Bioaccumulation and toxicity of 4-nonylphenol (4-NP) and 4-(2-dodecyl)-benzene sulfonate (LAS) in Lumbriculus variegatus (Oligochaeta) and Chironomus riparius (Insecta)

Aquatic Toxicology 77 (2006) 329–338

Bioaccumulation and toxicity of 4-nonylphenol (4-NP) and 4-(2-dodecyl)-benzene sulfonate (LAS) in Lumbriculus variegatus (Oligochaeta) and Chironomus riparius (Insecta) K. M¨aenp¨aa¨ ∗ , J.V.K. Kukkonen Department of Biology, University of Joensuu, FIN-80101 Joensuu, Finland Received 24 October 2005; received in revised form 4 January 2006; accepted 5 January 2006

Abstract The discharge of surfactants, such as 4-nonylphenol (4-NP) and linear alkylbenzene sulfonates (LAS), into water bodies leads to accumulation of the chemicals in the sediments and may thus pose a problem to benthic organisms. To study the bioaccumulation of surfactants, Oligochaeta worm Lumbriculus variegatus was exposed to sediment-spiked, [14 C]-labeled 4-NP and 4-(2-dodecyl)-benzene sulfonate (C12-LAS) in three different sediments (S1–S3). The sediments were characterized for organic carbon (OC) content and particle size distribution. The acute toxicity was examined by exposing L. variegatus and three to four instar Chironomus riparius (Insecta) larvae in water-only exposure to 4-NP and LAS at different concentrations. After 48-h exposure, lethal water concentrations (LC50 ) and lethal body residues (LBR50 ) were estimated using liquid scintillation counting. Chronic toxicity was evaluated in two different sediments by exposing first instar C. riparius larvae to sediment-spiked chemicals at different concentrations. After 10 days, the sublethal effects of surfactants were observed by measuring wet weight and head capsule length. Finally, another 10-day test was set up in order to measure the LAS body residues associated with sublethal effects in C. riparius in S2 sediment. The bioaccumulation test revealed that the bioaccumulation of both 4-NP and LAS increased as the sediment organic matter content decreased. It is assumed that the chemical binding to organic material decreased chemical bioavailability. The acute toxicity tests showed that L. variegatus was more tolerant of 4-NP, and C. riparius was more tolerant of LAS when based on water exposure concentration. The LBR-estimates revealed, however, that L. variegatus tolerated clearly higher tissue residues of both chemicals compared with C. riparius. Both chemicals had sublethal effects on C. riparius growth in sediment exposure, reducing larvae wet weight and head capsule size. 4-NP, however, showed an irregular dose–response pattern. The characteristics of the exposure media affected the bioaccumulation potential of both chemicals. Thus, exposure concentrations offered no prediction of body residue, and therefore it is proposed that organism body residue offered a more accurate dose-metric for chemical exposure than the chemical concentration of the environment. © 2006 Elsevier B.V. All rights reserved. Keywords: Sediment; Lumbriculus variegatus; Chironomus riparius; Surfactant; Critical body residue

1. Introduction Nonylphenols (NPs), such as 4-nonylphenol, are degradation products of alkylphenol polyethoxylates (APEs), which are common environmental contaminants widely used in industry and in domestic products (Naylor et al., 1992; Talmage, 1994; Canadian Council of Ministers of the Environment, 2002; ∗ Corresponding author. Department of Biology, University of Joensuu, P.O. Box 111 (Yliopistokatu 7), FIN-80101 Joensuu, Finland. Tel.: +358 13 251 3545; fax: +358 13 251 3590. E-mail address: [email protected] (K. M¨aenp¨aa¨ ).

0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.01.002

Bettinetti et al., 2002). Linear alkylbenzene sulfonates (LAS) are group of synthetic organic surfactants, varying in their alkyl chain length (Cx-LAS where x is the chain length). LAS are produced in large quantities and also used in industry and in household cleaning and personal care products (Larson and Woltering, 1995; Tolls et al., 1997; International Programme on Chemical Safety, 1996). Both chemicals are released into the environment, usually with treated wastewater, where NPs are commonly sequestered in the sediments and have adverse effects on benthic communities (Schmude et al., 1999). LAS have been shown to be highly biodegradable in aerobic conditions, but are also removed by adsorption, e.g. onto suspended particles, there-

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fore accumulating in sediments (Larson and Woltering, 1995; International Programme on Chemical Safety, 1996; Tabor and Barber, 1996). Both chemicals have been under ecotoxicological examination, but only a few studies have focused on bioaccumulation or the effects of sediment-originating 4-NP (Bettinetti et al., 2002; Bettinetti and Provini, 2002; Schmude et al., 1999) and LAS (Casellato et al., 1992) on freshwater benthic organisms. Croce et al. (2005) exposed sediment dwelling Oligochaeta worm Lumbriculus variegatus to sediment-spiked 4-NP and observed that no steady state was reached over the 56-day study period. They suggested that ingested sediment was an important accumulation route for 4-NP in the L. variegatus tissues. Bioaccumulation and the effects of sediment-originating NP and C12-LAS on aquatic organisms need thorough investigation, since sediment sequesters the chemicals (Westall et al., 1999; Federle and Ventullo, 1990). The characteristics of the environment, such as sediment organic carbon content, are known to affect the bioavailability of chemicals, especially lipophilic ones (Westall et al., 1999; Ying et al., 2003; Koelmans et al., 2005). Thus, McCarty and Mackay (1993) proposed that the toxic effect of a chemical is related to body residue rather than to environmental concentration. Application of the critical body residue (CBR) approach to surfactant chemicals, such as 4-NP and C12-LAS, needs evaluation. So far, there have been only a few studies on body residues related to defined toxic effect in 4-NP and LAS (Fay et al., 2000; Hwang et al., 2003). The objectives of this study were: (1) to determine bioaccumulation of sediment associated surfactants, 4-nonylphenol (4-NP) and 4-(2-dodecyl)-benzene sulfonate (C12-LAS), in L. variegatus (Oligochaeta), (2) to analyze the sediments for carbon content and particle size fractions that may affect chemical bioavailability, (3) to examine the acute toxicity of surfactants in L. variegatus and Chironomus riparius (Insecta) larvae, (4) to examine the chronic toxicity of sediment-spiked surfactants in C. riparius larvae, and (5) to determine critical body residues for lethal and sublethal endpoints, and to consider whether the tested chemicals support the CBR approach. 2. Materials and methods 2.1. Test organisms The C. riparius Meigen (Insecta) and L. variegatus M¨uller (Oligochaeta) used in the experiments originated from cultures maintained at the Department of Biology, University of Joensuu. The experimental animals were reared in the laboratory at a constant temperature 20 ± 2 ◦ C and under a light regime of 16 h light and 8 h dark. L. variegatus were cultured in artificial fresh water (pH 7, hardness 1.0 mM as Ca + Mg) in 5 L tanks with constant aeration. As a substrate, the tanks contained a 2-cm layer of shredded and presoaked paper towels. The C. riparius culturing aquarium contained a layer of natural sediment a few centimeters thick with 4 L of artificial fresh water on top. For both test organisms, the water was renewed once a week and the organisms were fed a few drops

