Growth retardation and altered isotope composition as delayed effects of PCB exposure in Daphnia magna

Transcription

1 Supporting Information Growth retardation and altered isotope composition as delayed effects of PCB exposure in Daphnia magna Caroline Ek a, *, Zandra Gerdes a, Andrius Garbaras b, Margaretha Adolfsson-Erici a and Elena Gorokhova a a Department of Environmental Science and Analytical Chemistry, Stockholm University, Svante Arrhenius väg 8, SE Stockholm, Sweden b Mass Spectrometry Laboratory, Center for Physical Science and Technology, Savanoriu 231, LT Vilnius, Lithuania *Corresponding author: Caroline Ek a Department of Environmental Science and Analytical Chemistry, Stockholm University, Svante Arrhenius väg 8, SE Stockholm, Sweden caroline.ek@aces.su.se Contents: 13 pages, 8 Tables, 1 Figure S1

2 Table S1. Contribution of individual PCB congeners to the spiked Spirulina powder on a mass fraction basis. PCB18 PCB40 PCB128 PCB209 Σ Contribution S2

3 Table S2. PCB internal concentration at the end of the accumulation phase (juveniles, day 3) and depuration phase (egg-bearing adults, day 7); the congener-specific and total values are shown as mean (SD). a Accumulation phase PCB18 PCB40 PCB128 PCB209 Total PCB mean (SD) 3.25 (0.46) 6.52 (0.86) 3.23 (0.64) 0.27 (0.54) (6.33) Depuration phase mean (SD) 0.19 (0.04) 0.51 (0.15) 0.53 (0.14) 0.09 (0.05) 1.32 (0.29) a Concentrations are expressed in µg PCB g DW Daphnia -1. S3

4 Table S3. Table of summarized data for the accumulation (juveniles) and depuration (adults) phase, values are shown as mean and SE. a Start Control PCB %C %N DW δ 15 N δ 13 C BL Fecundity Neonates Mean SE Juveniles Mean SE Adults Mean SE Juveniles Mean SE Adults Mean SE a %C, carbon content; %N, nitrogen content; DW, dry weight; BL, total body length; fecundity, number of eggs and embryos per female. S4

5 Quality Assurance for PCB analysis The limit of quantification was defined as a peak height 5 the chromatographic noise, estimated to be µg g -1 DW Daphnia and algae -1. In the PCB analyses of Spirulina powder and Daphnia magna, blanks were included in all runs and showed no peaks. Surrogate standards were used and accounted for in the results of the samples. The recovery was 65±19% for 13 C-PCB128 and 102±32% for 13 C-PCB209. A series (n=10) of spiked daphnids were also analysed with the following recovery values: PCB18: 81±9.1%, PCB40: 82±7.9%, PCB128: 72±14%, and PCB209: 59±12%. The extraction efficiency was estimated to be satisfactory. Table S4. Number of PCB samples analysed for PCB-spiked Spirulina powder prior to the experiment and juvenile and adult D. magna from the control and exposed group during the experiment. a Control PCB Spirulina Juveniles Adults 0 16 a PCB analyses were done after the accumulation phase and after the depuration phase, separately. For each occasion, 1 solvent control and 1 blank was analysed together with test samples. Samples were pooled by beaker (7-10 ind. sample -1 ). S5

6 Quality Assurance for stable isotope ratio analysis and elemental composition The analytical accuracy for the stable isotope ratio analysis was derived using a laboratory standard (EMA P2, true value = relative to IEAE-600). For analytical precision of δ 13 C and δ 15 N, fine-grained fish muscle (Esox luscious) originating from a single individual was used as an internal standard. For elemental composition (%C and %N), the internal standard was a set of samples, each containing a single individual of Daphnia magna collected from a culture (size µg). Results are presented in Table S5. Table S5. The analytical accuracy (EMA P2; δ 13 C) and analytical precision (fish muscle and zooplankton; δ 13 C, δ 15 N, %C and %N) for the stable isotope analysis. Values are presented as mean (SD). a n δ 13 C δ 15 N %C %N Analytical accuracy EMA-P Analytical precision Esox lucius (0.1) 12.1 (0.1) Daphnia magna (0.009) 8.7 (0.002) a The analytical accuracy was calculated as the difference between the average test result for a series of measurements and its true value. Analytical precision was calculated as the standard deviation of a series of measurements. Values are expressed in for δ 13 C and δ 15 N; and in % for %C and %N. S6

7 Table S6. Total number of samples analysed for stable isotopes ratios (δ 15 N and δ 13 C) during the experiment for D. magna and the diet. a Control PCB Diet Neonates 3 P. subcapitata 6 (4)* Juveniles 5 (4)* 5 Spirulina powder 2 Adults a Daphnia samples were taken at the start (neonates), after the accumulation phase (juveniles) and the depuration phase (adults); see Fig. 2 in the paper. Diet isotope samples were taken for P. subcapitata (double samples) each new feed and for freeze-dried Spirulina powder once at the end (double samples). * Outlier detected for δ 15 N, new sample size for δ 15 N in brackets. S7

