Earthworms comprise up to 60 to 80% of total soil biomass and play an important role in maintaining soil health because they are primary consumers of organic matter. Soil contact and consumption of soil may lead to the bioaccumulation of soil contaminants in earthworms 1. Thus, earthworms are important organisms when studying the ecotoxicological effects of contaminants in soil. Agrochemicals are often used during the vegetation period to combat several pests and diseases simultaneously 2. In addition, a large number of industrial chemicals are released into the environment and may be added to soils by applying sewage sludge from wastewater treatment systems 3. Hence, contaminants can be found in soils as mixtures; consequently, soil organisms are exposed to multiple toxicants.
Environmental risk assessment methods often do not address the effects of contaminant mixtures and frequently evaluate mixtures using a chemical-by-chemical approach. As the toxicity in natural ecosystems is a result of the integrated effects of all contaminants, such an approach may underestimate or overestimate actual effects 4. It is therefore important to study the impact of chemicals entering the soil ecosystem using more complex experimental designs, including mixtures of chemicals. Only a few studies have been conducted on mixture toxicity to earthworms 5–8.
Furthermore, it is not only the number of chemicals, but also the duration of exposure that may affect an organism's response to chemical stress. Soil organisms are exposed to contaminants over multiple generations due to repeated applications of pesticides or chronic exposure to industrial chemicals. It is of high interest to record effects on more than just one generation to assess the true risks these contaminants pose to the living environment. Indeed, the response of a population to chemicals is not static but rather varies over time. Increasing internal concentrations in filial generations can increase exposure and may lead to amplifying adverse effects or cumulative damages may occur 9. On the other hand, populations may also become resistant to certain chemicals when being exposed over time. Therefore, single-generation exposure tests might be inappropriate to predict effects on natural populations with multiple generations over time 9. Few attempts have been made to extend chronic toxicity tests of organic chemicals to multiple generations of invertebrate organisms, including earthworms 9–12, and studies on transgenerational effects of mixtures are scarce 13, 14.
Two pesticides, the strobilurin fungicide Acanto (DuPont, active ingredient picoxystrobin; hereafter referred to as picoxystrobin) and the synthetic pyrethroid ester insecticide esfenvalerate were chosen for study as they are used commonly in crop production in Norway and many other countries. In addition, the antimicrobial agent triclosan, which is used in many consumer products, was included. Triclosan accumulates onto activated sludge used to remove chemicals from wastewater and thus enters the soil from amendments with biosolids. The three biocides have different modes of action on their target organisms: picoxystrobin inhibits mitochondrial respiration in fungi 15; esfenvalerate disrupts the nervous system of insects by acting as a sodium channel agonist 16; and triclosan inhibits fatty acid synthesis in bacteria 17. The biocides were used in single- and mixture exposure tests with the earthworm Eisenia fetida. Mixture toxicity results obtained in the present study were compared with predictions made by the commonly used concepts of concentration addition and independent action. The concept of concentration addition usually yields best predictions for mixtures of similar acting components 18, whereas independent action is a better predictor for mixtures of dissimilar acting chemicals 19. When the specific mode of action is unknown, both concentration addition and independent action can be used as being equally valid reference models when assessing mixture toxicity 6. Because chemicals with different modes of action were studied, it was expected that independent action could be a better predictor for the toxicity of the mixture than concentration addition.
The aims of the present study were first to determine if the toxicity of a mixture comprising picoxystrobin, esfenvalerate, and triclosan on E. fetida could be explained by the toxicity of the single substances using the concepts of concentration addition and independent action. Second, we sought to determine whether acute and chronic toxicity differs between two consecutive generations of E. fetida exposed to the mixture of biocides. To the knowledge of the authors, the present study is the first attempt to investigate the toxic effects of a mixture to more than one earthworm generation.
MATERIAL AND METHODS
Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol, purity ≥97.0%) was purchased from Sigma-Aldrich and esfenvalerate (cyano-3-phenoxybenzyl (S)-2-(4-chlorophenyl)-3-methylbutyrate, purity ≥97.0%) was purchased from Chiron AS. Acanto (active ingredient 250 g picoxystrobin (methyl (αE)-α-(methoxymethylene)-2-[[[6-(trifluoromethyl)-2-pyridinyl]oxy]methyl]benzeneacetate) L−1 Acanto) was purchased directly from DuPont. Beside picoxystrobin, Acanto contains inert ingredients and <10% propylene glycol, which is degraded very rapidly and has a low toxicity (http://www2.mst.dk/udgiv/publications/2001/87-7944-596-9/pdf/87-7944-597-7.pdf). Accordingly, it is unlikely that the formulation dramatically changes the toxicity of picoxystrobin to earthworms. Thus, effects can be principally attributed to the active ingredient picoxystrobin.
The Ap-horizon of an agricultural soil from Norderås, Norway (59°68′′N, 10°77′′E) was used for the experiments. This represents a typical agricultural soil in Norway (based on the database of the Norwegian monitoring program on soil containing more than 1,400 A and B horizons of agricultural soils, Skog og Landskap, Ås, Norway). The soil properties are listed in Table 1. Prior to starting the test, the soil was air-dried and sieved (≤2 mm).
