The bioaccumulation of estrone by Daphnia magna was determined. Direct uptake via the aqueous medium occurred within the first 16 hours. A bioconcentration factor of  228 was established over all temporal periods. Ingestion via Chlorella vulgaris gave a partitioning factor of 24 which may approximate to a biomagnification factor assuming steady state conditions. These preliminary results indicate that the partitioning to Daphnia magna via the food source, Chorella vulgaris is less significant than bioconcentration

Birkett, JW

Deacon, HE

Gomes, RL

Lai, KM

Lester, JN

Scrimshaw, MD

Environmental Toxicology and Chemistry

English

Brunel University Research Archive

105Environmental Toxicology and Chemistry, Vol. 23, No. 1, pp. 105–108, 2004q 2004 SETACPrinted in the USA0730-7268/04 $12.00 1 .00Short CommunicationAN ASSESSMENT OF THE BIOACCUMULATION OF ESTRONE IN DAPHNIA MAGNARACHEL L. GOMES,† HANNAH E. DEACON,† KA M. LAI,‡ JASON W. BIRKETT,† MARK D. SCRIMSHAW,† andJOHN N. LESTER*††Environmental Processes and Water Technology Research Group, Department of Environmental Science and Technology, Imperial CollegeLondon, Exhibition Road, London SW7 2BP, United Kingdom‡Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115, USA(Received 12 December 2002; Accepted 18 May 2003)Abstract—The bioaccumulation of estrone by Daphnia magna was determined. Direct uptake via the aqueous medium occurredwithin the first 16 h. A bioconcentration factor of 228 was established over all temporal periods. Ingestion via Chlorella vulgarisgave a partitioning factor of 24, which may approximate to a biomagnification factor assuming steady state conditions. Thesepreliminary results indicate that the partitioning to Daphnia magna via the food source, C. vulgaris is less significant thanbioconcentration.Keywords—Estrone Daphnia magna Chlorella vulgaris Bioconcentration BiomagnificationINTRODUCTIONSteroid estrogens have been identified as the key chemicalsresponsible for endocrine disruption in fish and are prioritypollutants in the United Kingdom aquatic environment [1,2].Current studies have been limited to fish and the accumulationof estrogens in organisms at the lower trophic levels has notbeen assessed. Invertebrates constitute 95% of the faunal spe-cies and are key components of all ecosystems. However, rel-atively little is known about their endocrine system or theirsusceptibility to environmental endocrine disruption. The pau-city of research into the impact of endocrine disrupting sub-stances on invertebrates has recently been highlighted [3,4].The determination of the bioaccumulation properties of allestrogens is essential to fully assess their impact and risk inthe aquatic environment [5,6].The presence of estrone (E1), 17b-estradiol, estriol and17a-ethinylestradiol in the aqueous environment have beenattributed to incomplete removal from sewage treatment works[7,8]. Interconversion between the steroids E1 and 17b-estra-diol occurs, favoring E1, and partly explains why concentra-tions of E1 are generally the greatest of all steroids in aqueoussamples [9]. Levels of E1 in sewage effluents have been de-tected up to 0.220 mg/L, though are typically in the range of0.005 to 0.02 mg/L [10]. Dilution, sorption, and biodegradationprocesses in surface waters decrease these concentrations tothe low ng/L range. E1 has been detected up to 0.014 mg/L[10] in surface waters and at 11.8 mg/kg in sediment [11].The uptake and accumulation of steroid estrogens by thealgae Chlorella vulgaris have been studied and a bioconcen-tration factor (BCF) of 27 established for E1 over 48 h underlight and dark conditions [12]. Daphnia magna is a primaryconsumer of algae and represents the subsequent step in thefood chain. The assessment of bioaccumulation through boththe direct route (via the aqueous medium) and via the trophicroute will determine the significance of E1 biomagnificationfor D. magna and indicate the level of exposure.* To whom correspondence may be addressed(j.lester@ic.ac.uk).MATERIALS AND METHODSTest materialIndividual stock solutions of E1 with a purity of .99%(Sigma-Aldrich, Poole, UK) of 1000 mg/mL were prepared inhigh-performance liquid chromatography grade acetone (Rath-burn, Walkerburn, UK). The stock solution was diluted in 50:50 acetonitrile (Rathburn) and ultrapure water (Maxima Ul-trapure water generator, USF Elga, Bucks, UK) to create work-ing solutions from 1 mg/L to 10,000 mg/L. Analysis was bynegative ionization, electrospray liquid chromatography/massspectrometry (LC/MS).Test culturesInitial cultures of C. vulgaris and D. magna were purchasedfrom the Culture Collection of Algae and Protozoa (Winderm-ere, UK). Chorella vulgaris was grown in Jaworski’s Mediumwith constant aeration and maintained at 17 6 18C under con-stant cool fluorescent light at 300 lx provided by Osram L58W/23 tubes (Langley, UK). Daphnia magna was culturedin 4-L polypropylene containers (Whitefurze, UK) containingsynthetic pond water and