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Use of the freshwater macroinvertebrate Chironomus riparius (Diptera: Chironomidae) in the assessment of sediment toxicity
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Pergamon
Waf. Sci. Tech. Vol. 34, No. 7-8, pp. 101-107, 1996. Copyright © 1996 fA WQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved. 0273-12 23/96 $15'00 + 0·00
PH: S0273-1223(96)00730-5
USE OF THE FRESHWATER MACROINVERTEBRATE CHIRONOMUS RIPARIUS (DIPTERA: CHIRONOMIDAE) IN THE ASSESSMENT OF SEDIMENT TOXICITY M. M. Watts and D. Pascoe Box 915, School of Pure and Applie d Biology, University of Wales, Cardiff, P.o. Cardiff, CF13T L, UK
ABSTRACT Meigen were used to evaluate the The growth and emergence responses of the midge Chironomus riparius obtained from polluted sites or either were which toxicity of natural and formulated reference sediments was only observed in one of the growth larval in n spiked with copper in the laboratory. A significant reductio r, copper spiking resulted in Howeve d. discusse are two polluted field sediments tested. The reasons for this e sediments at copper referenc ted formula two the significant growth reductions of larvae exposed to e sediment was not referenc natural in exposed concentrations of 3 and 4 mg Cu 2+ I-I. Growth of larvae rations. significantly affected at equivalent concent t compared to formulated reference The emergence of adults was adversely affected in polluted field sedimen ) were significantly greater in the sediment. Male, female and total adult median emergence times (EmT50 sediment contamination. In addition, former treatment indicating a delay in emergence, attributable to g in a lower percentage of adults survivorship of the larvae in the polluted sediment was reduced, resultin of this study illustrate the usefulness of emerging compar ed to the formulated reference sediment. The results es of C. riparius, in the assessment of chronic response criteria, incorporating the more sensitive life-stag Ltd. sediment toxicity. Copyright © 1996 IAWQ. Published by Elsevier Science
KEYWORDS ion; natural sediment; sediment Chironomus riparius; emergence; formulated sediment; growth; inhibit toxicity; survivorship. INTRODUCTION ial effects of dissolved pollutants on Traditionally, the study of aquatic toxicology has centred on the potent and fish forming the basic regulatory pelagic organisms, with acute toxicity data for algae, crustaceans table to the underlying substrate, framework. However, these data fail to take into account effects attribu organic and inorganic contaminants which can greatly influence the fate of a given pollutant. Hydrophobic sediments which consequently act as a that enter the aquatic ecosystem may rapidly become associated with than those in the water column 'sink', accumulating concentrations several orders of magnitude higher by polluted sediments, appropriate (Ingersoll and Nelson, 1990). In order to evaluate the threat posed JWST 34: 7/8-E
101
M. M. WATTS and D. PASCOE
102
organisms which are actually exposed to contaminants in this phase, should be incorporated into any f~rm of testing programme (Pascoe and Edwards, 1989). A suitable test species within this ca.tegory I~ t~e macroinvertebrate Chironomus riparius Meigen, a member of the diptera n family ChironomIdae, WhICh IS the most widely distributed and frequently most abundant group of insects in freshwater (Cranston, 199~) .. In addition to the functionally important benthic niche occupied by the species, several other charactenstIcs lend weight to its suitability (Taylor et al., 1993), including the existen ce of an established method for laboratory culture (McCahon and Pascoe, 1988). The species has been w.idely utilis.ed in b~t~ .acute ~d chronic testing thereby providing a large database of tolerance levels, rangmg from hIgh sensIt~v~ty of f~rst instar larvae to high tolerance of the fourth instars (Williams et aI., 1986). In order that tOXICIty testmg continues to playa role in the protection of the aquatic environment, standa rdised sediment assessment tests, incorporating the appropriate species, need to be designed and introd uced as soon as possible. The adult emergence and larval growth of C. riparius are responses which should help to explain the mechanisms of sediment toxicity and provide a means of quantifying the toxic effects . METHODS Test sediments. Natural sediments were collected from two polluted and one 'clean' site in South Wales. Five to eight replicate samples (the number being dependent on the nature of the substrate) were obtained at each site using a fIxed handle grab, and pooled in accordance with SETA C (1993) recommendations. Samples were stored at 4°C ± laC in the dark and sieved to 1 mm prior to use in tests which were initiated within 14 days of collection. Two formulated reference sediments were prepared in the laboratory. The fIrst was based on OECD (1981) test guideline no. 207 and comprised, on the basis of dry weight: 10% sphagnum peat (visible plant debris removed, air dried and finely ground), 20% kaolin clay and 70% fine sand (:5 0.25 mm). The three fractions were thoroughly blended and formed into a thick paste by addition of dechlorinated mains water prior to use in tests. The second sediment was formulated in the light of Suedel and Rodgers' (1994) work to more accurately reflect the natural environment and compr ised, on the basis of dry weight: 5% kaolin clay, 15 or 30% *Shamrock® peat-free compost (large debris removed, air dried) and 80 or 65% *Shamrock horticultural grade silver sand (divided into coarse: 2.0-0.5 mm, medium: 0.5-0.25 mm and fine: :5 0.25 mm). Growth tests. Two separate tests were performed using changes in growth to indicate pollutant stress. The first test involved a comparison of growth in three natural sedime nts ('clean' and 2 types of polluted sediment) with that in a formulated reference sediment. In the second test, growth of C. riparius was evaluated in sediments ( natural and formulated) spiked with copper . All tests were carried out at 20°C ± laC with a 16 hour light photoperiod and 8 hours dark, with dechlorinated mains water (hardness, 114 mg 1-1 as CaC0 3 ) as diluent. In all cases, 25 individual second instar larvae were exposed to I g of sediment with 5 ml of water in 6 ml repIi dish wells. Overlying solutions were replac ed daily, thus maintaining dissolved oxygen at :5 80% of the air saturation value (Taylor et ai., 1991). Food was also administered daily and comprised a 500 ml dose of a suspension of Tetramin® fish flake (1 g in 1 litre of dechlorinated water). FollOWing 10 days exposure, individual larval wet weights were record ed using a sensitive, non-destructive technique as described by Blockwell et ai. (in press). Samples of overlying solutions were taken daily, passed through a 0.45 J.lm filter and acidified to 1% (v Iv) with ARIST AR® nitric acid. The sediment was removed from each compartment at the end of the test and prepared for analysis as outlined by Taylor et al. (1994). Copper concentrations in the water and sediment samples were measured against suitable standards (0.2-4.0 mg 1-1) by flame atomic absorption spectrophotometry on an Instrumentation Laboratory Model 457, using standard operating procedures. Emergence tests. The emergence of adult C. riparius was emplo yed to assess the toxicity of one of the polluted field sediments used in the first growth test, compared with a non-contaminated formulated reference sediment. Ten exposure containers allocated to each sediment type all received sediment to a depth ?f 1 cm and 400 ml of dechlorinated water (hardness, 114 mg 1-1 as CaC0 3) prior to the addition of 25 first mstar larvae. The water column was continually aerated ensuring that dissolved oxygen did not fall to less
* Two reference sediments were formulated with differing % organic matter and sand levels.