Table 1. Properties of the Norderås (Ås, Norway) soil used for 28-d toxicity tests with the earthworm Eisenia fetida
Organic carbon (g 100g−1 dry wt)
Physical and chemical properties
Water-holding capacity (% of dry wt)
Cation-exchange capacity (nmolc(+) kg−1)
Base saturation (%)
Exchangeable acidity (nmolc(+) kg−1)
The compost worm E. fetida (Annelida: Lumbricidae. Savigny, 1826) was used as the test organism. It is an epigeic species that can be cultivated easily in the laboratory; therefore, it is used commonly in ecotoxicological testing. Eisenia fetida has a short reproductive cycle, which facilitates studies on generational effects. Its reproductive success is high, with two to five cocoons per individual in a week with up to four juveniles per cocoon. The mean incubation time of cocoons is three weeks, and it takes another four to six weeks until hatchlings reach maturity 20. Mature individuals can be reproductively active for more than 500 d 21. The life span of E. fetida is approximately three years (http://www.bodenkunde-online.de/bodenkunde/index2.php?option=com_content&do_pdf=1&id=22). For the toxicity tests performed, earthworms of 300 to 500 mg with a visible clitellum were obtained from an unsynchronized culture of our laboratory in Ås, Norway. The earthworm cultures are kept in moist potting soil at 20 ± 1°C and are fed ground raw vegetables once a week. Water was replenished as required. Cultures were renewed regularly to maintain a maximum age of one year for earthworms.
First, chronic toxicity tests (28-d exposure) with all three single biocides were performed on one E. fetida generation to obtain data for the mixture toxicity predictions. Subsequently, two consecutive generations (F0 and F1) of E. fetida were exposed to soil spiked with a mixture of picoxystrobin, triclosan, and esfenvalerate.
The chosen test parameters were mortality and the inhibition of cocoon production of adult earthworms, as well as the bodyweight of F1 juveniles. Three replicates per treatment and six controls with unspiked soil were used in each experiment.
Single exposure experiments
Reproduction tests for all three single compounds were conducted according to an adapted version of Organisation for Economic Co-operation and Development (OECD) guideline 222 for chemical testing 22. Deviations from the guideline are mentioned in subsection Single exposure experiments. Acetone was used as a solvent for spiking the soil with esfenvalerate and triclosan. Twenty percent of the soil was spiked, and the acetone was allowed to evaporate under a fume hood overnight to minimize solvent effects on biological soil processes. The spiked soil was then mixed thoroughly with the remaining clean soil. Because Acanto is a water-soluble formulation, it was dissolved in distilled water and added to the soil. Test vessels were filled with 500 g (air-dried) spiked Norderås soil adjusted to 60% of its water holding capacity. For each of the three compounds, the following nominal concentrations were used: 0.3, 0.6, 1.1, 2.3, 4.6, 9.1, and 18.2 µmol (0.1–6.7 mg kg−1) picoxistrobin kg−1 dry soil; and 2.4, 6.0, 11.9, 23.8, 47.6, 95.3, 142.9, and 190.5 µmol (1.0–80 mg kg−1) esfenvalerate kg−1 dry soil; and 0.2, 0.4, 0.9, 1.7, 3.5, and 4.1 µmol (0.07–1.2 mg kg−1) triclosan kg−1 dry soil.
Adult earthworms with a well-developed clitellum were cleaned with tap water and left on wet filter paper in Petri dishes for 24 h to depurate. They were cleaned again and softly blotted on absorbent paper to remove excess water. Ten earthworms were weighed and introduced to each test vessel. All test vessels were incubated for 28 d at 20 ± 1 °C in a 16:8 h light:dark regime. During the experimental period, earthworms were fed ground horse manure (dried and frozen to eliminate fly eggs) mixed with water. Approximately 20 g of food were added every second week to each container. Once a week, the test vessels were weighed and water loss was compensated. At the end of the test period, adult earthworms were removed from all test vessels and the number of surviving individuals was recorded. The adult earthworms were reweighed after depuration for 24 h. The cocoons were extracted by washing the soil with tap water through a 2 mm mesh and then counted.
Mixture experiment on two consecutive generations
For the mixture experiment, a fixed-ratio design was chosen 4, 18. While the mixture ratio was kept constant, the total concentration of the mixture varied so that a complete concentration–response relationship of the mixture could be obtained. This experimental approach leads to equitoxic mixtures and is used commonly to assess joint actions of mixtures 23. The three compounds were mixed in the proportion to their median effective concentration (EC50) values. This resulted in the following nominal fractions of the components in the mixture: 74% esfenvalerate, 19% picoxystrobin, and 7% triclosan. The nominal mixture concentrations used to spike the soil were 9.7, 14.6, 21.9, 32.9, 49.3, and 73.9 µmol kg−1 dry soil. The experimental procedure and conditions for the mixture exposure of the F0 generation were identical to the procedures described for the single exposures. The soil was spiked with triclosan and esfenvalerate in acetone, and the acetone was allowed to evaporate overnight under a fume hood. The next day, the soil was spiked with the corresponding picoxystrobin concentrations and the water holding capacity was adjusted to 60% using distilled water. Ten earthworms with a well-developed clitellum were added to each replicate. For each test vessel containing spiked soil and 10 earthworms, an additional replicate with soil contaminated with the same mixture concentration and without earthworms was incubated concurrently with the other test vessels (hereafter, worm-free vessels).
