Bioaccumulation and toxicity of sediment associated herbicides (ioxynil, pendimethalin, and bentazone) in Lumbriculus variegatus (Oligochaeta) and Chironomus riparius (Insecta)

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Ecotoxicology and Environmental Safety 56 (2003) 398–410

Bioaccumulation and toxicity of sediment associated herbicides (ioxynil, pendimethalin, and bentazone) in Lumbriculus variegatus (Oligochaeta) and Chironomus riparius (Insecta) Kimmo A. Ma¨enpa¨a¨,
Arto J. Sormunen, and Jussi V.K. Kukkonen Department of Biology, University of Joensuu, P.O. Box 111, 80101 Joensuu, Finland Received 28 May 2002; received in revised form 17 January 2003; accepted 20 January 2003

Abstract The benthic macroinvertebrates Lumbriculus variegatus and Chironomus riparius were used in toxicity and bioaccumulation tests to determine the toxic concentrations and accumulation potential of sediment associated herbicides. The tested chemicals were ioxynil, bentazone, and pendimethalin. The bioaccumulation tests with L. variegatus were performed in four different sediments, each having different characteristics. Water-only LC50 tests were performed with both L. variegatus and C. riparius. A sublethal effect of model compounds in sediments was assessed by a C. riparius larvae growth-inhibition test. Of the model compounds, ioxynil appeared to be the most toxic, with LC50 values 1.79 and 2.79 mg L1 for L. variegatus and C. riparius, respectively. The LC50 water concentrations for bentazone were 79.11 and 62.31 mg L1 for L. variegatus and C. riparius, respectively. Similarly, ioxynil revealed the highest bioaccumulation potential in bioaccumulation tests. The most important characters affecting chemical fate in the sediment seemed to be the organic matter content and the particle size fraction. The sediments with low organic material and coarse particle size consistently showed high bioaccumulation potential and vice versa. In C. riparius growth tests bentazone had a statistically significant effect on larval growth at sediment concentrations of 1160 and 4650 mg kg1 (Po0:05). It is noteworthy that standard deviations tend to be greater at high chemical concentrations, which addresses the fact that part of the individuals started to suffer. Ioxynil had an effect on the larval growth in other test sediment at the highest concentration (15.46 mg kg1 dw), in which head capsule length correlated with larval weight, decreasing toward higher exposure concentrations. The current results show the importance of sediment organic matter as a binding site of xenobiotics. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Bentazone; Bioaccumulation; Chironomus riparius; Ioxynil; Lumbriculus variegatus; Pendimethalin; Sediment toxicity

1. Introduction Benthic organisms are exposed to all detrital, inorganic, and organic particles eventually settling on the bottom of a body of water. The sediment material often binds chemicals that enter a water ecosystem. Consequently, the role that benthic systems play in sequestering contaminants is well recognized (e.g., Burton, 1992). The bioavailability of sediment-sorbed chemicals has risen in concern because sediments can serve as both sinks and sources of contaminants. Sediment-inhabiting organisms are therefore exposed to sediment-bound pollutants. There are three potential sources/paths for contaminants to reach benthic organisms: the sediment itself


Corresponding author. Fax: +358-13-251-3590. E-mail address: kimmo.maenpaa@joensuu.fi (K.A. M.aenp.aa. ).

(ingestion), overlying water, and interstitial water (across body wall) (Power and Chapman, 1992). The largest volume of sediment is generally occupied by interstitial water (pore water). The inorganic phase includes rock and shell fragments and mineral grains. Organic matter occupies a low volume, but is important because it often regulates the sorption and bioavailability of many contaminants. Sediments are a relatively heterogeneous matrix in terms of physical, chemical, and biological characteristics (Power and Chapman, 1992). Organic sediments act as a sink for many hydrophobic xenobiotics, losing their binding affinity with decreasing organic content. Thus, organisms in low total organic carbon (low-TOC) sediment may show mortality at concentrations that would be tolerated in high-TOC sediment. Conversely, many of the more water-soluble compounds are less persistent and generally are not strongly sorbed to sediments. In either

0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(03)00010-1

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case, physical parameters such as partition coefficients (e.g., Kow ) have been valuable in predicting how and where these chemicals will partition into the environment (Lydy et al., 1990a). Determination of the potential toxicity of sediment-associated contaminants by aquatic organisms is a critical component of the exposure assessment process (Ankley et al., 1992). Sediment provides a habitat for many aquatic organisms, thus a wide variety of benthic invertebrates have been used extensively to assess bioaccumulation and toxicity of xenobiotics in controlled laboratory studies (e.g., Brunson et al., 1998; Burton et al., 1992; Egeler et al., 1997; Leppa¨nen and Kukkonen, 1998a, b). The selection of test species depends on the goals of study. In general, the species tested should be selected based on their behavior in sediment (e.g., habitat, feeding habits), sensitivity to test materials, ecological relevance, wide geological distribution, good availability, ease of culturing, adequate tissue mass for chemical analysis, possibility of conducting long-term tests without extra nourishment, wide tolerance of natural geochemical sediment characteristics such as grain size, and incorporation of all relevant routes of exposure (Brunson et al., 1998; Burton et al., 1992). In a freshwater environment, oligochaete worms and chironomids fulfill most of these criteria. Lumbriculus variegatus is the most common oligochaete species in evaluations of freshwater toxicity (Leppa¨nen, 1999), and it has been used in toxicity testing over 20 years (Bailey and Liu, 1980). L. variegatus occurs in shallow oxygenrich aquatic environments throughout the Northern hemisphere and can be found in both oligotrophic and mesotrophic waters (Dermott and Munawar, 1992). In Finland, L. variegatus lives in shallow waters in southern and central Finland (Laakso, 1967). The chironomid Chironomus riparius (midge) is also recognized as a useful tool in sediment toxicity testing (Ristola et al., 1999b). Both larval growth and adult emergence have been used as sublethal, chronic response criteria to investigate sediment toxicity. Midges have a cosmopolitan distribution, are important in aquatic food chains, and have a large database on the effects of toxic chemicals (Lydy et al., 1990a). Several authors have reported a reduction in larval growth in response to sediment contamination (Watts and Pascoe, 1996). Sediment contamination is to be expected if a chemical is found from the environmental samples. Ioxynil, pendimethalin and bentazone are commercially marketed and extensively used herbicides for agriculture purposes and subsequently found from the environment. Ioxynil and pendimethalin belong to the classes of nitrile herbicides and dinitroaniline herbicides, respectively. Bentazone is an unclassified herbicide (Tomlin, 1994). Ioxynil is applied all over the world as a postemergence herbicide, mainly on crops, vegetable and cotton

