applications in the Social Science. Sage Publications Inc., Newbury Park, California, USA. Altenburger, R., Nendza, M., Schu¨u¨rmann, G., 2003. Mixture toxicity and its modeling by quantitative structure–activity relationships. Environmental Toxicology and Chemistry 22, 1900–1915.
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effects with polychlorinated biphenyls on reproductive processes in Japanese medaka (Oryzias latipes). Environmental Toxicology and Chemistry 18, 717–721. Norberg-King, T.J., Mount, D.I., Amato, J.R., Jensen, D.A., Thompson, J.A., 1992. Toxicity identification evaluation: Characterisation of chronically toxic effluents, Phase I, EPA/600/6-91/005F, Environmental Research Laboratory, Duluth, MN. ¨ stersjo¨ (1994–2003) Yearly Reports from the Marine Research O Center of Stockholm University, Stockholm. (In Swedish, English summary). Phillips, B.M., Anderson, B.S., Hunt, J.W., Thompson, B., Lowe, S., Hoenicke, R., Tjeerdema, R., 2003. Causes of sediment toxicity to Mytilus galloprovincialis in San Francisco Bay, California. Archives of Environmental Contamination and Toxicology 45, 492–497. Rosenberg, R., 1976. Benthic faunal dynamics during succession following pollution abatement in a Swedish estuary. Oikos 27, 414– 427. Sasson-Brickson, G., Burton Jr., G.A., 1991. In situ and laboratory sediment toxicity testing with Ceriodaphnia dubia. Environmental Toxicology and Chemistry 10, 201–207. Segerstra˚le, S.G., 1970. Light control of the reproductive cycle of Pontoporeia affinis (Crustacea, Amphipoda). Journal Experimental Marine Biology and Ecology 5, 272–275. Segerstra˚le, S.G., 1971. Light and gonad development in Pontoporeia affinis. In: Crisp, D.J. (Ed.), 4th European Marine Biology Symposium. Cambridge University Press, London, UK, pp. 573–581. Sepu´lveda, M.S., Quinn, B.P., Denslow, N.D., Holm, S.E., Gross, T.S., 2003. Effects of pulp and paper mill effluents on reproductive success of largemouth bass. vol. 22, pp. 205–213. Sepu´lveda, M.S., Gallagher, E.P., Wiesser, C.M., Gross, T.S., 2004. Reproductive and biochemical biomarkers in largemouth bass sampled downstream of a pulp and paper mill in Florida. Ecotoxicology and Environmental Safety 57, 431–440. Sibley, P.K., Legler, J., Dixon, D.G., Barton, D.R., 1997. Environmental health assessment of the benthic habitat adjacent to a pulp mill discharge. I. Acute and chronic toxicity of sediments to benthic macroinvertebrates. Archives of Environmental Contamination and Toxicology 32, 274–284. Simpson, S.L., Apte, S.C., Batley, G.E., 2000. Effect of short-term resuspension events on the oxidation of cadmium, lead, and zinc sulfide phases in anoxic estuarine sediments. Environmental Science and Technology 34, 4533–4537. ˚ kerman, G., Grunder, K., Ishaq, R., Sundberg, H., Tja¨rnlund, U., A Hammar, T., Broman, D., Balk, L., 2005. The distribution and relative toxic potential of organic chemicals in a PCB contaminated bay. Marine Pollution Bulletin 50, 195–207. Sundelin, B., 1983. Effects of cadmium on Pontoporeia affinis (Crustacea: Amphipoda) in laboratory soft-bottom microcosms. Marine Biology 74, 203–212. Sundelin, B., 1988. Effects of sulfate pulp mill effluents on soft bottom organisms a microcosms study. Water Science and Technology 20, 175–177. Sundelin, B., 1989. Ecological affect assessment of pollutants using Baltic benthic organisms. Ph.D. thesis. Stockholm University, Sweden. Sundelin, B., Eriksson, A.K., 1996. Effect monitoring in pulp mill areas: response of the meiofauna community to altered process technique. In: Servos, M.R., Munkittrik, K.R., Carey, J.H., Van Der Kraak, G.J. (Eds.), Environmental Fate and Effects of Pulp and Paper Mill Effluents. St Lucie Press, Del Ray Beach, FL, pp. 483–494. Sundelin, B., Eriksson, A.K., 1998. Malformations in embryos of the deposit-feeding amphipod Monoporeia affinis, in the Baltic Sea. Marine Ecology Progres Series 171, 165–180. Sundelin, B., Bo¨rjesson, T., Eriksson Wiklund, A.K., Ha˚kansson, E., 2003. Vitma¨rlans reproduction I Va¨nern och Va¨ttern 2002. Va¨tterns vattenva˚rdsfo¨rbund Jo¨nko¨ping Sweden (in Swedish).
