- Email: [email protected]
Toxicity of 4-nonylphenol in spiked sediment to three populations of Chironomus riparius
Chemosphere 46 (2002) 201±207
www.elsevier.com/locate/chemosphere
Toxicity of 4-nonylphenol in spiked sediment to three populations of Chironomus riparius R. Bettinetti
a,*
, D. Cuccato a, S. Galassi b, A. Provini
a
a
b
Biology Department, University of Milan, Via Celoria 26, Milan 20133, Italy Biotechnology and Bioscience Department, University of Milan Bicocca, Via Emanueli 12 bis, Milan 20126, Italy Received 22 November 2000; accepted 9 April 2001
Abstract Nonylphenols (NPs) are the primary stable metabolites of alkylphenol polyethoxylates (APEs), a family of compounds widely used in industry and in some domestic products. As NPs accumulate in sediments in aquatic environments, the risk to benthic organisms needs to be assessed. In this study 4NP-spiked sediments were tested on larvae of the dipteran Chironomus riparius. First instar larvae obtained from populations at three dierent sources were used. To spike the sediments, an equilibration procedure between water and sediment was adopted to avoid the use of solvents. Lower 10-d LC50 values were determined for two populations of C. riparius from clean environments (315±465 and 315± 350 lg g 1 d.w., respectively) than those of a strain deriving from a population collected in a polluted river (600±680 lg g 1 d.w.). Larval growth always decreased with increasing 4NP concentration but without any de®ned trend. The results of this study suggest that tolerance to the toxicant can be developed in populations of polluted environments and that testing procedures should be standardised. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: 4-Nonylphenol toxicity; Chironomids; Sediment spiking
1. Introduction Alkylphenol polyethoxylates (APEs) are non-ionic surfactants widely used in the formulation of industrial detergents. It has been estimated that the net worldwide production of APEs exceeds 300,000 t annually (Chemical Manufacturers Association, 1994). Once discharged into the environment, their degradation leads mainly to the formation of metabolites such as alkylphenols, alkylphenol monoethoxylates and alkylphenol diethoxylates, well known for their high aquatic toxicity (Chemical Manufacturers Association, 1994). In most APEs the alkyl chain is a nonyl, and nonylphenols (NPs) therefore represent the main stable metabolites of these
*
Corresponding author. Fax: +39-02-26604361. E-mail address: [email protected] (R. Bettinetti).
commercial products. NPs have been used as additives in plastics, household and personal care products, lubricating oils, agrochemicals, latex coatings and adhesive, paint, pulp and paper production and textile manufacturing (Liber et al., 1999). NPs are hydrophobic and accumulate in sediments, where they can persist in excess of 400 d (Heinis et al., 1999). Consequently, sediments are able to act as a sink for NP and represent a risk to aquatic organisms. To date, several tests have been done to detect the acute and chronic toxicity of NPs to a number of aquatic species (Comber et al., 1993; Kahl et al., 1997; Schmude et al., 1999). However, variable 4NP toxicity values are reported, probably because of errors associated with the low solubility of NP in water, dierences in sensitivity among strains used and the varying organic carbon content of sediments. Kahl et al. (1997), applying NP to the overlying water and using sand as substrate for Chironomus tentans, found an LOEC value of 91 lg l 1
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 1 1 4 - X
202
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
2. Material and methods
ugation. Spiked sediments for use in tests were obtained by mixing this stock sediment with the reference sediment. They were mixed ®rst manually for 10 min and subsequently with a mechanical mixer for 30 min. Each spiked sediment was then subdivided into ®ve replicate beakers. Spring water (pH 7.8; hardness 106 mg l 1 CaCO3 ) was then added and the sediment left to settle before the animals were added, as recommended in the test procedures described by Day et al. (1994) and OECD (1998). The water content of the sediment was evaluated as weight dierence after wet sediments had been dried at 105°C for 12 h. Organic matter content (OM%) was estimated as the weight loss after the dried sediment had been heated at 550°C for 8 h. At the start of each test, the sediment dry weight (d.w.), OM% and the actual concentration of 4NP in the sediment were determined. Analyses were performed in each replicate beaker and, in some cases, also in subsamples from the same beaker.
2.1. Collection and preparation of reference sediment
2.3. Toxicant analysis
Preliminary tests to evaluate the best larval growth conditions of C. riparius were carried out using both natural sediments sampled in dierent unpolluted areas, and arti®cial sediments (Suedel and Rodgers, 1994). The best development of larvae was observed with unpolluted sediments from Lake Monate (Northern Italy). After a chemical analysis showing that 4NP was not present and the other parameters were in the range of concentrations suggested by Salomons and F orstner (1984), this sediment was selected as reference sediment. Sediments were collected with an Ekman grab at a lake depth of about 10 m. In the laboratory they were wet sieved through a 250-lm mesh using dechlorinated tap water to remove indigenous macrofauna and coarse particles (Day et al., 1995). After 24 h of settling, the overlying water was removed and the settled sediments centrifuged. Sediments were then stored in the dark at 4°C. Before each test, the sediment was mixed to homogeneity in a 1 l glass beaker with a mechanical mixer.
The 4NP concentration was analytically determined both in sediments and in water. The complete extraction of 4NP for analysis was obtained by mixing approximately 2.3 g w.w. of sediment from each test concentration with 50 ml of ultrapure methanol, stirred for 1 h with a mechanical agitator; the methanol was then separated by centrifugation. This procedure was repeated three times and the total extract (150 ml) ®ltered with 0.45-lm glass micro®bre ®lters (Whatmanâ ). The extracts were analysed for 4NP according to Kahl et al. (1997) with a Jasco HPLC equipped with a ¯uorescence detector. Preconcentration of the extract was unnecessary, except for analysis of the reference sediment. The detector excitation and emission wavelengths were set at 230 and 290 nm, respectively. 50 ll of sample was injected directly into a Lichrospher 100 RP-18 5 lm column (4 125 mm; Merck) and eluted with a water± methanol gradient that varied from 20:80 to 0:100 in 11 min. The external standard method was used for quanti®cation. Each analysis was carried out in duplicate. The detection limit was 5 lg 4NP g 1 of sediment (d.w.). Overlying water was preconcentrated 50 times using LiChrolut ENâ cartridges eluted with ultrapure methanol and analysed with the same procedure adopted for sediments. The detection limit was 3 lg l 1 .
in a 20-d test, while Schmude et al. (1999) reported an LOEC of 243 41 lg l 1 for chironomids exposed in littoral enclosures. The present study represents a ®rst attempt to determine the toxicity of 4NP-spiked sediments to dierent strains of the benthic larvae of C. riparius Meigen 1804. This chironomid is the most widely distributed and abundant insect of natural freshwater macrobenthic communities in Europe (Thienemann, 1954) and is frequently adopted for sediment testing (Rosenberg, 1992). Since the various methods of spiking sediments with compounds that are not readily water soluble usually involve the use of a solvent at some stage (Murdoch et al., 1997), this article also attempts to de®ne a reproducible protocol for spiking sediments with hydrophobic chemicals without the use of a carrier, which might interfere with the ecotoxicological response.