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cultivation, and it inhibits photosynthesis and uncouples oxidative phosphorylation (Tomlin, 1994). Pendimethalin is a selective herbicide absorbed by roots and leaves, inhibiting cell division and cell elongation (Tomlin, 1994). Pendimethalin is detected in stormwater runoff samples in England (Thomas et al., 2001) and Mississippi Delta surface water in the United States (Zimmerman et al., 2000). Bentazone is a postemergence herbicide used for selective control of broadleaf weeds and sedges, and it also inhibits photosynthesis (Tomlin, 1994). Bentazone is reported to occur in surface water samples in Denmark (Spliid and K^ppen, 1998), France (Jeannot et al., 2000), and Italy (Lagana` et al., 2002). In spite of the widespread occurrence of these herbicides, the consequences they may cause to, e.g., aquatic organisms are not thoroughly studied. Therefore, more research is needed to understand the effects of these chemicals in the environment. The objectives of this study were, first, to estimate LC50 water concentrations and the critical tissue concentrations for three herbicides (ioxynil, bentazone, and pendimethalin) in L. variegatus and C. riparius larvae; second, to determine availability of sediment associated herbicides to L. variegatus in four different sediments in order to see an effect of sediment characteristics; third, to determine subchronic toxicity of sediment associated herbicides to the midge larvae and evaluate the effect of sediment characteristics on the toxicity.

2. Materials and methods 2.1. Test organisms C. riparius and L. variegatus Mu¨ller (Oligochaeta), used in the experiments, originated from cultures reared in the laboratory at a constant temperature of 20721C and under a light regime of 16 h light and 8 h dark. L. variegatus worms were cultured in artificial fresh water (pH 7, hardness 1.0 mmol L1 as Ca+Mg) in 5-L tanks with constant aeration. The tanks contained approximately a 2-cm layer of shredded and presoaked paper towels as a substrate. The midge-culturing aquarium contained a few centimeters of natural sediment and 2 L of artificial fresh water on top. For both test organisms, the water was renewed once a week and they were fed twice a week with a few drops of Tetramin fish food (Tetrawerke, Melle, Germany). 2.2. Chemicals Radiolabeled [14C]ioxynil (specific activity 1 0.07236 mCi mg , radiochemical purity of 98.5%) was obtained from Aventis CropScience UK Ltd., Essex, England. [14C]pendimethalin (specific activity

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0.0554 mCi mg1, chemical purity of 499%, radiochemical purity of 99.22%) was obtained from Cyanamid, Princeton, USA. Radiolabeled [14C]bentazone (specific activity 0.333 mCi mg1, chemical and radiochemical purity of 495%) was obtained from the Institute of Isotopes Co., Ltd., Budabest, Hungary. All the radiolabeled chemicals were dissolved in acetonitrile. Nonlabeled chemicals were obtained from DHI Water & Environment, Department of Ecotoxicology (Danish Academy of Technical Sciences). Nonlabeled chemicals were in the form of powders, and when necessary, acetone-based stock solutions were prepared for experiments. Ioxynil, however, was dissolved in artificial fresh water due to its moderate solubility to the common solvents. Chemical and physical characteristics of the compounds are shown in Table 1. 2.3. Exposure medium (sediment and artificial water) Tests were performed using aerated artificial freshwater as overlying water, pH adjusted to 6.5 with hydrogenchloride (1 M HCl). Artificial fresh water was prepared with deionized water, yielding 0.5 mM inorganic salt concentrations. The following inorganic salts were added: MgSO4  7H2O 24.65 g L1, KCl 1.15 g L1, NaHCO3 12.95 g L1, and CaCl2  H2O 58.80 g L1. The water was buffered against pH alteration by adding phosphate buffer (Na2HPO4  2H2O) yielding a concentration of 0.5 mM. Sediments (S1, S2, S3, and S4) were used in growth and bioaccumulation experiments. S1 (Lake Mekrija¨rvi), S3 (Lake Kuorinka), and S4 (Lake Ho¨ytia¨inen, Varparanta) sediments were grab-sampled (Ekman grab sampler) at depths of 2, 8, and 2.5 m, respectively. S2 (Lake Ho¨ytia¨inen, southern end) sediment was pump-collected at a depth of 19 m. The sediments used in the current experiments were characterized by particle size, organic matter, and carbon and nitrogen content. Lake Ho¨ytia¨inen sediment (S2 and S4) has been analyzed previously and contains only

low levels of contaminants (Ristola et al., 1996, 1999a). The two other sediments were collected from similar remote areas. Therefore, possible trace amounts of pollutants were not expected to have an influence on the experiments. In the laboratory, sediments were sifted through a 2-mm sieve and homogenized by stirring. Triplicate subsamples were analyzed for percentage of dry matter in an oven (Memmert UE 400, OY Tamro Ab, Vantaa, Finland) at 1051C until constant weight and loss of ignition (Naber 2804 L47, Lilienthal, Bremen, Germany) at 5501C for 2 h. Particle size distribution was measured by sifting triplicate sediment samples, 50–80 g, through a series of sieves including 400-, 125-, 63-, 37-, and 20-mm mesh sizes. The particles remaining at each sieve were rinsed into a vial and dried at 1051C for 24 h. The next day, dry weight was taken, and total carbon (C) and total nitrogen (N) content were measured from fractions of sizes 63–37 and 37–20 mm (Elemental Analyzer Model 1106, Carlo Erba Strumentazione, Milano, Italy). 2.4. Acute toxicity experiments Acute toxicity of ioxynil, bentazone, and pendimethalin was determined from water-only exposure for both L. variegatus and C. riparius. Medium-sized L. variegatus and fourth instar C. riparius larvae were used in the experiments. The exposure time was 48 h and previously described artificial fresh water was a test medium. The water was spiked with 14C-labeled test compounds to measure critical tissue residue using the known ratio of labeled and nonlabeled compound. The stock solutions of unlabeled chemicals were used to spike the water to the desired concentrations. In the test with bentazone, total acetone concentration of 0.67% was used to conduct LC50 tests. The test chambers were placed in a cabin incubator at +41C for 2 days to let the acetone evaporate. Acetone controls were done for highest amount of acetone used. Three

Table 1 The characteristics of the model compounds ioxynil, pendimethalin, and bentazone Ioxynil Molecular weight Mol. formula log KOW Solubility in water (mg L1) pKa a

Tomlin (1994). Tomlin (1997). c Linders et al. (1994). d Nicholls (1994). e Andersson and Wenell (1993). f Gustafsson (1989). b

a

370.9 C7H3I2NOa 0.89b, 3.4c,d 1.8c,d, 50 (251C)a 3.96a

Pendimethalin a

281.3 C13H19N3O4a 5.18a, 5.2e 0.3 (201C)a —

Bentazone 240.3a C10H12N2O3Sa 0.77 (pH 5), 0.46 (pH 7), 0.55 (pH 9)a 500f, 570 (pH 7, 201C)a 3.3 (241C)a