of Tetramin® fish food (Tetrawerke, Melle, Germany) twice a week. 2.2. Chemicals The unlabeled and [14 C]-radiolabeled 4-nonylphenol (4-NP) and 4-(2-dodecyl)-benzene sulfonate (C12-LAS) were provided by Water & Environment, Department of Ecotoxicology, Danish Academy of Technical Sciences, Denmark. The specific activities of [14 C]-labeled chemicals were 0.107 and 1.029 mCi/mmol for 4-NP and LAS, respectively. The [14 C]-labeled stock solutions were prepared by dissolving 4-NP in 99.5% ethanol and C12-LAS in 90% ethanol. The unlabeled chemicals were in the form of a powder; and when necessary, ethanol based stock solutions were prepared for the experiments. 2.3. Exposure media The sediments (S1–S3) used in the bioaccumulation and toxicity experiments originated from fresh water lakes situated in North Carelia, Finland. Lake Mekrij¨arvi (S1) and Lake Kuorinka (S3) sediments were grab sampled (Ekman grab sampler) at depths of 2 and 8 m, respectively. Lake H¨oyti¨ainen (S2) sediment was collected by pump at a depth of 19 m. In the laboratory, the sediments were sieved through a 2-mm sieve and homogenized by stirring. The sediments were characterized according to dry weight, particle size, organic matter (loss of ignition), and carbon and nitrogen content (Elemental Analyzer Model 1106, Carlo Erba Strumentazione, Milan, Italy). The sediments were not treated to remove carbonates prior to carbon content analysis because the inorganic carbon content is negligible in this type of sediments (Pajunen, 2004). For percentage of dry matter and loss of ignition, triplicate subsamples were first dried in an oven (Memmert UE 400, OY Tamro Ab, Vantaa, Finland) at 105 ◦ C until they reached constant weight, and then ignited at 550 ◦ C for 2 h (Naber 2804 L47, Lilienthal, Bremen, Germany). Particle size distribution was measured by sieving triplicate 50–80 g ww sediment samples through a series of sieves including mesh sizes of 400, 200, 125, 63, 37, and 20 ␮m. The particle size fractions of S2 and S3 sediments were further analyzed, and the particles remaining in each sieve were rinsed into a vial and dried at 105 ◦ C for 24 h. The next day, dry weight was determined, and carbon and nitrogen contents were measured for each fraction. Artificial freshwater was prepared from deionized water yielding 0.5 mM (0.1 mmol Mg/L, 0.4 mmol Ca/L) inorganic salt concentrations. The following salts were added: MgSO4 , KCl, NaHCO3 and CaCl2 . Finally, the pH was adjusted to 6.5. 2.4. Bioaccumulation experiment The bioaccumulation of 4-NP and C12-LAS in L. variegatus was studied in the three different sediments, spiking a single compound to a single sediment at a time. The test compound was spiked with the sediment in one large (4 L) beaker. The [14 C]-labeled chemical was added to give a final concentration of 10,000 DPM per gram of dry sediment for S1 and S2, and

K. M¨aenp¨aa¨ , J.V.K. Kukkonen / Aquatic Toxicology 77 (2006) 329–338

5000 DPM/g dw for S3. Stock solutions of [14 C]-labeled chemicals were added dropwise to sediment while it was mixed. It was assumed that a homogenous distribution of radioactivity was achieved in the sediment after mixing the sediment with a rotating metal blade for 2 h at room temperature. The control sediment was treated similarly by adding only solvent. The experimental unit was a 200 mL glass jar (height 10 cm, Ø 6 cm) containing 50 g wet weight sediment and 100 mL artificial fresh water. To avoid sediment disturbance, water was cautiously added to the top of the sediment. To allow the sediment materials to descend and the sediment–chemical interactions to take place, the jars were stored in a cabin incubator at 4 ◦ C for 2 days after spiking. After 2 days, the jars were allowed to warm up and 10 test organisms were placed in each beaker. The bioaccumulation experiments were conducted at room temperature (20 ± 1 ◦ C) with a 16/8 h light/dark regime using a yellow light source (>500 nm). Evaporated water was replaced daily by adding aerated deionized water (DI-water). For each sampling time (0, 8, 24, 48, 72, 169, and 240/264 h), three replicate test chambers were chosen and sampled for pH, oxygen content, and chemical concentration of overlying water, sediment and animals. A water sample (6 mL) was mixed with 6 mL of liquid scintillation counter (LSC) cocktail (InstaGel, Packard BioScience B.V., Groningen, The Netherlands) and shaken until a gel formed. The pH of the overlying water was then measured, after which the overlying water was removed by vacuum and the pH of the sediment was determined. Next, to measure a chemical concentration in the sediment, a sediment sample (0.1–0.2 g wet weight) collected with a plastic spoon was weighed and mixed with 1 mL of tissue solublizer (Lumasolve, Packard BioScience B.V., Groningen, The Netherlands) and dissolved for 24 h at room temperature. Next day, 12 mL of LSC cocktail (Ultima Gold, Packard BioScience B.V., Groningen, The Netherlands) was added and the vials were shaken. The sediment dry weight was determined for each replicate by weighing 0.5–1 g wet weight of sediment and drying it in an oven at 105 ◦ C for 24 h. Finally, the worms from each test unit were sieved, blotted dry, weighed, and placed in a 6-mL LSC vial with 0.5 mL tissue solublizer and dissolved for 24 h at room temperature. Next day, 5 mL of LSC cocktail (UltimaGold) was added and the vials were shaken. The samples were analyzed with a Wallac WinSpectral Liquid Scintillation Counter (LSC) (Wallac Finland OY, Turku, Finland). The radioactivity counts of the samples were corrected for background and quench by applying external standards. Pore-water pH was determined for triplicate control chambers at the beginning (0 h) and at the end (240 h) of the experiment. The pore water was extracted by centrifuging the sediment for 30 min at 1620 × g. 2.5. Acute toxicity The acute toxicity of 4-NP and C12-LAS were tested as water-only exposure for both L. variegatus and C. riparius. Previously described artificial fresh water was the test medium, with the exception that the water was buffered against a change in