2.2. Sediment spiking and analysis 4NP (Sigma-Aldrich, UK) is a mixture of ring and chain isomers. Since 4NP has very low solubility in water and high lipophylicity, with a log Kow value of 4.48 (Ahel and Giger, 1993), saturation concentration in the water medium is not sucient to ensure a high enough concentration for toxicity tests to be performed, after sediment equilibration. Therefore 1 g of 4NP was directly added to 250 g of wet sediment (water content about 50%) and mixed with 250 ml of water. The slurry was then stirred for 20 h and left to settle for 72 h. The sediment was then separated from the water by centrif-
2.4. Chironomid cultures Egg masses of C. riparius were initially obtained from a population collected in the River Lambro (L larvae), a river polluted by civil and industrial discharges from the northern part of Milan (Italy), as well as from cultures
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
maintained at RIZA, Lelystad, The Netherlands (R larvae), and at the University of Leuven, Belgium (B larvae). The last two cultures were from clean environments. The larvae were bred at 21 1°C under the daily photoperiod in 40 l aquaria with Lake Monate reference sediment (3 cm deep) as substrate. An 8-cm deep column of dechlorinated tap water (hardness: 320 mg l 1 CaCO3 ) was maintained over the sediment. Cultures were fed weekly with 1 g Tetraminâ ®sh food per tank. The ®rst larval instars for use in each experiment were obtained by transferring egg ropes from the culture to glass vessels containing spring water. The time required to hatch the ®rst instar larvae was about 3 d at 21 1°C. The animals were then transferred to each test beaker with a glass pipette, putting them below the water surface to avoid trapping them in the surface ®lm. 2.5. Bioassay design Four acute tests (96 h) with K2 Cr2 O7 dissolved in water as reference substance were carried out to verify that laboratory-bred chironomids were suitable for toxicity tests and that their sensitivity remained constant (RIZA, 1996). 4NP toxicity tests were performed according to the guidelines set out by Day et al. (1994) with minor modi®cations. One day before the addition of the animals, 250-ml glass beakers were ®lled with 60 g of spiked wet sediment (water content about 50%) and 200 ml of water gently added to the sediment. 3.5 ml of a 4 g l 1 water suspension of ®sh food, corresponding to 14 mg d.w. Tetraminâ , was put into each beaker. The contents of the beakers, covered with a plastic Petri dish, were allowed to settle in the dark at 21 1°C without aeration. Five replicate beakers were prepared for each nominal concentration, including the control. At the start of the test, the overlying water of each beaker was gently aerated for 2 h and 10 ®rst instar larvae chosen at random were transferred to each beaker. Tests were performed under 16:8 h light:dark photoperiod for 10 d. Every three days the animals were fed with 3.5 ml of Tetraminâ suspension and the water lost through evaporation was replaced with spring water. Temperature, pH, conductivity and dissolved oxygen were measured in all the beakers before and after the tests. Total survival was noted and the individual larval wet weight of each larva recorded using the sensitive, non-destructive technique described by Blockwell et al. (1996). Final larval weights were considered because they represent an integration of physiological responses which are known to be aected by the environment and by pollutant stress (Kosalwat and Knight, 1987). Four tests were performed with the L larvae, two with the B larvae and two with the R larvae.
203
2.6. Data analysis Data on the survival of C. riparius were analysed using the EPA Probit Analyses Program for calculating the LC50 values. The t-test for independent samples was used to determine if the weights of control larvae from clean and polluted environments were similar. One-way ANOVA and the post hoc Tukey test for unequal samples (Spjotvoll±Stoline test, a level 0.05) were performed to evaluate dierences between treatments with the STATISTICA 4.5 package (StatSoft). 3. Results and discussion The ®rst instar larvae of C. riparius used in this study showed a constant and acceptable sensitivity to K2 Cr2 O7 toxicity. The 96-h LC50 values for the L larvae were 13:3 mg l 1 (c.l.: 11.7±14.7) and 14:8 mg l 1 (c.l.: 12.7± 16.5), and for the R larvae 14:8 mg l 1 (c.l.: 12.6±16.8) and 12.3 (c.l.: 5.2±17.3). These values were smaller than the sensitivity range indicated by RIZA (1996) for second instar larvae. The organic matter content of the reference sediment was 2.3%. The 4NP concentration of the stock spiked sediment was between 6 and 6:6 mg g 1 d.w. Analytical concentrations determined in sediment prepared by dilution of stock spiked sediments were generally within 20±25% of the nominal concentrations (Table 1). The proposed spiking method produced a homogeneously dosed sediment, since the 4NP concentration in the replicate beakers was comparable (5%). Moreover, 4NP concentration measured in subsamples of the same beaker was the same. No concentration decrease was measured within 28 d. This is in agreement with the high persistence of 4NP in sediments reported in both laboratory studies and littoral enclosures (Heinis et al., 1999). During the experiments, pH dierences among the beakers were minimal (0.2 pH units) and the oxygen saturation values were always more than 50%. Conductivity values were more variable mainly because of sediment resuspension and the dierent survival of the organisms in each beaker. At equilibrium, 4NP concentration in the overlying water was between 5 and 20 lg l 1 , depending on its concentration in the sediment. The 10-d 4NP LC50 value obtained with the L larvae was within a range of 600±680 4NP lg g 1 d.w., whereas the corresponding values obtained for the larvae collected in unpolluted areas (R and B larvae) were in the range of 315±465 4NP and 315±350 4NP lg g 1 d.w., respectively (Table 2). This observed variability could be due to genetic dierences in cultures deriving from different natural populations, and to a development of tolerance to 4NP and related organic toxicants in the L
204
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
Table 1 Nominal and measured 4NP concentrations (lg g spiked sediments
1
d.w.) in
Table 2 The 10-d LC50 4NP values obtained in tests conducted with three C. riparius larvae (L Lambro; B Belgio; R RIZA)
Nominal concentration
Measured concentration
Test
LC50 (4NP lg g 1 d.w.)