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150-mL replicates per concentration were spiked separately and mixed well for homogenous distribution of a herbicide in the water. Aerated deionized water was added daily to replace evaporated water. The concentration of herbicide in the water was assessed by taking triplicate 6-mL samples for a Wallac WinSpectral Liquid Scintillation Counter (LSC) (Wallac Finland OY, Turku, Finland) after spiking the water in the beginning (0 h) and at the end (48 h) of the experiment. Water pH and oxygen content were measured (Multiline P4, WTW, Weilheim, Austria) in the beginning and at the end of the experiment. Ten test organisms were exposed in each replicate test chamber. At the end of the experiment the count of survivors was taken and surviving organisms were carefully blotted dry, weighed (Sartorius 4503, Go¨ttingen, Germany), and placed in a 6-mL LSC vial with 0.5 mL tissue solubilizer (LumaSolve) for tissue residue analysis. The next day, 5 mL of LSC cocktail (UltimaGold) was added and sample 14 C activity was measured after 2 weeks. The LSC data were corrected for background and quench by external standards. Preliminary experiments with pendimethalin revealed that mortality cannot be achieved in the range of its water solubility (0.3 mg L1 at 201C). In addition, mortality of midges was not seen when S2 sediment was spiked with very high concentrations (410 mg g1 dw) of pendimethalin. The spiking was done either by adding acetone-based stock solutions or by mixing powder into the sediment. After negative findings, nonlabeled pendimethalin was reanalyzed to assure the correctness of the chemical. 2.5. Bioaccumulation experiments The bioaccumulation of ioxynil, pendimethalin, and bentazone in L. variegatus was studied in a sediment system. The bioaccumulation was determined by spiking a single compound in a sediment at a time. The sediment was spiked with the test compound in one large (4-L) beaker. The 14C-labeled chemical was added to give a final concentration of approximately 10,000 DPM for S1 and S2, and 5000 DPM per gram of dry sediment for S3 and S4. The chemical, dissolved in 1-mL acetone, was added dropwise in 1100 g of sediment while mixing. The sediments were mixed with a rotating metal blade for 3 h at room temperature to give a homogenous distribution of the chemical in the sediment. Acetone control was used to determine the possible effect of acetone on the test organism. An experimental unit was a 200-mL glass jar (height 10 cm, +6 cm) containing 50 g (ww) of sediment and 100 mL of artificial fresh water. Water was cautiously added on top of the sediment to avoid sediment disturbance. Control jars were loaded with acetone-only spiked sediment. Test chambers were stored in a cabin

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incubator at +41C for 2 days after spiking to let the sediment–chemical interactions to take place. After 2 days, 10 medium-sized L. variegatus (ww app. 42.5 mg) were placed in each beaker, yielding a ratio of 1:70, 1:20, 1:20, and 1:12 animal wet weight:sediment organic matter in S1, S2, S3, and S4 sediments, respectively. The bioaccumulation experiments were conducted at room temperature (+2070.51C) with 16/8 h light/dark periods using a cold fluorescent light source (4500 nm), which averts possible photodegradation of chemicals. Evaporated water was replaced daily by adding aerated deionized water (DI-water). Water oxygen content was detected at the beginning and the end of the experiment and no aeration was provided. For each sampling time (0, 8, 24, 48, 72, 169, and 240 h) three replicate test chambers were destroyed and sampled for overlying water, sediment, and animals. The water sample (6 mL) was mixed with 6 mL of LSC cocktail (InstaGel) and shaken vigorously until it formed a gel. The overlying water was removed by vacuum and a sediment sample (0.1–0.2 g) collected with a spoon was mixed with 12 mL of LSC cocktail (UltimaGold) and sonicated (Sonics et Materials Inc., Danbury, Connecticut, USA) for 2 min. The sediment dry weight was determined for each replicate by weighing approximately 0.5–1 g (ww) of sediment and drying it in an oven (Memmert UM500, OY Tamro Ab, Vantaa, Finland) at 1051C for 24 h. After sieving from the sediment, worms from a test unit were carefully blotted dry, divided into two samples, weighed (Sartorius 4503, Go¨ttingen, Germany), and placed in a 6-mL LSC vial with 0.5 mL tissue solubizer (Lumasolve) and dissolved for 24 h at room temperature. The next day, 5 mL of LSC cocktail (UltimaGold) was added and the vials were shaken vigorously. To avoid pseudoreplication, the average of the divided samples from a test unit was used in result analysis. The pH was determined for triplicate control test chambers at the beginning (0 h) and at the end (240 h) of the experiment (Multiline P4, WTW, Weilheim, Austria). The pH was measured for water, sediment, and pore water individually. The sediment was centrifuged (Jouan B4i, Unterhaching, Germany) for pore water pH analysis for 30 min at 1620 g. 2.6. Ten-day growth test The effect of chemicals on the growth and development of C. riparius larvae was tested in the S2 and S3 sediments. Preliminary range-finding experiments were performed to determine the test concentrations. Sediment was spiked with the test compound in one 4-L beaker. The labeled and unlabeled bentazone dissolved in acetone was added dropwise to sediment while mixing. The unlabeled ioxynil was dissolved in artificial fresh water and added into the sediment. The

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radiolabeled solution was added to give a final concentration of approximately 10,000 DPM for S2 and 5000 DPM per gram of dry sediment for S3. The sediments were mixed with a rotating metal blade for 3 h at room temperature to give a homogenous distribution of radioactivity in the sediment. Fifteen replicate 50-mL glass beakers with approximately 6 g spiked wet sediment and 25 mL of artificial fresh water per treatment were prepared. The larvae were fed with fish food (TetraMin, Tetrawelke, Germany) using feeding level of 0.12 mg larva1 d1 (Ristola et al., 1999b). The total amount of food was added in a jar after the spiking of the sediment. The sediments were allowed to settle 2 days at 41C before the experiment started. As in the acute toxicity tests, it was assumed that radiolabeled and nonlabeled compounds have an equal bioaccumulation potential. Control sediments containing the maximum volume of solvent used for the exposure concentrations were prepared. The total acetone volume added in the sediment did not exceed 0.23%. A 10-day growth test were performed with C. riparius. The midges mated in cages and laid their eggs into beakers containing clean artificial fresh water. Three to five egg masses of similar age laid into beakers were then collected for the experiments. First instar (o3 day from hatching) larvae were used in the growth tests. The experiments were carried out under the same temperature and light conditions as bioaccumulation experiments. After 2 days of settling, 15 first-instar larvae were carefully pipetted into each beaker in random order and gentle aeration was started. Water lost by evaporation was replaced daily by adding aerated DI-water. Temperature and dissolved oxygen in the overlying water were monitored at the beginning (0 h) and the end (240 h) of the experiment. At the end of the growth test, larvae were separated from the sediment by sieving (63-mm mesh size). The survived larvae were counted and growth and development stage was determined by measuring head capsule length under a stereomicroscope (Nikon SMZ 800). Larvae were blotted dry and wet weight was taken (Sartorius 4503 Micro, Go¨ttingen, Germany). After weighing, the larvae were grouped to samples with five larvae in each, placed in 6-mL LSC vials with 0.5 mL of tissue solubilizer (Lumasolve), and let dissolve overnight at a room temperature. In some of the highest exposure concentrations survival of larvae was low and thus only one or two samples were constructed for LSC analysis. The next day, 5 mL of LSC cocktail (UltimaGold) was added and the vials were shaken cautiously. Sediment and water samples were assessed as in bioaccumulation experiments. Water, sediment, and tissue sample 14C activity were measured after a settling period of 2 weeks. The total concentrations of herbicides in the samples were calculated based on the ratio