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Toxicity evaluation by using intact sediments and sediment extracts Ann-Kristin Eriksson Wiklund *, Brita Sundelin Dag Broman Department of Applied Environmental Science, Stockholm University, SE-10691 Stockholm, Sweden
Abstract The toxicity of intact sediments and sediment extracts, from both an uncontaminated site and a site contaminated by pulp-mill effluents, was tested in a five months study. The deposit-feeding amphipod Monoporeia affinis was exposed in soft-bottom flowthrough water microcosms. To examine potential toxicity a set of reproduction endpoints was used including fecundity and different embryo aberrations such as malformed eggs. Among extracts, the aliphatic/monoaromatic and diaromatic fractions along with the total extract were shown to cause the highest toxicity measured as malformed eggs, while the polyaromatic fraction caused toxicity at background levels. A comparison between sediment extracts and pulp mill contaminated intact sediment, however, showed no toxicity of the intact sediment. Thus, the extraction procedure seems to increase bioavailability and subsequently toxicity as compared to the intact sediments in situ. In toxicity testing using fractionated extracts of sediments in a toxicity identification evaluation (TIE) procedures, caution should therefore be taken when assessing bioavailable contaminants in contaminated areas. This should be taken in account both in determining remediation priorities as well as in ecological risk assessments. 2005 Elsevier Ltd. All rights reserved. Keywords: Toxicity; Sediment extracts; Intact sediments; TIE; ERA
1. Introduction The pulp and paper industry was one of the main sources of organic contaminants in the Bothnian Sea in the 20th century (Jonsson, 2003). The most obvious effect found in early field studies was an increase in organic loading of the receiving waters resulting in high oxygen demand in both water column and sediments (Rosenberg, 1976). Reduced discharges of oxygen consumptive substances have led to recovered health status in both fish and sediment-living bottomfauna. Discriminating between effects of various bleaching processes has been the major issue in both field studies and laboratory experiments during the last decades. Severely deformed skeletons in fish was one of the first reported serious toxic effects of bleached kraft mill effluents *
Corresponding author. E-mail address: [email protected] (A.-K. Eriksson Wiklund). 0025-326X/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2005.02.030
(BKME) (Bengtsson, 1988), but in the last few years the focus has changed to reproduction disorders due to endocrine disrupting chemicals. The recent papers on reproduction effects of pulp mill effluent have mainly been concerned with fish (McMaster et al., 1996; Munkittrick et al., 2003; Jenkins et al., 2003; Sepu´lveda et al., 2003, 2004), while there are few studies on invertebrates, e.g. Sibley et al. (1997). Among those are laboratory experiments with pulp and paper mill effluents and sediments as well as field studies in recipients having demonstrated toxic effects on amphipod reproduction (Sundelin, 1988, 1989; Sundelin and Eriksson, 1998). When examining sediment toxicity, benthic species in whole sediment exposures are considered to be the best indicators (Burton et al., 1992) due to their direct contact with sediment and interstitial water. Among benthic species, amphipods are considered as particularly sensitive since they are the first to disappear from contaminated sediments (Swartz et al., 1982). The usefulness of amphipods is well established and they are often used
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in toxicity assessments in fresh, estuarine and marine waters (Chapman et al., 1998). The high lipid content of the amphipod Monoporeia affinis makes this species important in the transfer of organic chemicals from sediments upwards in the food chain as well as in the transfer of organic chemicals from parent to offspring (Eriksson Wiklund et al., 2003). Thus, we consider M. affinis as a relevant test species in bioassays for sediment toxicity in the Baltic. Toxicity identification and evaluation (TIE) procedures were developed by the US-EPA (Mount and Andersson-Carnahan, 1988, 1989; Norberg-King et al., 1992; Mount and Norberg-King, 1993). These procedures were originally designed for aqueous samples but methods for sediment testing are continuously being developed (Burgess et al., 2000). This strategy has grown increasingly popular since the 1990s, especially when pinpointing substances in multiple endocrine disrupting mixtures (Burnison et al., 2003). The TIE procedures are mainly used in connection with effluent discharge regulation, but are also used in environmental risk assessment (ERA) as well as in remediation work. From the TIE procedures, several bioassay/effect directed analyses (BDA/EDA) concepts have being developed. When examining sediment toxicity by these methods, sediment elutriates or sediment extracts in aqueous test procedures are normally used (e.g. Brack et al., 1999; Burgess et al., 2000; Phillips et al., 2003). 