95% Con®dence limits
L1
50 120 200 400 800
60 100 180 350 680
L1 L2 L3 L4
673 603 674 ±a
526±1067 539±676 486±1309
L2
220 350 600
180 440 750
B1 B2
350 314
± 270±355
230 290 350 400 500 650 750
290 365 430 475 490 560 650
R1 R2
465 315
393±623 268±363
L4
150 200 300 350 400 450
115 229 238 309 325 345
B1
150 250 450
200 310 400
B2
250 350 600 950
230 280 500 800
R1
350 400 550
270 350 480
R2
300 500
230 440
L3
larvae. The River Lambro, where the larvae originated, receives the discharges of textile industries and 4NP concentrations ranging from 1 to 4:7 lg g 1 have been found (Polesello, S., personal communication). Genetic dierences leading to altered growth and reproduction have been noted in Tubifex tubifex (Anlauf, 1994) and more recently Sturmbauer et al. (1999) identi®ed dierent genetic lineages in populations of T. tubifex diering in cadmium resistance. In the present study we hypothesise that natural selection facilitates increased tolerance to 4NP in the L population originating in the most contaminated area, and that the midge population
a
Not possible to calculate.
could maintain this adaptation for several generations. This hypothesis needs to be con®rmed by studying a larger number of Chironomus populations but, if it is true, care should be taken in the choice of the test organism to be used in toxicity bioassays. The post hoc Tukey test indicated that the survival of the L larvae was not aected by 4NP up to a concentration of 350 lg g 1 d.w. in sediment. At higher 4NP concentrations mortality increased and zero survival was noted at 1500 lg g 1 d.w. The numbers of both R and B larvae surviving were already decreasing at a 4NP concentration of 230 lg g 1 d.w., and no survival was observed at 800 lg g 1 d.w. The growth response shows that after 10 d the L larvae developed in control beakers had an average wet weight of 3:8 1:2 mg and the R and B larvae were signi®cantly smaller 2:8 1:3 mg (t-test, P < 0:000). The mean wet weight of the control larvae was always higher than that of the exposed organisms, with the exception of tests L1 and L3, where the mean wet weights of the larvae, at 4NP concentration of 60 and 290 lg g 1 , respectively, were similar to that of the control (Fig. 1). After 10 d of exposure the mean wet weight showed a signi®cant P < 0:01 decrease with increasing 4NP concentrations (Fig. 1). Liber et al. (1996) observed that when larval weight increased, the mean emergence time decreased and the mean successful emergence increased. This reduced growth rate due to 4NP toxicity could have long-term consequences on adult emergence success, aecting population density in natural environments. In Fig. 1 the weights of exposed larvae which are signi®cantly dierent P < 0:001 from those of the control are indicated by an asterisk. It is not possible to state with certainty what concentrations cause weight dierences in the L larvae, while critical levels for the B and R larvae can be set at 230 and 440 lg g 1 d.w., respectively.
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
205
Fig. 1. Relationship between 4NP concentration in sediment and larval wet weight (S.D.) of C. riparius larvae (L Lambro; B Belgio; R RIZA). The weights signi®cantly dierent from the control are indicated with P < 0:001.
Previous studies have determined an LOEC for NP on Chironomus survival of 91 lg l 1 (Kahl et al., 1997) and 243 lg l 1 (Schmude et al., 1999). In both these tests, NP was dissolved in the water column, and the subsequent concentrations were much higher than those measured in this study in the water overlying NPpolluted sediments, which never exceeded 20 lg l 1 . Therefore, adverse eects on larvae living in sediment should be ascribed to direct uptake from polluted sediments. Recent monitoring studies have found NPs to occur in natural environments. In the UK, Blackburn and Waldock (1995) recorded NP concentrations of 15 lg g 1 d.w. in the sediments and 180 lg l 1 in the water of the River Aire, which receives high inputs of surfactants from textile processing mills. In Switzerland, nonylphenolic compounds were the main organic pollutants of the Glatt River (Ahel et al., 1994) with a 4NP concentration of 13.1 lg g 1 d.w. Marcomini and Giger (1987) reported an NP concentration of about 1 lg g 1
in the Rhine sediments, and higher levels, ranging from 6.8 to 40:2 lg g 1 , were found in marine sediments in the Netherlands (de Voogt et al., 1997). In the Venice Lagoon (Italy), particularly in an area heavily aected by industrial pollution, an NP concentration of up to 42 ng g 1 d.w. (Marcomini et al., 1990) was recorded. The LC50 values determined in the present study on C. riparius are much higher than the concentrations so far measured in sediments. However, the concentration measured in River Aire water (Blackburn and Waldock, 1995) exceeds the concentration measured in the water in our experiments, indicating that much higher sediment contamination may exist. As a consequence, there is probably a risk to benthic organisms from sediment NP, since steady-state conditions can be reached between the sediment and the water column. Moreover, recent studies have pointed out that NPs and related compounds aect the endocrine functions in vertebrates (Jobling and Sumpter, 1993) and interfere with the development of some invertebrates (Brown
206
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
et al., 1999). As our research involved only short-term experiments, we were not able to examine this possibility of interference with the endocrine system of chironomids; this could be evaluated only through long-term bioassays, at concentrations lower than those causing mortality and close to those found in some aquatic environments. 4. Conclusions Laboratory tests with benthic chironomids are commonly used to assess the potential toxicity of a contaminant in sediment. The present research reveals the need to standardise current test procedures in terms of animal stock selection. In order to avoid the experimental variability deriving from the use of dierent stocks of chironomids, preliminary intercalibration tests between dierent laboratories should be performed with at least two reference chemicals, such as a metal and an organic micropollutant. Moreover, when dealing with pure substances which have low water solubility, it is important to choose a sediment spiking method which will guarantee the success and the reproducibility of the tests. In this case the test chemical cannot be mixed directly with the sediment, but must usually be dissolved in a water-miscible organic solvent before being added to the sediment. However, the solvent might interact with the test substance and modify the ecotoxicological response. In this respect, the spiking method adopted in the present research and applied to NP seems to be reliable, avoiding any possible interference and being reproducible. As for 4NP's properties as an endocrine disrupter, further investigations are required of the emergence success of Chironomus, sex ratio, fecundity of adults and egg viability in long-term experiments. Acknowledgements We would like to thank Stefano Polesello who provided 4NP concentrations in the River Lambro sediments. Giovanna Meregalli and Davide Vignati supplied the larvae of clean environments.
References Ahel, M., Giger, W., 1993. Partitioning of alkylphenols and alkylphenol polyethoxylates between water and organic solvents. Chemosphere 26, 1471±1478. Ahel, M., Giger, W., Schaner, C., 1994. Behaviour of alkylphenol polyethoxylate surfactants in the aquatic environment-II. Occurence and transformations in rivers. Water Res. 28, 1143±1152.