Use of the freshwater macroinvertebrate
103
than 80% of the air saturation value. Measures of conductivity and pH of the overlying solutions were obtained using hand-held meters and found to lie within the following ranges: conductivity, 268-348 ms cm• 1 and pH, 6.8-7.2. The bioassays were carried out at a temperature of 20°C ± 1°C with 16 hours light photoperiod and 8 hours dark. The larvae were fed on alternate days with 290 ml of a suspension of Tetramin® fish flake (1.75 mg flake in 20 ml of dechlorinated water). Daily feeding was then introduced when the larvae were seen to have reached the third instar stage. Emerged adults were counted, sexed and removed from the system daily until 100% emergence was achieved or alternatively a period of 10 days had elapsed. Data analysis Growth tests. Data from the growth tests were analysed using one-way ANOVA on the Minitab (version 9) statistical package, to determine differences in final larval weights, followed by multiple comparisons (Tukey-Kramer) to locate differences between treatments. Final larval weights were used since individual growth rates represent an integration of physiological responses which are known to be affected by the environment (Gauss et ai., 1985) and by pollutant stress (Kosalwat and Knight, 1987a). Emergence tests. Median emergence times (EmT50) of C. riparius for males, females and all adults were calculated and compared for each treatment using a FORTRAN programme developed in this laboratory and based on the time response analysis methods of Litchfield (1949). Percentage emergence data were analysed using the Minitab statistical package. The data were arcsine transformed prior to one-way ANOVA testing for differences in adult numbers, followed by multiple comparisons (Tukey-Kramer) to locate differences between treatments. RESULTS AND DISCUSSION Growth tests. The comparative growth, expressed as mean wet weight, of larvae exposed to three natural and one formulated reference sediment is shown in Table 1. Table 1. Mean wet weight of C. riparius larvae exposed to natural and formulated reference sediments for 10 days Sediment Type DECD Reference Sediment (Control) Natural Sediment 1- Clean Natural Sediment 2- Polluted Natural Sediment 3-Polluted
Mean larval weight (mg) S.E. shown in parentheses 7.25 mg (0.254) 8.24 mg (0.344) 7.90 mg (0.318) 6.50 mg (0.290)
The results (ANOVA) indicate that the different sediments tested, produced a significant inhibition (P = 0.001) of growth in the larvae compared to the control but multiple comp~iso?s \p ::; 0.05) r~vealed that only sediment 3 was responsible for this effect. This could be interpreted as 1OdIcat1Og that sedIment 3 was more polluted than sediment 2, or alternatively that any contaminants present were bioavail~ble to the l~ae in the former but not in the latter polluted sediment type. The weights of larvae exposed 10 the remam10g sediments displayed no significant difference from those of the control. The distribution and fate of copper within three different sediment / water systems and its effect on growth of C. riparius larvae are shown in Table 2. The data exhibit variation in larval weights between treatments, indicating that the presence of copper adversely affected norm~ growth and de.velopment. In the natural clean sediment, no significant difference from the control arose 10 final ~arval ~eI~ht, (P ~ 0.178) at an~ of the copper concentrations, although a gradual reduction in weight was eVIdent WIth mcreasmg con~entratlOn. Larvae exposed to copper in both of the formulated sediments were significantly smaller than theIr controls
M. M. WAlTS and D. PASCOE
104
(P = 0.001) as indicated by multiple comparison (P ~ 0.05), at the 3 and 4 mg Cu 2+ I-I concentrations.
Growth of larvae at 2 mg Cu 2+ 1-1, although reduced compared to control, was not significantly inhibited.
Table 2. Mean wet weight of C. riparius larvae exposed to natural and formulated reference sediments spiked with copper for 10 days Sediment Type / Initial copper dose
Mean larval weight (mg) S.E. shown in parentheses
Copper conc of overlying water at 240 hr (mg r l )
Total sediment R copper conc at 240 hr (Ilg kg-I)
9.797 (0.520) 9.750 (0.593) 9.460 (0.283) 8.580 (0.455)
0.097 0.114 0.139
4660 7200 7530
8.530 (0.393) 7.595 (0.339) 5.108 (0.409) 1.195 (0.124)
0.119 0.150 0.200
3630 5170 5230
9.213 7.355 2.765 2.316
0.148 0.238 0.264
2760 6300 7840
Clean Natural sediment Control 2 mg cu 2+r l 3 mg Cu 2+ r l 4 mg Cu2 + r l Formulated sediment (15% organic matter) Control 2 mg Cu2+ r l 3 mg Cu2+ r l 4 mg Cu 2+ r l Formulated sediment (30% organic matter) Control 2 mg Cu 2 + r l 3 mg Cu2 + r l 4 mg Cu 2+ r l
R
(0.338) (0.384) (0.307) (0.276)
80
70
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emergenc~ patterns of adult C. riparius from formulated reference and polluted field sediments. Data are the total from 10 replicates.