between measured concentration of radiolabeled and known nominal concentration of nonlabeled herbicides at each concentration. 2.7. Data analysis and kinetic models LC10 and LC50 values and critical body residues were calculated using probit analysis (SPSS Corporation, Chicago, IL). Bioaccumulation data were fitted for least-squares nonlinear regression to describe the bioavailability of sediment-associated herbicides (SigmaPlot 5.0, SPSS Corporation, Chicago, IL). The data were fitted to a two-compartment, first-order kinetic model, in which the equation takes the decrease of sediment chemical concentration into consideration (Landrum, 1989), ðt¼0Þ

Ca ¼

ðks Cs Þ lt ðe  eke t Þ; ðke  lÞ

where Ca is the concentration of a chemical in the animal (mmol kg1 ww), ks is the uptake clearance coefficient (mmol kg1 h1), ke is the conditional elimination rate coefficient (h1), Cs is the concentration of a chemical in sediment (mmol kg1), and t is the time (h). The lambda value (l) is the slope of the linear regression of sediment chemical concentration. The lambda value is suggested to include the factors that decrease bioavailability of the chemical in the sediment (Landrum, 1989). The bioavailability of the chemicals was estimated by calculating bioaccumulation factors (BAF) (Table 5). The factors were determined in three different ways: (1) h) BAF=ks/ke, (2) BAF=C(t=240 mmol kg1 sediment a (t=0 h) 1 dw/Cs mmol kg tissue ww, (3) BSAF=BAF normalized to lipid content in the organism and the organic carbon content (OC%) in the sediment. Worm lipid content (1.23% ww; dw:ww ratio 0.16) used in BSAF calculations was examined earlier for the same L. variegatus culture as was employed in the current experiments (Leppa¨nen and Kukkonen, 2000). In the C. riparius growth tests the statistical comparisons of larval growth among treatments were made by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test when variances among treatments were equal and by the Games–Howell post-hoc test when variances among the treatments were not equal. Levene’s test was used to check homogeneity of variances.

3. Results 3.1. Characteristics of the sediments The organic carbon (OC) content in the test sediments ranged from 24.28% in the S1 to 0.5% in the S4

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Table 2 The characteristics of the test sediments (average7SD)

Dry weight % LOI % OC % N% C:N Fraction o20 mm % Fraction 20–37 mm % Fraction 37–63 mm % Fraction 63–125 mm % Fraction 125–400 mm % Fraction 4400 mm % OC % (fraction 37–63 mm) N % (fraction 3763 mm) OC % (fraction o37 mm) N % (fraction o 37 mm)

S1

S2

S3

S4

10.11 (0.00) 41.39 (0.06) 24.28 (0.17) 1.26 (0.02) 19.3 32.9 (5.2) 8.1 (1.3) 13.3 (3.4) 17.5 (1.8) 24.7 (1.1) 3.5 (0.3) 22.18 (0.30) 1.15 (0.02) 12.93 (0.07) 0.79 (0.06)

19.34 (0.01) 8.64 (0.04) 3.20 (0.02) 0.23 (0.01) 13.9 68.7 (1.3) 4.7 (0.3) 5.6 (1.4) 7.2 (0.5) 10.7 (0.2) 3.2 (0.5) 3.89 (0.03) 0.30 (0.04) 2.68 (0.01) 0.20 (0.02)

54.11 (0.09) 3.15 (0.01) 1.64 (0.11) 0.07 (0.01) 23.4 62.4 (2.8) 3.3 (0.7) 12.2 (2.3) 15.7 (1.6) 4.5 (1.0) 1.9 (0.5) 3.00 (0.12) 0.19 (0.02) 2.64 (0.04) 0.16 (0.03)

65.55 (0.04) 1.56 (0.01) 0.54 (0.03) 0.01 (0.01) 54.0 27.4 (0.8) 2.7 (0.3) 7.1 (0.1) 30.7 (0.8) 21.0 (0.6) 11.0 (0.2) 1.18 (0.05) 0.08 (0.02) 0.89 (0.05) 0.05 (0.03)

LOI %=percent loss in ignition. OC %=percent carbon content. N %=percent nitrogen content. C:N=carbon to nitrogen ratio.

Table 3 Lethal concentrations (LC-10, LC-50, and 95% confidence limits) for C. riparius and L. variegatus after water-borne exposure to ioxynil and bentazone for 48 h Water exposure (mg L1)

Water exposure (mmol L1)

Tissue residue (mmol kg1 ww)

LC-10

LC-50

LC-10

LC-50

LC-10

LC-50

Ioxynil Chironomus riparius Lumbriculus variegatusa

1.71 (1.25–1.93) 1.60 (1.41-1.69)

2.79 (2.50–3.69) 1.79 (1.70-1.86)

4.62 (3.38–5.20) 4.32 (3.79–4.55)

7.52 (6.74–9.96) 4.82 (4.58–5.01)

61.6 (4.45–90.6) 178.8

249 (170–3418) 267.1

Bentazone Chironomus riparius Lumbriculus variegatus

34.4 (25.9–41.2) 63.2 (53.0–69.4)

62.3 (54.3–70.9) 79.1 (73.0–84.0)

143 (107–171) 263 (220–289)

259 (226–295) 329 (304–349)

982 (635–1277) 1783 (1341–2172)

3676 (2653–6991) 2849 (2351–3551)

a

The 95% confidence limits for tissue residue were not provided by probit analysis.

(Table 2). The OC content in the fine-grained particle fraction (o63 mm) show similar trends from 35.11% in S1 to 2.07% in S4. The organic matter (OM) content in the bulk sediment measured by loss of ignition varied from 41.39% in S1 to 1.56% in S4. The visual detection during the particle size analysis showed that in S1 large particle size fraction consisted of high amount of undecomposed plant material. In S3 and S4 the smallest particle fraction (o20 mm) contained clay material indicating the inorganity of this fraction. Sediments S2 (Ho¨ytia¨inen) and S3 (Kuorinka) are fine grained consisting 79.0% and 77.9% of particles less than 63 mm in diameter, respectively. S4 is a coarse grained sediment having 62.7% of particles greater than 63 mm in diameter. In S1 the particles are distributed somewhat evenly between the fine and coarse classes.