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pH by adding a phosphate buffer (Na2 HPO4 /NaH2 PO4 ), which yielded a concentration of 0.25 mmol/L. The exposure time was 48 h and the temperature and light conditions were the same as in the bioaccumulation experiments. To measure the tissue residue, the water was spiked with [14 C]-labeled test compounds using a known ratio of labeled and unlabeled compound. The exposure concentrations for L. variegatus were 0.003–0.011 and 0.003–0.017 mmol/L for 4-NP and C12-LAS, respectively. For C. riparius the exposure concentrations were 0.001–0.009 and 0.001–0.014 mmol/L for 4-NP and C12-LAS, respectively. The water was spiked to the desired concentrations with a stock solution of unlabeled chemical, and at least five different exposure concentrations were used. In every experiment, a solvent (ethanol) control was prepared with the maximum concentration of solvent used along with the water-only control. First, [14 C]-labeled test compound was spiked in a large beaker and mixed for half an hour with a magnetic stirrer. The water was then divided into 500 mL beakers and an unlabeled compound was added to each beaker to set exposure concentrations. Then, water was mixed for an additional half hour. Finally, spiked water of each exposure concentration was divided into three 150 mL replicate beakers, 100 mL water in each. Then, 10 organisms were carefully placed in each replicate jar. The jars were covered with a cap and aerated through a Pasteur pipette. The concentration of a surfactant in the water was assessed by taking triplicate 6 mL samples for LSC at the beginning (0 h) and the end (48 h) of the experiment. Aerated deionized water was added daily to replace the evaporated water. Water pH, oxygen content and conductivity were measured (Multiline P4, WTW, Weilheim, Austria) in the beginning and at the end of the experiment. At the end of the experiment, survivors were counted, and the organisms were prepared for LSC analysis as described above. 2.6. Chronic toxicity 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. The sediment was spiked with a test compound in one 4 L beaker. The exposure concentrations were prepared with chemical dissolved in 96% ethanol (4-NP) or 50% ethanol (C12-LAS), and added dropwise to the sediment during mixing. It was assumed that a homogenous distribution of radioactivity was achieved in the sediment after mixing the sediment with a rotating metal blade for 2 h at room temperature. Fifteen replicate 50-mL glass beakers with 6.0 g (±0.1) wet weight of spiked sediment and 25 mL (±1.0 mL) of artificial fresh water per treatment were prepared for each exposure concentration. The larvae were fed with fish food, using a feeding level of 0.12 mg/larva/day (Ristola et al., 1999). The total amount of food was added to the jar at the same time as the sediment was spiked. The sediment was allowed to settle for 2 days at 4 ◦ C before the larvae were exposed. Chemical-free and solvent-only treatments were prepared similarly for comparison. Three egg ropes of similar age laid in the culture were collected for the experiments. First instar (<3 days from hatching)

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larvae were used in the growth tests. Temperature and light conditions were the same as those applied in the bioaccumulation experiments. After the 2-day settling period, one first instar larva was carefully and randomly pipetted into each beaker, and gentle aeration was started. Water lost by evaporation was replaced daily by adding aerated DI-water. At the end of the 10-day growth test, the larvae were separated from the sediment by sieving (63 ␮m mesh size), the survivors were counted and the head capsule length was measured under a stereomicroscope. Then, the larvae were blotted dry and wet weight was taken. The pH, oxygen content and conductivity of the overlying water were measured in three replicate jars in the beginning and at the end of the experiment. The pH of the sediment was measured in the beginning and at the end of the experiment. Finally, a similar 10-day test was set up with S2 sediment spiked with [14 C]-labeled C12-LAS in order to measure critical body residues in larvae associated with measured effects. 2.7. Data analysis and kinetic models

y = min +


To describe the bioavailability of sediment-associated 4-NP and C12-LAS, the bioaccumulation data were fitted with the least squares nonlinear regression method, using SigmaPlot (5.0, SPSS corporation, Chicago, IL). The data for each accumulation experiment were fitted to a two-compartment, first-order kinetic model (Eq. (1)). The C12-LAS concentration in S2 sediment decreased and was therefore fitted to Eq. (2), in which the decrease in sediment chemical concentration is taken into consideration (Landrum, 1989): Ca = Ca =

ks Cs0 (1 − e−ke t ) ke (ks Cs0 ) (ke − λ)

(e−λt − e−ke t )