1.1. Aim and strategy Several field studies performed 5–10 years before this study have demonstrated toxicity in the pulp mill area in terms of a changed structure of the meiofauna community and an increased level of malformed embryos of M. affinis (Sundelin and Eriksson, 1996, 1998). Large industrial efforts in altering and improving processes have since then been performed, i.e. changing the bleaching process from chlorine to elemental chlorine free (ECF) bleaching as well as substantially reducing effluents. In this study the sediment-dwelling amphipod M. affinis was exposed to five experimental treatments. Three treatments were based on samples from a pulp mill recipient using natural intact sediments, unfractionated and fractionated sediment extracts of varying aromaticity, while two treatments were based on samples from a reference site using natural intact and unfractionated sediment extracts. To simulate the mother system in situ and accordingly achieve ecologically relevant experimental conditions, we used the strategy of mixing the sediment extracts into a sediment matrix. The aims were to identify and compare the toxicity of the different sediment extracts and to examine whether the toxicity is found in the fractions or remains in the unfractioned extract (total extract). Furthermore, we wanted to examine the possibility of assessing bioavailable and toxic com-
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pounds and making field predictions by comparing the toxicity of untreated intact sediment with unfractionated sediment extract.
2. Material and methods 2.1. Test species M. affinis is found both in the reference area and in the vicinity of the contaminated area, thus fulfilling the requirement of ecological significance addressed by Batley et al. (2002) and underlined by Chapman (2002). M. affinis has been the dominating soft bottom species of the Baltic, further strengthening its relevance as a test species. It is a deposit feeder using the surface sediment as its main food source (Byre´n et al., 2002). The spring phytoplankton bloom is the most important food contribution but the amphipods continue to accumulate lipids throughout the summer months. The reproduction cycle starts in the late summer/early autumn with gonad maturation (oogenesis and spermatogenesis) triggered by the reduction of light (Segerstra˚le, 1970, 1971). Mating occurs in November, followed by three months of embryo development. The method used in this paper is a method for biomonitoring recommended by ICES and applied for effect monitoring of contaminated sediments in the Swedish ¨ stersjo¨, 1994–2003; Marine National Programme (O Bottniska viken, 1994–2000). The method includes different reproduction variables, e.g. the embryonic development in M. affinis. The variable Ômalformed embryosÕ has proven to be a powerful contaminant monitor in both laboratory experiments and field studies, while the variable Ôfemales carrying dead broodsÕ is a monitor of hypoxia tested in both laboratory experiments and field studies (Eriksson Wiklund and Sundelin, 2001, 2004). 2.2. Microcosm system The microcosms used were two-litre Erlenmeyer flasks containing approximately 4 cm of sediment (Sundelin, 1983). The microcosms were connected to a continuous water flow of 2.2 l h1. The incoming water was collected at 37 m depth and followed the natural temperature regime of 2–8 C during the experiment. Light was adjusted to natural day length. 2.3. Sampling Reference sediment, to be mixed with extracts or covered with intact pulp mill sediments, was collected with a modified Ockelman dredge, mesh size 450 lm, (Blomqvist and Lundgren, 1996) on a reference site within the Swedish National Monitoring Programme at position 5847 0 4700 N, 1743 0 9300 E. Intact contaminated
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sediments and sediments for extraction and fractionation were collected with a Kajak-corer close to the pulp mill at position 6137 0 N, 1708 0 E. Amphipods were collected at position 5850 0 5000 N, 1747 0 1600 E. To collect amphipods sediment was sieved through a, 1.0 mm mesh size net, while the reference sediment was sieved through a 0.5 mm mesh size net to avoid predators. 