3.2. Acute toxicity experiments The pH of the artificial fresh water remained constant throughout the experiments. The pH of the test medium remained constant throughout the experiments, average (7SD, n ¼ 12) being 6.62 (0.02). Water oxygen concentration remained high, 9.20 (0.52) mg L1, and water temperature stayed at 201C (70.5). Ioxynil appeared to be from highly to moderately toxic, being the most effective of the three model compounds, with LC50 values 1.79 and 2.79 mg L1 for L. variegatus and C. riparius, respectively (Table 3). The LC50 values for bentazone were 79.1 and 62.3 mg L1 for L. variegatus and C. riparius, respectively. Pendimethalin did not show toxicity, even though reexamination of the herbicide ensured the correctness of the chemical.

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3.3. Bioaccumulation experiments No mortality of L. variegatus occurred during the experiments. The pH measured from water, sediment,

Fig. 1. Bioaccumulation of ioxynil, pendimethalin, and bentazone in L. variegatus exposed in different sediments.

and pore water varied among the sediments: pH tended to be higher in S3 and S4 and lower in S1 and S2 sediments, especially for pore water and sediment. Mean (7SD) (n ¼ 9) pH for overlying water was 6.05 (0.03), 6.27 (0.05), 6.58, and 6.37 for S1, S2, S3, and S4 sediments, respectively. Mean (7SD) (n ¼ 9) pH for sediment was 5.94 (0.16), 5.75 (0.07), 6.37 (0.02), and 6.2 (0.06) for S1, S2, S3, and S4 sediments, respectively. Finally, mean (7SD) (n ¼ 9) pH for pore water was 5.69 (0.01), 5.59 (0.04), 6.21 (0.02), and 6.5 (0.02) for S1, S2, S3, and S4 sediments, respectively. Water oxygen concentration was sufficient, 7.05 (70.41) mg L1 (n ¼ 12) throughout the experiments, and water temperature remained at 201C (70.5). The concentration of the contaminant in the sediment decreased during the experiment. Decreases in concentration seemed to be compound-specific rather than sediment-specific. Ioxynil concentration decreased from 75% to 90% during the 240 h. Pendimethalin remained the best in the sediment, concentrations decreasing from 22% to 41% during the experiments. Bentazone concentrations decreased from 59% to 72%. The bioaccumulation of each compound was rather fast, and in many cases a steady state was achieved within 72 h. However, ioxynil and bentazone accumulated throughout the test period in the S3 and S4 sediments, leveling off slowly (Fig. 1). The uptake clearance coefficients (ks ) are the lowest for S1 sediment, increasing in S2 and being the highest in S3 and S4 sediments for all the chemicals. In contrast, elimination rate coefficients (ke ) S4 sediment has the lowest values (Table 4). As bioaccumulation factors (BAF, BSAF) in Table 5 show, ioxynil had the highest bioaccumulation potential

Table 4 Toxicokinetic parameters for L. variegatus exposed to ioxynil, pendimethalin and bentazone contaminated sediments (values ks and ke are obtained by fitting the equation (given in text) to the organism wet-weight-normalized data) Sediment dosea

Ioxynil, S1 Ioxynil, S2 Ioxynil, S3 Ioxynil, S4 Pendimethalin, Pendimethalin, Pendimethalin, Pendimethalin, Bentazon, S1 Bentazon, S2 Bentazon, S3 Bentazon, S4

S1 S2 S3 S4

(mg kg1 dw)

(nmol kg1 dw)

0.393 (0.015) 0.224 (0.011) 0.144 (0.001) 0.106 (0.002) 0.262 (0.014) 0.254 (0.014) 0.119 (0.001) 0.112 (0.003) 1.20 (0.044) 1.14 (0.032) 0.496 (0.008) 0.371 (0.014)

1.06 (0.041) 0.604 (0.030) 0.388 (0.004) 0.285 (0.005) 0.931 (0.049) 0.903 (0.049) 0.424 (0.005) 0.397 (0.012) 4.98 (0.185) 4.73 (0.134) 2.06 (0.034) 1.54 (0.057)

Lambda valueb

ks c (mmol kg1 h1)

ke d (h1)

r2 e

0.009 0.006 0.006 0.009 0.002 0.001 0.002 0.002 0.004 0.004 0.004 0.005

0.056 0.090 0.511 0.444 0.019 0.122 0.398 0.388 0.009 0.019 0.050 0.050

0.005 0.010 0.002 0.002 0.251 0.053 0.162 0.038 0.011 0.013 0.007 0.003

0.97 0.86 0.99 0.99 0.95 0.91 0.84 0.96 0.96 0.94 0.77 0.98

Mean 7SD sediment concentration at time 0 h. Linear regression slope for change in sediment chemical concentration. c Uptake clearance coefficient 7SD (mmol dry sediment kg1 wet organism h1). d Elimination rate coefficient 7SD (h1). e Coefficient of determination for nonlinear regression. a

b

(0.005) (0.015) (0.033) (0.031) (0.007) (0.030) (0.196) (0.053) (0.001) (0.002) (0.014) (0.004)

(0.001) (0.003) (0.001) (0.001) (0.087) (0.015) (0.085) (0.006) (0.002) (0.002) (0.004) (0.001)

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in each sediment. Bentazone and pendimethalin produced equal bioaccumulation factors. The sediments had a significant effect on bioaccumulation potential revealing an ascending trend from S1 to S4.

Table 5 The bioaccumulation factors for L. variegatus exposed to ioxynil, pendimethalin, and bentazone contaminated sediments (see text for details)

Ioxynil, S1 Ioxynil, S2 Ioxynil, S3 Ioxynil, S4 Pendimethalin, Pendimethalin, Pendimethalin, Pendimethalin, Bentazone, S1 Bentazone, S2 Bentazone, S3 Bentazone, S4

S1 S2 S3 S4

BAFa (ks/ke)

BAFa (Ca/Cs)

BSAFb

11.9 9.5 300.4 277.3 0.1 2.3 2.5 10.3 0.8 1.5 6.8 14.6

2.8 4.0 50.4 34.5 0.1 1.4 1.1 7.4 0.5 0.9 3.7 4.4

83.9 15.7 101.7 22.9 3.0 5.5 2.2 4.9 14.9 3.5 7.5 2.9

a

BAF=Bioaccumulation factor. BSAF=BAF normalized to organism lipid content and sediment organic carbon content. b

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3.4. Midge growth experiments 3.4.1. Ioxynil In the 10-day growth tests using sediment S2, C. riparius larva mortality increased toward higher exposure concentrations and showed 47% survival at the highest sediment exposure concentration (50.4 mg kg1 dw) (Table 6). In contrast to S2, when larvae were exposed to sediment S3 mortality remains negligible at all the exposure concentrations, except at 10.3 mg kg1 dw, in which 67% survival was detected. Although the doses were designed to produce significant mortality, no mortality was observed at the highest dose (15.5 mg kg1 dw) by the end of the experiment. Therefore, no LC50 values could be determined. In the S2 the initial sediment chemical concentrations decreased 40– 65%. In the sediment S3 ioxynil concentrations at the end of the experiment were only 21–54% of the initial concentrations. Exposing to ioxynil showed no significant effect on the larval weight or head capsule length in the sediment S2 (Table 6). In contrast, larvae exposed to ioxynil in sediment S3 revealed a statistically significant difference (Po0:01) in larval weight at the highest exposure concentration (15.5 mg kg1 dw) when compared to control exposure. Head capsule length and larval wet