The C. riparius larvae growth experiment data were tested for normality (Kolmogorov–Smirnov test) and the Levene median test was used to determine homogeneity of variances within groups. When the data were normally distributed and variances were equal, the statistical differences among treatments were analyzed by one-way analysis of variance (ANOVA, SigmaStat 2.0 or SPSS 11.0, SPSS corp., Chicago, IL, USA), and Tukey’s test was used for multiple comparisons between control treatment and exposed groups. When the data were not normally distributed or variances were unequal, Kruskal–Wallis One Way Analysis of Variance on Ranks was used. Dunnet’s or Dunn’s post hoc test was used for multiple comparisons. Estimations of lethal water concentrations (LC50 ) and lethal body residues (LBR50 ) for 50% mortality in acute toxicity tests were made by probit analysis (SPSS 11.0, SPSS corp., Chicago, IL). The LBR estimates are from surviving organisms. The CBRs for lethal and sublethal effects in the C. riparius growth test with [14 C]-LAS were estimated using the built-in four parameter logistic function in Sigmaplot software (SPSS corp., Chicago, IL): (max − min)  Hillslope  1 + ECx50

(3)

where y is the measured effect (e.g. larva biomass), min the minimum response plateau, max the maximum response plateau, x is the C. riparius body residue (mmol/kg wet weight) and Hillslope is related to slope of curve. The critical body residues were determined for 50% inhibition of effect compared to control treatments. 3. Results

(1) 3.1. Sediment characterization (2)

In the equations, Ca is the concentration of a chemical in the animal (␮mol/kg wet weight), ks the uptake clearance coefficient (kg dry sediment/kg organism wet weight/h), ke the elimination rate coefficient (1/h), Cs0 the concentration of a chemical in sediment (␮mol/kg dry weight) at the beginning of the exposure, t the time (h), and ␭ is the rate constant for the chemical to become biologically unavailable (1/h). The contaminant dilution due to organism growth was not taken into account in fitting the data. The bioaccumulation of the chemicals was estimated by calcusteady state lating bioaccumulation factors (BAF): BAF = Ca /Cs0 . BAFs were estimated also from kinetic factors (ks /ke ) according to Bailer et al. (2000). Biota-to-sediment accumulation factors (BSAF) were estimated similarly by fitting the kinetic data after normalizing the chemical concentrations for sediment organic carbon content and the organisms’ lipid content. The lipid content of the worms (1.23% ww) used in the BSAF calculations were taken from the literature for the same L. variegatus culture as was used in the current experiments (Lepp¨anen and Kukkonen, 2000).

The characteristics of the test sediments are given in Table 1. In general, the characteristics imply that the organic carbon (OC) content of the test sediments decreases from S1 to S3. Organic carbon content in the test sediments ranged from 24.28% in S1 to 1.64% in S3. The organic matter (OM) content in the bulk sediment measured by loss of ignition varied from 41.39% in S1 to 3.15% in S3. Examination of the particle size fractionation showed that S2 is the most fine-grained sediment, followed by S1, with S3 having the coarsest composition. The OC content was measured for each fraction in S1 and S2 sediments. In the S1 sediment, OC seems to be distributed fairly evenly among the fractions, unlike the S2 sediment, in which almost all of the OC is in the smallest particle fraction (0–20 ␮m) of sediment. 3.2. Bioaccumulation The experiments showed that both chemicals, 4-NP and C12LAS, bioaccumulated in L. variegatus tissues (Fig. 1). The uptake clearance coefficients (ks ) increased in similar order in both chemicals, being: S1 < S2 < S3 (Table 2). C12-LAS was

K. M¨aenp¨aa¨ , J.V.K. Kukkonen / Aquatic Toxicology 77 (2006) 329–338

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Table 1 The characteristics of the test sediments (average, standard deviation, n = 3) Parameter

Sediment S1

S2

S3

Dry wt.% Lost of ignition% Organic carbon% Nitrogen%

9.99 (0.02) 41.39 (0.06) 24.28 (0.17) 1.26 (0.02)

22.89 (1.41) 8.64 (0.04) 3.20 (0.02) 0.23 (0.01)

55.87 (0.01) 3.15 (0.01) 1.64 (0.11) 0.07 (0.01)

Fraction <20 ␮ma Organic carbon%b

49.0 (3.0) 4.8 (0.3)

79.3 (0.7) 2.8 (0.0)

15.9 (0.6)

Fraction 20–37 ␮ma Organic carbon%b

5.6 (1.4) 16.8 (0.6)

2.4 (0.1) 2.8 (0.0)

5.1 (0.4)

Fraction 37–63 ␮ma Organic carbon%b

9.7 (0.8) 25.9 (0.1)

4.1 (4.6) 3.5 (0.1)

10.4 (0.9)

Fraction 63–125 ␮ma Organic carbon%b

11.1 (0.8) 32.0 (0.2)

4.6 (0.9) 3.7 (0.0)

38.1 (1.0)

Fraction 125–200 ␮ma Organic carbon%b

12.1 (0.4) 28.9 (0.4)

5.5 (0.7) 3.5 (0.1)

15.5 (0.5)

Fraction 200–400 ␮ma Organic carbon%b

10.1 (1.0) 30.6 (0.7)

3.7 (0.7) 2.9 (0.1)

7.0 (0.8)

Fraction 400–2000 ␮ma Organic carbon%b

2.5 (0.5) 27.8 (0.3)

0.4 (0.2) 2.8 (0.2)

8.1 (0.6)

a b

% of fraction of the bulk sediment. Organic carbon content of the fraction.

eliminated (ke ) distinctly faster than 4-NP, which produced higher tissue residues and bioaccumulation factors for 4-NP than for C12-LAS (Table 3). No clear steady state was achieved for 4-NP tissue residue after 264 h exposure, in contrast to C12-LAS tissue residue, which reached steady state after about 150 h of exposure. No mortality was observed during the experiments. However, the number of the worms increased by reproduction, while the mass of individual worms decreased. In the S2 and S3 sediments whole sample wet weight remained about the same, but in the S1 sediment sample wet weight decreased 20% in 4-NP exposure and 17% in C12-LAS exposure. The number of organisms at the end of the experiments had increased from the initial 10 in each sediment: the average number of individuals was 12.3, 15 and

Fig. 1. Sediment-spiked 4-NP and C12-LAS bioaccumulation in L. variegatus in three different sediments (S1, S2 and S3). Each mark with error bars indicates average and standard deviation of three replicate samples.