Anlauf, A., 1994. Some characteristics of genetic variants of Tubifex tubifex in laboratory cultures. Hydrobiologia 278, 1±6. Blackburn, M.A., Waldock, M.J., 1995. Concentrations of alkylphenols in rivers and estuaries in England and Wales. Water Res. 29 (7), 1623±1629. Blockwell, S.J., Pascoe, D., Taylor, E.J., 1996. Eects of lindane on the growth of the freshwater amphipod Gammarus pulex (L). Chemosphere 32, 1795±1803. Brown, R.J., Conradi, M., Depledge, M.H., 1999. Long-term exposure to 4-nonylphenol aects sexual dierentiation and growth of the amphipod Corophium volutator (Pallas, 1766). Sci. Total Environ. 233 (1±3), 77±88. Chemical Manufacturers Association, 1994. Alkylphenol Ethoxylates in the Environment. Alkylphenol and Ethoxylates Panel of the CMA, Washington, DC. Comber, M.H.I., William, T.D., Stewart, K.M., 1993. The eects of nonylphenol on Daphnia magna. Water Res. 27, 273±276. Day, K., Kirby, R.S., Reynoldson, T.B., 1995. The eect of manipulations of freshwater sediments on responses of benthic invertebrates in whole-sediment toxicity tests. Environ. Toxicol. Chem. 14 (8), 1333±1343. Day, K.E., Scott, R., Reynoldson, T.B., 1994. Sexual dimorphism in Chironomus riparius: impact on interpretation of growth in whole sediment toxicity tests. Environ. Toxicol. Chem. 13, 35±39. de Voogt, P., de Beer, K., van der Wielen, F., 1997. Determination of alkylphenol ethoxylates in industrial and environmental samples. Trend Anal. Chem. 16 (10), 584±595. Heinis, L.J., Knuth, M.L., Liber, K., Sheedy, B.R., Tunell, R.L., Ankley, G.T., 1999. Persistence and distribution of 4-nonylphenol following repeated application to littoral enclosures. Environ. Toxicol. Chem. 18 (3), 363±375. Jobling, S., Sumpter, J.P., 1993. Detergent components in sewage euent are weakly estrogenic to ®sh: an in vitro study using rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat. Toxicol. 27, 361±372. Kahl, M.D., Makynen, E.A., Kosian, P.A., Ankley, G.T., 1997. Toxicity of 4-nonylphenol in a life-cycle test with the midge Chironomus tentans. Ecotoxicol. Environ. Saf. 38, 155± 160. Kosalwat, P., Knight, A.W., 1987. Chronic toxicity of copper to a partial life cycle of the midge Chironomus decorus. Arch. Environ. Contam. Toxicol. 16, 275±282. Liber, K., Call, D.J., Dawson, T.D., Whiteman, F.W., Dillon, T.M., 1996. Eects of Chironomus tentans larval growth on adult emergence and ovipositing success: implications for interpreting freshwater sediment bioassays. Hydrobiologia 323, 155±167. Liber, K., Knuth, M.L., Stay, F.S., 1999. An integrated evaluation of the persistence and eects of 4-nonylphenol in an experimental littoral ecosystem. Environ. Toxicol. Chem. 18, 357±362. Marcomini, A., Giger, W., 1987. Simultaneous determination of linear alkylbenzene sulfonates, alkylphenol polyethoxylates and nonylphenol by high-performance liquid chromatography. Anal. Chem. 59, 1709±1715. Marcomini, A., Pavoni, B., Sfriso, A., Orio, A.A., 1990. Persistent metabolites of alkylphenol polyethoxylates in the marine environment. Mar. Chem. 29, 307±323.
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207 Murdoch, M.H., Chapman, P.M., Norman, D.M., Quintino, V.M., 1997. Spiking sediment with organochlorines for toxicity testing. Environ. Toxicol. Chem. 16 (7), 1504±1509. OECD, 1998. OECD guidelines for the testing of chemicals ± Chironomid toxicity test using spiked sediment. Proposal for a new guideline. Draft document, p. 16. RIZA, 1996. Protocol for testing of substances in chronic sediment bioassays with the freshwater dipteran Chironomus riparius. RIZA Protocol, 94.096X. Rosenberg, D.M., 1992. Freshwater biomonitoring and Chironomidae. Neth. J. Aquat. Ecol. 26, 101±122. Salomons, W., F orstner, U., 1984. In: Metals in Hydrocycle. Springer, Berlin, p. 349. Schmude, K.L., Liber, K., Corry, T.D., Stay, F.S., 1999. Eects of 4-nonylphenol on benthic macroinvertebrates and insect emergence in littoral enclosures. Environ. Toxicol. Chem. 18 (3), 386±393. Sturmbauer, C., Opadiya, G.B., Niederstatter, H., Riedmann, A., Dallinger, R., 1999. Mitochondrial DNA reveals cryptic Oligochaete species diering in cadmiun resistance. Mol. Bio. Evol. 16 (7), 967±974. Suedel, B.C., Rodgers, J.H., 1994. Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem. 13 (7), 1163±1175. Thienemann, A., 1954. Chironomus. Leben, Verbreitung und wirtschaftliche Bedeutung der Chironomiden. Binnengew asser 20, 1±834.
207
Dr. Roberta Bettinetti has a Master's degree in Biology (1994) (subject: Ecology, Limnology) and a Ph.D. in Invertebrate Biology (1999). Dr. Bettinetti is at present in a post-doctorate position at the University of Milan. The author's main research ®elds are Ecotoxicology and Paleoecology of sediments. Dr. Bettinetti has authored scienti®c papers published in conference proceedings, reports, national and international journals. Dr. Debora Cuccato has a Master's degree in Biology (1998) on the bioaccumulation of trace metals by ®sh. Dr. Cuccato has had practical training on sediment toxicity assessment at the University of Milan. The author has attended the ``International Summer School for Xenobiotics and Human Health'', University of Siena (1999). Dr. Cuccato is at present studying the eects of long-term glucocorticoid therapy on human beings. Prof. Silvana Galassi has a Master's degree in Biology (1973) from the University of Milan. Prof. Galassi is a Full Professor of Ecology at the University of Insubria, Como, Italy. The author's main research ®elds are risk assessment of organic micropollutants making use of ecotoxicological tests and predictive models. Prof. Alfredo Provini is a Full Professor of Ecology at the University of Milan. the author's main research ®elds are organic micropollutants in waters and sediments to detect toxic factors and to evaluate the environmental hazards associated with their transfer through the trophic chains. Prof. Provini has authored more than 110 scienti®c papers.