Table 6 The toxic responses of C. riparius after exposure to sediment (S2 and S3) associated ioxynil in 10-day chronic experiment (average7SD) Exposure

Tissue conc. (mmol kg1 ww)

mg kg1 dw

mmol kg1 dw

S2 0.00 6.19 12.4 18.6 24.7 31.0 34.5 42.4 50.4

(0.00) (0.22) (0.45) (0.64) (0.86) (1.07) (0.95) (1.17) (1.39)

0.00 (0.00) 16.7 (0.58) 33.4 (1.20) 50.1 (1.73) 66.7 (2.31) 83.5 (2.88) 92.9 (2.57) 114 (3.16) 136 (3.75)

0.00 5.68 11.2 16.1 22.2 32.0 39.0 41.7 59.9

S3 0.00 0.22 0.80 1.60 2.39 3.19 3.93 4.91 5.89 10.3 12.9 15.5

(0.00) (0.00) (0.01) (0.01) (0.02) (0.03) (0.43) (0.55) (0.65) (0.82) (1.03) (1.23)

0.00 0.59 2.16 4.31 6.45 8.60 10.6 13.2 15.9 27.8 34.7 41.7

0.00 (0.00) 1.03 (0.15) 3.53 (1.16) 5.30 (0.56) 7.54 (3.43) 9.14 (0.89) 18.45 (14.70) 19.19 (8.53) 22.70 (10.20) 42.87 (32.60) 72.94 (48.00) 125.64 (74.80)

(0.00) (0.01) (0.02) (0.03) (0.05) (0.07) (1.17) (1.47) (1.76) (2.21) (2.77) (3.32)

(0.00) (0.63) (1.52) (2.87) (3.03) (7.71) (2.46)

Larval weigth (mg ww)

Head capsule length (mm)

Instar II

III

IV

Survival (%)

n

1.54 1.96 1.71 1.56 1.72 1.74 0.97 1.43 1.63

(0.51) (0.65) (0.52) (0.67) (0.59) (0.72) (0.45) (0.44) (0.60)

0.61 0.63 0.64 0.61 0.60 0.60 0.54 0.63 0.58

(0.13) (0.10) (0.04) (0.11) (0.11) (0.10) (0.13) (0.09) (0.13)

— — — — — — — — —

6 3 1 4 — 3 4 1 2

23 12 13 11 15 10 4 10 5

97 100 93 100 100 87 53 73 47

30 15 15 15 15 15 15 15 15

2.05 1.76 2.11 2.75 2.70 2.24 1.83 1.71 2.21 1.79 1.60 1.15

(0.86) (0.61) (0.78) (0.71) (0.66) (1.00) (1.22) (0.49) (1.09) (0.73) (0.43) (0.55)


0.68 0.67 0.64 0.69 0.66 0.62 0.58 0.65 0.62 0.62 0.62 0.58

(0.05) (0.06) (0.09) (0.06) (0.05) (0.09) (0.18) (0.04) (0.08) (0.10) (0.12) (0.11)

— — — — — — 2 — — — — —

— — 1 — — 1 1 — — 2 1 8

45 15 14 15 14 14 12 14 15 8 11 7

100 100 100 100 93 100 100 93 100 67 80 100

45 15 15 15 15 15 15 15 15 15 15 15

The probability level of Po0:05 (
) was used to indicate statistical significance relative to the control treatment (one-way ANOVA, Tukey/ Games–Howell post-hoc test).

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weight were slightly decreasing in the both sediments, but showed no significant differences to the control treatment because of the high standard deviations within treatment. Similarly, no clear trend was seen in larval development stage in S2 sediment. In S3 sediment, however, the highest exposure concentration produced significantly more third instar larvae. In addition, the larval survival was high, which supposedly means that larvae had a clear sublethal response to the chemical. The measured tissue concentration of ioxynil generally increased with increasing sediment dose. As supposed on the basis of the bioaccumulation tests, ioxynil bioavailability followed the trend of sediment organity; thus BAFs remained larger in S3 than in S2 sediment. Average BAF (standard deviation) for the exposure concentrations were 0.37 (0.04) and 1.60 (0.56) for S2 and S3 sediments, respectively. 3.4.2. Bentazone The toxic responses of C. riparius larvae to bentazone are given in Table 7. In the growth test using sediment S2 and bentazone, survival of larvae was high at concentrations below (4650 mg kg1 dw), but lowered sharply at higher exposures, being 67% at 7004 and 100% at 9269 mg bentazone kg1 sediment dw. In the sediment S3 mortality was not detected at concentrations below 320 mg kg1 dw, but at concentration 778 mg kg1 dw survival was 67%, and there were no survivors at the highest exposure concentration. It is possible that only the initial concentrations were at toxic level. In the end of the growth test bentazone

concentrations in the sediment S2 were only from 36% to 47% of the initial concentrations. Correspondingly, in S3 from 68% to 80% of the chemical was found in the sediment at the end of the exposure period. Exposure to bentazone decreased significantly larval wet weight at exposure concentrations 1160 and 4650 mg kg1 dw (one-way ANOVA, Po0:05) in S2. Average wet weight of the larvae in the exposure 7004 mg kg1 dw was 68% of that in the control sediment in S2, and respectively 55% at the exposure 778 mg kg1 dw in the sediment S3. At the exposure 778 mg kg1 dw (S3) statistical testing did not show significant differences from the control, because of high standard deviations (Table 7). It is noteworthy that variation in response tends to be greater in the higher chemical concentrations. Therefore, it can be presumed that only some of the individuals showed toxic response, causing large variation. Head capsule length showed statistically significant (one-way ANOVA, Po0:05) difference in S2 sediment when exposed animals were compared to control treatment. The head capsule was shorter in all, except the lowest exposure (465 mg kg1 dw). In the sediment S3 bentazone also had a significant effect on the length of head capsule. Only at exposure concentration of 118 mg kg1 dw the difference was not significant compared to control exposure. At the exposure 778 mg kg1 dw difference was not statistically significant (P40:05) because of high standard deviations. However, retarded development of the larvae can be seen when studying average instar level (Table 7).