13.3 in 4-NP-spiked S1, S2 and S3 sediments, respectively, and 14.3, 17.7 and 17.7 in C12-LAS-spiked S1, S2 and S3 sediments, respectively. The pH measured from water, sediment and pore water varied among the sediments, and the pH did not change significantly during the experiments. Thus, the pHs are given as a single value, i.e. the sum of all the measurements for the same sediment. The average (standard deviation, n = 36) pH for overlying water was 5.7 (0.06), 6.3 (0.03) and 5.8 (0.04) for S1, S2 and S3

Table 2 Toxicokinetic parameters for L. variegatus exposed to 4-NP- and C12-LAS-spiked sediments Sediment

Cs a (mmol/kg dw)

4-NP S1 S2 S3

0.013 (0.001) 0.009 (0.000) 0.010 (0.001)

C12-LAS S1 S2 S3

0.006 (0.000) 0.050 (0.000) 0.004 (0.000)

␭b – – – – −0.0016 –

ks c (kg dw/kg ww/h)

ke d (1/h)

r2

0.011 (0.001) 0.053 (0.006) 0.074 (0.013)

0.006 (0.001) 0.008 (0.002) 0.002 (0.002)

0.99 0.98 0.96

0.009 (0.001) 0.045 (0.004) 0.139 (0.040)

0.017 (0.002) 0.012 (0.001) 0.035 (0.012)

0.99 0.99 0.90

Values ks and ke are obtained by fitting Eq. (1) or (2) to the organism wet-weight-normalised tissue chemical concentration (n = 3). a The chemical concentration in the sediment (average, standard deviation, n = 3). b The slope of linear regression of log transformed sediment chemical concentration. c The uptake clearance coefficient (coefficient, standard error, n = 3). d The elimination rate coefficient (coefficient, standard error, n = 3).

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Table 3 The bioaccumulation factors (BAF) and biota-to-sediment accumulation factors (BSAF) (standard error) for L. variegatus exposed to 4-NP- and C12-LAS-spiked sediments Sediment

BAF (Cass /Cs0 )

BAF (ks /ke )

BSAF (ks OC/lipid /ke OC/lipid )

4-NP S1 S2 S3

1.5 (0.1) 5.9 (0.6) –

1.8 (1.2) 6.5 (1.3) 33.6 (2.8)

55.4 (1.2) 14.1 (1.2) 34.8 (2.3)

C12-LAS S1 S2 S3

0.5 (0.03) 2.7 (0.1) 4.7 (0.2)

0.5 (1.1) 3.7 (1.1) 4.0 (1.6)

35.5 (1.3) 19.3 (2.1) 5.7 (1.7)

Cass = Chemical concentration in organism at steady state. Cs0 = Chemical concentration in sediment at the beginning of the test. ks = Uptake clearance coefficient. ke = Elimination rate coefficient. ks OC/lipid = Uptake clearance coefficient for the normalized data for sediment organic carbon (OC) content and organism lipid content. ke OC/lipid = Elimination rate coefficient for the normalized data for sediment organic carbon (OC) content and organism lipid content.

sediments, respectively. The average (n = 36) pH for sediment was 5.7 (0.05), 5.7 (0.09) and 6.1 (0.05) for S1, S2 and S3 sediments, respectively. Finally, the average (n = 12) pH for pore water was 5.4 (0.05), 5.8 (0.03) and 5.7 (0.11) for S1, S2 and S3 sediments, respectively. The concentration of oxygen in water decreased during the experiments from 7.1 (0.27) at 0 h to 5.3 (0.13) mg/L at 240/264 h. The water temperature remained at 20 ◦ C (±1.0). The concentrations of chemicals in the sediment decreased during the experiments. The decrease in concentration seemed to be compound-specific rather than sediment-specific. 4-NP remained in the sediment longer, the concentration decreasing from 12.7 to 13.9% during the 264 h of the experiment. The concentration of C12-LAS in sediment decreased by 23.2% and 19.7% in S1 and S3 sediments, respectively. In the S2 sediment C12-LAS concentration decreased by 58.8%, which was taken into account when accumulation kinetic factors (ks ) were defined. 3.3. Acute and chronic toxicity The acute toxicity tests revealed that L. variegatus was more tolerant to waterborne 4-NP exposure than three to four instar C. riparius (Table 4) as far as exposure concentration (LC50 ) Table 4 Lethal water concentrations (LC50 ) and lethal body residues (LBR50 ) in L. variegatus and C. riparius three to four instar larvae exposed to waterborne 4-NP and C12-LAS for 48 h Organism

LC50 (95% CL), ␮mol/L

LBR50 (95% CL), mmol/kg ww

4-NP L. variegatus C. riparius

6.264 (5.831–6.699) 3.969 (3.059–4.934)

11.555 (9.890–15.281) 3.742 (3.102–4.442)

C12-LAS L. variegatus C. riparius

5.648 (5.408–5.917) 11.425 (9.705–13.992)

14.263 (6.877–20.497) 0.816 (0.680–1.124)

95% CL = 95% confidence limits.