After completing the 28-d mixture exposure, surviving adults and cocoons were removed from the soil. The cocoons of each replicate were transferred to the corresponding worm-free vessel and incubated for another 28 d for hatching. Hatched juveniles (F1 generation) were removed from the soil using forceps. Ten randomly picked juveniles of each replicate were rinsed, weighed, and then transferred back to the worm-free vessels and incubated until complete maturation of all juveniles (indicated by a well-developed clitellum). The earthworms were fed every second week, and evaporated water was replenished weekly. During the maturation period, the earthworms of each vessel were extracted once a week and weighed and the development of the clitella assessed visually. In some of the test vessels, more than 10 individuals were observed during the first three weeks. In these cases, the excess earthworms per vessel found after extracting 10 individuals were removed.
When the F1 earthworms of all test containers were mature, they were used for another 28-d reproduction test. New soil was spiked with the same mixture concentrations as were used in the experiment with the F0 generation. The adult F1 individuals were rinsed in water, depurated over 24 h, weighed, and transferred to the freshly spiked soil. Cocoon production and adult survival were assessed after 28 d at 20 ± 1 °C and a 16:8 h light:dark regime using the previously used methods.
Soil sample measurements
The total concentrations of the applied chemicals in the soil were measured at the start and after completion of the experiments. Five grams of moist soil were transferred to a 50 ml polytetrafluoroethylene tube, and 5 g of anhydrous MgSO4 were added to dry the sample. Acetonitrile (10 ml) was used for extraction, and triphenyl phosphate (1µg ml−1) was added as internal standard. The samples were shaken end over end for 16 h, centrifuged at 7741 g for 10 min, and an aliquot was taken for analysis by gas chromatography–mass spectrometry (GC–MS; Agilent 6890 GC with PTV-injector and Agilent 5973 MSD). The limit of quantification for the compounds was 0.02 µg ml−1, and the average recovery for spike levels was 70 to 120%. Another 20 g of the soil of each replicate was dried at 105 °C for 12 h to determine the water content of the samples. All concentrations were related to dry soil weight.
The concentration–response relationships of the single substances and the mixture were calculated by fitting log-logistic and Weibull models to the toxicity data using the analysis of dose-response curves package DRC 24 in the statistical software R 25. Average measured initial concentrations were used in all models. The Model Selection function in R was used to find the best model. This function selects the best fit model by comparing the maximum log-likelihood value, the Akaike's Information Criteria, the estimated residual variance, and the p value from a lack-of-fit test of all models. The 2-parametric Weibull model with the scale and shape parameters b and e, and the measured concentration x was superior for all data sets. An exception was the relationship between the ternary mixture and the reproduction of the F0 generation, which was best explained by the 2-parametric log-logistic model . The best model for each data set was used to calculate the respective EC50 and median lethal concentration (LC50).
Because the 2-parameter Weibull model fit the data of the single compounds best, calculations of mixture toxicity predictions according to the concepts of concentration addition and independent action were based on this model. Mixture toxicity predictions were calculated using the concept of concentration addition and independent action. The concept of concentration addition is based on the idea that the single components of a mixture have a similar mode of action and all components can serve as a dilution to each other. Mathematically, concentration addition can be expressed as where ECxMix is the total concentration of the mixture causing x% effect, pi the proportion of the ith component in the mixture, ECxi the concentration of the ith compound that causes x% effect when applied singly, and n is the number of mixture components 26. The measured proportions of the mixture components and the ECx values obtained from the concentration–response models for each mixture component were used as input variables.
If the components of a mixture have dissimilar modes of action but contribute to a common response, the mixture toxicity is expected to follow independent action. According to this concept, the mixture effect is the sum of the individual effects minus the proportion of the population in which sensitivities overlap. The mathematical expression of independent action is with E(cmix) being the expected effect of the mixture, and E(ci) the effect of the ith component in the mixture when applied alone 27, 28.
To quantitatively compare the observed and predicted mixture toxicity, model deviation ratios (MDR) were used. The MDR is defined as the ratio between the expected effective concentration predicted by the perspective concept and the observed effective concentration obtained from toxicity testing 26. These ratios give information about the general use of the concepts of concentration addition and independent action. Model deviation ratios smaller than one indicate antagonism, and MDRs higher than one indicate synergism to the prediction concept. In the present study, the EC50 and LC50 were used as the effective concentration. Student's t test (5% significance level) was used to determine if the effects between the F0 and F1 generation were significantly different from each other.
All of the toxicity tests passed the validity criteria of the test protocols (>30 juveniles per control replicate, adult mortality <10%).
The water content of the soil samples varied between 16 and 20%. The recovery of the internal standard varied between 88 and 117% for all measured samples. The actual concentrations of the three compounds in the soil of the single compound experiments directly after spiking, corrected for the recovery of the internal standard, were 84 ± 15% (average ± standard deviation, picoxystrobin), 101 ± 27% (triclosan), and 94 ± 15% (esfenvalerate) of the nominal concentrations. After the 28-d exposures, a decrease in concentrations was observed. This was expected because all three substances are subject to microbial degradation 15–17. The measured concentrations ranged then from 56 ± 17% (picoxystrobin), 54 ± 15% (triclosan), and 6 ± 6% (esfenvalerate) of the lowest initial concentrations to 68 ± 20% (picoxystrobin), 75 ± 22% (triclosan), and 11 ± 2% (esfenvalerate) of the highest initial concentrations.
For the mixture experiments, the actual concentrations of the substances directly after spiking were 102 ± 6% (picoxystrobin), 79 ± 18% (triclosan), and 90 ± 4% (esfenvalerate) of the nominal concentrations. After 28 d, the actual concentrations ranged from 43 ± 22% (picoxystrobin), 4 ± 7% (triclosan), and 106 ± 14% (esfenvalerate) of the lowest initial concentrations to 73 ± 25% (picoxystrobin), 36 ± 20% (triclosan), and 89 ± 19% (esfenvalerate) of the highest initial concentrations. There was no difference in degradation of the substances between the F0 and F1 experiments.