maintained under natural light. Thewater temperature was measured by thermometer and main-tained at 18 6 28C in a temperature-controlled environment.The synthetic pond water was made to the following com-position NaHCO3 (192 mg/L), CaSO4·2H2O (120 mg/L),MgSO4 (120 mg/L), and KCl (8 mg/L) using ultrapure water(SCIENTO Culture instructions, Culture Collection of Algaeand Protozoa). The dry weight of the C. vulgaris was calcu-lated by measuring the optical density (OD680nm) using a spec-trometer (SP6–200 Spectrometer, Unicam, Cambridge, UK)and converted to dry weight using 1.0769 mg/mL/OD [12].Bioconcentration of estroneIn a 400-ml Pyrex glass beaker (VWR International,Poole, UK), 200 ml of synthetic pond water was spiked to400 mg/L of E1 (quantified by LC/MS). Fifty adult D. magna(over 10 d old), which had not been fed for 24 h, were placedin the pond water containing E1. Uptake of E1 by the D. magna106 Environ. Toxicol. Chem. 23, 2004 R.L. Gomes et al.Table 1. Estrone (E1) concentration in Daphnia magna and theaqueous medium after different exposure times and calculatedbioconcentration factors (BCFs). Values are determined fromthree replicatesDuration ofexperiment(hours)Average E1concentration inD. magna (mg/g)Average E1concentration inaqueous medium(mg/mL) BCF4416244.24 6 2.9665.00 6 5.6463.58 6 20.8256.80 6 13.050.018 6 0.0050.234 6 0.0320.278 6 0.1200.343 6 0.012241278229165was measured at 4, 16, and 24 h and the final concentrationof E1 in the pond water was analyzed by LC/MS at each timepoint. The experiment was repeated at a lower concentrationof E1 (40 mg/L) and uptake in the D. magna and concentrationof E1 in the water was determined after 4 h. All bioconcen-tration experiments were carried out in triplicate. On deter-mining the concentration of E1 in the D. magna and the finalpond water, the BCF was calculated using Equation 1.E1 concentration in D. magmaBCF 5 (1)E1 concentration in pond waterBiomagnification of estroneChorella vulgaris was spiked to 2.5 mg/L of E1 and rotaryshaken in the dark for 6 h. Dark conditions were used as algaetransforms E1 to 17b-estradiol in the light [12]. The algaewere filtered through a 0.22 mm membrane filter (Scheicherand Schuell, London, U.K.) and rinsed off the filter paper intofresh pond water. The concentration of E1 in the algal extractspiked to 2.5 mg/L was previously determined to be 0.055 mg/g(dry wt) [12]. The suspended algae were diluted to 200 mLwith pond water. Sixty D. magna which had been starved forthe previous 24 h, were placed in the new medium with thesuspended algae for a period of 48 h. All biomagnificationexperiments were carried out in triplicate. On determining theE1 concentration in D. magna, the partitioning factor wascalculated using Equation 2.E1 concentration in D. magmapartitioning factor 5 (2)E1 concentration in C. vulgarisEstrone determination in D. magnaThe D. magna were removed from the pond water anddipped into clean water to rinse off excess spiked pond water.The D. magna were then placed in a 20-mL screw-cap glasscontainer containing 10 mL acetone and shaken vigorously for10 min. The sample was filtered through a 0.22-mm Duraporemembrane filter (Millipore, Watford, UK), transferred to around bottom flask, and rotary evaporated. The recovery ofE1 for the acetone extraction was determined to be 88.94 65.56% (n 5 3) and results were adjusted to account for therecovery. Final samples were made up to 1 mL in 50:50 ace-tonitrile:water. The pond water filtrate was collected at eachtime point and 1-mL subaliquots were analyzed by LC/MS todetermine the final concentration of E1 in the aqueous medium.The filtered D. magna were dried at 908C for 24 h and mea-sured to determine the dry weight. A control experiment wasconducted to assess whether extraction of D. magna with ac-etone for 10 min affects the subsequent determined dry weight.The dry weight after acetone extraction was 90.5% (n 5 6)of the dry weight after shaking with water under the sameconditions. After acetone extraction, the subsequent dry weightwas adjusted to account for the recovery. Dividing the E1concentration in D. magna by the dry weight of the D. magnaallowed for comparison between samples.LC/MS analysisAll E1 analysis in this study was by LC/MS, a Perkin-Elmer Series 200 LC System (Bucks, UK) connected to aSCIEX API 150 EX with TurboIonSpray interface (AppliedBiosystems, Warrington, Cheshire, UK). Liquid chromato-graphic separation was achieved using a Hypersil BDS C18column (100 3 2.1 mm, 5 mm) with an injection volume of10 mL. The mobile phases, ultrapure water, and high-perfor-mance liquid chromatography grade acetonitrile were deliv-ered at 200 mL/min on a 50:50 gradient. Analysis of E1 wasin single ion monitoring at mass 269.4 [M-H2], eluting at aretention time of 8.59 min. Detection and quantification limitsof E1 were 10 pg and 50 pg on column, respectively.RESULTS AND DISCUSSIONBioconcentration of estroneThe results of the temporal study indicate that