Table 5 Development and survival of C. riparius larvae after 10-day exposure to sediment spiked 4-NP and C12-LAS at various concentrations Exposurea (mmol/kg dw)

Survival (%)

Head capsuleb , length (mm)

Larvae wetb , weight (mg)

4-NP/S2 0.00 2.75 4.40 5.92 7.62 9.23

80.0 93.3 86.7 86.7 80.0 60.0

0.42 (0.11) 0.42 (0.08) 0.38 (0.02) 0.38 (0.01) 0.42 (0.10) 0.40 (0.08)

0.74 (0.21) 0.59 (0.10) 0.43 (0.14)** 0.54 (0.17) 0.66 (0.14) 0.60 (0.27)

4-NP/S3 0.00 0.36 0.78 1.19 1.61 2.04

80.0 80.0 93.3 80.0 80.0 66.7

0.46 (0.10) 0.36 (0.02)* 0.36 (0.03)* 0.37 (0.02)* 0.41 (0.07) 0.38 (0.07)

0.81 (0.23) 0.40 (0.15)*** 0.31 (0.11)*** 0.62 (0.19) 0.61 (0.14)* 0.49 (0.14)***

C12-LAS/S2 0.00 0.26 0.66 1.06 1.46 1.86

80.0 86.7 100.0 93.3 53.3 33.3

0.40 (0.03) 0.40 (0.06) 0.42 (0.08) 0.39 (0.08) 0.32 (0.08) 0.24 (0.08)***

0.49 (0.11) 0.49 (0.16) 0.61 (0.14) 0.38 (0.19) 0.18 (0.10)*** 0.09 (0.07)***

C12-LAS/S3 0.00 0.45 1.01 1.57 2.13 2.71

100.0 93.3 86.7 66.7 33.3 0.0

0.40 (0.06) 0.38 (0.09) 0.39 (0.13) 0.36 (0.05) 0.27 (0.10) –

0.53 (0.32) 0.50 (0.17) 0.48 (0.26) 0.38 (0.21) 0.17 (0.16)* –

Statistically significant differences in head capsule length and wet weight of exposed groups to control treatment is shown with an asterisk (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). a Nominal sediment exposure. b Average and standard deviation.

or body residue (LBR50 ) are concerned. For C12-LAS, C. riparius had a higher LC50 value than L. variegatus, which on the other hand tolerated a higher tissue residue. Curiously, the C12-LAS body residues in L. variegatus tend to be higher in exposure groups with low mortality, compared with exposure groups with high mortality, where body residues were lower. This effect was not seen in C12-LAS-exposed C. riparius. The growth inhibition experiments with unlabeled chemicals revealed that both chemicals have an adverse effect on larval growth and survival of C. riparius (Table 5). A decrease in larval wet weight appeared to be a more sensitive indicator of stress caused by both tested chemicals than head capsule length. 4NP exposure had contradictory effects when comparing S2 and S3 sediments: intermediate exposure concentration significantly decreased larval wet weight in S2 sediment, but in S3 sediment the wet weight returned to the initial value. 4-NP reduced larval head capsule length only at the three lowest exposure concentrations in S3 sediment. Survival was clearly decreased only in the highest exposure concentration in 4-NP-spiked sediments. The no-observed-effect-concentration (NOEC) for larval growth in

K. M¨aenp¨aa¨ , J.V.K. Kukkonen / Aquatic Toxicology 77 (2006) 329–338

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Table 6 Body residue, development and survival of C. riparius larvae after 10-day exposure to S2 sediment-spiked mixture of [14 C]-labeled and unlabeled C12-LAS at various concentrations Exposure (mmol/kg dw)

Body residue (mmol/kg ww)

Survival (%)

Head capsule, length (mm)

Larvae wet, weight (mg)

0.0 (0.0)a 0.0 (0.0)b 0.306 (0.000) 1.062 (0.000) 1.686 (0.000) 2.503 (0.000) 3.034 (0.000)

0.0 (0.0) 0.0 (0.0) 0.053 (0.011) 0.087 (0.030) 0.251 (0.036) 0.361 (0.066) –

93.33 93.33 85.72 93.33 78.57 13.33 0.00

0.67 (0.03) 0.67 (0.04) 0.64 (0.03) 0.64 (0.09) 0.46 (0.13)*** 0.37 (0.03)*** –

1.55 (0.42) 1.96 (0.29) 1.80 (0.39) 1.86 (0.60) 0.86 (0.45)** 0.78 (0.49)** –

Statistically significant differences in head capsule length and wet weight among exposure groups are marked with an asterisk (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). a Control treatment. b Solvent control treatment.

S2 sediment was 2.75 mmol 4-NP/kg sediment dry weight, while NOEC was not reached in the S3 sediment. Lowest-observedeffect-concentrations (LOEC) were 4.40 and 0.36 mmol 4NP/kg dw for the S2 and S3 sediments, respectively. C12-LAS-spiked sediments produced statistically significant effects on larval growth and reduced survival at the highest exposure concentrations in both sediments (Table 5). In the S3 sediment, however, the great variation as to how individual larvae responded to C12-LAS exposure prevented the emergence of statistically significant differences other than in wet weight at the highest exposure concentration. In S3 sediment generally, larval growth decreased and mortality increased with increasing exposure concentration, and no survivors were found at the highest exposure concentration. NOECs for sublethal effects were 1.06 and 1.57 mmol C12-LAS/kg dw for S2 and S3 sediments, respectively. LOECs were 1.46 and 2.13 mmol/kg dw for S2 and S3 sediments, respectively. In the experiment with radiolabeled C12-LAS, a significant reduction in larval growth was encountered at similar exposure concentrations as in the experiment with unlabeled C12-LAS (Tables 5 and 6). These experiments were carried out independently, which attests to the reliability of the results. NOEC and LOEC for sublethal effects were 1.06 and 1.69 mmol/kg sediment dry weight, respectively. NOEC and LOEC for sublethal effects as body residues were 0.087 and 0.251 mmol/kg larval wet weight, respectively. Survival was clearly lower at and above the LOEC value. Lethal body residue (LBR50 ) for 50% mortality was 0.290 (0.007) mmol/kg ww. CBR50 values for sublethal effect were 0.256 (0.065) and 0.172 (0.045) mmol/kg ww for larvae head capsule length and wet weight, respectively. The larval wet weight and head capsule length remained smaller in the experiments carried out with an unlabeled chemical compared to experiments where a radiolabeled chemical was used. This was probably caused by the fact that the larvae exposed to the labeled chemical were fed with Tetramin after hatching before exposure to C12-LAS-spiked S2 sediment. The larvae fed with Tetramin may therefore have started growth sooner and grown at a faster rate, reaching a larger size compared to larvae that were not fed immediately after hatching. In the acute tests water pH remained constant at 6.88 (standard deviation 0.05, n = 6) throughout the 24 h test period. In