Cross comparison of sediment treatments revealed no significant difference between the weight of control larvae. However, larvae exposed to copper in the natural sediment were significantly heavier (P = 0.006)
Use of the freshwater macroinvertebrate
105
than their c?unte~arts in both formulated preparations, with subsequent multiple comparison testing (P :::; 0.05) reveahng a dIfference at all the concentrations tested. Emergence tests. Comparison of the adult emergence data indicates that the polluted (sediment 3 in the first ~rowth test) and formulated reference sediments (Fig. 1), produced a bimodal emergence pattern, with a peak In male emergence f~llowed by the corresponding female peak for both sediments. However, emergence from the polluted sedIment was delayed and total adult numbers reduced in comparison to the formulated reference sediment. Statistical analyses of the median emergence times (EmT50) confirm the trends suggested in Fig.1 with male, female and total adult emergence significantly delayed in the polluted sediment (Table 3). In addition, one-way ANOVA demonstrated that significantly more adults (P = 0.001) emerged from the formulated as opposed to the polluted sediment, with survivorship in the former reaching 100% compared to 87% in the latter. Table 3. Summary of median adult emergence data from polluted and formulated reference sediments. Data are the total from 10 replicates Test Statistic
Reference Sediment (Control)
Polluted Sediment
Male EmT50 Female EmT50 Total Adult EmT50 No. Males No.Females No. Adults (From 250 individuals)
15.69 Days 17.09 Days 16.02 Days 148 117 265*
21.67 Days 23.37 Days 22.43 Days 126 92 218
* An excess of 15 individuals was recorded, due to the difficulty of counting first instar larvae when setting up the study. Both the growth and emergence responses of C. riparius are known to be sensitive indicators of chronic pollutant stress (Macek et ai., 1976; Giesy et ai., 1988; Pascoe et ai., 1989; Taylor et ai., 1991; Maund et ai., 1992). The results described confirm this, with effects on growth and emergence indicating a need for further investigation into one of the polluted field sediments tested. In addition, larval growth in copper spiked sediments demonstrated the modifying influence of sediment / contaminant complexing and subsequent effects on bioavailability and toxicity. Taylor et ai. (1991) quoted the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for aqueous copper as 16.9 and 25.3 mg I-I, respectively, levels which are exceeded in the overlying water of all three sediments types, highlighting the importance of the partitioning of copper between the aqueous and substrate phase. The extremely high copper loading of all sediments and the limited toxicity at these concentrations would appear to agree with the findings of Ankley et ai. (1994), that pollutants bound in the sediment are non• bioavailable. In addition, Pascoe et ai. (1990) demonstrated that the toxicity of cadmium to C. riparius larvae was reduced in the presence of artificial sediment. Copper is known to be more toxic in the aqueous as opposed to the substrate phase (Kosalwat and Knight, 1987b), however, the level of growth inhibition seen in this study, is far less than would be predicted based on species' tolerance, thereby discounting the water column as the primary route of exposure. This may be explained by the fact that aqueous copper can exist in non-toxic complexes with dissolved organic matter, (Meador, 1991). Such complexes may account for 25-98% of the soluble copper present (Stiff, 1971). The remaining route of exposure and in all probability, the bulk of bioavailable metal, is associated with the pore-water contained within the sediment, supporting evidence for which is provided by Swartz et ai. (1985). The differences in larval weights between sediments could be attributed to the different physico-chemical properties of the three sediment types, which influence the proportion of bioavailable copper, with growth inhibition due to copper in the pore-water affecting normal growth metabolism, presumably by incorporation into and derangement of, the complex series of reactions involved in this process.
M. M. WATTS and D. PASCOE
106
The polluted sediment which produced a reduction in larval weight in the first C. riparius growth experiment. also resulted in deleterious effects on adult emergence, emphasising the sensitivity of the two response criteria, and the consistency of the sediment in bringing about a toxic effect. The fact that emergence is delayed by such a wide margin and adult numbers are significantly lower in comparison to the formulated reference sediment would imply that the polluted sediment exerted an inhibitory effect on larval survival and development, a result previously reported by Wentsel et al. (1978). Pollutants present in the sediment must be bioavailable to the larvae, presumably via the pore-water, to bring about the adverse effects noted. The exact nature of the toxicity observed cannot, however, be established from this experiment. The reduction in total adult numbers may be due to mortality amongst the larvae, especially during the sensitive first and second instar stages, which would agree with the results of the acute toxicity tests of Williams et al. (1986). Alternatively, the larvae may be able to tolerate the pollutants present through the larval stages, with mortality occurring at the delicate pupation stage (Thornton and Wilhm, 1975) where massive morphogenetic change occurs prior to adult emergence. The delay in adult emergence may be due to the increased energy cost of pollutant tolerance which would slow down the normal development process, an effect similar to that of larval growth inhibition, previously noted in this sediment. Both the decline and delay in emergence of chironomids from the polluted sediment could have far reaching consequences at the population level. with low adult numbers affecting mating success (Pascoe et al., 1989) and possibly leading to a reduction in genetic fitness of the population as a whole. CONCLUSIONS The use of natural (polluted and 'clean') and formulated reference sediments and the differences in larval responses to sediment type illustrate the complex nature of sediment toxicity. The physico-chemical parameters of sediments which can exert a major influence on toxicity by affecting contaminant partitioning and bioavailability, vary greatly between sediments and further research is required to fully understand the complex interactions involved. The two response criteria tested, however, go some way in helping to explain sediment toxicity and are useful as a means of quantifying its effects for regulatory purposes. ACKNOWLEDGEMENTS The authors thank Zeneca Agrochemicals for financial support and Mr M O'Reilly and Mrs 0 James for their technical assistance. REFERENCES Ankley, G. T .. Thomas, N. A., Di Toro, D. M., Hansen, D. J., Mahony, J. D., Berry, W. 1., Swartz, R. C., Hoke, R. A., Garrison, W. A., Allen, H. E. and Zarba, C. S. (1994). Assessing potential bioavailability of metals in sediments: A proposed approach. Environ. Management, 18(3), 331-337. Blockwell, S. J., Pascoe, D. and Taylor, E. 1. (in press). Effects of lindane on the growth of the freshwater amphipod Gammarus pulex (L). Chemosphere. Cranston, P. S. (1 ?95). Introduction to the Chironomidae. In: The Chironomidae: The Biology and Ecology of non-biting Midges, P D ArmItage, P S Cranston and L C V Pinder (Eds.), Chapman and Hall, UK, pp, 1-7. Gauss, J. D., Woods, P. E., Winner, R. W. and Skillings, J. H. (1985). Acute toxicity of copper to three life stages of Chironomus . tentans as affected by water hardness-alkalinity. Environ. Poll. Series A., 37, 149-157. Glesy, 1. P.,. Grane~, R. L., Newsted, ~. L., Ros~u, .C. 1., B~nda, A., Kreis, R. G. and Hovarth, F. J. (1988). Comparison of three sedIment bIoassay methods USIng DetrOIt nver sedIments. Environ. Toxicol. Chem., 7, 483-498. Ingersoll, C.. G. and ~elson,~. K. ~1990). Testin.g sediment toxicity with Hyalella azteca (Amphipoda) and Chironomus riparius (Dlptera). In. ~quatlc Tox~cology art! RISk ~ssessment, W G Landis and W H van der Schalie (Eds.), ASTM STP 1096, Amencan SocIety for TestIng Matenals, PhIladelphia. Vol 13, 93-109. Kosalwat, P..and Knight, A. W. (1987a). Chronic toxicity of copper to a partial life cycle of the midge Chironomus decorus. Arch. EnVIron. Contam. Toxico/., 16, 283-290. Kosalwat, P. and ~ight, A. W. (198~b). Acute toxicity of aqueous and substrate bound copper to the midge Chironomus decorus. Arch. EnVIron. Contam. Taxlcol.. 16, 275-282.