Soil samples were also taken during the maturation period. At the end of the maturation period of the F1 juveniles, the actual concentrations ranged from 0% (picoxystrobin and triclosan) and 27 ± 15% (esfenvalerate) of the lowest initial concentrations to 6 ± 1% (picoxystrobin), 0% (triclosan), and 39 ± 8% (esfenvalerate) of the highest initial concentrations. In the picoxystrobin experiment, 8 ± 8% of the nominal initial concentration of picoxystrobin was measured at the end of the maturation period. The degradation of the mixture components from the start of the 28-d exposure of the F0 generation until the end of the maturation period is depicted in Figure 1A through C. Average measured start concentrations were used to calculate effect concentrations.
Toxicity of single compounds
All three chemicals caused adult mortality and inhibited the reproduction of E. fetida in the single-substance exposure experiments, and effect concentrations (LC50s and EC50s) could be determined. In the 28-d experiment with triclosan-spiked soil, adult mortality was too low to derive a complete dose–response curve. Lethal concentrations of triclosan were therefore calculated from the range-finding test results. Note that the test duration of the range-finding test was 14 d only, and even in the highest concentrations, 100% mortality was not achieved. The concentration–response curves of the three single compounds of picoxystrobin, triclosan, and esfenvalerate are shown in Figure 2. The LC50s were 10.0 (95% confidence interval [CI], 8.0–12.0) µmol kg−1 dry soil for picoxystrobin; 1,907 (1,537–2,277) µmol kg−1 dry soil for triclosan, and 85.8 (79.3–92.33) µmol kg−1 dry soil for esfenvalerate. The EC50s for reproduction were 5.4 (4.2–6.6) µmol kg−1 dry soil for picoxystrobin, 3.0 (1.6–4.5) µmol kg−1 dry soil for triclosan, and 33.5 (29.9–37.2) µmol kg−1 dry soil for esfenvalerate. The lowest concentrations at which a significant (p < 0.05, student's t test) decrease in cocoon production occurs (LOEC), were 3.8 µmol kg−1 dry soil for picoxystrobin, 3.2 µmol kg-1 for triclosan, and is 25.9 µmol kg−1 for esfenvalerate.
Measured fractions of the single components in the mixture were used to calculate the predictions according to concentration addition and independent action. Because a considerable but different degradation of the compounds in the mixture occurred in the course of the experiment, the predictions were calculated once using fractions based on measured start concentrations and once based on fractions in the lowest measured concentrations at the end of the exposure period (28 d). The fractions at the start of the experiment were 76% esfenvalerate, 20% picoxystrobin, and 4% triclosan (hereafter, start fractions) and were thus similar to nominal fractions. At the end of the experiment, the fractions in the highest mixture concentration did not change, whereas in the lowest mixture concentrations the fractions were, on average, 95% esfenvalerate, 5% picoxystrobin, and 0% triclosan (hereafter, low-end fractions). Thus, in the lowest exposure concentrations, the mixture changed from being ternary to binary. To allow us to use the same model calculations as those used with the start fractions, triclosan concentration cannot be 0. We therefore used a dummy fraction with the value of 0.01% for triclosan (and respectively 94.99% for esfenvalerate).
Adult mortality of the F0 generation exposed to the mixture and the predictions made by the concepts of concentration addition and independent action are depicted in Figure 3A and B. The LC50 of the mixture was 39.9 (37.0–42.9) µmol kg−1 dry soil and thus coincides with the LC50s determined for the single compounds. The data followed the concentration addition curve rather well at high mixture concentrations when using the start fractions (Fig. 3A), whereas at low concentrations, adult mortality data followed the concentration addition curve nicely when using the low-end fractions (Fig. 3B). The observed LC50 of the mixture fell between the two conceptual predictions when using start fractions: concentration addition overestimated and independent action underestimated the LC50 of the mixture. When using the low-end fractions for calculating predictions, both concepts underestimated the LC50 (Table 2).
Table 2. Predicteda and measured effect concentrations of the ternary mixture comprising Acanto (DuPont, active ingredient picoxystrobin), esfenvalerate, and triclosan on adult mortality (LC50) and the reproduction (EC50) of two consecutive generations (F0 and F1) of Eisenia fetidab
Predicted by CA and IA based on start fractions/end fractions. See text for further explanation.
Effect concentrations are given in µmol kg−1 dry soil and are obtained from 28-d exposures in sandy loam soil at 20 ± 1 °C with three replicates per concentration and six controls. Predictions are calculated from single exposure experiments on a F0 generation. F1 earthworms grew up in the treated soil of the F0 generation.
LC50 = median lethal concentration; EC50 = median effective concentration; CA = concentration addition; IA = independent action; MDR = model deviation ratio
The toxicity of the mixture on the reproduction of the F0 generation and the concentration addition and independent action predictions are shown in Figure 3C and D. The EC50 of the mixture was 28.1 (25.5–30.6) µmol kg−1 dry soil and thus higher than the EC50 of picoxystrobin and triclosan, but lower than the EC50 of esfenvalerate. The sublethal mixture data followed the independent action curve rather well at low concentrations when using the low-end fractions, whereas at high mixture concentrations, deviations from either of the two different independent action curves were small. The observed EC50 was closest to predictions calculated with the low-end fractions and was between the two conceptual predictions: concentration addition overestimated and independent action underestimated the EC50 slightly (Table 2).