Table 7 The toxic responses of C. riparius after exposure to sediment (S2 and S3) associated bentazone in 10-day chronic experiment (average7SD) Exposure

Tissue conc. (mmol kg1 ww)

Larval weight (mg ww)

Head capsule (mm)

mg kg1 dw

mmol kg1 dw

S2 0.00 (0.00) 465 (26.2) 1160 (65.4) 2354 (132) 4650 (262) 7004 (394) 9269 (222)

0.00 (0.00) 1935 (109) 4827 (272) 9796 (551) 19350 (1089) 29146 (1640) 38572 (923)

0.00 (0.00) 211 (27.7) 603 (133) 995 (327) 2169 (915) 3373 (977) —

2.65 2.62 1.92 2.76 1.21 1.81 —

(0.63) (1.17) (0.70)
(0.93) (0.40)

(0.74)

0.69 0.65 0.56 0.62 0.50 0.56 —

S3 0.00 (0.00) 20.9 (0.17) 53.1 (0.43) 118 (0.87) 218 (1.73) 320 (2.62) 778 (61.2) 1042 (82.0)

0.00 (0.00) 87.0 (0.71) 221 (1.79) 490 (3.62) 908 (7.20) 1333 (10.9) 3237 (255) 4338 (341)

0.00 (0.00) 96.1 (74.3) 238 (118) 894 (710) 1723 (1666) 1720 (1337) 2364 (191) —

2.79 1.74 1.79 1.98 2.13 1.52 1.55 —

(0.73) (0.30)

(0.41)

(0.55)

(0.57)
(0.54)

(1.51)

0.68 0.59 0.62 0.64 0.61 0.59 0.54 —

Instar

Survival (%)

n

II

III

IV

(0.07) (0.08) (0.07)

(0.06)
(0.11)

(0.10)



— — — — — — —

— — 1 — 4 1 —

29 16 15 15 9 9 —

97 100 100 100 87 67 0

30 16 16 15 15 15 15

(0.06) (0.04)

(0.07)
(0.06) (0.05)

(0.04)

(0.18)

— — — — — — 1 —

— — 1 — — — 5 —

25 15 14 15 15 15 4 —

96 100 100 100 100 100 67 0

26 15 15 15 15 15 15 15

The probability level of Po0:05 (
) and Po0:01 (

) was employed to indicate statistical significance of a treatment to the control treatment (one-way ANOVA, Tukey/Games–Howell post-hoc test).

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Based on LSC results, the concentration of bentazone in the larval tissue after 10-day exposure correlated with the sediment concentration, being highest at S2 concentration 7004 mg kg1 sediment dw inducing 3373 mmol kg1 tissue ww. In the S3 at the highest exposure concentration in which larvae survived, the larval tissue reached 2364 mmol bentazon kg1 tissue ww. The bioaccumulation factors (BAF=tissue concentration ww/sediment concentration dw) showed bentazone to be more bioavailable in S3 sediment. Average BAFs (standard deviation) were 0.11 (0.01) and 1.32 (0.46) for S2 and S3 sediments, respectively. The LC50 concentrations for sediment exposure and tissue residue were estimated as in the acute toxicity tests. LC50 sediment concentration for S2 was 6688 mg kg1 dw and 5330 mg kg1 dw for sediment S3. The LC50 tissue concentrations were 5312 and 2818 mmol kg1 ww for sediments S2 and S3, respectively.

4. Discussion 4.1. Acute toxicity Ioxynil showed high to moderate toxicity in the acute toxicity experiments. The LC50 values (2.79 and 1.79 mg L1 for L. variegatus and C. riparius) obtained for ioxynil fall approximately in the same range with literature data reported earlier: 3.30 (ioxynil octanoate) and 4.00 (ioxynil-sodium) mg L1 for harlequin fish (Tomlin, 1994). The LC50 tissue residues (249 and 267 mmol kg1 ww for C. riparius and L. variegatus, respectively) obtained in the acute toxicity experiments have quite large 95% confidence limits. However, assuming that avian and invertebrate ecotoxicological data are comparable, the values are rather similar to oral LD50 of 202 and 539 mmol kg1 reported for pheasant and hen, respectively (Tomlin, 1994). Acute toxicity experiments revealed bentazone to be virtually nontoxic, showing low bioaccumulation potential, presumably due to its high water solubility. The water LC50 toxicity values obtained (79.1 and 62.3 mg L1 for L. variegatus and C. riparius) are lower than LC50 (96 h) values reported for fish, rainbow trout and bluegill sunfish 4100 mg L1, invertebrate, Daphnia sp. EC50 (48 h) 125 mg L1, and green algae (Ankistrodesmus) EC50 (72 h), 62.0 mg L1 (Tomlin, 1994; Weed Science Society of America, 1994; US Environmental Protection Agency, 1985). The current estimated tissue LC50 residues (3676 and 2849 mmol kg1 ww for C. riparius and L. variegatus, respectively) again fall close to literature values reported for avian species: acute oral bentazone LD50 for bobwhite quail and mallard duck range from 2996 to

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8323 mmol kg1 (Tomlin, 1994; Weed Science Society of America, 1994). Pendimethalin is bioavailable and accumulated by L. variegatus, as was measured in the bioaccumulation tests, but it remains unclear why toxic effects were not encountered in the current experiments. Literature data, however, argue that pendimethalin would be highly toxic to aquatic invertebrates, being the most toxic of the three model compounds studied in the current experiments (Weed Science Society of America, 1994; US Environmental Protection Agency, 1985). 4.2. Herbicide bioaccumulation It has been proposed that the bioavailability of hydrophobic organic compounds in the sediments is based on thermodynamic balance between sorbed (particles, DOM) and desorbed (interstitial water) phases (Di Toro et al., 1991). Composition of a sediment, especially quantity and quality of an organic material, greatly influence the fate and bioavailability of the chemicals (DePaolis and Kukkonen, 1997; Gunnarsson et al., 1996; Maloney, 1996). As discussed by several authors (e.g., Landrum and Robbins, 1990; Lydy et al., 1990b), sediment organic material and pore water dissolved organic material (DOM) have an affinity to chemicals, the binding efficiency depending on the hydrophobicity of the chemical. When bound, the chemical is removed to the less bioavailable fraction and results in decreased body burden. In the current experiments, L. variegatus bioaccumulation factors and accumulation rate coefficients were significantly reduced along the sediments’ increasing organic material content. Similarly, in the C. riparius growth tests, the bioavailabilities of the chemicals were measured to be greater in the sediment containing less organic material. Therefore, we suggest that sediment organic material had the most pronounced effect on the herbicide bioavailability in the current experiments. The calculated BAFs (bioaccumulation factors) directly show the potential availability of the chemical in the sediment. Thus, the current results affirm the importance of xenobiotic binding potential to sediment organic material. The characters of the three test compounds may explain the differences in bioaccumulation. Pendimethalin has a high Kow value and thus relatively low water solubility, which may have resulted in strong sorption to sediment and low bioaccumulation. On the other hand, high water solubility and low Kow value may explain the low bioaccumulation potential of bentazone (Barron, 1990). The literature offers competing values for ioxynil water solubility and Kow (Table 1). However, the results show that ioxynil does bioaccumulate well, suggesting moderate lipophilicity of this chemical.