Lilchfiel~o~. T.
(1949). A method for the rapid graphic solution of time-percentage effect curves. J. Pharm. Exp. Ther., 97,399-
Macek, Kfi" hBuxton, K.. Derr, S., Dean, J. a~d Saunter, S. (1976). Chronic toxicity of lindane to selected aquatic invertebrates and IS es. U.S. EnVIronmental ProtectIOn Agency. Eco. Res. Ser. EPA-600 / 3-76-046.
Use of the freshwater macroinvertebrate
107
Maund, S. J., Peither, A, Taylor, E. 1., Ji.ittner, 1., Beyerle-Pfnur, R., Lay, 1. P. and Pascoe, D. (1992). Toxicity of lindane to freshwater insect larvae in compartments of an experimental pond. Ecotox. Environ. Sa!. 23, 76-88. Meador, J. P. (1991). The interaction of pH, dissolved organic carbon, and total copper in the determination of ionic copper and toxicity. Aquat. Tox., 19,13-32. McCahon, C. P. and Pascoe, D. (1988). Culture techniques for three freshwater macroinvertebrate species and their use in toxicity tests. Chemosphere, 17,2471-2480. OECD (1981). Guideline for testing of chemicals. Earthworm, acute toxicity test. OECD, Paris. Guideline 207, 1-9. Pascoe, D. and Edwards, R. W. (1989). Single species toxicity tests. In: Aquatic Ecotoxicology: Fundamental Concepts and Methodologies, A Boudou and F Ribeyre (Eds.), CRC Florida, 11,93-126. Pascoe, D., Williams, K. A and Green, D. W. J. (1989). Chronic toxicity of cadmium to Chironomus riparius Meigen - effects upon larval development and adult emergence. Hydrobiologia, 175, 109-115. Pascoe, D., Brown, A E, Evans, B. M. J. and McKavanagh, C. (1990). Effects and fate of cadmium during toxicity tests with Chironomus riparius - the influence offood and artificial sediment. Arch. Environ. Contam. Toxicol., 19, 872-877. SETAC (1993) Guidance document on sediment toxicity tests and bioassays for freshwater and marine enviroments. I R Hill, P Matthiessen and F Heimbach (Eds.). From the "Workshop on sediment toxicity assessment." Renesse, The Netherlands (1993). Stiff, M. J. (1971). Copper / bicarbonate equilibria in solutions of bicarbonate ion at concentrations similar to those found in natural water. Wat. Res., 5, 171-176. Suedel, B. C. and Rodgers, J. H. (1994). Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem., 13(7), 1163-75. Swartz, R. c., Ditsworth, G. R., Schults, D. W. and Lamberson, 1. O. (1985). Sediment toxicity to a marine infaunal amphipod: Cadmium and its interaction with sewage sludge. Marine Environ. Res., 18, 133-153. Taylor, E. J., Maund, S. J. and Pascoe, D. (1991). Evaluation of a chronic toxicity test using growth of the insect Chironomus riparius Meigen. In: Bioindicators and Environmental Management, D W Jeffrey and B Madden, (Eds.), Proc 6th lOBS Symposium, Dublin, 1990. Academic Press Limited, London. pp, 343-352. Taylor, E. J., Blockwell, S. J., Maund, S. J. and Pascoe, D. (1993). Effects of lindane on the life-cycle of a freshwater macroinvertebrate Chironomus riparius Meigen (Insecta: Diptera). Arch. Environ. Contam. Toxicol., 24, 145-150. Taylor, E. J., Rees, E. M. and Pascoe, D. (1994). Mortality and drift-related response of the freshwater amphipod Gammarus pulex (L) exposed to natural sediments, acidification and copper. Aquat. Tox., 29, 83-101. Thornton, K. W. and Wilhm, J. L. (1975). The use of life tables in demonstrating the effects of pH, phenol and NaCI on Chironomus attenuatus populations. Environ. Ent., 4, 325-328. Wentsel, R., McIntosh, A. and McCafferty, W. P. (1978). Emergence of the midge Chironomus tentans when exposed to heavy metal contaminated sediment. Hydrobiologia, 57(3) 195-196. Williams, K. A, Green, D. W. J., Pascoe, D. and Gower, D. E. (1986). The acute toxicity of Cadmium to different larval instars of Chironomus riparius (Diptera:Chironomidae) and its ecological significance for pollution regulation. Oceologia (Berlin), 70, 362-366.