The toxicity of the ternary mixture was tested on two consecutive E. fetida generations (F0 and F1). The average weight increase per week was higher for the F1 juveniles in treated soil than in the controls (Table 3). This trend was statistically significant for the mixture concentration of 27.6 µmol kg−1 dry soil (Student's t test, p = 0.0013 and p = 0.0492) and was marginally significant for the mixture concentrations of 12.4 and 19.2 µmol kg−1 dry soil (p < 0.1) after eight and 14 weeks. It took almost 24 weeks for all juveniles (F1) to reach maturity. The F1 individuals exposed to the two highest mixtures matured earlier than the individuals in the lower concentrations and in the controls (Table 3). In the second highest mixture concentration (27.6 µmol kg−1 dry soil), the F1 juveniles reached maturity after 15 ± 1.7 weeks, whereas it took 22 ± 1.5 weeks for control worms to mature. This difference was statistically significant (p = 0.0281). At the mixture concentration of 41.7 µmol kg−1, no F1 juveniles were found in two of three replicates; hence, no statistical test could be applied (Table 3). Body weight of earthworms at the time of complete maturation did not differ significantly between the exposure concentrations (Student's t test, p > 0.05). Furthermore, earthworms reaching maturity earlier were not significantly heavier (p > 0.05) when the toxicity test with the F1 generation was started. Hence, effects of body weight at the start of the F1 experiment on the outcome of the toxicity test are unlikely.
Table 3. Development of the Eisenia fetida F1 generation from hatching to adult stage in sandy loam soil at 20 ± 1 °C with three replicates per concentration and six controlsa
Mixture (µmol kg−1)
Weight of 10 earthworms (% of control)
Average growth week−1 (g)
F1 earthworms grew up in the treated soil of a 28-d experiment with the F0 generation exposed to a mixture comprising Acanto (DuPont, active ingredient picoxystrobin), esfenvalerate, and triclosan. The average weight of 10 individuals as percentage of increase/decrease in relation to the control (0 µmol kg−1) is given. For the control, average weights in g are given in brackets.
No earthworms survived in two of the three replicates.
When all earthworms of the respective concentration had reached maturity.
The results of the effects of the mixture on adult mortality and reproduction are depicted in Figure 4. The distribution of the observed adult mortality data along the mixture concentration range was very similar for both generations (Fig. 4A). No statistical generational differences in median adult mortality were observed in earthworms exposed to the mixture. The mixture LC50 for the F0 generation (39.9 [37.0–42.9] µmol kg−1 dry soil) was not statistically different from the LC50 for the F1 generation (43.0 [39.0–47.1] µmol kg−1 dry soil; Student's t test, p = 0.1162). Yet, generational differences in sublethal toxicity were observed (Fig. 4B). The slope of the sublethal concentration–response curve for the F1 generation was flatter than for the F0 generation, and the EC50 of the mixture was 1.5 times higher for the F0 generation (28.1 [25.5–30.6] µmol kg−1 dry soil) than for the F1 generation (19.0 [15.2–22.7] µmol kg−1 dry soil). This difference was highly significant (Student's t test, p < 0.0001). Also the LOEC for cocoon production was considerably higher for the F0 generation (27.6 µmol kg−1) than for the F1 generation (9.1 µmol kg−1). A comparison of measured and predicted mixture effect concentrations are given in Table 2.
Degradation of mixture components
The degradation of the three biocides differed between the single exposure and the mixture exposure experiments. Whereas esfenvalerate degraded strongly in all concentrations in the single exposures, degradation of that compound was quite limited in the mixture exposure. The degradation of picoxystrobin did not differ between the single and the mixture exposure, yet a weak dose dependency occurred in both exposures with a slightly stronger degradation at lower concentrations. A dose-dependent degradation was also found for triclosan in both exposure scenarios. It is possible that the degradation of esfenvalerate in the mixture and of triclosan and picoxystrobin at the high mixture and single compound concentrations were hampered by the toxic effects of the three compounds to soil microbes. This has been observed, for example, for phenanthrene in soil 9. Degradation of triclosan at all concentrations was considerably stronger in the mixture exposure compared to the single exposure. It is difficult to explain this difference, because the precise removal processes of triclosan in soil are not known. Yet, the literature reports quite different degradation times for triclosan, indicating that slight differences in conditions affect degradation of triclosan considerably 3.
For picoxystrobin, the European Commission states an acute LC50 (14-d exposure) of 3.4 mg kg−1 (9.3 µmol kg−1) for E. fetida (http://sitem.herts.ac.uk/aeru/footprint/en/Reports/527.htm), which is very similar to the LC50 found in the present study (10.0 µmol kg−1 dry soil). Another study determined a much higher LC50 of 7.2 mg kg−1 soil (19.7 µmol kg−1) for E. fetida in artificial OECD-soil 29. The influence of soil organic matter on the availability of organic compounds to living matter may explain differences in the determined LC50s. Furthermore, lethal and sublethal effects occurred in a narrow concentration range, and concentration–response curves were consequently very steep. The determination of EC50s and LC50s, therefore, bears corresponding uncertainty. The mode of action of picoxystrobin on E. fetida is currently not known. The acute-to-chronic ratio (ACR) may be used to assess whether a chemical acts specifically or by narcosis on an organism. The ACR is commonly calculated as the ratio of the LC50 and a sublethal endpoint, such as the LOEC or EC50. In the present study, we used the LOEC for cocoon production, in order to compare ratios with ACRs calculated in a study by Roex and co-workers. 30. Using the threshold ACR for baseline toxicity proposed by Roex et al., to distinguish between narcotic (ACR < 2.58) and specific (ACR ≥ 2.58) mode of action 30, reveals that the ACR for picoxystrobin was at the threshold for narcosis (ACR = 2.6). In this case, the ACR does not clearly indicate whether or not picoxystrobin acts specifically on E. fetida.