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Besides being classified by organic content, sediments are typically categorized as to size of particles. It is proposed that there are two fractions dividing sediment particle distribution to fine and coarse fractions: particles greater than 62 mm in diameter, a coarse fraction, and particles less than 62 mm, a fine fraction (Power and Chapman, 1992). It is argued that the coarse particle fraction does not bind contaminants as efficiently as the fine particle fraction, and thus, it is not generally associated with chemical contamination. The fine particle fraction consists of particles with a relatively large surface-area-to-volume ratio, and consequently, surface electric charges cause these particles to be more chemically and biologically reactive than larger particles (Power and Chapman, 1992). In the current experiments, S2 and S3 sediments have the highest surface-to-volume ratios (79% and 77.9% particles o63 mm, respectively), which could in part explain why bioaccumulation of the model compounds remains low in these sediments. In contrary, S4 sediment has the lowest surface-to-volume ratio (37.2% particles o63 mm), which presumably leaves more chemical in the dissolved state. S1 has the fine particle fraction (particles o63 mm) 54.3%, which would indicate higher bioaccumulation than is seen. In this sediment, however, other factors such as feeding rate and organic matter content presumably take a more important part in explaining the degree of bioaccumulation. Indeed, the fine particle fraction OC% was clearly the highest in S1 sediment and the lowest in S4 sediment (Table 2). In conclusion, the particle size distribution may probably affect bioavailability from the model compounds aside from the organic material. The sediments having a fine particle structure and high organic material content in the fine particle fraction may adsorb chemicals in a high degree, showing low bioaccumulation in benthic animals. 4.3. Midge growth experiments According to our knowledge, ioxynil and bentazone have not been used in chronic exposures to benthic invertebrates earlier. Many other chemicals, however, have been used in chronic exposures. For example, exposure to surfactant ABS or pesticide lindane is discovered to decrease chironomid emergence in wholelife-cycle tests (Takamura, 1992, Taylor et al., 1993). Of the two herbicides studied in the 10-day growth experiments, bentazone only affected C. riparius larvae growth. Moreover, both ioxynil and bentazone were more bioavailable in the S3 than S2 sediment that was observed in the bioaccumulation tests as well. Exposure to ioxynil significantly decreased larval weight only of the highest chemical concentration in the S3 sediment, but had no sublethal effect on larvae in S2 sediment even though the sediment exposure was

much higher than in S3 sediment. The accumulated tissue concentration remained lower in S2 sediment in spite of the sediment exposure concentration, which may explain why no sublethal effect was seen. However, despite the chemical residue in tissue, larval survival clearly decreased in S2 sediment but not in S3 sediment. Similarly, Ristola et al. (1999b) recorded no sublethal effects in C. riparius larvae exposed to 2,4,5-trichlorophenol in 10-day chronic tests. In their experiment, the chemical concentration decreased and they suggested that only the initial sediment concentrations were at toxic level and that the survivors recovered with reducing chemical concentration during the experiment, and therefore grew equally well in all the treatments. Ioxynil caused significant mortality in S2 sediment even though the sediment concentration acutely decreased, which may indicate that the initial loading was high enough to cause mortal effect and larvae did not recover despite the sediment chemical concentration decreased. DT50-value (degradation time when 50% of chemical has degraded) for ioxynil in soil is approximately 10 days and it is degraded by hydrolysis and deiodination to less toxic substances such as hydroxybenzoic acid (Tomlin, 1994). Strong degrading of ioxynil has probably happened during the current experiment, since only approximately 31.9% (721.0, n ¼ 3) of the total initial count of DPMs was measured in the end of the experiment. However, approximately half of the larvae (survivors) reached third or fourth instar level during the test, which may signify that a proportion of the larvae are more vulnerable to certain chemicals than others. For example, the respective physical state of the individual at a certain moment may affect the sensitivity to the chemicals. First instar chironomid larvae has been suggested to be the most sensitive stage of the chironomid lifecycle (Kosalwat and Knight, 1987; Pascoe et al., 1989). Thus, the larvae that survived through this stage may have recovered and shown no difference in physical status from control animals. Overall, environmental trace amounts of ioxynil do not seem to cause significant threat to benthic animals. The chronic toxicity experiments with bentazone showed a statistically significant effect on C. riparius larval weight and head capsule length in both sediments. Moreover, survival of the larvae decreased toward the highest concentrations used in the both sediments. The exception is the highest concentration the animals were exposed to. High standard deviation caused no statistical differences to be detected from the control treatment. However, instar level was determined to be lower, which indicates bentazone’s sublethal effect on larvae. As discussed, bentazone had a significant effect on larval growth, but only in high sediment application rates (41160 and 20.9 mg kg1 dw for S2 and S3, respectively). The high sediment exposure concentrations were probably needed because of the hydrophilic

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nature of bentazone. Due to bioaccumulation tests, approximately 76.3% (77.0, n ¼ 3) of bentazone was found from the water column by the end of the test. Another reason for the low toxicity of bentazone is that bacteria and fungi degrade it rather rapidly and its halflife in soil is reported to be less than two weeks (Huber and Otto, 1994; Wauchobe et al., 1992). Degradation of the parent compound could have happened by sediments containing bacteria, even though metabolites were not analyzed. In companion with literature data the results of the current experiments indicate that bentazone forms no significant hazard to benthic invertebrates.

5. Conclusions The experiments that were completed have revealed that sediment characteristics had an effect on bioavailability of the model compounds. It is difficult, however, to precisely predict the fate and actions of the chemical in the sediment system even when the chemical and sediment characteristics are well known. However, the characteristics of sediment give valuable information about the possible behavior of a chemical. Based on the current results, the most important characters affecting chemical fate in the sediment seems to be the organic matter content and the particle size fraction. The sediments with low organic material and coarse particle size constantly show high bioaccumulation potential and vice versa. The nutritional status of the sediment may also have an influence on bioaccumulation. The current results may indicate that the pesticides are bioaccumulated whether or not they are bound to sediment particles. The hydrophilic chemicals are bioaccumulated through integument from pore water and to some extent from overlying water, where hydrophobic chemicals are bioaccumulated through intestine as an animal digests the sediment particles. Water solubility has a strong effect on sorption and bioaccumulation of a chemical. High water solubility of bentazone indicated moderate toxicity and low bioaccumulation potential, as was experimentally established in the current results. The relatively high lipophilicity could suggest high toxicity and high bioaccumulation potential, as was seen in the case of ioxynil. Pendimethalin, however, needs more detailed studies to clarify the characters affecting its behavior. High initial sediment exposure concentrations of ioxynil and bentazone are required to produce sublethal effects or mortality, as was seen in C. riparius growth tests. The herbicides may also be degraded to less toxic substance rather rapidly. Therefore, it is assumed that the herbicides tested do not create a potential risk to benthic invertebrates in the environment. Finally, toxicity of the chemicals in the sediments has proved

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to be difficult to test because the bioavailable fraction of the chemicals varies in different sediments. Acknowledgments The work was funded by NordTest (Project 1522-01) and the Academy of Finland (Project 73166). We thank Matti Leppa¨nen and Jani Honkanen for the critical review of the article.

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