Toxicity of 17a-ethynylestradiol and norethindrone, constituentsof an oral contraceptive pill to the swimming and reproduction of

cladoceran Daphnia magna, with special reference to theirsynergetic effect

Takashi Goto *, Juro Hiromi

Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-8510, Japan

Abstract

Toxicity of 17a-ethynylestradiol (EE2) and norethindrone (NOR), constituents of low dosage oral contraceptives, was assessedfor the freshwater cladoceran Daphnia magna. Acute toxicity tests showed that 5 ppm of EE2, the highest concentration in this

study, never inhibited swimming, whereas NOR inhibited swimming at >3 ppm: 48 h EC50 for NOR was 6.41 ppm. Chronic toxicitytests were carried out for 25 days by measuring the number of offspring, moltings and sex ratios of neonates at 20, 100 and 500 ppb.

EE2 > 100 ppb significantly decreased the number of offspring to 75% of the control; however, no effect was observed in moltingand sex ratios at <500 ppb. NOR did not affect reproduction and sex ratios at <500 ppb. Mixture of EE2 (5.88 ppb) and NOR(94.12 ppb) also significantly decreased the number of offspring to 57% of the control. This result indicates the importance of

examining synergetic effects of chemicals in the context of natural environments which face exposure to myriad chemicals.

� 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Norethindrone; 17a-ethynylestradiol; Daphnia magna; Chronic toxicity; Synergetic effect of chemicals

1. Introduction

‘‘The pill’’ is typically a low dosage oral contraceptive

composed of EE2 (17a-ethynylestradiol) and NOR

(norethindrone). Prohibition of its use ended in the USAin the 1960s and in Japan in 1999. According to Barton

et al. (2002), the pill is now used by more than 100

million women around the world; nevertheless, there

have been few reports to describe actual contamination

of EE2 and NOR in environmental waters. Both EE2

and NOR affect mammals as estrogenic and progeste-

ronic hormones, respectively; however, no reports have

assessed their toxicity to freshwater animals, exceptHarries et al. (1999). They reported that the most

probable cause of testiculus atrophy in rainbow trout

observed in England was EE2.

The present study aims to evaluate toxicity of EE2

and NOR to a freshwater cladoceran, Daphnia magna,

that connects phytoplankton to fishes; particular atten-

tion is given to the mixed effects of the two chemicals.

2. Materials and methods

2.1. Cultures of Daphnia magna

D. magna (Clone 5) were individually placed in 100

ml glass beakers containing 50 ml of a culture medium,

aerated distilled water, for two days. Cultured daphnids

were fed on the green alga Scenedesmus sp. at concen-

tration of 5� 104 cells/ml every second day; they weremaintained at 20� 1 �C in an 8 h light/16 h dark

photoperiod regime. During four weeks before toxicity

tests, generated neonates were discarded after checking

their number and sex every second day.

2.2. Preliminary experiment on toxicity of solvents

For dissolving EE2 and NOR in test solutions, 550and 5 ppm of DMSO (dimethyl sulfoxide) were used,

*Corresponding author. Tel./fax: +81-466-84-3686.

E-mail addresses: utogo@mac.com (T. Goto), jhiromi@brs.nihon-u.

ac.jp (J. Hiromi).

0025-326X/03/$ - see front matter � 2003 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0025-326X(03)00052-3

www.elsevier.com/locate/marpolbul

Marine Pollution Bulletin 47 (2003) 139–142

respectively. The 48 h LC50 value of DMSO for D.

magna was 25,000 ppm and DMSO less than 5 ppm

showed NOEC (no-observed-effect concentration) for

swimming activity, total number of offspring, repro-duction frequency, number of moltings, sex ratio of

offspring, and resting egg production in preliminary

tests.

2.3. Acute toxicity test

Acute toxicity tests were performed in accordance to

the standard protocol for D. magna acute tests (OECD,1984). Twenty neonates aged less than 24 h were indi-

vidually transferred into 10 ml glass beakers filled with

10 ml of test solution and incubated at 20� 1 �C in thedark for 48 h. Exposure concentrations of single com-

pound EE2 and NOR (Wako Pure Chemical Ind., Ltd.,

Osaka, Japan) were 1–5 and 2–25 ppm, respectively.