the chronic experiments, S2 sediment pH at the beginning of the tests was 7.2 (0.06, n = 6) and that of overlying water was 7.3 (0.02, n = 3), while at 240 h time point sediment pH was 5.6 (0.07, n = 3) and that of overlying water was 5.7 (0.04, n = 6). In the S3 sediment pH at 0 h was 5.9 (0.03, n = 3) and overlying water pH was 6.0 (0.05, n = 3), while at 240 h, sediment pH was 4.5 (0.20, n = 3) and overlying water pH was 4.5 (0.06, n = 3). 4. Discussion 4.1. Bioaccumulation The bioaccumulation experiments revealed that both the surfactants, C12-LAS and 4-NP were bioaccumulated by L. variegatus. The bioaccumulated fraction found in L. variegatus tissues increased for both chemicals as follows: S1 < S2 < S3. The characteristics of the test sediments differed in a similar order by dry wt.%, and in the reverse order by loss of ignition%, OC content and nitrogen content. In conclusion, the more organic the sediment, the lower the bioaccumulation of chemical, which suggested that a fraction of the chemicals was sequestered in a non-bioavailable pool in the sediment. It is concluded that organic material lowers the bioavailability of both 4-NP and C12-LAS. The sediment organic material was found to reduce 4-NP bioaccumulation in three estuarine amphipods (Hecht et al., 2004), and it has been shown that 4-NP has a strong tendency to sorb into sediment (Ying et al., 2003). The bioaccumulation factors (BAF) for amphipods varied from 149 to 1074, depending on the species and exposure media, being clearly higher than that estimated in the current study for L. variegatus. Biota-to-sediment accumulation factors (BSAF) were, however, close to those estimated in the current study. BAFs for 4-NP evened out when converted to BSAFs, which may indicate that 4-NP is mainly bound in sediment organic material. The BSAF values remained large when equilibrium partitioning theory is considered (Spacie et al., 1995). This may indicate that other factors besides the lipophilicity of the chemical play a role in the distribution. The contaminant distribution and movement are likely affected also by characteristics of the organisms and the sediments. Further, possible biotransformation of the contaminants (discussed below) may affect on BSAF values.

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It is reported that detritus and sediment OC are a significant site for LAS removal (Westall et al., 1999; Federle and Ventullo, 1990), reducing their bioavailability. Westall et al. (1999) found that LAS sorption into five different sediments increased with the increasing OC content of the sediment. In the current study, S1 had the highest BSAF value while S3 had the lowest value. This may indicate that the type of organic matter differed among the sediments, which affected the binding affinity for C12-LAS. It has been shown that contaminant binding affinity varies among the different organic matter fractions in soil and sediment (Kohl and Rice, 1998; Kubicki and Apitz, 1999). Organic material in the S3 sediment may have bound C12-LAS more effectively compared to S2 and especially S1 sediment. The chemical distribution among the different sediment fractions may vary and thus may affect the bioavailability of contaminants in general (Kukkonen and Landrum, 1996). A high proportion of fine sediment particles in S1 and S2 sediment may explain the lower bioavailability of 4-NP and C12-LAS, compared to S3 sediment, which clearly had less of the fine particles that offer numerous binding sites for contaminant. The importance of the amount of organic matter in the fine particle fractions is, however, unclear. When S1 and S2 sediments are compared, the higher total amount of organic matter in the S1 sediment may play a more important role in offering binding sites for chemicals. 4.2. Toxicity It has been suggested that both LAS and 4-NP act as nonpolar narcotics (Matozzo et al., 2003; Matozzo et al., 2004; Hwang et al., 2003), and thus similar lethal body residues (LBR50 = body residue at 50% mortality) were expected (McCarty and Mackay, 1993) for both chemicals in both test organisms in the acute tests. In the organism, the organic chemicals are assumed to partition in the lipids (Spacie et al., 1995). Based on literature, the lipid content of the C. riparius larvae varies from 0.6 to 1.53% of wet weight (Harkey et al., 1994; Looser et al., 1998; Hwang et al., 2001). Therefore, the lipid content of C. riparius was probably similar to that of L. variegatus (1.23% ww) in our study. Unexpectedly, C. riparius was more sensitive to both tested chemicals than L. variegatus on a body residue basis. It is concluded, however, that there is high variability of CBRs among nonpolar narcotics (Barron et al., 2002), and it is thus difficult to say whether the findings in this paper are in accordance with the hypothesis of the CBR-approach. Nevertheless, it is noteworthy that if only LC50 values had been concerned, no information about true exposure (i.e. body residue) would have been gathered. Differences in species characteristics (structure, function) associated with the expression of toxic effects may explain the different critical body residues between the species. The body residue analysis was based on a radiolabeled chemical and possible biotransformation was not measured. There is evidence that C. riparius has a better biotransformation capability for some organic chemicals than L. variegatus (Verrengia Guerrero et al., 2002). It is suggested, however, that C. riparius is not capable of biotransforming LAS (Hwang et al., 2003). No information