The lethal toxicity of esfenvalerate was rather low, with a high LC50 of 85.8 µmol kg−1 dry soil. The European Health Commission states a LC50 of 10.6 mg kg−1 substrate (25 µmol kg−1) for E. fetida (http://sitem.herts.ac.uk/aeru/iupac/269.htm). It is difficult to explain this large difference, as exposure scenario, test duration, and soil type of the study are not known. In the present study, even the EC50 for reproduction (33.5 µmol kg−1 dry soil) was higher than the LC50 found in the literature. The ACR for esfenvalerate in the present study is 3.3 and may thus indicate a specific mode of action on E. fetida. Esfenvalerate is designed to target the sodium channels of the nervous system of arthropods. Arthropods and annelids share a synapomorphic ladder-like nervous system. The mode of action of esfenvalerate may therefore be the same on arthropods and annelids.
The LC50 for triclosan in the present study was 1,907 µmol kg−1 dry soil. Even higher was the LC50 for adult mortality of Eisenia andrei (866 mg kg−1 (2990 µmol kg−1) as determined by Amorim and coworkers 31. Noteworthy is that neither the study by Amorim et al. 31 nor in the present study did the exposure concentrations evoke 100% mortality. Hence, estimations of LC50 values are imprecise. The reported EC50 for reproduction in the closely related species E. andrei is 4 mg triclosan kg−1 (13.8 µmol kg−1) 31, which is more than four times greater than the EC50 determined for E. fetida in the present study (3.0 µmol kg−1). Again, differences in soil properties and among species may explain the differing EC50 values. Given the very high ACR of 602, it can be assumed that triclosan acts specifically on E. fetida. A recent study showed that triclosan alters enzyme activity and causes oxidative stress and DNA damages in E. fetida32.
According to the results of the present study, the maximum spray doses of both Acanto (1000 ml per hectare) and Sumi-Alpha (DuPont, 500 ml with 50 g esfenvalerate L−1 Sumi-Alpha per hectare) are not expected to evoke any measurable effect on the reproduction or survival of one E. fetida generation (<EC1 estimated from the single exposure results in the present study) when applied singly. Triclosan concentrations in sewage sludge of up to 3.3 mg kg−1 (11.4 µmol kg−1) have been reported in sludge in Norway (http://www.klif.no/no/Publikasjoner/Publikasjoner/2010/April/Undersokelse-av-miljogifter-ved-fire-norske-renseanleggPFOA-Bisfenol-A-Triklosan-Siloksan-D5-Dodecylfenol-og246-Tri-tertbetylfenol/). Soil concentrations, however, are lower than sludge concentrations as the application of sludge to agricultural sites results in a dilution of triclosan in large soil volumes. Hence, triclosan concentrations in field soil will probably be below concentrations provoking effects on reproduction or survival.
The mixture used in the present study consisted of anthropogenic biocides with specifically designed modes of action toward three different organism groups. It was therefore expected that the concept of independent addition could describe mixture toxicity better than the concept of concentration addition. This assumption was supported by the ACRs calculated in the present study, which indicate that esfenvalerate and triclosan may act specifically and dissimilarly, whereas it remains unclear whether picoxystrobin acts specifically or by nacosis on E. fetida.
Commonly, when calculating mixture toxicity predictions using concentration addition and independent action, the fractions of the mixture components at the start of the exposure period are used. In the present study, this resulted in an overestimation of both the observed lethal and sublethal toxicity at low concentrations by both concepts, whereas concentration addition was a good predictor of lethal toxicity and independent action of sublethal toxicity at high mixture concentrations. Without taking the predictions based on the low-end fractions into account, one would conclude that dose–dependent deviations occur with antagonism at low-mixture concentrations. This is not uncommon, and examples can be found in the literature. For example, a study on the escape behavior of E. andrei exposed to a binary mixture comprising the pesticides dimethoate and spirodiclofen reported antagonism at low and synergism at high mixture concentrations 5. A study on the joint effects of glyphosate and spirodiclofen on the escape behavior of Porcellionides pruinosus also showed dose–dependent deviations from the mixture toxicity predicted by independent action, with synergistic effects at low and antagonistic effects at high concentrations 33. It has been stated that dose–dependent deviations from model predictions often occur when mixtures comprise only a few components and when higher organization level endpoints such as reproduction are used 6, 34. Deviations may indicate interactions, because both the concept of concentration addition and independent action assume non-interaction as a default 4. However, in the present study, an additional and probably the main factor driving deviations from model predictions is the strong and different degradation of the mixture components because the antagonism at low-mixture concentrations disappeared when recalculating concentration addition and independent action predictions using the low-end fractions. Yet, both concepts then strongly underestimated lethal toxicity at high-mixture concentrations. This is because the predictions assumed a mixture comprising 94.99% esfenvalerate, 5% pixocystrobin, and 0.01% triclosan at all concentrations. These fractions represent the mixture composition at the end of the experiment in the low-mixture concentrations. In the highest-mixture concentrations, however, the fractions of the components did not change considerably over the 28-d exposure. The single exposure experiments showed that triclosan provoked adult mortality only at very high concentrations, whereas picoxystrobin was very toxic to survival and probably the main driver of adult mortality: at the LC50 of the mixture, picoxystrobin is present at its EC34, whereas esfenvalerate and triclosan at concentrations provoking less than 2 and 1% mortality, respectively, when applied singly. While the mixture comprised 14 to 19% picoxystrobin in the highest mixture concentrations at the end of the experiment, the predictions with the low-end fractions assumed only 5% picoxystrobin at all mixture concentrations over the complete exposure period. Consequently, the predicted mortality at high concentrations reflects mainly the lethal toxicity of esfenvalerate. Hence, much lower mortality was predicted for earthworms exposed to high-mixture concentrations than observed. Thus, at high-mixture concentrations the predictions of lethal mixture toxicity based on the start fractions are more realistic.