8 Results of the immobilization and feeding inhibition tests Task. To establish the exposure concentration that allowed for measurable PCB body burden while not generating high mortality ( 10 %), a Daphnia sp. Acute Immobilisation Test (OECD 2004) was performed. In addition to mortality, effects on feeding were assessed using a post-exposure feeding inhibition test. The tested dilution series (0-32 µg PCB tot L -1 ) was based on reported body concentrations for Daphnia pulicaria exposed to PCB54 (Lynn et al. 2007). Method. Daphnids (<24h, 10 ind. replicate -1, 3 replicates per concentration) were incubated in 500 ml M7 media and fed PCB-spiked Spirulina powder and P. subcapitata at saturation level (8 µg C ml -1 ) for 24 h. Five individuals per replicate were used for PCB analysis and the remaining five were used in the post-exposure feeding inhibition test. In the latter test, 5 individuals per replicate were transferred to glass beakers with 50 ml M7 and 1.5 µg C ml -1 P. subcapitata and incubated during 24 h. Incubation units were kept at 22 C in darkness to limit algal growth. The feeding rate was calculated as the reduction rate of the algal concentration measured with a 10AU Field Fluorometer (Turner designs, Sunnyvale, California, US). Results and conclusions. Concentration 3.2 µg PCB L -1 resulted in 10% mortality and a body burden sufficient for quantitative PCB analysis. Moreover, none of the tested concentration affected the feeding rate compared to control, thus 3.2 µg PCB L -1 was chosen as a target exposure concentration. Results are presented in Table S7. S8

9 Table S7. Total PCB body burden and DW of juvenile D. magna after 48 h exposure to a PCB gradient (0-32 µg PCBtot L -1 ), mortality observed in the Acute Immobilisation Test, and the post-exposure feeding rate. Values are shown as mean (SD). a PCB tot concentration DW ΣPCB Mortality Feeding rate (0.11) NF 0% (0) 0.71 (0.01) (0.07) 1.9 (0.65) 3% (5) 0.77 (0.02) (0.05) 5.37 (0.01) 10% (9) 0.74 (0.03) (0.10) 6.15 (1.46) 10% (0) 0.79 (0.01) (0.07) 9.58 (1.53) 10% (0) 0.74 (0.01) (0.06) (17.77) 20% (8) 0.72 (0.01) a DW, dry weight in mg; body burden in µg PCB g -1 DW; feeding rate, measured as individual removal rate of carbon µg C h -1 ; NF, not found. S9

10 Length-weight relationship for D. magna As only BL values were available for the animals used for PCB measurements, it was necessary to obtain DW estimates that could be used for the calculation of the PCB concentrations using the body burden (total amount of the PCBs per individual) and body mass. For transformations of body length (BL, mm) to dry weight (DW, µg), a culturespecific length-weight regression (BL-DW regression) was established. By using compiled measurements of daphnid BL and DW from the culture as well as from previous experiments with similar growth conditions (only control groups), we created a data set consisting of 99 samples (1-40 ind. sample -1 depending on individual size) in the age range of 0-11 days. Individual BL (from the eye to the base of the apical spine) was measured using images taken with CanoScan 8800F 13.0 and analysed using either ImageJ version 1.48 (± 0.05 mm) or Vermessung, an in-house software created by Thomas Preuss (± 0.05 mm). For small individuals the samples were pooled. All samples were dried at 60 C for 24 h and weighed using a Sartorius M5P electronic microbalance to obtain DW (± 7 µg). The data was analysed using R version ( ). The power function DW= was fit to the data (see Table S8) using nonlinear least square regression, package stats version Residual standard error was on 97 degrees of freedom. To account for the observed fecundity on day 7, we derived the DW based on the BL and corrected for the reproductive DW by adding the DW calculated from mean fecundity average embryo DW (assumed to be equal the neonate DW). This method was validated by subtracting egg mass from the observed DW of the egg-bearing females on day 7 and evaluating the position on the biplot relative to the BL-DW regression line (see Figure S1). Table S8. Nonlinear least square regression for =. a variable estimate SE t p value DW a b a DW, dry weight (µg); BL, body length (mm). S10

11 Figure S1. Established clone-specific length-weight regression that was used for transforming BL to DW in Daphnia magna. The power function DW= (red dotted line) was fit to the data compiled from culture and experimental measurements (observed in culture and controls, black dots, n=99). The observed DW-BL relationships was plotted (orange dots, n=5) to evaluate the fit of the function to the current observed data. Corrections for egg mass in the observed DW was verified by subtracting the egg/embryo DW from the individual DW (corrected DW-BL, purple dots that represent only somatic DW) and evaluating the fit on the regression line. The total biomass used for calculating PCB concentrations in the experimental S11

12 animals was then calculated as the sum of the somatic DW value and the reproductive DW (treatment-specific fecundity neonate DW). S12

13 REFRENCES 1. Lynn, S. G.; Price, D. J.; Birge, W. J.; Kilham, S. S. Effect of nutrient availability on the uptake of PCB congener 2, 2, 6, 6 -tetrachlorobiphenyl by a diatom (Stephanodiscus minutulus) and transfer to a zooplankton (Daphnia pulicaria). Aquat. Toxicol. 2007, 83 (1), OECD. OECD 202 Guideline for testing of chemicals. Daphnia sp., acute immobilisation test S13