Journal of Environmental Science and Health Part A (2007) 42, 1–9 Copyright C Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934520601015255 Effects of cadmium and tributyltin on development and reproduction of the non-biting riparius (Diptera)—baseline experiments for future multi-generation studies

CHRISTIAN VOGT, DANIELA BELZ, SIMONE GALLUBA, CARSTEN NOWAK, MATTHIAS OETKEN and JORG¨ OEHLMANN Johann Wolfgang Goethe-University Frankfurt am Main, Department of Aquatic Ecotoxicology, Frankfurt am Main, Germany

In this study the effects of tributyltin (TBT) and cadmium (Cd) were tested on the freshwater (Diptera) in life-cycle experiments. To this end, the OECD guideline 218 was extended with reproduction relevant parameters (e.g. number of fertile egg masses per female). Based on these reproduction data the number of larvae for next generation and the population growth rate were calculated. Experiments were performed using environmentally relevant concentrations of the test substances in the sediment (nominal ranges: 50–200 µg Sn/kg and 0.2–2.1 mg Cd/kg, on a dry weight basis). Quartz sand was used as sediment in order to develop a test system for a following research project, focusing on the effects of TBT and Cd as model stressors on C. riparius populations in multi-generation studies. Both model stressors caused significant effects on development and reproduction of C. riparius. Larval mortality proved to be a sensitive parameter and a clear concentration-response relationship was observed with a significant increase (P < 0.001) at the highest TBT concentration and at a Cd concentration of 1.17 mg Cd/kg dw (P < 0.05). The two highest TBT concentrations emergence was significantly (P < 0.05) delayed compared to the solvent control. For Cd a clear prolonged average main emergence interval was observed, but not for TBT. TBT and Cd inhibited also oviposition. These effects resulted in a concentration-depended reduction of the population growth rate. Keywords: Chironomus riparius,Tributyltin, Cadmium, Life-cycle experiments, Multi-generation studies.

Introduction native field sediments with larvae being exposed to a mix- ture of native and man-made substances.[1−3] Furthermore, Various studies with C. riparius have shown that this or- two guidelines were developed for the routine testing and ganism is suitable for the testing of sediment toxicity in assessment of chemicals, wherein the larvae were exposed the laboratory. The main advantages of chironomids as test to substances via water[4] or via sediment.[5] These guide- species are that they (I) are easy and cheap to culture in lines are well established in ecotoxicological research and the laboratory; (II) spend a large part of their life-cycle standard testing, because various life-cycle parameters can as a larva in the sediment; (III) are model organisms for be observed to detect possible impacts of substances on the a widespread and species-rich taxonomic order in aquatic development and reproduction of chironomids.[6−8] ecosystems; (IV) are sensitive to pollutants; (V) have a short Therefore, we used C. riparius as a model organism for life-cycle with completing 10 or more generations per year aresearch project, in which the relation between pollution under standard conditions and (VI) have a sexual mode of and effects on the genetic diversity of exposed populations reproduction. Thus, C. riparius is becoming more popular is investigated. In this project, effects of model substances as a standard organism for bioassays. Several monitoring like tributyltin (TBT) and cadmium (Cd) on ecotoxicologi- studies were performed for the assessment of the toxicity of cal and genetic endpoints are analysed by performing multi- generation experiments according to the OECD guideline [5] Address correspondence to Christian Vogt, Johann Wolfgang 218. The guideline was extended with reproduction rele- Goethe-University Frankfurt am Main, Department of Aquatic vant parameters such as the number of fertile egg masses per Ecotoxicology, Siesmayerstrasse 70, D-60323 Frankfurt am female, the clutch size and the hatchability of egg masses. Main, Germany. E-mail: [email protected] Furthermore, the sediment described in the OECD guide- Received May 26, 2006. line was modified and a sediment free of organic matter 2 Vogt et al. containing only pure quartz sand was used. This modified into 24-microwell plates for hatching (2 mL reconstituted sediment was selected to avoid fungi or bacterial infections water per cavity). Newly hatched larvae from different egg and to reduce the risk of a possible extinction of the chi- ropes were combined to randomly select 20 and 50 first ronomid population during the multi-generation studies. instar larvae for the TBT and Cd experiments respectively. The aim of the baseline experiments reported here was The larvae were transferred to each test vessel using a to determinate suitable test concentrations for TBT and Pasteur glass pipette and a stereo microscope (Olympus