www.elsevier.com/locate/chemosphere
Toxicity of 4-nonylphenol in spiked sediment to three populations of Chironomus riparius R. Bettinetti
a,*
, D. Cuccato a, S. Galassi b, A. Provini
a
a
b
Biology Department, University of Milan, Via Celoria 26, Milan 20133, Italy Biotechnology and Bioscience Department, University of Milan Bicocca, Via Emanueli 12 bis, Milan 20126, Italy Received 22 November 2000; accepted 9 April 2001
Abstract Nonylphenols (NPs) are the primary stable metabolites of alkylphenol polyethoxylates (APEs), a family of compounds widely used in industry and in some domestic products. As NPs accumulate in sediments in aquatic environments, the risk to benthic organisms needs to be assessed. In this study 4NP-spiked sediments were tested on larvae of the dipteran Chironomus riparius. First instar larvae obtained from populations at three dierent sources were used. To spike the sediments, an equilibration procedure between water and sediment was adopted to avoid the use of solvents. Lower 10-d LC50 values were determined for two populations of C. riparius from clean environments (315±465 and 315± 350 lg g 1 d.w., respectively) than those of a strain deriving from a population collected in a polluted river (600±680 lg g 1 d.w.). Larval growth always decreased with increasing 4NP concentration but without any de®ned trend. The results of this study suggest that tolerance to the toxicant can be developed in populations of polluted environments and that testing procedures should be standardised. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: 4-Nonylphenol toxicity; Chironomids; Sediment spiking
1. Introduction Alkylphenol polyethoxylates (APEs) are non-ionic surfactants widely used in the formulation of industrial detergents. It has been estimated that the net worldwide production of APEs exceeds 300,000 t annually (Chemical Manufacturers Association, 1994). Once discharged into the environment, their degradation leads mainly to the formation of metabolites such as alkylphenols, alkylphenol monoethoxylates and alkylphenol diethoxylates, well known for their high aquatic toxicity (Chemical Manufacturers Association, 1994). In most APEs the alkyl chain is a nonyl, and nonylphenols (NPs) therefore represent the main stable metabolites of these
*
Corresponding author. Fax: +39-02-26604361. E-mail address: [email protected] (R. Bettinetti).
commercial products. NPs have been used as additives in plastics, household and personal care products, lubricating oils, agrochemicals, latex coatings and adhesive, paint, pulp and paper production and textile manufacturing (Liber et al., 1999). NPs are hydrophobic and accumulate in sediments, where they can persist in excess of 400 d (Heinis et al., 1999). Consequently, sediments are able to act as a sink for NP and represent a risk to aquatic organisms. To date, several tests have been done to detect the acute and chronic toxicity of NPs to a number of aquatic species (Comber et al., 1993; Kahl et al., 1997; Schmude et al., 1999). However, variable 4NP toxicity values are reported, probably because of errors associated with the low solubility of NP in water, dierences in sensitivity among strains used and the varying organic carbon content of sediments. Kahl et al. (1997), applying NP to the overlying water and using sand as substrate for Chironomus tentans, found an LOEC value of 91 lg l 1
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 1 1 4 - X
202
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
2. Material and methods
ugation. Spiked sediments for use in tests were obtained by mixing this stock sediment with the reference sediment. They were mixed ®rst manually for 10 min and subsequently with a mechanical mixer for 30 min. Each spiked sediment was then subdivided into ®ve replicate beakers. Spring water (pH 7.8; hardness 106 mg l 1 CaCO3 ) was then added and the sediment left to settle before the animals were added, as recommended in the test procedures described by Day et al. (1994) and OECD (1998). The water content of the sediment was evaluated as weight dierence after wet sediments had been dried at 105°C for 12 h. Organic matter content (OM%) was estimated as the weight loss after the dried sediment had been heated at 550°C for 8 h. At the start of each test, the sediment dry weight (d.w.), OM% and the actual concentration of 4NP in the sediment were determined. Analyses were performed in each replicate beaker and, in some cases, also in subsamples from the same beaker.
2.1. Collection and preparation of reference sediment
2.3. Toxicant analysis
Preliminary tests to evaluate the best larval growth conditions of C. riparius were carried out using both natural sediments sampled in dierent unpolluted areas, and arti®cial sediments (Suedel and Rodgers, 1994). The best development of larvae was observed with unpolluted sediments from Lake Monate (Northern Italy). After a chemical analysis showing that 4NP was not present and the other parameters were in the range of concentrations suggested by Salomons and F orstner (1984), this sediment was selected as reference sediment. Sediments were collected with an Ekman grab at a lake depth of about 10 m. In the laboratory they were wet sieved through a 250-lm mesh using dechlorinated tap water to remove indigenous macrofauna and coarse particles (Day et al., 1995). After 24 h of settling, the overlying water was removed and the settled sediments centrifuged. Sediments were then stored in the dark at 4°C. Before each test, the sediment was mixed to homogeneity in a 1 l glass beaker with a mechanical mixer.
The 4NP concentration was analytically determined both in sediments and in water. The complete extraction of 4NP for analysis was obtained by mixing approximately 2.3 g w.w. of sediment from each test concentration with 50 ml of ultrapure methanol, stirred for 1 h with a mechanical agitator; the methanol was then separated by centrifugation. This procedure was repeated three times and the total extract (150 ml) ®ltered with 0.45-lm glass micro®bre ®lters (Whatmanâ ). The extracts were analysed for 4NP according to Kahl et al. (1997) with a Jasco HPLC equipped with a ¯uorescence detector. Preconcentration of the extract was unnecessary, except for analysis of the reference sediment. The detector excitation and emission wavelengths were set at 230 and 290 nm, respectively. 50 ll of sample was injected directly into a Lichrospher 100 RP-18 5 lm column (4 125 mm; Merck) and eluted with a water± methanol gradient that varied from 20:80 to 0:100 in 11 min. The external standard method was used for quanti®cation. Each analysis was carried out in duplicate. The detection limit was 5 lg 4NP g 1 of sediment (d.w.). Overlying water was preconcentrated 50 times using LiChrolut ENâ cartridges eluted with ultrapure methanol and analysed with the same procedure adopted for sediments. The detection limit was 3 lg l 1 .
in a 20-d test, while Schmude et al. (1999) reported an LOEC of 243 41 lg l 1 for chironomids exposed in littoral enclosures. The present study represents a ®rst attempt to determine the toxicity of 4NP-spiked sediments to dierent strains of the benthic larvae of C. riparius Meigen 1804. This chironomid is the most widely distributed and abundant insect of natural freshwater macrobenthic communities in Europe (Thienemann, 1954) and is frequently adopted for sediment testing (Rosenberg, 1992). Since the various methods of spiking sediments with compounds that are not readily water soluble usually involve the use of a solvent at some stage (Murdoch et al., 1997), this article also attempts to de®ne a reproducible protocol for spiking sediments with hydrophobic chemicals without the use of a carrier, which might interfere with the ecotoxicological response.
