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The effects of 4-nonylphenol and ethanol on acute toxicity, embryo development, and reproduction in Daphnia magna
Ecotoxicology and Environmental Safety 55 (2003) 330–337
The effects of 4-nonylphenol and ethanol on acute toxicity, embryo development, and reproduction in Daphnia magna L. Zhang,a R. Gibble,b and K.N. Baerb,* a
The McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-8591, USA b Department of Toxicology, College of Pharmacy, The University of Louisiana at Monroe, Monroe, LA 71209-0470, USA Received 27 February 2002; accepted 8 July 2002
Abstract The mean 48-h EC50 (n ¼ 3) of 4-nonylphenol (NP) using ethanol as the carrier solvent was 155 mg/L, compared to a mean 48-h EC50 (n ¼ 3) of 281 mg/L without ethanol. The 96-h EC50’s for embryo lethality (arrested egg development) and deformities (curved or unextended shell spines and undeveloped second antennae) were 738 and 263 mg/L, respectively. Reproduction studies were conducted using conditions that stimulate male production (i.e., reduced photoperiod and food levels). An increase in neonate deformities was observed at 50 mg/L (without ethanol), but no changes were observed in fecundity or sex ratios. A decrease in sex ratios was observed at 25 and 50 mg/L (with ethanol) compared to the ethanol control. However, an increase in sex ratios was observed in the ethanol control compared to media controls. The use of ethanol as a solvent carrier confounds the effects of 4-NP on acute toxicity and male production in daphnids. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Daphnia; Reproduction; Sex ratio; 4-nonylphenol; Ethanol
1. Introduction Daphnia magna is one of the most important freshwater species employed in ecotoxicity testing throughout the world. Adequate survival, growth, and reproduction of daphnids are crucial for success in the environment, but more important is the ability of the species to survive adverse environmental conditions during winter. Most daphnids are cyclic parthenogenetic species capable of both asexual and sexual reproduction (Dodson and Frey, 1991). The survival strategy in sexual reproduction is to produce sufficient numbers of resting or winter eggs known as ephippia. Resting eggs require the presence of males for fertilization and development. These eggs then enter a diapause stage and hatch in spring as parthenogenetic females. The switch to male offspring production and subsequent sexual reproduction appears to be triggered naturally by environmental cues such as photoperiod, temperature, density, and food availability (Hobc! k and Larsson, 1990). The timing *Corresponding author. Fax: +1-318-342-1901. E-mail address: [email protected] (K.N. Baer).
of sexual reproduction and the optimal ratio of male to female offsprings appear critical for adequate ephippia production. Exposure to endocrine disrupting chemicals may disrupt this process. Nonylphenol (NP), a major degradative product of NP ethoxylates, has recently drawn attention because of its intrinsic estrogenic potential. Nonylphenol concentrations of 54.3 mg/L have been demonstrated to induce the formation of vitellogenin and inhibit testicular growth in male trout (Jobling et al., 1996). Similar effects attributed to NP exposure have been reported in other fish species (Christiansen et al., 1998; Kinnberg et al., 2000). Nonylphenol has been measured in sewage effluent and river water in the UK at concentrations of 330 and 180 mg/L, respectively (Blackburn and Waldock, 1995). However, concentrations in US rivers generally range from o0.11 to 0.64 mg/L (Naylor et al., 1992; reviewed in Bennie, 1999). Nonylphenol has been demonstrated to be acutely toxic to daphnids; however, at concentrations substantially higher than those normally found in the environment. For example, acute EC50 values in Daphnia
0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(02)00081-7
L. Zhang et al. / Ecotoxicology and Environmental Safety 55 (2003) 330–337
neonates range from 104 to 440 mg/L (Monsanto, 1985; Huls, . 1992; Brooke, 1993; Comber et al., 1993). Recent studies have focused on the toxicity of NP to developing embryos and larvae, perhaps a more sensitive invertebrate life stage to these environmental contaminants. Nonylphenol concentrations of 100 mg/L caused mouthpart deformities in chironomid larvae, Chironomus riparius (Meregalli et al., 2001). However, in D. magna, LeBlanc et al. (2000) observed developmental retardation via maternal exposure to 100 and 200 mg/L NP, but not during direct embryo exposure. Chronic studies with NP have also been conducted to assess survival, growth, and asexual reproduction in daphnids. The no-observed-effect concentration (NOEC) for reproduction ranges from 10 to 116 mg/L, still higher than most environmental concentrations (Huls, . 1992; Comber et al., 1993; Brooke, 1993; Baldwin et al., 1997; Shurin and Dodson, 1997). However, studies have demonstrated that sexual reproduction may be more sensitive to certain toxicants than asexual reproduction. For example, changes in Daphnia sex ratio were observed to be one to two orders of magnitude more sensitive to atrazine than survivorship or fecundity (Dodson et al., 1999). The influence of NP on sex ratios in D. magna is not clear. Several studies have not observed changes in sex ratios following NP exposure (Shurin and Dodson, 1997), whereas previous studies in our laboratory indicate a possible interaction between carrier solvents and NP on sex ratios (Baer and Owens, 1999; Zhang and Baer, 2000). These interactions need to be clarified before interpretations can be made concerning NP exposure on sex ratios in daphnids. In recent studies, carrier solvents routinely employed in ecotoxicity testing have produced some surprising effects. Dodson et al. (1999) observed a 40% increase in the sex ratio of D. pulicaria in acetone controls (45 ml/L) compared to water controls. However, under different conditions, acetone (80 ml/L) increased female offspring in D. galeata mendotae while having no effect on male production (Shurin and Dodson, 1997). The increase in fecundity was not observed in D. magna at an acetone concentration of 90 ml/L (Comber et al., 1993). Studies in our laboratory indicate that acetone (100 ml/L) and ethanol (10 ml/L) caused a significant increase of fecundity at low food levels while the effects on sex ratio varied depending on experimental conditions (Zhang and Baer, 2000). The possibility that carrier solvents may be modulating reproductive endpoints under certain experimental conditions may have farreaching implications for ecotoxicity testing protocols. In view of these findings, the objective of the present study was to investigate the effects of 4-NP and ethanol on acute toxicity, embryo development, and reproduction in D. magna.