Concentration of EE2 > 5 ppm could not be preparedbecause of precipitation in the test solution. The 48 hEC50 value was determined for swimming inhibition of

daphnids by the probit method recommended by the

OECD.

2.4. Chronic toxicity test

Chronic toxicities of EE2 and NOR were assessed in

a semi-static test according to the standard protocol forD. magna reproduction test (OECD, 1998). Daphnids

were exposed to three different concentrations of EE2 or

NOR: 20, 100 and 500 ppb. Synergetic effects of EE2

mixed with NOR were also tested; the mixture ratio was

1:17 in weight based on the mixture ratio of both

chemicals in the pill. Concentrations of EE2 in these

two mixtures were 5.88 and 29.41 ppb. During the

chronic toxicity test, the green alga Scenedesmus sp.was supplied with the concentration of 5� 104 cells/mlevery second day. The number of offspring, reproduc-

tion frequency, number of moltings, sex ratio of off-

spring, and presence of a resting egg were checked as

endpoints.

3. Results

3.1. Acute toxicity

No swimming inhibition was observed in the control.

The EE2 (1–5 ppm) did not inhibit swimming of neonate

daphnids. Fig. 1 shows the interrelationship between

swimming inhibition rates and NOR concentrations.

Swimming inhibition was enhanced with increased NORconcentration from 2 to 11.2 ppm. The 48 h EC50 value

was calculated to be 6.41 ppm based on the regression

equation which was obtained as:

Immobilisation ð%Þ ¼ 35:87� lnNOR� 16:61ðR2 ¼ 0:89; P < 0:05; n ¼ 153Þ

3.2. Chronic toxicity

Resultant reproductive features of D. magna in con-

trols were as follows: the mean total number of offspring

was 106.8, sex ratio of offspring was 0.24 (ratio of thenumber of males/total number of offspring), molting

frequency was 10.24, and no resting egg was produced

(Table 1, Figs. 2 and 3). The number of offspring of

daphnids was distinctly decreased by EE2 at 100 and

500 ppb (Fig. 2). Resultant total number of offspring at

100 and 500 ppb were 75% of the control after 25 days.

NOR (20–500 ppb) did not affect the number of off-

spring. Molting frequency, sex ratio of offspring, and thenumber of resting eggs were scarcely affected by EE2

and NOR. The total number of offspring generated by

one female daphnid for 25 days was further reduced by

exposure to the two-chemical mixture to 76% of EE2 at

500 ppb and 57% of controls (Fig. 3).

4. Discussion

4.1. Acute toxicity

Notably, NOR�s function in vertebrates as a follicle-stimulating hormone (FSH) showed an acute toxic effect

in inhibiting swimming activity of D. magna with a 48 h

EC50 value of 6.41 ppm. Immobilisation of the animals

leads to their death because of the inherent impossibilityof capturing food. Although the mechanism of the lethal

effect in D. magna remains unknown, present results

suggest that NOR released into environmental waters,

such as groundwater, should be addressed critically in

future considering that NOR is a major constituent of

Fig. 1. Acute toxicity: Relationship between immobilization (%) of D.

magna and concentrations of Norethindrone (NOR). Vertical bars

mean a standard deviation.

140 T. Goto, J. Hiromi / Marine Pollution Bulletin 47 (2003) 139–142

typical oral contraceptives (>90% in weight) and that itsuse will increase world wide.