2.4. Chemical fractionation The wet sediment samples were placed in preextracted cellulose thimbles and Soxhlet extracted with toluene for 24 h. The apparatus was equipped with a Dean–Stark trap for collecting water. After toluene evaporation (<10 Torr; 35–40 C), the residue was cleaned on a silica column with n-hexane as a mobile phase. To fractionate the extracts we used a HPLCsystem (Zebu¨hr et al., 1989) consisting of a MERCKHitachi L-6200 pump, Rheodyne 7125 valve injector, Rheodynhe 7067005 automatic valve station and switching valves and MERCK-Hitachi 1-4200 UV–VIS detector. A semipreparative Bondpak amino column (300 · 7.8 mm, Waters) was used with n-hexane as the mobile phase. Three fraction were collected: aliphatics/monoaromatics, diaromatics and polyaromatics. At a rate of 3 ml min1, the aliphatics/monoaromatics were eluted first, followed by the diaromatics in a forward direction (Zebu¨hr et al., 1989). The column was then back flushed (5 ml min1) and the polyaromatics eluted as one narrow peak. The compounds were monitored at 254 nm. Low-boiling aliphatics/monoaromatics were probably lost during the evaporation steps. 2.5. Experimental design The amphipods were exposed to nine different series (five replicates of each), including an aliphatic/monoaromatic fraction, a diaromatic fraction, a polyaromatic fraction, a recombination of those three fractions, a total unfractionated extract, an intact contaminated sediment, a total reference unfractioned extract, an intact reference sediment, and a blank extract. Sediment extract originating from 204 g dry weight sediment (corresponding to the amount of added intact sediment, see below) was dissolved in 25 ml acetone before being slowly blended in 550 ml of continuously stirred reference sediment. The sediment-extract mixture was stirred for an additional hour and then allowed to settle for one week. To each microcosm, 100 ml sediment extract mixture or 100 ml intact sediment was added on top of 300 ml reference sediment and allowed to settle in the aquaria for two days before the water flow of 2.2 l h1 was started. To avoid effects of acetone the test organisms (50 preadults) were added after one more day. The experiment started in the middle of August and ended in the middle of January.
2.6. Biological analyses Adults and embryos of M. affinis were examined for fertilisation rate, fecundity, egg developmental stage and different aberrations from normal development such as malformed embryos, dead and undifferentiated eggs and females carrying a dead brood according to Sundelin and Eriksson (1998). Variables studied in females were fertilisation success, fecundity (eggs/female), and dead brood. Embryos were examined for the developmental stage, percentage of malformed embryos, unfertilised/ undeveloped (henceforth called undifferentiated) eggs and dead eggs and embryos (for further information, see Sundelin and Eriksson, 1998). 2.7. Statistical evaluation The statistical evaluations were performed by logit analyses (embryo variables and dead brood) (Aldrich and Nelson, 1984; Demaris, 1992). Analysis of variance was used to test the non-dichotomous data, egg development and fecundity. Significance of a < 0.05 was applied.
3. Results Different types of malformations were identified, several of them caused by malfunction of the inner vitellin membrane, resulting in leakage of stored lipids into the space between egg and vitellin membranes. Other less frequently occurring malformations were enlarged embryos, shortened extremities appendages, deformed midguts and eyespots. In the statistical analysis the different types of embryo malformations have been merged into one category. There was no difference in toxicity, measured as percentage of malformed embryos, between the extracted reference sediment and the intact reference sediment or the blank extract. In all further comparisons the extracted reference sediment will be used as reference. Large differences in toxicity were found between experimental sediment series, the aliphatic/monoaromatic fraction, diaromatic fraction and the total unfractionated extract from pulp mill recipient exerting the largest toxicity with approximately 11%, 14% and 12% malformed embryos respectively. All extracts from the contaminated site exerted toxicity as compared to the sediment extract from the reference site (Fig. 1a, Table 1). The contaminated unfractionated total extract was more toxic than the polyaromatic fraction but did not differ from the aliphatic/monoaromatic fraction and the diaromatic fraction. The toxicity of the recombined extract was significantly lower than that of the single fractions. The polyaromatic fraction exerted lower toxicity than both the aliphatic/monoaromatic fraction and the diaromatic fraction, which did not differ in
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Fig. 1. Results from 5 months of exposure of the amphipod Monoporeia affinis to series of fractionated and unfractionated sediment extracts as well as intact sediments. The bars represent mean values of five replicates + standard error. (a) malformed embryos, (b) undifferentiated embryos, (c) dead eggs.