Pergamon
Waf. Sci. Tech. Vol. 34, No. 7-8, pp. 101-107, 1996. Copyright © 1996 fA WQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved. 0273-12 23/96 $15'00 + 0·00
PH: S0273-1223(96)00730-5
USE OF THE FRESHWATER MACROINVERTEBRATE CHIRONOMUS RIPARIUS (DIPTERA: CHIRONOMIDAE) IN THE ASSESSMENT OF SEDIMENT TOXICITY M. M. Watts and D. Pascoe Box 915, School of Pure and Applie d Biology, University of Wales, Cardiff, P.o. Cardiff, CF13T L, UK
ABSTRACT Meigen were used to evaluate the The growth and emergence responses of the midge Chironomus riparius obtained from polluted sites or either were which toxicity of natural and formulated reference sediments was only observed in one of the growth larval in n spiked with copper in the laboratory. A significant reductio r, copper spiking resulted in Howeve d. discusse are two polluted field sediments tested. The reasons for this e sediments at copper referenc ted formula two the significant growth reductions of larvae exposed to e sediment was not referenc natural in exposed concentrations of 3 and 4 mg Cu 2+ I-I. Growth of larvae rations. significantly affected at equivalent concent t compared to formulated reference The emergence of adults was adversely affected in polluted field sedimen ) were significantly greater in the sediment. Male, female and total adult median emergence times (EmT50 sediment contamination. In addition, former treatment indicating a delay in emergence, attributable to g in a lower percentage of adults survivorship of the larvae in the polluted sediment was reduced, resultin of this study illustrate the usefulness of emerging compar ed to the formulated reference sediment. The results es of C. riparius, in the assessment of chronic response criteria, incorporating the more sensitive life-stag Ltd. sediment toxicity. Copyright © 1996 IAWQ. Published by Elsevier Science
KEYWORDS ion; natural sediment; sediment Chironomus riparius; emergence; formulated sediment; growth; inhibit toxicity; survivorship. INTRODUCTION ial effects of dissolved pollutants on Traditionally, the study of aquatic toxicology has centred on the potent and fish forming the basic regulatory pelagic organisms, with acute toxicity data for algae, crustaceans table to the underlying substrate, framework. However, these data fail to take into account effects attribu organic and inorganic contaminants which can greatly influence the fate of a given pollutant. Hydrophobic sediments which consequently act as a that enter the aquatic ecosystem may rapidly become associated with than those in the water column 'sink', accumulating concentrations several orders of magnitude higher by polluted sediments, appropriate (Ingersoll and Nelson, 1990). In order to evaluate the threat posed JWST 34: 7/8-E
101
M. M. WATTS and D. PASCOE
102
organisms which are actually exposed to contaminants in this phase, should be incorporated into any f~rm of testing programme (Pascoe and Edwards, 1989). A suitable test species within this ca.tegory I~ t~e macroinvertebrate Chironomus riparius Meigen, a member of the diptera n family ChironomIdae, WhICh IS the most widely distributed and frequently most abundant group of insects in freshwater (Cranston, 199~) .. In addition to the functionally important benthic niche occupied by the species, several other charactenstIcs lend weight to its suitability (Taylor et al., 1993), including the existen ce of an established method for laboratory culture (McCahon and Pascoe, 1988). The species has been w.idely utilis.ed in b~t~ .acute ~d chronic testing thereby providing a large database of tolerance levels, rangmg from hIgh sensIt~v~ty of f~rst instar larvae to high tolerance of the fourth instars (Williams et aI., 1986). In order that tOXICIty testmg continues to playa role in the protection of the aquatic environment, standa rdised sediment assessment tests, incorporating the appropriate species, need to be designed and introd uced as soon as possible. The adult emergence and larval growth of C. riparius are responses which should help to explain the mechanisms of sediment toxicity and provide a means of quantifying the toxic effects . METHODS Test sediments. Natural sediments were collected from two polluted and one 'clean' site in South Wales. Five to eight replicate samples (the number being dependent on the nature of the substrate) were obtained at each site using a fIxed handle grab, and pooled in accordance with SETA C (1993) recommendations. Samples were stored at 4°C ± laC in the dark and sieved to 1 mm prior to use in tests which were initiated within 14 days of collection. Two formulated reference sediments were prepared in the laboratory. The fIrst was based on OECD (1981) test guideline no. 207 and comprised, on the basis of dry weight: 10% sphagnum peat (visible plant debris removed, air dried and finely ground), 20% kaolin clay and 70% fine sand (:5 0.25 mm). The three fractions were thoroughly blended and formed into a thick paste by addition of dechlorinated mains water prior to use in tests. The second sediment was formulated in the light of Suedel and Rodgers' (1994) work to more accurately reflect the natural environment and compr ised, on the basis of dry weight: 5% kaolin clay, 15 or 30% *Shamrock® peat-free compost (large debris removed, air dried) and 80 or 65% *Shamrock horticultural grade silver sand (divided into coarse: 2.0-0.5 mm, medium: 0.5-0.25 mm and fine: :5 0.25 mm). Growth tests. Two separate tests were performed using changes in growth to indicate pollutant stress. The first test involved a comparison of growth in three natural sedime nts ('clean' and 2 types of polluted sediment) with that in a formulated reference sediment. In the second test, growth of C. riparius was evaluated in sediments ( natural and formulated) spiked with copper . All tests were carried out at 20°C ± laC with a 16 hour light photoperiod and 8 hours dark, with dechlorinated mains water (hardness, 114 mg 1-1 as CaC0 3 ) as diluent. In all cases, 25 individual second instar larvae were exposed to I g of sediment with 5 ml of water in 6 ml repIi dish wells. Overlying solutions were replac ed daily, thus maintaining dissolved oxygen at :5 80% of the air saturation value (Taylor et ai., 1991). Food was also administered daily and comprised a 500 ml dose of a suspension of Tetramin® fish flake (1 g in 1 litre of dechlorinated water). FollOWing 10 days exposure, individual larval wet weights were record ed using a sensitive, non-destructive technique as described by Blockwell et ai. (in press). Samples of overlying solutions were taken daily, passed through a 0.45 J.lm filter and acidified to 1% (v Iv) with ARIST AR® nitric acid. The sediment was removed from each compartment at the end of the test and prepared for analysis as outlined by Taylor et al. (1994). Copper concentrations in the water and sediment samples were measured against suitable standards (0.2-4.0 mg 1-1) by flame atomic absorption spectrophotometry on an Instrumentation Laboratory Model 457, using standard operating procedures. Emergence tests. The emergence of adult C. riparius was emplo yed to assess the toxicity of one of the polluted field sediments used in the first growth test, compared with a non-contaminated formulated reference sediment. Ten exposure containers allocated to each sediment type all received sediment to a depth ?f 1 cm and 400 ml of dechlorinated water (hardness, 114 mg 1-1 as CaC0 3) prior to the addition of 25 first mstar larvae. The water column was continually aerated ensuring that dissolved oxygen did not fall to less
* Two reference sediments were formulated with differing % organic matter and sand levels.