SZ Cd for the following multi-generation studies. In this paper 40, Hamburg, Germany). The aeration was stopped for 24 the concentration-response curve for TBT and Cd in an hinorder to let the larvae dig into the sediment. The larvae extended life-cycle test according to the modified OECD were fed with a ground TetraMin suspension (Tetra GmbH, guideline 218 is shown. Because endpoints determined on Melle, Germany) according to OECD guideline 218. As the individual level have an insufficient power to extrapolate from the 12th experimental day on, the number of dead for population effects of pollutants,[9−11] an integrated ap- larvae and pupae, as well as the number of emerged imag- proach of single parameters was applied, which facilitated ines were counted once a day. Successfully emerged the calculation of the population growth rate. were collected with an exhauster and transferred into a breeding container, which consisted of a glass aquarium (30 × 20 × 20 cm) closed with a stainless steel gaze (mesh Materials and methods size 0.5 mm). Each breeding container contained a plastic dish (11.5 × 11.5 × 5.5 cm), filled with 400 mL of recon- Test organisms stituted water for oviposition. The egg ropes were removed daily out of the breeding container. The number of eggs per All Chironomus riparius larvae used in the experiments were egg mass was counted and each egg mass was placed in a obtained from our in-house mass culture, which was es- tube of a 24-microwell plate. After 3 days the hatchability tablished in 2004 with egg ropes of 11 different labora- was determined. Finally, the number of dead larvae and tory stocks from 7 countries (Bulgaria, Germany, France, dead pupae in the sediment was counted. At the end of the Finland, Great Britain, the Netherlands and the United TBT experiment (at day 32), dead imagines were collected States of America). The culture was maintained in a cli- ◦ ◦ and dried at 36 Cfor 3 days. The dry body weight of each mate room with a temperature of 20 ± 1 C, 70% relative was determined with a sensitive electronic balance humidity and a light:dark rhythm of 16:8 hours. The test (Sartorius 4401, Gottingen,¨ Germany) to the nearest µg. conditions were equal to the culturing conditions. The number of eggs per clutch were recorded, based on the method of Benoit et al.[13] for Chironomus tentans with the modification that the number of spirals per egg mass Experimental procedures wasmultiplied with the number of eggs from the middle In this study, static life-cycle experiments were conducted spiral (ring counting method). Prior to the experiment the according to the OECD Guideline 218.[5] Midges were ex- estimation was evaluated using a calibration curve. For this posed to the test substances via sediment starting with the purpose the number of eggs from 76 clutches was first calcu- first larval stage until emergence. For establishing a suitable lated according to the described method and then the same test design for the following multi-generation studies two egg masses were dissolved by using 40% sulphuric acid and different glass dishes with different numbers of larvae per all eggs from each clutch were counted separately. Very few beaker and sediment:water ratio were used. First, 600 mL egg masses did not show a regular spiral-shape so that their glass beakers (Simax, Czech Republic) containing 100 g egg number could not be estimated with the ring counting quartz sand (grain size 0.1–0.4 mm, Quick Mix Group, Os- method. nabruck,¨ Germany), 400 mL reconstituted water (pH-value 7.9–8.4; conductivity 540 µS/cm)[12] and 20 were Chemicals and residual analyses used for the TBT experiment. Secondly, 2 L crystallising dishes (Schott, Mainz, Germany, ∅ 19 cm) filled with 100 g Prior to the insertion of the larvae, the sediment was spiked quartz sand and 1 L reconstituted water were conducted with tributyltin (TBT-Cl, CAS-number 1461-22-9, Merck, for the cadmium experiment. In this experiment 50 animals Darmstadt Germany, > 97% purity, nominal concentra- per crystallising dish were exposed. Each exposure group tions: 50, 80, 100 and 200 µg Sn/kg dw) or cadmium ∗ consisted of five (TBT) or two (Cd) replicates. Prior to the (CdSO4 8H2O, CAS-number 7790-84-3, Merck, Darm- experiment the vessels were prepared with sediment and stadt Germany, > 99% purity, nominal concentrations: 0.2, water and finally covered with gaze (mesh size 0.75 mm). 