toxicity. Unlike the extracted pulp mill sediment, the intact sediment from the contaminated site did not exert any significant toxicity (4% malformed embryos) compared to the reference sediment extract. The toxicity of the intact reference sediment and the blank extract did not differ significantly from that of the extracted reference sediment. Undifferentiated eggs were significantly more abundant in the intact reference sediment and the recombined extract of pulp mill sediment compared to the extracted reference sediment (Table 1, Fig. 1b). Other sediments did not differ from the extracted reference sediment. A lower frequency of dead eggs was found in the recombined fractionated extracts, unfractionated extract and intact contaminated sediment compared to the extracted reference sediment (Table 1, Fig. 1c). In the diaromatic and polyaromatic fractions and the intact reference
sediment, however, a higher frequency of dead eggs was found.
4. Discussion 4.1. Toxicity The major part of the toxicity in this pulp mill recipient study is found in the aliphatic/monoaromatic fraction and the diaromatic fraction of the sediment. In other parts of the Baltic Sea the major part of the toxicity has been found in the polyaromatic fraction (Sundberg et al., 2005). The total extract, on the other hand, does not show any additional toxicity. Knowledge about specific compounds in the aliphatic/monoaromatic fraction is limited, but a relatively low persistence of these
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Table 1 Statistical parameters from the logit analyses Dependent variable
Treatment
Regression SE coefficient
p
Malformed
Intact ref sediment Aliphatic extract Diaromatic extract Polyaromatic extract Recombined extract Total extract Intact recipient Blank extract
0.41 1.35 1.73 0.69 0.85 1.40 0.21 0.29
0.21 0.20 0.16 0.17 0.17 0.15 0.21 0.19
ns <0.001 <0.001 <0.001 <0.001 <0.001 ns ns
Dead
Intact ref sediment Aliphatic extract Diaromatic extract Polyaromatic extract Recombined extract Total extract Intact recipient Blank extract
0.93 0.97 0.96 0.67 0.52 0.88 0.91 0.92
0.22 0.56 0.22 0.21 0.29 0.31 0.38 0.21
<0.001 <0.001 0.01 <0.001 ns 0.05 <0.001 <0.001
Undifferentiated
Intact ref sediment Aliphatic extract Diaromatic extract Polyaromatic extract Recombined extract Total extract Intact recipient Blank extract
1.14 0.41 0.26 0.06 0.92 0.10 0.14 0.15
0.17 0.34 0.24 0.20 0.16 0.18 0.21 0.21
<0.001 ns ns ns <0.001 ns ns ns
compounds has been recorded in an earlier study (Zebu¨hr pers. comm.). In the diaromatic fraction compounds like PCBs, PCDDs and PCDFs will be retained (Zebu¨hr et al., 1989). Results from an earlier survey showed increased sediment concentrations of PCDD/F in the vicinity of the pulp mill (Na¨f et al., 1992). PAHs, expected to be retained in the polyaromatic fraction, are not related to pulp mill effluents. This is reflected in the low toxicity found in this fraction. There are several possible explanations of the difference in toxicity between fractions. One possible explanation is a higher toxicity or higher concentrations of toxic compounds in the low aromatic fractions. A lower binding capacity to the sediment, making these substances more bioavailable, is another possible explanation. Complex contaminant mixtures may not exert an additive effect but act both synergistically and/or antagonistically (Altenburger et al., 2003). In this experiment two of the fractions showed a higher toxicity than the recombination of fractions, indicating antagonistic behaviour. Nirmala et al. (1999) observed antagonistic effects on embryological survival in Japanese medaka, while Nasci et al. (2000) observed antagonistic effects in clams exposed to natural sediments including complex contaminant mixtures. Bosveld and Vandenberg (1994) reported that several PCDD/Fs in combination with PCBs might cause both synergistic and antagonistic effects. There is a large difference in toxicity between the recombined and the unfractionated total extract. The
unfractionated extract contains polar substances extracted by toluene that are omitted in the fractions and in the recombination of the fractions by a cleanup process on silica-column. The unfractionised extract might contain substances like phenols and chloroguaiacols. Bleached kraft mill effluents affect the reproductive physiology of fish by reducing concentrations of plasma sex steroids, gonadosomatic index and expression of secondary sex characteristics (Sepu´lveda et al., 2003). The mechanisms behind the effects in invertebrates are unknown. Both laboratory experiments and field studies have shown malformed embryos to be a powerful tool for monitoring effects of contaminants (Sundelin, 1983, 1988; Sundelin and Eriksson, 1998), a conclusion that was further confirmed