2.2. Sediment spiking and analysis 4NP (Sigma-Aldrich, UK) is a mixture of ring and chain isomers. Since 4NP has very low solubility in water and high lipophylicity, with a log Kow value of 4.48 (Ahel and Giger, 1993), saturation concentration in the water medium is not sucient to ensure a high enough concentration for toxicity tests to be performed, after sediment equilibration. Therefore 1 g of 4NP was directly added to 250 g of wet sediment (water content about 50%) and mixed with 250 ml of water. The slurry was then stirred for 20 h and left to settle for 72 h. The sediment was then separated from the water by centrif-
2.4. Chironomid cultures Egg masses of C. riparius were initially obtained from a population collected in the River Lambro (L larvae), a river polluted by civil and industrial discharges from the northern part of Milan (Italy), as well as from cultures
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
maintained at RIZA, Lelystad, The Netherlands (R larvae), and at the University of Leuven, Belgium (B larvae). The last two cultures were from clean environments. The larvae were bred at 21 1°C under the daily photoperiod in 40 l aquaria with Lake Monate reference sediment (3 cm deep) as substrate. An 8-cm deep column of dechlorinated tap water (hardness: 320 mg l 1 CaCO3 ) was maintained over the sediment. Cultures were fed weekly with 1 g Tetraminâ ®sh food per tank. The ®rst larval instars for use in each experiment were obtained by transferring egg ropes from the culture to glass vessels containing spring water. The time required to hatch the ®rst instar larvae was about 3 d at 21 1°C. The animals were then transferred to each test beaker with a glass pipette, putting them below the water surface to avoid trapping them in the surface ®lm. 2.5. Bioassay design Four acute tests (96 h) with K2 Cr2 O7 dissolved in water as reference substance were carried out to verify that laboratory-bred chironomids were suitable for toxicity tests and that their sensitivity remained constant (RIZA, 1996). 4NP toxicity tests were performed according to the guidelines set out by Day et al. (1994) with minor modi®cations. One day before the addition of the animals, 250-ml glass beakers were ®lled with 60 g of spiked wet sediment (water content about 50%) and 200 ml of water gently added to the sediment. 3.5 ml of a 4 g l 1 water suspension of ®sh food, corresponding to 14 mg d.w. Tetraminâ , was put into each beaker. The contents of the beakers, covered with a plastic Petri dish, were allowed to settle in the dark at 21 1°C without aeration. Five replicate beakers were prepared for each nominal concentration, including the control. At the start of the test, the overlying water of each beaker was gently aerated for 2 h and 10 ®rst instar larvae chosen at random were transferred to each beaker. Tests were performed under 16:8 h light:dark photoperiod for 10 d. Every three days the animals were fed with 3.5 ml of Tetraminâ suspension and the water lost through evaporation was replaced with spring water. Temperature, pH, conductivity and dissolved oxygen were measured in all the beakers before and after the tests. Total survival was noted and the individual larval wet weight of each larva recorded using the sensitive, non-destructive technique described by Blockwell et al. (1996). Final larval weights were considered because they represent an integration of physiological responses which are known to be aected by the environment and by pollutant stress (Kosalwat and Knight, 1987). Four tests were performed with the L larvae, two with the B larvae and two with the R larvae.
203
2.6. Data analysis Data on the survival of C. riparius were analysed using the EPA Probit Analyses Program for calculating the LC50 values. The t-test for independent samples was used to determine if the weights of control larvae from clean and polluted environments were similar. One-way ANOVA and the post hoc Tukey test for unequal samples (Spjotvoll±Stoline test, a level 0.05) were performed to evaluate dierences between treatments with the STATISTICA 4.5 package (StatSoft). 3. Results and discussion The ®rst instar larvae of C. riparius used in this study showed a constant and acceptable sensitivity to K2 Cr2 O7 toxicity. The 96-h LC50 values for the L larvae were 13:3 mg l 1 (c.l.: 11.7±14.7) and 14:8 mg l 1 (c.l.: 12.7± 16.5), and for the R larvae 14:8 mg l 1 (c.l.: 12.6±16.8) and 12.3 (c.l.: 5.2±17.3). These values were smaller than the sensitivity range indicated by RIZA (1996) for second instar larvae. The organic matter content of the reference sediment was 2.3%. The 4NP concentration of the stock spiked sediment was between 6 and 6:6 mg g 1 d.w. Analytical concentrations determined in sediment prepared by dilution of stock spiked sediments were generally within 20±25% of the nominal concentrations (Table 1). The proposed spiking method produced a homogeneously dosed sediment, since the 4NP concentration in the replicate beakers was comparable (5%). Moreover, 4NP concentration measured in subsamples of the same beaker was the same. No concentration decrease was measured within 28 d. This is in agreement with the high persistence of 4NP in sediments reported in both laboratory studies and littoral enclosures (Heinis et al., 1999). During the experiments, pH dierences among the beakers were minimal (0.2 pH units) and the oxygen saturation values were always more than 50%. Conductivity values were more variable mainly because of sediment resuspension and the dierent survival of the organisms in each beaker. At equilibrium, 4NP concentration in the overlying water was between 5 and 20 lg l 1 , depending on its concentration in the sediment. The 10-d 4NP LC50 value obtained with the L larvae was within a range of 600±680 4NP lg g 1 d.w., whereas the corresponding values obtained for the larvae collected in unpolluted areas (R and B larvae) were in the range of 315±465 4NP and 315±350 4NP lg g 1 d.w., respectively (Table 2). This observed variability could be due to genetic dierences in cultures deriving from different natural populations, and to a development of tolerance to 4NP and related organic toxicants in the L
204
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
Table 1 Nominal and measured 4NP concentrations (lg g spiked sediments
1
d.w.) in
Table 2 The 10-d LC50 4NP values obtained in tests conducted with three C. riparius larvae (L Lambro; B Belgio; R RIZA)
Nominal concentration
Measured concentration
Test
LC50 (4NP lg g 1 d.w.)