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.1 mg Cd/kg dw). Because Moreover, test vessels were gently aerated through Pasteur of the low water solubility of tributyltin, ethanol (Merck glass pipettes, which were attached to an air compressor (40 Schuchardt Chemicals, Darmstadt, Germany, > 99.5% pu- A, Die Pumpe, Holm, Germany) via PVC tubes. Three days rity) was used as solvent and thus a solvent control was before the start of each experiment, freshly laid (≤ 24 hour) included in this experiment. For the comparison of results, egg ropes were taken from the mass culture and transferred ethanol was also used in the cadmium experiment. Spiking Cadmium and TBT effects on midge reproduction 3 of the sediment was performed with a solvent volume of 60% of all midges emerged per gender. Differences in the sex 300 mL ethanol/kg dw and the solvent was evaporated. ratio (expressed as female fraction) were tested with Fisher’s Furthermore, a control group with reconstituted water (300 exact test. For the number of egg ropes (total and fertile) mL/kg dw) was included in the TBT experiment to assess per female no statistical test was possible, because only one potential effects of the solvent. breeding container was used per treatment. The number of The residual analyses for TBT were performed by eggs per egg mass was determined based on clutches that GALAB laboratories Geesthacht according to the DIN were countable with the ring counting method described guideline 19744. The detection limit for TBT was 1 µg/kg before. Non-countable egg masses were neglected for the dw. TBT was analysed after solvent evaporation and after determination of the clutch size. For the calculation of the a test duration of 14 days. Based on the TBT loss after number of larvae for the next generation, it was assumed solvent evaporation (87%) and the measured TBT concen- that egg masses with an observed hatching consisted exclu- tration after 14 days the time-weighted mean concentration sively of fertile eggs. Non-countable egg masses were con- was calculated according to OECD guideline 211.[14] For Cd sidered for that parameter with an assumed value of 500 the residual analyses were performed by the International eggs per egg mass. Graduated School (IHI) Zittau according to DIN EN ISO Furthermore, the population growth rate (PGR) was 17294-2:2005-02. Sediments were analysed after the full test calculated based on the sex ratio, larval mortality, num- duration of approximately 50 days and corrected with the ber of eggs per egg mass (± SD), mean emergence time background concentration of the quartz sand. Again time- of females (± SD), population starting size and the num- weighted mean concentrations were determined according ber of fertile egg masses per female according to Forbes to OECD guideline 211.[14] Because of the chemical prop- and Cold[9] and Sibley et al.[10] The significance level was erties of Cd the spiked nominal concentrations were set set at P < 0.05 and is indicated in the graphs by asterisks for concentration on day 0. For both experiments all ef- (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). fect data are referred to calculated time weighted mean concentrations. Results

Statistical analysis Residual analyses Statistical analyses were done with the software program The calculated time-weighted mean concentrations are GraphPad Prism, Version 4.03 (GraphPad Software Inc., shown in Table 1. For TBT, time-weighted mean concen- San Diego, CA, USA). All data were tested for normality by trations were lower than expected with only approximately the Kolmogorov–Smirnov test. Normally distributed data 6% of the nominal concentrations. In contrast to this, for were checked for significant differences using one-way anal- Cd time-weighted mean concentrations nearly represented ysis of variances (ANOVA) followed by Dunnett’s post hoc test, otherwise the Kruskal–Wallis test followed by Dunns post hoc was used. For the cadmium experiment the num- Table 1. Time-weighted mean concentration for the TBT and the [14] ber of replicates was to low to perform a normality test Cd experiment according to OECD Guideline 211 and thus data were analysed with an one-way analysis of Nominal Time-weighted mean variances (ANOVA). Based on the time and the number of concentration concentration emerged imagines the mean emergence time (EmT50)was calculated for each gender and replicate. This parameter Tributyltin [µg Sn/kg dw] could be determined only for replicates with a mortality Control 0 below 80%, because only in those cases meaningful values Solvent control 0 50 2.79 were generated. For calculating the EmT it was neces- 50 80 4.46 sary to transform the data. The natural logarithm of time 100 5.58 (in days) was taken and the number of emerged midges was 200 11.2 cumulated and normalised to percentages for each replicate Cadmium [mg/kg dw] and gender. The normalisation was performed to correct for Solvent control 0 non-substance mediated differences in emerged midges be- 0.20 0.17 tween replicates within a treatment. After that, non-linear 0.50 0.39 regression was performed using the logistic curve that con- 0.80 0.61 1.00 0.78 tains the EmT50 as a parameter. 1.20 0.76 Furthermore, the time when 20% (EmT20) and 80% (EmT )ofthe midges have been emerged was calculated for 1.50 0.91 80 1.80 1.39 each gender and replicate. Those two parameters were used 2.10 1.17 to determine the average main emergence interval, wherein 4 Vogt et al. the chosen nominal concentrations with recoveries between 55% (2.1 mg Cd/kg dw) and 85% (0.2 mg Cd/kg dw).