Use of the freshwater macroinvertebrate
103
than 80% of the air saturation value. Measures of conductivity and pH of the overlying solutions were obtained using hand-held meters and found to lie within the following ranges: conductivity, 268-348 ms cm• 1 and pH, 6.8-7.2. The bioassays were carried out at a temperature of 20°C ± 1°C with 16 hours light photoperiod and 8 hours dark. The larvae were fed on alternate days with 290 ml of a suspension of Tetramin® fish flake (1.75 mg flake in 20 ml of dechlorinated water). Daily feeding was then introduced when the larvae were seen to have reached the third instar stage. Emerged adults were counted, sexed and removed from the system daily until 100% emergence was achieved or alternatively a period of 10 days had elapsed. Data analysis Growth tests. Data from the growth tests were analysed using one-way ANOVA on the Minitab (version 9) statistical package, to determine differences in final larval weights, followed by multiple comparisons (Tukey-Kramer) to locate differences between treatments. Final larval weights were used since individual growth rates represent an integration of physiological responses which are known to be affected by the environment (Gauss et ai., 1985) and by pollutant stress (Kosalwat and Knight, 1987a). Emergence tests. Median emergence times (EmT50) of C. riparius for males, females and all adults were calculated and compared for each treatment using a FORTRAN programme developed in this laboratory and based on the time response analysis methods of Litchfield (1949). Percentage emergence data were analysed using the Minitab statistical package. The data were arcsine transformed prior to one-way ANOVA testing for differences in adult numbers, followed by multiple comparisons (Tukey-Kramer) to locate differences between treatments. RESULTS AND DISCUSSION Growth tests. The comparative growth, expressed as mean wet weight, of larvae exposed to three natural and one formulated reference sediment is shown in Table 1. Table 1. Mean wet weight of C. riparius larvae exposed to natural and formulated reference sediments for 10 days Sediment Type DECD Reference Sediment (Control) Natural Sediment 1- Clean Natural Sediment 2- Polluted Natural Sediment 3-Polluted
Mean larval weight (mg) S.E. shown in parentheses 7.25 mg (0.254) 8.24 mg (0.344) 7.90 mg (0.318) 6.50 mg (0.290)
The results (ANOVA) indicate that the different sediments tested, produced a significant inhibition (P = 0.001) of growth in the larvae compared to the control but multiple comp~iso?s \p ::; 0.05) r~vealed that only sediment 3 was responsible for this effect. This could be interpreted as 1OdIcat1Og that sedIment 3 was more polluted than sediment 2, or alternatively that any contaminants present were bioavail~ble to the l~ae in the former but not in the latter polluted sediment type. The weights of larvae exposed 10 the remam10g sediments displayed no significant difference from those of the control. The distribution and fate of copper within three different sediment / water systems and its effect on growth of C. riparius larvae are shown in Table 2. The data exhibit variation in larval weights between treatments, indicating that the presence of copper adversely affected norm~ growth and de.velopment. In the natural clean sediment, no significant difference from the control arose 10 final ~arval ~eI~ht, (P ~ 0.178) at an~ of the copper concentrations, although a gradual reduction in weight was eVIdent WIth mcreasmg con~entratlOn. Larvae exposed to copper in both of the formulated sediments were significantly smaller than theIr controls
M. M. WAlTS and D. PASCOE
104
(P = 0.001) as indicated by multiple comparison (P ~ 0.05), at the 3 and 4 mg Cu 2+ I-I concentrations.
Growth of larvae at 2 mg Cu 2+ 1-1, although reduced compared to control, was not significantly inhibited.
Table 2. Mean wet weight of C. riparius larvae exposed to natural and formulated reference sediments spiked with copper for 10 days Sediment Type / Initial copper dose
Mean larval weight (mg) S.E. shown in parentheses
Copper conc of overlying water at 240 hr (mg r l )
Total sediment R copper conc at 240 hr (Ilg kg-I)
9.797 (0.520) 9.750 (0.593) 9.460 (0.283) 8.580 (0.455)
0.097 0.114 0.139
4660 7200 7530
8.530 (0.393) 7.595 (0.339) 5.108 (0.409) 1.195 (0.124)
0.119 0.150 0.200
3630 5170 5230
9.213 7.355 2.765 2.316
0.148 0.238 0.264
2760 6300 7840
Clean Natural sediment Control 2 mg cu 2+r l 3 mg Cu 2+ r l 4 mg Cu2 + r l Formulated sediment (15% organic matter) Control 2 mg Cu2+ r l 3 mg Cu2+ r l 4 mg Cu 2+ r l Formulated sediment (30% organic matter) Control 2 mg Cu 2 + r l 3 mg Cu2 + r l 4 mg Cu 2+ r l
R
(0.338) (0.384) (0.307) (0.276)
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emergenc~ patterns of adult C. riparius from formulated reference and polluted field sediments. Data are the total from 10 replicates.