accumulationof E1 into the D. magna occurred within the first 16 h. Withinthe closed system, the BCF decreased after 24 h (Table 1).This may be attributed to an increase in depuration or deg-radation of E1 by the D. magna, which may involve an ac-climation lag phase. The downward trend may also be attri-buted to changes in solution due to increased concentration ofdissolved organic carbon in the aqueous phase, similar to thatobserved for steroid estrogens when binding to sediment [13].The relationship between E1 concentration in D. magna tothat in the aqueous phase over all temporal points was r2 50.75. However, the significant positive correlation increasedto 0.93 when results from the 24-h experiments were excluded(Fig. 1). The equation of the line (y 5 243.09x) in Figure 1also equates to BCF.After 4 h of exposure to E1, the average BCF in D. magnawas 272 (n 5 6). This compares to a recorded BCF of 37under similar conditions, for the uptake of E1 in C. vulgaris[12]. The BCFs for E1 are relatively small compared to otherendocrine disrupting substances, such as nonylphenol and itsmono- and di-ethoxylates [14]. However, due to the high po-tency of estrogens, low levels of bioaccumulation potentiallycan have significant effects. Previous studies have determinedthat bioconcentration increases at higher trophic levels re-ported to be due to increased lipid content and decreased chem-ical elimination efficiency up the food chain [15]. A log BCFof 2.36 was determined in the present study. This comparesto a modeled log BCF of 1.77 for daphnids and 2.31 and 2.56for nonspecies-specific data [16]. The log BCFs having beenderived from physico-chemical equations using the log octan-ol/water partition coefficient (Kow) of 3.43 for E1.The acute median lethal concentration value for E1 on D.magna has not been determined. However, for the syntheticestrogen 17a-ethinylestradiol, the effective concentration inDaphnia (species not stated) was found to be 5700 mg/L (effectnot stated) with a no-observed-effects concentration of 10 mg/L[17]. In the current study, D. magna were exposed to E1 con-centrations ranging from 363 mg/L to 15 mg/L. Due to thelimitations of current detection limits, the test concentrationsAssessing the bioaccumulation of estrone in Daphnia magna Environ. Toxicol. Chem. 23, 2004 107Fig. 1 Relationship between estrone (E1) concentrations in the Daph-nia magna and the aqueous medium for the 4- and 16-h exposureexperiments. The equation of the line is y 5 243.09x, which alsoapproximates to bioconcentration factors.used here are a minimum of 2000 times in excess of thosefound in the environment. Thus extrapolation to the aquaticenvironment is not without the attendant risk of error.Biomagnification of estroneDaphnia magna were maintained in culture with C. vul-garis for 48 h, with the algae containing an average E1 con-centration of 0.055 mg/g. E1 was detected in all D. magnasamples, with an average concentration of 1.32 6 0.21 mg/g(n 5 3). Uptake via a food source will depend on the feedingrate; the concentration of the toxicant in the food and the ratethat the food is processed; and the amount absorbed comparedto the amount excreted [18]. Using Equation 2, the averagepartitioning from food source to D. magna was calculated tobe 23.98 6 3.79. The system had not been assessed to deter-mine a steady state and the possible desorption of E1 fromthe algae (resulting in bioconcentration into the D. magna asopposed to biomagnification) was not eliminated as a possi-bility. This is a first estimation of partitioning between thefood source, C. vulgaris, and D. magna and may approximateto the biomagnification factor assuming steady state condi-tions.The data presented here indicate that the uptake via thetrophic route is likely to be less significant compared to bio-concentration from the aqueous medium; this agrees with an-other study [19]. Even if the concentration in the food sourceis greater than that of the water, uptake from the water is moresignificant. This is a consequence of the volume (quantity) offood ingested being much smaller than the volume of waterpassing through the gills [20].CONCLUSIONAccumulation of E1 from the aqueous medium into the D.magna occurs within the first 16 h with a decrease in BCFbeing observed after 24-h exposure. The log BCF of 2.36obtained from the present study is slightly greater than pre-dicted by the Kow equations for Daphnids but is similar to thenon-species specific physico-chemical equations. A linear re-lationship between the concentration of E1 in the aqueousmedium to that in the D. magna was obtained (r2 5 0.93) forthe 4- and 16-h temporal experiments.Biomagnification of E1 in D. magna via the algae, C. vul-garis, is nine times less than the bioconcentration from theaquatic environment, with a partitioning factor of 24. 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Binding of waterborne steroid estrogens to solid phases in river and estuarine systems.