Validation criteria of the experiments All experiments fulfilled the validity criteria for mortality and emergence according to OECD guideline 218.[5] The observed mortality in the control groups was always below 30% and emergence occurred between day 12 and 23. The pH values, which were measured at the start and the end of the experiment, ranged from 5.5 to 8.4, temperature was 20 ± 1◦C, conductivity was 540 µS/cm at the start of the experiment, and dissolved oxygen level was always above 60% of air saturation volume.

Potential solvent effects and test design To assess the impact of the solvent ethanol on develop- ment and reproduction of C. riparius a control with re- constituted water was included in the TBT experiment. No significant differences between the solvent control and the control group were observed. Furthermore, two dif- ferent test designs, with different numbers of larvae per beaker and sediment:water ratio were tested for possible impacts on development and reproduction. No significant differences between the endpoints of both test designs were determined.

Mortality and sex ratio Fig. 1. Mean mortality (± SD, in %) of Chironomus riparius ex- Figure 1 shows the mean mortality of C. riparius for the posed to tributyltin (A) and cadmium (B). Significant differences TBT (Fig. 1A) and the Cd (Fig. 1B) experiment. The expo- are calculated with one-way analysis of variances (ANOVA, Dun- sure to both substances resulted in a clear concentration- nett’s post hoc test, nTBT = 4–5, nCd = 1–2). Asterisks indicate sig- ∗ ∗∗∗ response curve and a significant correlation was calculated nificant differences to solvent control ( P < 0.05, P < 0.001). = = (Pearson correlation, P < 0.05). A significantly (P < 0.001) C control, SC solvent control (ethanol). LC50 are calculated using non-linear regression (sigmoidal dose-response curve, vari- enhanced larval mortality was observed at the highest TBT = µ ± µ able slope, solid line). LC50 TBT 8.27 g Sn/kg dw ( 95% CI concentration (11.2 g Sn/kg dw) with a mean of 75%. µ = = µ ± 6.56–10.4 g Sn/kg dw, n 22) and LC50 Cd 0.85 mg/kg dw Hence, a LC50 value of 8.27 g Sn/kg dw ( 95% CI, (± 95% CI 0.62–1.18 mg/kg dw, n = 16). 6.56–10.4 µg Sn/kg dw) was calculated. Increasing Cd concentrations in the sediment led to an EmT50 and main emergence interval increasing larval mortality of C. riparius (Fig. 1B) and a significantly increased (P < 0.05) mortality was determined Figure 2 shows the effect of the test substances on the at aCdconcentration of 1.17 mg Cd/kg dw with a mean EmT50 of both gender. In all treatments females emerged mortality of 79%. One exception of this clear concentration- later than males. Furthermore, both substances affected response curve was observed at the highest Cd concentra- males and females similarly, since EmT50 values were highly tion, whereby the mortality was 52%. The LC50 value for correlated (Pearson correlation, P < 0.001). For TBT, a Cd was calculated with 0.85 mg Cd/kg dw ( ± 95% CI, concentration-response curve was detected (Fig. 2A) with 0.62–1.17 mg Cd/kg dw). No remarkable number of dead a significant delay in emergence of both gender at the two fourth instar larvae and pupae mortalities were observed. highest concentrations (P < 0.05, P < 0.01). The highest The maximum was detected at the 1.17 mg Cd/kg dw treat- EmT50 of males with 30 days and of females with 32 days ment with 6% in sum. The emergence success of pupae of was observed at the highest TBT concentration. For the both gender was not affected by exposure. main emergence interval no significant effects of the TBT In the experiments no effect of the test substances were treatment were observed (Fig. 3A). In the solvent control detected on the sex ratio (expressed as female fraction). This animals emerged during a period of approximately 5 days. parameter ranged between 0.38 (0.76 and 1.17 mg Cd/kg A similar main emergence interval was observed under TBT dw) and 0.57 (SC of the Cd experiment). exposure. Cadmium and TBT effects on midge reproduction 5

Fig. 2. Mean emergence time (EmT50 ± SEM, in days) of Chirono- mus riparius males (closed circles) and females (open squares) ex- posed to tributyltin (A) and cadmium (B). Significant differences Fig. 3. Average main emergence interval ( ± SEM, in days) of are calculated using one-way analysis of variances (ANOVA, Chironomus riparius males (closed circles) and females (open Dunnett’s post hoc test, nTBT = 4–5, nCd = 1–2) compared to squares) exposed to tributyltin (A) and cadmium (B). Signifi- the solvent control (∗P < 0.05, ∗∗P < 0.01). C = control, SC = cant differences are calculated using one-way analysis of variances solvent control (ethanol). Values with index 1 represent one repli- (ANOVA, Dunnett’s post hoc test, nTBT = 4–5, nCd = 1–2) com- cate due to mortality >80% in the other replicate. pared to the solvent control (P > 0.05). C = control, SC = solvent control (ethanol). Values with index 1 represent one replicate, due to mortality >80% in the other replicate. Furthermore, no differences between males and females were observed for this parameter. For Cd, the calculated the solvent control (ca. 5 days). Males showed this prolon- EmT50 and the main emergence interval were rather vari- able due to the lower statistical power (2 replicates per con- gation of emergence as well and notable differences were centration). At 0.91 and 1.17 mg Cd/kg dw, the calculated observed at 0.78, 0.91 and 1.17 mg Cd/kg dw. EmT50 and the main emergence interval were based on only one replicate, due to the high mortality in the other repli- Reproduction cate (> 80%). Cadmium significantly delayed the EmT50 of both gender in a concentration-depended manner (Fig. 2B, In both experiments the number of egg masses (total and Pearson correlation, P < 0.05). The maximum effect was fertile) per female and the number of eggs per egg mass observed at 0.76 mg Cd/kg dw, whereby the EmT50 was were assessed (Table 2). In the 2.79 µg Sn/kg dw treatment 36 days for males and 40 days for females. In this group of the TBT experiment and in the SC of the Cd experiment emergence was delayed by 60% when compared with the females laid more than 1.0 egg mass per female with 1.03 solvent control. Furthermore, significant effects of the Cd and 1.04, respectively. exposure were detected for the main emergence interval For the TBT experiment an inhibition of reproduction (Fig. 3B), whereby a positive correlation was calculated for was detected at the two highest concentrations. At the high- females (Pearson correlation, P < 0.05). Thus, increasing est TBT concentration only one egg mass was produced, concentrations resulted in longer main emergence intervals, resulting in a reduced reproduction with 0.13 egg masses although the maximum value for females was detected al- per female. Hatchability of the egg masses was not affected ready at 0.76 mg Cd/kg dw. At this concentration the main and ranged between 74% (5.58 µg Sn/kg dw) and 100% emergence interval (ca. 10 days) was 2 times longer than in (11.2 µg Sn/kg dw). Cadmium also tended to result in an 6 Vogt et al.

Table 2. Egg-masses per female (total and fertile), eggs per egg-mass, number of non-countable egg-masses (total and fertile) and estimated number of larvae for the next generation for the tributyltin and cadmium experiment

Egg-masses/female Treatment [time weighted Eggs/egg-mass (median with Number of non-countable Estimated number of mean-concentrations] Total Fertile 25/75% percentile; n) egg-masses (total and fertile) larvae for next generation

Tributyltin [µg/kg dw] Control 0.80 0.68 504 (218/593; 25) 5/2 11,246 Solvent control 0.90 0.80 518 (300/608; 28) 6/2 14,068 2.79 1.03 1.00 530 (263/630; 26) 8/7 15,478 4.46 0.80 0.60 455 (340/545; 13) 9/5 8,138 5.58 0.65 0.48 418 (320/495; 14) 4/2 6,814 11.2 0.13 0.13 495 (−/−; 1)0 495 Cadmium [mg/kg dw] Solvent control 1.04 0.89 678 (457/819; 42) 7/6 26,606 0.17 0.96 0.84 605 (361/693; 40)∗∗ 7/4 21,643 0.39 0.96 0.77 625 (329/748; 24) 1/0 13,167 0.61 0.92 0.81 586 (373/661; 24)∗ 0 10,777 0.76 0.40 0.33 551 (351/645; 6)0 2,537 0.78 0.75 0.63 595 (442/656; 11)∗ 1/0 5,225 0.91 0.83 0.78 534 (240/635; 14)∗∗ 1/1 7,176 1.17 0.63 0.25 512 (434/638; 5)0 1,209 1.39 0.73 0.55 418 (382/570; 16)∗ 0 6,008

Concentrations are expressed as time-weighted averages, significant differences for eggs per egg mass were calculated using Kruskal–Wallis test (followed by Dunns post hoc test, ∗P < 0.05, ∗∗P < 0.01). inhibited reproduction at 0.76, 1.17 and 1.39 mg Cd/kg dw tively. However, due to the high linear regression coefficient in a non-monotonic concentration response relationship, (r2 = 0.84) this effect was neglected. For TBT, no effects on because reproduction was not affected in the 0.78 and 0.91 the number of eggs per clutch were observed (Table 2). In mg Cd/kg dw treatment. The hatchability of egg masses was contrast, Cd exposure led to a decreased clutch size. Thus, also disturbed. At 1.17 mg Cd/kg dw only 40% of clutches for 0.17, 0.61, 0.78, 0.91 and 1.39 mg Cd/kg dw treatments were fertile in comparison to 85% in the SC. a significantly (P < 0.05, P < 0.01) lower number of eggs Furthermore, the number of eggs per clutch was esti- per clutch were found in comparison to the control group, mated based on the countable egg masses. The results of whereas concentration response relations were often non- the calibration curve are shown in Figure 4. The number monotonic. Furthermore, the numbers of non-countable of estimated eggs per clutch was highly correlated with the egg masses (total and fertile) are shown in Table 2. Such counted number of eggs per