The concept of independent action described sublethal toxicity at low-mixture concentrations much better when using the low-end fractions, where almost no triclosan was assumed to be present in the mixture. In contrast, at high-mixture concentrations the predictions based on start concentrations were slightly more accurate. This leads to the assumption that triclosan, which appears to be very toxic to the reproduction of E. fetida when applied singly, may cause sublethal toxicity first after some time of exposure. This assumption is supported by a study on the biochemical and genetic toxicity of triclosan on E. fetida conducted by Lin et al. 32. The study reported that the catalase activity of earthworms exposed to triclosan was higher than in the controls after a 2-d exposure, returned to control levels after a 7-d exposure, and finally decreased under control levels after a 14-d exposure. Catalase is an antioxidant enzyme that targets reactive oxygen species and thus indirectly takes part in the metabolism of contaminants. Lin et al. 32 suggest that E. fetida may tolerate oxidative stress at the first stage of exposure, while the antioxidant defense system is damaged after longer exposure. Hence, in low mixture concentrations, triclosan may be degraded before it could provoke effects, and the measured inhibition of reproduction is caused by the remaining two mixture components, mainly esfenvalerate. Comparing EC values of esfenvalerate when applied singly with the EC values of the mixture at concentrations up to the EC50, shows that mixture concentrations were only minimally higher than esfenvalerate concentrations.
At high-mixture concentrations, the concept of independent action fits the observed sublethal toxicity better when start fractions are used. This is because triclosan is less degraded in higher than in lower mixture concentrations, and the start fraction accords better with the measured fractions at these concentrations. Thus, the observed antagonism at lower concentrations may probably be tracked back to the concentration–dependent degradation of triclosan and picoxystrobin and to the time-dependent effects of triclosan.
When combining the predictions based on the start and the low-end fractions, concentration addition describes lethal mixture toxicity rather well, while independent action is the better predictor for the inhibition of reproduction. It has been suggested that the toxicity of mixtures comprising dissimilar compounds is higher (i.e., closer to concentration addition predictions) on mortality than on the inhibition of reproduction. The proposed explanation is that the inhibition of reproduction by a mixture is commonly based on more specific modes of action in comparison with mortality. Hence, concentration addition is more often observed when the endpoint mortality is studied, and independent action for the inhibition of reproduction 35. This is consistent with the results of the present study.
In the present study, a positive effect of the mixture on maturation time and growth were observed, while the reproduction of the F1 generation was more strongly inhibited than of the F0 generation. Brunninger et al. 12 reported positive effects of the herbicide terbuthylazine on the growth and cocoon production of E. andrei F1 individuals and a correlation of these two traits. They suggested that the increased growth was a result of increased metabolism due to chemical stress. It is likely that chemical stress caused an increase in metabolism also in the F1 individuals exposed to high-mixture concentrations in the present study, resulting in a faster growth and consequently earlier maturation than the F1 individuals in the controls. However, as the growth and maturation of F0 juveniles was not assessed in the present study, it cannot be concluded that this is a transgenerational effect but a general effect of the mixture on E. fetida. Yet, it can be assumed that the energy expense for growth and maturation led to a greater exhaustion of the F1 individuals, resulting in a lower cocoon production than in individuals of the F0 generation exposed to the same mixture concentrations. This assumption is supported by results of tests conducted by international laboratories for the purpose of inter-laboratory calibration (ring tests), which showed an inverse relationship between reproduction and body weight changes 36.
The mixture appeared to be more toxic to the reproduction of the F1 generation than the F0 generation. Mixture concentrations that did not provoke any effects on the F0 generation led to measurable toxic effects on the F1 generation, which is a result of the different slopes of the concentration–response curves for the two concentrations. This change in slope may indicate a change in mode of action. The ACR of the mixture increased from 1.45 for the F0 generation to 4.7 for the F1 generation in the mixture experiment. A study on the effects of phenanthrene to multiple generations of the springtail F. candida also showed a change in ACRs (LC50/EC50) from 1.4 in the F0 generation to 3 and 2.3 in the F1 and F2 generation, respectively, suggesting a shift from narcosis to specific effects 9. The mode of action of the mixture is a combination of the modes of action of the single substances and further investigation is needed to understand whether the increase in the mixture's ACR value from F0 to F1 indicates a shift toward more specific mechanisms. The increasing sublethal and unchanging lethal toxicity indicate however that stronger specific effects may be provoked in the F1 generation than in the F0 generation.