Cross comparison of sediment treatments revealed no significant difference between the weight of control larvae. However, larvae exposed to copper in the natural sediment were significantly heavier (P = 0.006)
Use of the freshwater macroinvertebrate
105
than their c?unte~arts in both formulated preparations, with subsequent multiple comparison testing (P :::; 0.05) reveahng a dIfference at all the concentrations tested. Emergence tests. Comparison of the adult emergence data indicates that the polluted (sediment 3 in the first ~rowth test) and formulated reference sediments (Fig. 1), produced a bimodal emergence pattern, with a peak In male emergence f~llowed by the corresponding female peak for both sediments. However, emergence from the polluted sedIment was delayed and total adult numbers reduced in comparison to the formulated reference sediment. Statistical analyses of the median emergence times (EmT50) confirm the trends suggested in Fig.1 with male, female and total adult emergence significantly delayed in the polluted sediment (Table 3). In addition, one-way ANOVA demonstrated that significantly more adults (P = 0.001) emerged from the formulated as opposed to the polluted sediment, with survivorship in the former reaching 100% compared to 87% in the latter. Table 3. Summary of median adult emergence data from polluted and formulated reference sediments. Data are the total from 10 replicates Test Statistic
Reference Sediment (Control)
Polluted Sediment
Male EmT50 Female EmT50 Total Adult EmT50 No. Males No.Females No. Adults (From 250 individuals)
15.69 Days 17.09 Days 16.02 Days 148 117 265*
21.67 Days 23.37 Days 22.43 Days 126 92 218
* An excess of 15 individuals was recorded, due to the difficulty of counting first instar larvae when setting up the study. Both the growth and emergence responses of C. riparius are known to be sensitive indicators of chronic pollutant stress (Macek et ai., 1976; Giesy et ai., 1988; Pascoe et ai., 1989; Taylor et ai., 1991; Maund et ai., 1992). The results described confirm this, with effects on growth and emergence indicating a need for further investigation into one of the polluted field sediments tested. In addition, larval growth in copper spiked sediments demonstrated the modifying influence of sediment / contaminant complexing and subsequent effects on bioavailability and toxicity. Taylor et ai. (1991) quoted the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for aqueous copper as 16.9 and 25.3 mg I-I, respectively, levels which are exceeded in the overlying water of all three sediments types, highlighting the importance of the partitioning of copper between the aqueous and substrate phase. The extremely high copper loading of all sediments and the limited toxicity at these concentrations would appear to agree with the findings of Ankley et ai. (1994), that pollutants bound in the sediment are non• bioavailable. In addition, Pascoe et ai. (1990) demonstrated that the toxicity of cadmium to C. riparius larvae was reduced in the presence of artificial sediment. Copper is known to be more toxic in the aqueous as opposed to the substrate phase (Kosalwat and Knight, 1987b), however, the level of growth inhibition seen in this study, is far less than would be predicted based on species' tolerance, thereby discounting the water column as the primary route of exposure. This may be explained by the fact that aqueous copper can exist in non-toxic complexes with dissolved organic matter, (Meador, 1991). Such complexes may account for 25-98% of the soluble copper present (Stiff, 1971). The remaining route of exposure and in all probability, the bulk of bioavailable metal, is associated with the pore-water contained within the sediment, supporting evidence for which is provided by Swartz et ai. (1985). The differences in larval weights between sediments could be attributed to the different physico-chemical properties of the three sediment types, which influence the proportion of bioavailable copper, with growth inhibition due to copper in the pore-water affecting normal growth metabolism, presumably by incorporation into and derangement of, the complex series of reactions involved in this process.
M. M. WATTS and D. PASCOE
106
The polluted sediment which produced a reduction in larval weight in the first C. riparius growth experiment. also resulted in deleterious effects on adult emergence, emphasising the sensitivity of the two response criteria, and the consistency of the sediment in bringing about a toxic effect. The fact that emergence is delayed by such a wide margin and adult numbers are significantly lower in comparison to the formulated reference sediment would imply that the polluted sediment exerted an inhibitory effect on larval survival and development, a result previously reported by Wentsel et al. (1978). Pollutants present in the sediment must be bioavailable to the larvae, presumably via the pore-water, to bring about the adverse effects noted. The exact nature of the toxicity observed cannot, however, be established from this experiment. The reduction in total adult numbers may be due to mortality amongst the larvae, especially during the sensitive first and second instar stages, which would agree with the results of the acute toxicity tests of Williams et al. (1986). Alternatively, the larvae may be able to tolerate the pollutants present through the larval stages, with mortality occurring at the delicate pupation stage (Thornton and Wilhm, 1975) where massive morphogenetic change occurs prior to adult emergence. The delay in adult emergence may be due to the increased energy cost of pollutant tolerance which would slow down the normal development process, an effect similar to that of larval growth inhibition, previously noted in this sediment. Both the decline and delay in emergence of chironomids from the polluted sediment could have far reaching consequences at the population level. with low adult numbers affecting mating success (Pascoe et al., 1989) and possibly leading to a reduction in genetic fitness of the population as a whole. CONCLUSIONS The use of natural (polluted and 'clean') and formulated reference sediments and the differences in larval responses to sediment type illustrate the complex nature of sediment toxicity. The physico-chemical parameters of sediments which can exert a major influence on toxicity by affecting contaminant partitioning and bioavailability, vary greatly between sediments and further research is required to fully understand the complex interactions involved. The two response criteria tested, however, go some way in helping to explain sediment toxicity and are useful as a means of quantifying its effects for regulatory purposes. ACKNOWLEDGEMENTS The authors thank Zeneca Agrochemicals for financial support and Mr M O'Reilly and Mrs 0 James for their technical assistance. REFERENCES Ankley, G. T .. Thomas, N. A., Di Toro, D. M., Hansen, D. J., Mahony, J. D., Berry, W. 1., Swartz, R. C., Hoke, R. A., Garrison, W. A., Allen, H. E. and Zarba, C. S. (1994). Assessing potential bioavailability of metals in sediments: A proposed approach. Environ. Management, 18(3), 331-337. Blockwell, S. J., Pascoe, D. and Taylor, E. 1. (in press). Effects of lindane on the growth of the freshwater amphipod Gammarus pulex (L). Chemosphere. Cranston, P. S. (1 ?95). Introduction to the Chironomidae. In: The Chironomidae: The Biology and Ecology of non-biting Midges, P D ArmItage, P S Cranston and L C V Pinder (Eds.), Chapman and Hall, UK, pp, 1-7. Gauss, J. D., Woods, P. E., Winner, R. W. and Skillings, J. H. (1985). Acute toxicity of copper to three life stages of Chironomus . tentans as affected by water hardness-alkalinity. Environ. Poll. Series A., 37, 149-157. Glesy, 1. P.,. Grane~, R. L., Newsted, ~. L., Ros~u, .C. 1., B~nda, A., Kreis, R. G. and Hovarth, F. J. (1988). Comparison of three sedIment bIoassay methods USIng DetrOIt nver sedIments. Environ. Toxicol. Chem., 7, 483-498. Ingersoll, C.. G. and ~elson,~. K. ~1990). Testin.g sediment toxicity with Hyalella azteca (Amphipoda) and Chironomus riparius (Dlptera). In. ~quatlc Tox~cology art! RISk ~ssessment, W G Landis and W H van der Schalie (Eds.), ASTM STP 1096, Amencan SocIety for TestIng Matenals, PhIladelphia. Vol 13, 93-109. Kosalwat, P..and Knight, A. W. (1987a). Chronic toxicity of copper to a partial life cycle of the midge Chironomus decorus. Arch. EnVIron. Contam. Toxico/., 16, 283-290. Kosalwat, P. and ~ight, A. W. (198~b). Acute toxicity of aqueous and substrate bound copper to the midge Chironomus decorus. Arch. EnVIron. Contam. Taxlcol.. 16, 275-282.