clutch (Spearman correlation, clutches were detected in the controls as well as in most P < 0.001). The graph shows that the determination pro- treatments of both substances. cedure tends to result in an overestimation of small egg Beside these reproduction parameters the number of lar- masses and an underestimation of large egg masses, respec- vaefor the next generation was calculated. For TBT, the numbers were 11,246 and 14,068 for the control and the solvent control, respectively. A low concentration of TBT promoted the number of larvae for next generation and at 2.79 µg Sn/kg dw 15,478 larvae were produced. In con- trast, at the three highest concentrations a clear inhibition in a concentration dependent manner was observed. In the Cd experiment higher numbers of larvae for the next gen- eration were produced by the control group (26,606) than in the TBT experiment. With increasing Cd concentrations the number of larvae decreased significantly (Pearson cor- relation, P < 0.01) and the minimum was observed at a concentration of 1.17 mg Cd/kg dw with 1,209 animals.

Dry body weight of imagines Fig. 4. Chironomus riparius—counted and estimated number of eggs per clutch (Spearman correlation P < 0.001, n = 76, dotted In the TBT experiment the dry body weights of dead imag- line indicates linear regression with slope 1). ines were measured. The weights of males were normally Cadmium and TBT effects on midge reproduction 7 distributed following a Gaussian distribution, while females exposure did not reduce the PGR. This disturbance was body weights were not. Male imagines from the control also observed for some other endpoints (e.g., mortality and group had the highest average dry body weights with 0.404 EmT50). mg, whereas male imagines from the highest treated group were significantly (P < 0.05) lighter (0.335 mg) than solvent control males. The female median dry body weights varied Discussion between 0.382 (5.58 µg/kg dw) and 0.606 (11.2 µg/kg dw) mg and no significant effect of TBT exposure was detected In this study effects of tributyltin and cadmium on the de- regarding this endpoint. velopment and reproduction of Chironomus riparius were investigated. The experiments were performed as prelimi- nary tests for a following multi-generation study, in which Population growth rate λ all the validation criteria of the OECD guideline 218[5] were The calculated population growth rate (PGR) is shown in kept. That indicates that the sediment free of organic mat- Figure 5. This integrated parameter gradually declined dur- ter had no inhibiting effect on growth and development of ing both experiments with increasing TBT and Cd concen- the midges. Furthermore, none of the parameters showed trations, whereas a significant concentration-response rela- a significant difference between the solvent control and the tionship was observed (Pearson correlation, P < 0.05). The control group, thus an effect of ethanol on C. riparius under highest population growth rates were detected in the solvent the applied scheme can be excluded. The experiments were controls of both experiments with 1.22 per day for TBT also performed to establish a suitable test design for the fol- and 1.26 per day for Cd. In comparison, the highest TBT lowing multi-generation studies in order to find an optimal concentration led to a PGR of 1.06 per day. For cadmium number of larvae per beaker and an optimal sediment:water the lowest PGR was observed at 1.17 mg Cd/kg dw with ratio. Both test designs indicated similar results and none 1.06 per day. There was a disturbed concentration-response of the investigated endpoints were significantly different for in the highest Cd treatment, because a further increasing the solvent control group. Thus, the crystallising dishes (50 larvae per beaker) will be chosen for the multi-generation studies, because a larger population size can be maintained in the experiments. The results indicate that both test substances, trib- utyltin and cadmium, are able to inhibit the development and reproduction of C. riparius.For both experiments a nearly complete concentration-response range was de- scribed, whereas sublethal and acute toxicity were observed. Apparently, TBT had a higher toxicological potential than Cd, since the latter showed a 100-fold higher LC50 value than TBT (expressed on Sn as basis). This difference was expected, because TBT was designed as an effective biocide used in anti-fouling agents for boat paints and wood preser- vative. Other studies with Chironomids have shown clearly that TBT exhibits acute toxicity at the low microgram- per-litre-range.