95% Con®dence limits
L1
50 120 200 400 800
60 100 180 350 680
L1 L2 L3 L4
673 603 674 ±a
526±1067 539±676 486±1309
L2
220 350 600
180 440 750
B1 B2
350 314
± 270±355
230 290 350 400 500 650 750
290 365 430 475 490 560 650
R1 R2
465 315
393±623 268±363
L4
150 200 300 350 400 450
115 229 238 309 325 345
B1
150 250 450
200 310 400
B2
250 350 600 950
230 280 500 800
R1
350 400 550
270 350 480
R2
300 500
230 440
L3
larvae. The River Lambro, where the larvae originated, receives the discharges of textile industries and 4NP concentrations ranging from 1 to 4:7 lg g 1 have been found (Polesello, S., personal communication). Genetic dierences leading to altered growth and reproduction have been noted in Tubifex tubifex (Anlauf, 1994) and more recently Sturmbauer et al. (1999) identi®ed dierent genetic lineages in populations of T. tubifex diering in cadmium resistance. In the present study we hypothesise that natural selection facilitates increased tolerance to 4NP in the L population originating in the most contaminated area, and that the midge population
a
Not possible to calculate.
could maintain this adaptation for several generations. This hypothesis needs to be con®rmed by studying a larger number of Chironomus populations but, if it is true, care should be taken in the choice of the test organism to be used in toxicity bioassays. The post hoc Tukey test indicated that the survival of the L larvae was not aected by 4NP up to a concentration of 350 lg g 1 d.w. in sediment. At higher 4NP concentrations mortality increased and zero survival was noted at 1500 lg g 1 d.w. The numbers of both R and B larvae surviving were already decreasing at a 4NP concentration of 230 lg g 1 d.w., and no survival was observed at 800 lg g 1 d.w. The growth response shows that after 10 d the L larvae developed in control beakers had an average wet weight of 3:8 1:2 mg and the R and B larvae were signi®cantly smaller 2:8 1:3 mg (t-test, P < 0:000). The mean wet weight of the control larvae was always higher than that of the exposed organisms, with the exception of tests L1 and L3, where the mean wet weights of the larvae, at 4NP concentration of 60 and 290 lg g 1 , respectively, were similar to that of the control (Fig. 1). After 10 d of exposure the mean wet weight showed a signi®cant P < 0:01 decrease with increasing 4NP concentrations (Fig. 1). Liber et al. (1996) observed that when larval weight increased, the mean emergence time decreased and the mean successful emergence increased. This reduced growth rate due to 4NP toxicity could have long-term consequences on adult emergence success, aecting population density in natural environments. In Fig. 1 the weights of exposed larvae which are signi®cantly dierent P < 0:001 from those of the control are indicated by an asterisk. It is not possible to state with certainty what concentrations cause weight dierences in the L larvae, while critical levels for the B and R larvae can be set at 230 and 440 lg g 1 d.w., respectively.
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
205
Fig. 1. Relationship between 4NP concentration in sediment and larval wet weight (S.D.) of C. riparius larvae (L Lambro; B Belgio; R RIZA). The weights signi®cantly dierent from the control are indicated with P < 0:001.
Previous studies have determined an LOEC for NP on Chironomus survival of 91 lg l 1 (Kahl et al., 1997) and 243 lg l 1 (Schmude et al., 1999). In both these tests, NP was dissolved in the water column, and the subsequent concentrations were much higher than those measured in this study in the water overlying NPpolluted sediments, which never exceeded 20 lg l 1 . Therefore, adverse eects on larvae living in sediment should be ascribed to direct uptake from polluted sediments. Recent monitoring studies have found NPs to occur in natural environments. In the UK, Blackburn and Waldock (1995) recorded NP concentrations of 15 lg g 1 d.w. in the sediments and 180 lg l 1 in the water of the River Aire, which receives high inputs of surfactants from textile processing mills. In Switzerland, nonylphenolic compounds were the main organic pollutants of the Glatt River (Ahel et al., 1994) with a 4NP concentration of 13.1 lg g 1 d.w. Marcomini and Giger (1987) reported an NP concentration of about 1 lg g 1
in the Rhine sediments, and higher levels, ranging from 6.8 to 40:2 lg g 1 , were found in marine sediments in the Netherlands (de Voogt et al., 1997). In the Venice Lagoon (Italy), particularly in an area heavily aected by industrial pollution, an NP concentration of up to 42 ng g 1 d.w. (Marcomini et al., 1990) was recorded. The LC50 values determined in the present study on C. riparius are much higher than the concentrations so far measured in sediments. However, the concentration measured in River Aire water (Blackburn and Waldock, 1995) exceeds the concentration measured in the water in our experiments, indicating that much higher sediment contamination may exist. As a consequence, there is probably a risk to benthic organisms from sediment NP, since steady-state conditions can be reached between the sediment and the water column. Moreover, recent studies have pointed out that NPs and related compounds aect the endocrine functions in vertebrates (Jobling and Sumpter, 1993) and interfere with the development of some invertebrates (Brown
206
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207
et al., 1999). As our research involved only short-term experiments, we were not able to examine this possibility of interference with the endocrine system of chironomids; this could be evaluated only through long-term bioassays, at concentrations lower than those causing mortality and close to those found in some aquatic environments. 4. Conclusions Laboratory tests with benthic chironomids are commonly used to assess the potential toxicity of a contaminant in sediment. The present research reveals the need to standardise current test procedures in terms of animal stock selection. In order to avoid the experimental variability deriving from the use of dierent stocks of chironomids, preliminary intercalibration tests between dierent laboratories should be performed with at least two reference chemicals, such as a metal and an organic micropollutant. Moreover, when dealing with pure substances which have low water solubility, it is important to choose a sediment spiking method which will guarantee the success and the reproducibility of the tests. In this case the test chemical cannot be mixed directly with the sediment, but must usually be dissolved in a water-miscible organic solvent before being added to the sediment. However, the solvent might interact with the test substance and modify the ecotoxicological response. In this respect, the spiking method adopted in the present research and applied to NP seems to be reliable, avoiding any possible interference and being reproducible. As for 4NP's properties as an endocrine disrupter, further investigations are required of the emergence success of Chironomus, sex ratio, fecundity of adults and egg viability in long-term experiments. Acknowledgements We would like to thank Stefano Polesello who provided 4NP concentrations in the River Lambro sediments. Giovanna Meregalli and Davide Vignati supplied the larvae of clean environments.
References Ahel, M., Giger, W., 1993. Partitioning of alkylphenols and alkylphenol polyethoxylates between water and organic solvents. Chemosphere 26, 1471±1478. Ahel, M., Giger, W., Schaner, C., 1994. Behaviour of alkylphenol polyethoxylate surfactants in the aquatic environment-II. Occurence and transformations in rivers. Water Res. 28, 1143±1152.