Lilchfiel~o~. T.
(1949). A method for the rapid graphic solution of time-percentage effect curves. J. Pharm. Exp. Ther., 97,399-
Macek, Kfi" hBuxton, K.. Derr, S., Dean, J. a~d Saunter, S. (1976). Chronic toxicity of lindane to selected aquatic invertebrates and IS es. U.S. EnVIronmental ProtectIOn Agency. Eco. Res. Ser. EPA-600 / 3-76-046.
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Maund, S. J., Peither, A, Taylor, E. 1., Ji.ittner, 1., Beyerle-Pfnur, R., Lay, 1. P. and Pascoe, D. (1992). Toxicity of lindane to freshwater insect larvae in compartments of an experimental pond. Ecotox. Environ. Sa!. 23, 76-88. Meador, J. P. (1991). The interaction of pH, dissolved organic carbon, and total copper in the determination of ionic copper and toxicity. Aquat. Tox., 19,13-32. McCahon, C. P. and Pascoe, D. (1988). Culture techniques for three freshwater macroinvertebrate species and their use in toxicity tests. Chemosphere, 17,2471-2480. OECD (1981). Guideline for testing of chemicals. Earthworm, acute toxicity test. OECD, Paris. Guideline 207, 1-9. Pascoe, D. and Edwards, R. W. (1989). Single species toxicity tests. In: Aquatic Ecotoxicology: Fundamental Concepts and Methodologies, A Boudou and F Ribeyre (Eds.), CRC Florida, 11,93-126. Pascoe, D., Williams, K. A and Green, D. W. J. (1989). Chronic toxicity of cadmium to Chironomus riparius Meigen - effects upon larval development and adult emergence. Hydrobiologia, 175, 109-115. Pascoe, D., Brown, A E, Evans, B. M. J. and McKavanagh, C. (1990). Effects and fate of cadmium during toxicity tests with Chironomus riparius - the influence offood and artificial sediment. Arch. Environ. Contam. Toxicol., 19, 872-877. SETAC (1993) Guidance document on sediment toxicity tests and bioassays for freshwater and marine enviroments. I R Hill, P Matthiessen and F Heimbach (Eds.). From the "Workshop on sediment toxicity assessment." Renesse, The Netherlands (1993). Stiff, M. J. (1971). Copper / bicarbonate equilibria in solutions of bicarbonate ion at concentrations similar to those found in natural water. Wat. Res., 5, 171-176. Suedel, B. C. and Rodgers, J. H. (1994). Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem., 13(7), 1163-75. Swartz, R. c., Ditsworth, G. R., Schults, D. W. and Lamberson, 1. O. (1985). Sediment toxicity to a marine infaunal amphipod: Cadmium and its interaction with sewage sludge. Marine Environ. Res., 18, 133-153. Taylor, E. J., Maund, S. J. and Pascoe, D. (1991). Evaluation of a chronic toxicity test using growth of the insect Chironomus riparius Meigen. In: Bioindicators and Environmental Management, D W Jeffrey and B Madden, (Eds.), Proc 6th lOBS Symposium, Dublin, 1990. Academic Press Limited, London. pp, 343-352. Taylor, E. J., Blockwell, S. J., Maund, S. J. and Pascoe, D. (1993). Effects of lindane on the life-cycle of a freshwater macroinvertebrate Chironomus riparius Meigen (Insecta: Diptera). Arch. Environ. Contam. Toxicol., 24, 145-150. Taylor, E. J., Rees, E. M. and Pascoe, D. (1994). Mortality and drift-related response of the freshwater amphipod Gammarus pulex (L) exposed to natural sediments, acidification and copper. Aquat. Tox., 29, 83-101. Thornton, K. W. and Wilhm, J. L. (1975). The use of life tables in demonstrating the effects of pH, phenol and NaCI on Chironomus attenuatus populations. Environ. Ent., 4, 325-328. Wentsel, R., McIntosh, A. and McCafferty, W. P. (1978). Emergence of the midge Chironomus tentans when exposed to heavy metal contaminated sediment. Hydrobiologia, 57(3) 195-196. Williams, K. A, Green, D. W. J., Pascoe, D. and Gower, D. E. (1986). The acute toxicity of Cadmium to different larval instars of Chironomus riparius (Diptera:Chironomidae) and its ecological significance for pollution regulation. Oceologia (Berlin), 70, 362-366.