[15,16] In contrast, Cd was less toxic with a concentration-response range between 0.17 and 1.39 mg Cd/kg dw. For Cd LC50 values are reported for exposed sediments in a range from 1.64 to 39 mg Cd/kg dw, de- pending on the concentration of organic matter in the sed- [17] iment. Milani et al. calculated a LC50 of 39 mg/kg dw in a study with C. riparius larvae exposed to native sed- iment containing 3% organic matter. Furthermore, in an experiment with Cd-exposed midges using artificial sedi- ments according to OECD guideline 218 with 5% organic [18] matter, a LC50 value of 1.64 mg Cd/kg was found. In a third study performed by Watts and Pascoe[19] with arti- ficial sediments containing 15% organic matter, mortality wasabout 13% at a concentration of 2 mg Cd/kg dw. These Fig. 5. Population growth rate [d−1]ofChironomus riparius ex- investigations show that different exposure designs lead to posed to tributyltin (A) and cadmium (B). Error bars represent different mortality levels. The present study also indicates standard deviation. C = control, SC = solvent control (ethanol). that the most sensitive developmental stages are the first 8 Vogt et al. to third larval instar, because no increased mortality was The sex ratio of imagines did not deviate from the ex- observed for the fourth larval stage and the pupa phase pected 1:1 relationship in both experiments. This result was for both experiments. Higher mortalities at higher concen- confirmed by Watts and Pascoe[19] for C. riparius in a Cd trations must have occurred between first and third larval experiment. In this investigation, the sex ratio tended not to instar. be a sensitive indicator for pollutant stress for this species. Furthermore, the mean emergence time for both gen- Furthermore, the dry body weights of imagines were der was calculated. For the solvent control of both exper- measured in the TBT experiment, whereby males were sig- iments a similar mean emergence time for both sexes was nificantly (P < 0.001) lighter than females. This might be determined, while males emerged on day 20 and females due to the egg laying of females, because not all females on day 24. Generally, males tended to emerged earlier than oviposited and thus some of them still have some eggs in females. This phenomenon, called protandrous, represents the ovipositor after dying. The dry body weights of imagines the usual emergence behaviour[20] and was reported in var- were only affected at the highest TBT concentration with ious studies.[21,22] Clear effects on larval development were significantly (P < 0.05) reduced weights of males. Concern- determined at TBT treatments of 5.58 and 11.2 µg Sn/kg ing the chironomid larvae, weights also decline in contami- dw,whereas the exposure led to a significantly delayed emer- nated field sediments [25] and after cadmium exposure.[19,21] gence of both gender. This developmental inhibition was Conversely results were reported for the dry body weight of also observed in the Cd experiment. In contrast to the TBT C. riparius imagines after exposure to 14C-cypermethrin[24] experiment no significant differences were calculated for and Cd.[22] Thus, this parameter indicates no clear sub- EmT50 due to less statistic power of only two replicates. stance effect and must be discussed critically. This inhibition of larval development, and thus a delayed As an integrated measure for the determination of popu- emergence of the imagines, was observed for cadmium ex- lation relevant parameters the population growth rate was posure in several investigations.[17,21−23] calculated based on single parameters according to Forbes Furthermore, differences between both test substances and Cold[9] and Sibley et al.[10] Thus, conclusions for pos- regarding the main emergence interval were detected. For sible effects on the population level can be drawn. For the TBT, the animals emerged later, but no prolongation of the solvent controls population growth rates of 1.22 [d-1] and mean emergence period was detected. Thus, the decreased 1.26 [d-1] were calculated for TBT and Cd, respectively, and reproduction at higher treatments was not caused by this thus the population grows with an increase of 22% (TBT) fact. In contrast, Cd exposure led to a later EmT50 as well and 26% (Cd) per day. A similar population growth rate as to longer main emergence intervals. Animals emerged of 1.28 [d-1] was reported by Lopes et al.[11] for C. riparius during a longer period and an indirect substance mediated under control conditions using the DEBtox model. TBT effect on reproduction caused by smaller swarm size could promoted the PGR at a low concentration, while higher be assumed. concentrations led to a decreasing PGR. In the present study, the OECD guideline 218 was ex- For both substances a clear concentration-response curve tended with reproduction-related endpoints. Both sub- was determined, with the PGR declining significantly (Pear- stances tended to inhibit reproduction at higher concen- son correlation, P < 0.05) with increasing concentrations. trations. This phenomenon was also observed by Pedina[18] Similar results on population relevant parameters were also in a comparable study, in which clutch size declined from reported for various pesticides.