Bioaccumulation kinetics: Experimental data and modeling.

Bioaccumulation of the lipophilic metabolites of nonionic surfactants in freshwater organisms. Environ Pollut 79:243–248.

Biotransformation and bioconcentration of steroid estrogens by Chlorella vulgaris.

Biotransformationand bioconcentration of steroid estrogens by Chlorella vulgaris.

Characterization of estrogenic compounds in water samples collected from United Kingdom estuaries. Environ Chem Toxicol 20:2165–

Characterization of estrogenic compounds in water samples collected from United Kingdom estuaries. Environ Chem Toxicol20:2165–

Concentrations of persistent lipophilic compounds in ﬁsh are determined by exchange across the gills, not through the food chain.

Degradation of 17b-estradiol and its metabolites by sewage bacteria.

Effects of environmental hormones in prosobranch molluscs.

Effects of environmental hormones in prosobranchmolluscs.Proceedings, Second Status Seminar Endocrine Disrupters,

Endocrine disrupters in sewage treatment plants, receiving river waters, and sediments: Integration of chemical analysis and biological effects on feral carp.

Endocrine disrupters in sewage treatment plants,receivingriver waters, and sediments: Integration of chemical analysis and biological effects on feral carp.

Evidence for endocrine disruption in European wildlife, especially ﬁsh.

Holten 108 Environ.

Identiﬁcation and quantiﬁcation of estrogen receptor agonists in wastewater efﬂuents.

Identiﬁcation of estrogenic chemicals in STW efﬂuent. 1. Chemical fractionation and in vitro biological screening. Environ Sci Technol 32:1549–1558.

Modeling the bioconcentration of hydrophobic organic chemicals in aquatic organisms.

Occurrence, fate, and effects of pharmaceutical substances in the environment—A review.

Prediction of the bioaccumulation factors and body burden of natural and synthetic estrogens in aquatic organisms in the river systems.

Proposed predicted-no-effect-concentrations (PNECs) for natural and synthetic steroid oestrogens in surface waters.

Reproduction and development of Japanese medaka following an early life stage exposure to xenoestrogens. Aquat Toxicol 44:141–156.

The effects of natural and synthetic steroid estrogens in relation to their environmental occurrence. Crit Rev Toxicol 32:113–132.

Toxicological proﬁle of pollutants in surface water from an area in Taihu Lake, Yangtze Delta. Toxicology 166:71–78.

Trophic level differences in bioconcentration of chemicals: Implications in assessing environmental biomagniﬁcation.

An assessment of the bioaccumulation of estrone in Daphnia magna

An assessment of the bioaccumulation of estrone in Daphnia magna

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