is available concerning L. variegatus. 4-NP, on the other hand, is commonly biotransformed in fish (e.g. Hughes and Gallagher, 2004) by the glutathione-S-transferase (GST) enzyme, which is also found in invertebrates (Livingstone, 1998). Thus, 4-NP was most likely metabolized by C. riparius, and probably also by L. variegatus. Consequently, part of the [14 C]-label in the organism tissues may have belonged to metabolites, L. variegatus being less capable of excreting them. This may have caused higher radiolabel concentrations in L. variegatus tissue. Overall, the results emphasize the importance of analyzing both, parent chemical and metabolites, when linking toxic effects to chemical body residues. There are no studies concerning critical body residues (CBR) of 4-NP exposed freshwater benthic invertebrates. However, the lethal body residue (LBR50 ) for marine amphipod Ampelisca abdita of 1.1 mmol/kg wet tissue was measured after exposure of 10 days to sediment-spiked 4-NP (Fay et al., 2000). The acute LBR50 value estimated for C. riparius in the current experiments is higher, even though the lipid content of A. abdita was higher (2.38% ww). This may indicate that part of the 4-NP was metabolized by C. riparius. It has been proposed that nonionic surfactants are more toxic than anionic surfactants, based on external surfactant concentration (Cserh´ati et al., 2002; Warne and Schifko, 1999). In this study, however, in the acute toxicity experiments no clear difference was detected in LC50 values for nonionic 4-NP compared with anionic C12-LAS. There are several studies concerning 4-NP toxicity, but only a few focus on benthic freshwater invertebrates. The reported 96-h LC50 values for 4-NP in L. variegatus and snail Physella virgata were 1.552 and 3.512 ␮mol/L, respectively (Brooke, 1993). These values are similar to the acute LC50 values estimated for L. variegatus and C. riparius in this study. The reported chronic LC50 values are, however, clearly lower: 14-day LC50 for C. tentans was 0.54 ␮mol 4-NP/L (England and Bussard, 1993). As regards the acute toxicity of LAS, a 72-h LC50 of 6.313 ␮mol/L was reported for C11.8-LAS in newly hatched larvae of C. riparius (Pittinger et al., 1989), which is close the LC50 values estimated for L. variegatus and C. riparius in the current experiment. Some organisms may be more tolerant when exposed to LAS, as reported for water-exposed snail Physa acuta, which had a 24-h LC50 value of 0.048 mmol/L (Liwarska-Bizukojc et al., 2005). The sediment characteristics had a clear influence on 4-NP and C12-LAS toxicity in the C. riparius larval growth test. A distinctly lower sediment exposure concentration was needed in S3 sediment to cause decreased growth than in S2 sediment. This can be explained by the lower bioavailability of chemicals in S2 compared to S3 sediment as shown in the bioaccumulation experiments. Both chemicals can be expected to biodegrade in water or sediment by microbial action (Ekelund et al., 1993; Larson and Woltering, 1995), and the degradation is expected to increase the longer the experimental time. The effective concentrations may therefore have been overestimated. Several studies have dealt with the sublethal effects of 4-NP in benthic organisms in water exposure (England and Bussard, 1993; Kahl et al., 1997; Czech et al., 2001; Schmude et al.,

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1999; Meregalli et al., 2001). There have been fewer studies concerning the toxicity of sediment-originating 4-NP. The NOEC and LOEC values for 4-NP sublethal toxicity in the current experiments fall approximately in the same range as the values estimated in sediment-exposed organisms. Bettinetti et al. (2002) found that at a 4-NP concentration of 1.044 mmol/kg sediment dry weight (organic matter content 2.3%), the survival of the C. riparius larvae decreased, and at 3.630 mmol/kg dw no survivors were observed. Furthermore, at sediment concentrations of 1.044–1.815 mmol/kg dw the larval wet weight was significantly decreased compared to control treatment. The finding that 4-NP had no toxic effect at high exposure concentrations in S2 sediment remains unresolved. LAS has been tested extensively in both short- and long-term studies. Most of the toxicity testing, however, is acute and has been conducted in a water-only system. In general, a decrease in alkyl chain length or a more internal position of the phenyl group in the LAS molecule is accompanied by a decrease in toxicity (International Programme on Chemical Safety, 1996). Regarding the sublethal effects of LAS, C11.53-LAS is reported to inhibit the development, growth and reproduction of the soil-living collembolan Folsomia fimetaria. The 10% inhibition in effects was seen at soil (OC% 2.3) concentrations of 0.42–0.53 mmol/kg dw (Holmstrup and Krogh, 1996). When the earthworm Eisenia foetida was exposed to C11.36-LAS incorporated into soil at nominal concentrations of 0.181–2.87 mmol/kg dw, the NOEC on the basis of a body weights was 0.674 mmol/kg dw. In a second study C11.36and C13.13-LAS incorporated into sludge and applied to soil gave a NOEC based on worm weight of 1.759 mmol/kg dw (Mieure et al., 1990). The values are very close the NOEC and LOEC values estimated for a decrease in C. riparius growth in this paper. Hwang et al. (2003) reported slightly lower LOECbody residue for an increase in developmental time after 30day exposure to 2-phenyl isomer of dodecylbenzene sulfonate in C. riparius. The concentrations of 4-NP and LAS found in the environment are lower than the sediment-spiked concentrations needed to produce significant sublethal effects in the current experiments. For example, 0.05–1.2 ␮mol 4-NP/kg dw is reported in river sediment from southwest Germany (Bolz et al., 2001) and 0.05–0.7 ␮mol 4-NP/kg dw in the German Bight of the North Sea (Bester et al., 2001). Similarly, 49–129 ␮mol LAS/kg dw is reported in the Tsurumi River, Japan (International Programme on Chemical Safety, 1996) and 0.11–0.30 ␮mol LAS/kg dw in the German Bight of the North Sea (Bester et al., 2001). However, there are findings that surfactants may enhance the mobility of other toxicants in the environment (Krogh et al., 2003). In addition, 4-NP is a known endocrine disruptor (White et al., 1994; McCormick et al., 2005) and presumably has more sensitive sublethal chronic toxic effects than observed in the current study. 4-NP may also act as an agonist with other endocrine disruptors. More detailed examination of the toxic effects and mixture toxicity effects of these chemicals is therefore needed. Further, the effect of biotransformation in the environment and in organisms on the toxicity results needs to be evaluated.

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