[9,11,24] However, the PGR approximately 330 (control) to 200 eggs per clutch at a con- wasabove 1.0 [d-1] in both experiments and in spite of centration of 3 mg Cd/kg dw. In contrast, TBT showed high-stress conditions the population still grows. An expla- no effect on the number of eggs per clutch. Moreover, Cd nation for that might be the high number of eggs per egg exposure resulted in a lower hatchability of egg masses at mass for which the highest TBT treatment is a good exam- 1.17 mg Cd/kg dw, whereby 40% of the egg masses were fer- ple. In this treatment only one fertile clutch, containing 495 tile. For TBT, no effects on the hatchability of egg masses single eggs was produced. Thus, the next generation will be were observed. Similar results on the hatching success of founded from 495 single individuals in comparison to the the egg ropes were observed in experiments with C. ri- first generation used in the experiment, which started with parius at different larval densities and 14C-cypermethrin 100 first instar larvae. Presumably the chosen concentra- exposure.[24] For the number of non-countable egg masses tions are too low to induce a definitive extinction although with the ring counting method, no substance mediated ef- the risk of extinction clearly increases. fects were found, because such egg masses occurred also in the control and solvent controls. The number of offspring is important for the survival of populations. This param- Conclusion eter was expressed as larvae for the next generation and a decrease with increasing concentrations was determined In the present study baseline experiments were performed for both substances. Thus, populations face a higher risk of to determine the concentration-response of two substances extinction with increasing concentrations of TBT and Cd. (tributyltin and cadmium) for following multi-generation Cadmium and TBT effects on midge reproduction 9 studies with C. riparius.Tothis end, both substances were importance of exposure scenario. Environ. Toxicol. Chem. 2005, 24, investigated for the toxicological potential on the sediment 78–86. dwelling organism according to the OECD guideline 218. [10] Sibley, P.K.; Benoit, D.A.; Ankley, G.T. The significance of growth in Chironomus tentans sediment toxicity test: Relationship to repro- The OECD guideline was extended with reproduction rele- duction and demographic endpoints. Environ. Toxicol. Chem. 1997, vant parameters to calculate the population growth rate as 16, 336–345. an integrated measure. Both substances are able to interfere [11] Lopes, C.; Pery, A.R.R.; Chaumot, A.; Charles, S. Ecotoxicology with the development and reproduction of C. riparius at the and population dynamics: Using DEBtox models in a Leslie mod- chosen test design in a clear concentration related manner. eling approach. Ecol. Model. 2005, 188, 30–40. [12] Duft, M.; Schulte-Oehlmann, U.; Tillmann, M.; Markert, B.; This facilitates the selection of appropriate concentrations Oehlmann, J. Toxicity of triphenyltin and tributyltin to the freshwa- for the following multi-generation studies. ter mudsnail Potamopyrgus antipodarum in a new sediment biotest. Environ. Toxicol. Chem. 2003, 22, 145–152. [13] Benoit, D.A.; Sibley, P.K.; Juenemann, J.L.; Ankley, G.T. Chirono- Acknowledgments mus tentans life-cycle test: design and evaluation for use in assessing toxicity of contaminated sediments. Environ. Toxicol. Chem. 1997, We would like to thank Udo Hommen (IME Fraunhofer 16, 1165–1176. Institute Schmallenberg) for the development of the popu- [14] Organisation for Economic Development and Cooperation. Guide- line for Testing Chemicals No. 211. Daphnia Magna Reproduction lation growth model and Claudia Schmitt for the English re- Test, adopted September, 1998, Paris, France. vision. This research has been supported by the programme [15] Hahn, T.; Schultz, R. Ecdysteroid synthesis and imaginal disc de- “Environmental Quality and its Security and Sustainment” velopment in the midge Chironomus riparius as biomarkers for en- (BW-Plus) with funding of the state Baden-Wurttemberg¨ docrine effects of tributyltin. Environ. Toxicol. Chem. 2002, 21, (project number BWR 22018). 1052–1057. [16] Fargasova, A. Comparison of tributyltin compound effects on the alga Scenedesmus quadricauda and the benthic organisms Tubifex tubifex and .Exotoxicol. Environ. Saf. 1998, References 41, 222–230. [17] Milani, D.; Reynoldson, T.B.; Borgmann, U.; Kolasa, J. The relative [1] DeLange, H.J.; DeHaas, E.M.; Mass, H.; Peeters, E.H.T.M. 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