The F1 cocoons, and later the hatched juveniles, were exposed to the remaining concentrations of the three mixture components as the F1 generation grew up in spiked soil. Even though picoxystrobin and triclosan were degraded strongly in the lowest mixture concentrations during the maturation period, measurable concentrations of both components were found in higher mixture concentrations for up to 100 d, and esfenvalerate was not degraded completely in any of the mixtures over the complete maturation period. This means that the mixture components can affect juveniles at least for some time of the maturation period. As concluded from the results on the F0 generation in the present study and the findings of the study conducted by Lin et al. 32 triclosan is very toxic to sublethal endpoints when applied singly but may affect earthworms first after longer exposure time. Beside the biochemical effects of triclosan, Lin et al. 32 also studied the genotoxicity of triclosan to E. fetida by using comet assays. They found no severe DNA damages after 2-d exposures, while after 7-d and 14-d exposures a significant increase in DNA damages occurred in earthworms exposed to triclosan, including damages in the extreme damage class (>75% damage) 32. To our knowledge no studies have been published on potential genotoxic effects of picoxystrobin or esfenvalerate on earthworms or other soil-dwelling animals. But the evidence of triclosan inducing genotoxicity may lead to the assumption that certain DNA damages were evoked in the juvenile F1 earthworms, which then affected their reproduction when adult. This would explain deviations in the sublethal effects observed for the F0 and F1 generation.
Despite the rather fast degradation of the three biocides, the results of the present transgenerational study indicate that a repeated exposure to the investigated biocide mixture may lead to an increase in sensitivity in subsequent earthworm generations as a consequence of cumulative damages as reported in previous studies 9, 10. Acanto can be applied only once to a crop in the growing season, while up to three applications of Sumi-Alpha are permitted (1–2 leaf stage, 3–4 leaf stage, and when infected with fungi). A maximum application of 20 to 40 t of sludge per hectare and 10 years is permitted on grains in Norway (http://www.norvar.no/nv/Fag-prosjekter/Andre-fagressurser/Kunnskapsbase-slam/Spoersmaal-svar-om-slam/Bruk-av-avloepsslam-i-jordbruket-spoersmaal-og-svar). Consequently, the biocides may pose a risk to natural earthworm populations due to repeated exposures leading to damages being transferred to subsequent generations. Single generation exposure experiments are not capable of detecting such long-term risks, and their use in risk assessment practices may be questioned. Moreover, our results on the F0 generation show that predicting mixture toxicity using concentration addition and independent action is not always straightforward. Predicting the mixture toxicity to multiple generations is a more formidable challenge. The predictions made by concentration additions and independent actions are based on effects observed in the F0 generation and are thus only strictly valid for this generation. However, the purpose of using prediction models rather than assessing mixture toxicity in a chemical-by-chemical approach is to simulate realism, and predictions should therefore also be applicable to subsequent generations. Predicting safe exposure concentrations for a given population is a main goal of risk assessment practices. It has been suggested that concentration addition is the more conservative of the two models when the slopes of the individual toxicants' logistic concentration–response curves exceed 1.25 37, as it is the case for the sublethal toxicity in the present study. The concept of concentration addition may therefore be chosen to predict toxicity if consideration is given to the precautionary principle 19. The concept of concentration addition overestimates the EC50 towards the F0 generation regardless of which fractions are used in the model, and concentration addition can thus be ranged conservative for the F0 generation. For the F1 generation, however, the observed EC50 and the concentration addition predictions based on start fractions converge, while concentration addition predictions based on low-end fractions underestimate the EC50 for the F1 generation. Consequently, concentration addition is not strictly conservative for the F1 generation. This shows that even though concentration addition and independent action give more realistic predictions on mixture toxicity than chemical-by-chemical approaches, they are not adequate for reliably predicting mixture toxicity to natural populations that comprise multiple generations. The use of model predictions to assess long-term toxicity of the biocides can have severe consequences because the models do not account for transgenerational changes of effects.
Our results show that simulating more realism in ecotoxicological testing by considering multiple chemicals and generations adds valuable information for predicting the risk chemicals pose in natural systems. They also underline, however, the limitations of the commonly used prediction models for mixture toxicity. We therefore see the need of developing predictive tools that address these topics and thus allow more realistic predictions of mixture toxicity.
The present study attempted to explain the toxicity of a biocide mixture using the concepts of concentration addition and independent action. We concluded that concentration addition was a rather good predictor of lethal toxicity, and independent action of sublethal mixture toxicity in the F0 generation. However, the degradation of the mixture components and the generational changes in sensitivity indicate limitations of the pharmacological concepts. Especially at low and thus environmentally relevant concentrations, time-dependent changes in mixture composition considerably affected the accuracy of model predictions. Transgenerational changes in sensitivity question the applicability of concentration addition and independent action to natural populations comprising multiple generations. While sublethal toxicity predictions made by concentration addition were conservative for the F0 generation, this was not strictly valid for the F1 generation. We conclude, therefore, that more advanced prediction models are needed, which are capable of incorporating dose–dependent changes in mixture composition over time and account for changes of toxic effects on subsequent generations.
The authors thank H. Norli and colleagues from the Norwegian Institute for Agricultural and Environmental Research, Plant Health and Plant Protection Division for running the GC analyses, and H. Bergheim from the Norwegian Institute for Agricultural and Environmental Research, Soil and Environment Division for laboratory assistance.