in this study. Effects of pulp mill effluents on fish fecundity and embryo development have been reported (Sepu´lveda et al., 2003). Our experiment showed no similar effects on amphipod fecundity. Since the contaminant exposure in the fish experiments occurred after vitellogenesis, when lipophilic chemicals are transferred into oocytes, the authors hypothesised a sub-optimal timing as a possible cause of the lack of effects in their experiments (Sepu´lveda et al., 2003). The timing could not, however, cause the lack of effects on fecundity and embryo development in our experiments covering a complete reproductive cycle including lipid transfer into in the oocytes. In field studies a close relationship between food availability and lipid content and fecundity of amphipods has been shown (Sundelin et al., 2003). Possible toxic effects of contaminants on fecundity might thus be confounded by availability of food resources. Fecundity thus for this species must be considered as a non-optimal field monitor of toxicity (Eriksson Wiklund and Sundelin, 2004). 4.2. Comparison with in situ conditions The great difference in toxicity between the intact sediments and the fractionated sediments indicates increased bioavailability due to the extraction procedure. The strategy of identifying the toxicity in different fractions of extracted sediment is particularly valuable in sediments or wastewater contaminated by various unknown chemicals, where it may facilitate the search for responsible toxicants. This strategy has also been successful in examining the outcome of remediation work (Eriksson et al., 1996). Observing toxicity in one specific fractionated extract, however, does not make it possible to predict that this fraction is the most bioavailable in situ. Results must be validated by field measurements or bioassays using intact sediments (Anderson et al., 2001). Bioavailability and subsequent toxicity may differ between experiments based on intact sediments and
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sediments spiked with contaminants. Contaminants bound into natural intact sediments are generally more strongly sorbed to the sediment and thus more slowly desorbed than contaminants in unaged laboratory spiked sediment. Also, greater toxicity is generally shown in laboratory tests than in in situ processes (Sasson-Brickson and Burton, 1991). Several authors (e.g. Simpson et al., 2000) therefore call for caution when extrapolating results from laboratory experiments to natural conditions. Several authors have drawn attention to certain weaknesses inherent in the TIE procedures, i.e. increased bioavailability caused by chemical manipulations and difficulties in extrapolating laboratory results to field conditions (Desbrow et al., 1998; Ho et al., 2002). Also, the identification of a compound in a fraction (the confirmation step in the TIE) does not prove that this compound per se caused the effect (Brack, 2003). In traditional toxicity testing a common strategy is to spike sediments with contaminants, but in a procedure aiming at identifying potent chemicals in contaminated sediments, the fractionated extracts are generally tested in aqueous solutions or pore water. This methodology might be inappropriate for sediment living organisms, which are generally exposed to two contaminant sources, the solid phase that often constitutes their food and the interstitial and overlying water (Chapman et al., 2002). As far as we know this is the first study where sediment extracts are mixed into the sediments and compared to intact natural sediment. This process closely mimics natural conditions, thus providing high ecological relevance. The results clearly demonstrate that caution is needed before extrapolating results from TIE/ BRD/EDA procedures to natural conditions. Since these procedures are developed as tools in risk assessment, an overestimated risk based on a delusive degree of bioavailability might lead to unnecessary remediation work and restoration activities. Acknowledgments We would like to acknowledge the following persons for skilful performances: Carina Na¨f participated in the early phase of this project and is also a co-author of an earlier paper, Eva Ha˚kansson assisted during the amphipod exposure and Kerstin Grunder extracted the sediments and made the fractionations. References Aldrich, J.H., Nelson, F.D., 1984. Linear Probability, Logit and Probit Models. Series Quantitative Applications in the Social Science. Sage Publications Inc., Newbury Park, California, USA. Altenburger, R., Nendza, M., Schu¨u¨rmann, G., 2003. Mixture toxicity and its modeling by quantitative structure–activity relationships. Environmental Toxicology and Chemistry 22, 1900–1915.
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