4.2. Chronic toxicity

Endocrine disruption by NOR is known to develop

mammary glands in male mice (Skarda, 2001); however,this chemical rarely affected reproductive features of D.

magna. On the contrary, EE2 (>100 ppb) singly de-creased the total number of offspring of D. magna. It is

likely that decreased reproduction appeared as the result

of endocrine disruption by EE2, but this idea should be

confirmed by measuring vitellogenin density in the body

of Daphnia exposed to this chemical. It is also remark-

able that EE2 (5.88 ppb) mixed with NOR (94.12 ppb)effectively decreased the total number of offspring per

female during 25 days. Such synergetic effects seem to be

identical to combined action of NOR and EE2 in the

pill. Interrelationship of both chemicals in the pill is a

mutual aid: EE2 acts in humans as a progesterone like

chemical, it stimulates the pituitary gland and promotes

ovulation firstly stimulated by NOR; thus, ovulation is

promoted by lower dosage of EE2 when it is mixed withNOR. Future examination must address whether such a

process occurs in daphnids. Although synergetic effects

of chemical compounds are expected in animal bodies,

information remains scarce because chemical toxicity to

animals has usually been studied for single compounds.

Poter et al. (1999) reported results of five years of re-

search on interactive effects of three chemicals: aldicarb,

atrazine (commonest agrochemicals) and nitrate (fertil-iser) contaminating groundwater in the USA at detect-

able levels. They concluded that immune, endocrine and

behavior changes occurred in mice because of mixtures,

but rarely due to single compounds at the same con-

centration. They did not specify how and why these

pesticides became more active in combination with

nitrate. The reasons should be elucidated in future;

Table 1

Chronic toxicity of EE2, NOR and EE2+NOR on the total number of offspring, reproduction frequency, the total number of male offspring, number

of resting egg and the total number of molts of D. magna

Treatment Endpoints N

Compo. Conc. (ppb) Total number

of offspring

Reproduction

frequency

Total number of male

offspring

Resting eggs Total number

of molts

Control 106.8� 18.8 4.0� 0.4 25.2� 8.0 0 8.3� 0.5 13

EE2 20 96.9� 14.1 3.9� 0.5 26.2� 22.5 0 8.2� 0.3 10

100 79.9� 12.4a 3.9� 0.4 15.3� 17.2 0 8.3� 0.4 11

500 80.0� 10.3a 3.8� 0.3 20.5� 20.0 0 8.0� 0.5 11

NOR 20 94.2� 16.6 4.0� 0.3 24.5� 19.2 0 8.6� 0.2 10

100 90.7� 14.6 3.7� 0.4 28.2� 10.6 0 8.3� 0.3 11

500 91.8� 16.5 3.9� 0.1 33.2� 20.6 0 8.4� 0.4 12

EE2+NOR 20 97.1� 15.6 3.9� 0.2 20.4� 10.3 0 8.2� 0.1 12

100 69.4� 6.0a 4.0� 0.3 28.5� 22.7 0 8.4� 0.5 11

500 61.4� 8.4a 3.8� 0.5 29.8� 9.9 0 8.2� 0.4 11

Data are presented as the mean� SD.N means the number of measurements.a Significantly different from the control at p5 0:05 (ANOVA).

Fig. 2. Chronic toxicity: Effects of single compounds, EE2/NOR on the

number of offspring per one daphnid for 25 days. Vertical bars mean a

standard deviation. �Significant at P < 0:05 (ANOVA).

Fig. 3. Chronic toxicity mixture effects of EE2 and NOR on the

number of offspring per one daphnid for 25 days. Vertical bars mean a

standard deviation. �Significant at P < 0:05 (ANOVA).

T. Goto, J. Hiromi / Marine Pollution Bulletin 47 (2003) 139–142 141

however, it is also important to examine such synergetic

effects of chemicals through ecotoxicological study,

considering that natural environments face exposure to

myriad chemicals.

Acknowledgements

The authors thank Nihon University for financial

support. Thanks are also due to Japan Pulp & Paper

Research Institute, Inc. for providing the strain of D.

magna. The authors would like to thank Dr. K. Ara(Nihon University), Prof. T. Katase (Nihon Univer-

sity), and Dr. T. Okumura (Japan Sea National Fish-

eries Research Institute) for technical advice and

valuable comments. Finally, they express their grati-

tude to Ms. M. Yoshimura for collaboration in toxicity

tests.

References

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142 T. Goto, J. Hiromi / Marine Pollution Bulletin 47 (2003) 139–142