Anlauf, A., 1994. Some characteristics of genetic variants of Tubifex tubifex in laboratory cultures. Hydrobiologia 278, 1±6. Blackburn, M.A., Waldock, M.J., 1995. Concentrations of alkylphenols in rivers and estuaries in England and Wales. Water Res. 29 (7), 1623±1629. Blockwell, S.J., Pascoe, D., Taylor, E.J., 1996. Eects of lindane on the growth of the freshwater amphipod Gammarus pulex (L). Chemosphere 32, 1795±1803. Brown, R.J., Conradi, M., Depledge, M.H., 1999. Long-term exposure to 4-nonylphenol aects sexual dierentiation and growth of the amphipod Corophium volutator (Pallas, 1766). Sci. Total Environ. 233 (1±3), 77±88. Chemical Manufacturers Association, 1994. Alkylphenol Ethoxylates in the Environment. Alkylphenol and Ethoxylates Panel of the CMA, Washington, DC. Comber, M.H.I., William, T.D., Stewart, K.M., 1993. The eects of nonylphenol on Daphnia magna. Water Res. 27, 273±276. Day, K., Kirby, R.S., Reynoldson, T.B., 1995. The eect of manipulations of freshwater sediments on responses of benthic invertebrates in whole-sediment toxicity tests. Environ. Toxicol. Chem. 14 (8), 1333±1343. Day, K.E., Scott, R., Reynoldson, T.B., 1994. Sexual dimorphism in Chironomus riparius: impact on interpretation of growth in whole sediment toxicity tests. Environ. Toxicol. Chem. 13, 35±39. de Voogt, P., de Beer, K., van der Wielen, F., 1997. Determination of alkylphenol ethoxylates in industrial and environmental samples. Trend Anal. Chem. 16 (10), 584±595. Heinis, L.J., Knuth, M.L., Liber, K., Sheedy, B.R., Tunell, R.L., Ankley, G.T., 1999. Persistence and distribution of 4-nonylphenol following repeated application to littoral enclosures. Environ. Toxicol. Chem. 18 (3), 363±375. Jobling, S., Sumpter, J.P., 1993. Detergent components in sewage euent are weakly estrogenic to ®sh: an in vitro study using rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat. Toxicol. 27, 361±372. Kahl, M.D., Makynen, E.A., Kosian, P.A., Ankley, G.T., 1997. Toxicity of 4-nonylphenol in a life-cycle test with the midge Chironomus tentans. Ecotoxicol. Environ. Saf. 38, 155± 160. Kosalwat, P., Knight, A.W., 1987. Chronic toxicity of copper to a partial life cycle of the midge Chironomus decorus. Arch. Environ. Contam. Toxicol. 16, 275±282. Liber, K., Call, D.J., Dawson, T.D., Whiteman, F.W., Dillon, T.M., 1996. Eects of Chironomus tentans larval growth on adult emergence and ovipositing success: implications for interpreting freshwater sediment bioassays. Hydrobiologia 323, 155±167. Liber, K., Knuth, M.L., Stay, F.S., 1999. An integrated evaluation of the persistence and eects of 4-nonylphenol in an experimental littoral ecosystem. Environ. Toxicol. Chem. 18, 357±362. Marcomini, A., Giger, W., 1987. Simultaneous determination of linear alkylbenzene sulfonates, alkylphenol polyethoxylates and nonylphenol by high-performance liquid chromatography. Anal. Chem. 59, 1709±1715. Marcomini, A., Pavoni, B., Sfriso, A., Orio, A.A., 1990. Persistent metabolites of alkylphenol polyethoxylates in the marine environment. Mar. Chem. 29, 307±323.
R. Bettinetti et al. / Chemosphere 46 (2002) 201±207 Murdoch, M.H., Chapman, P.M., Norman, D.M., Quintino, V.M., 1997. Spiking sediment with organochlorines for toxicity testing. Environ. Toxicol. Chem. 16 (7), 1504±1509. OECD, 1998. OECD guidelines for the testing of chemicals ± Chironomid toxicity test using spiked sediment. Proposal for a new guideline. Draft document, p. 16. RIZA, 1996. Protocol for testing of substances in chronic sediment bioassays with the freshwater dipteran Chironomus riparius. RIZA Protocol, 94.096X. Rosenberg, D.M., 1992. Freshwater biomonitoring and Chironomidae. Neth. J. Aquat. Ecol. 26, 101±122. Salomons, W., F orstner, U., 1984. In: Metals in Hydrocycle. Springer, Berlin, p. 349. Schmude, K.L., Liber, K., Corry, T.D., Stay, F.S., 1999. Eects of 4-nonylphenol on benthic macroinvertebrates and insect emergence in littoral enclosures. Environ. Toxicol. Chem. 18 (3), 386±393. Sturmbauer, C., Opadiya, G.B., Niederstatter, H., Riedmann, A., Dallinger, R., 1999. Mitochondrial DNA reveals cryptic Oligochaete species diering in cadmiun resistance. Mol. Bio. Evol. 16 (7), 967±974. Suedel, B.C., Rodgers, J.H., 1994. Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem. 13 (7), 1163±1175. Thienemann, A., 1954. Chironomus. Leben, Verbreitung und wirtschaftliche Bedeutung der Chironomiden. Binnengew asser 20, 1±834.
207
Dr. Roberta Bettinetti has a Master's degree in Biology (1994) (subject: Ecology, Limnology) and a Ph.D. in Invertebrate Biology (1999). Dr. Bettinetti is at present in a post-doctorate position at the University of Milan. The author's main research ®elds are Ecotoxicology and Paleoecology of sediments. Dr. Bettinetti has authored scienti®c papers published in conference proceedings, reports, national and international journals. Dr. Debora Cuccato has a Master's degree in Biology (1998) on the bioaccumulation of trace metals by ®sh. Dr. Cuccato has had practical training on sediment toxicity assessment at the University of Milan. The author has attended the ``International Summer School for Xenobiotics and Human Health'', University of Siena (1999). Dr. Cuccato is at present studying the eects of long-term glucocorticoid therapy on human beings. Prof. Silvana Galassi has a Master's degree in Biology (1973) from the University of Milan. Prof. Galassi is a Full Professor of Ecology at the University of Insubria, Como, Italy. The author's main research ®elds are risk assessment of organic micropollutants making use of ecotoxicological tests and predictive models. Prof. Alfredo Provini is a Full Professor of Ecology at the University of Milan. the author's main research ®elds are organic micropollutants in waters and sediments to detect toxic factors and to evaluate the environmental hazards associated with their transfer through the trophic chains. Prof. Provini has authored more than 110 scienti®c papers.