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2. Materials and methods 2.1. Chemicals 4-Nonylphenol (B85% based on p-isomers) was purchased from Fluka Chemika (Milwaukee, WI). Ethanol (Absolute-200 Proof) was purchased from AAPER Alcohol and Chemical Company (Shelbyville, KY). High-performance liquid chromatography(HPLC) grade acetonitrile and methanol used in the HPLC system were purchased from Fisher Scientific (Houston, TX). 2.2. Animal cultures Daphnia magna Straus cultures (clone 5 obtained from the Academy of Natural Sciences of Philadelphia) were held in 1-L glass beakers containing B900 mL of H-H COMBO medium (Baer and Goulden, 1998). The temperature was maintained at 2072 C and a 16-h light:8-h dark photoperiod (illumination ranged between 300 and 450 lux) was employed. The medium was renewed and daphnids were fed a green algae species, Ankistrodesmus falcatus (15 106 cells/beaker), three times weekly (i.e., Monday, Wednesday, and Friday). These conditions allowed for continuous parthenogenetic reproduction in laboratory cultures. 2.3. Acute toxicity studies To evaluate the effects of ethanol as a carrier solvent on the 48-h acute toxicity of NP, randomly selected neonates from laboratory cultures (o24 h old, Xthirdbrood) were used. Nominal test concentrations of NP with and without ethanol as a carrier solvent were 39, 65, 108, 180, 300, and 500 mg/L. At each concentration, two replicates with five neonates per zeplicate were employed. Stock solutions of NP using a carrier solvent were prepared by dissolving the appropriate amount of NP into ethanol. The solvent limit in these test concentrations was p8 ml/L (ethanol control was 8 ml/ L). For studies without ethanol as the carrier solvent, a stock solution of NP (3.6 mg/L) was prepared by dissolving 2 mL NP into distilled water followed by ultrasonication for 1 h. A 100-mL syringe was loaded with NP from behind by a 1-mL syringe, then 2 mL NP was injected from the 100-mL syringe into a 1-L beaker containing 500 mL distilled water. The solubility and stability of the NP stock solution (3.6 mg/L) were measured by HPLC (see below). All studies were conducted in a Cryo-Fridge environmental chamber (Revco Scientific, Asheville, NC) with a constant temperature (20 72 C) and normal photoperiod (16-h light:8-h dark). Temperature, dissolved oxygen, and pH were monitored at 0, 24, and 48 h. Immobility (synonymous with death) was observed daily.
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2.4. Ex vivo embryo development study
2.6. Analytical methodology
Daphnid embryos are capable of normal development outside of the brood pouch and are therefore suitable for ex vivo studies. Adult females from normal laboratory cultures bearing more than 10 parthenogenetic eggs less than 6 h old (stage 1 as described by Kast-Hutcheson et al., 2001) were selected and placed in petri dishes containing H-H COMBO medium. The daphnids were placed under a dissecting microscope and the abdominal side of the carapace was held with the aid of fine forceps. Eggs were removed carefully from the brood chamber by gently swirling the body of the animal in the medium (Ohta et al., 1998). The isolated eggs were pooled and placed into 10 plastic cups containing 25 mL of NP test solutions (10 eggs/ cup). Nominal test concentrations using ethanol as the carrier solvent (8 ml/L) were 50, 200, 800, and 1000 mg/L. All test containers were covered and placed in the environmental chamber with a constant temperature (2072 C) and normal photoperiod (16-h light:8-h dark). Embryo development was observed at 24, 48, and 96 h. The live and/or deformed neonates were observed at 96-h.
The solubility and stability of NP stock solution in distilled water (3.6 mg/L; without ethanol) was measured at 0, 48, and 72 h according to methods previously reported (Ahel and Giger, 1985; Marcomini and Giger, 1987). Nominal NP concentrations used in the study were below the limit of detection for this method. Prior to HPLC analysis, samples were subjected to solid-phase extraction (SPE) using C-18 Bond Elut LRC extraction cartridges that were preconditioned with methanol. Nonylphenol stock solutions (n ¼ 4) were eluted with methanol, ultrasonicated, and analyzed by HPLC. Samples and standards (prepared in methanol) were analyzed on a P2000 SpectraSYSTEM gradient pump equipped with a SCM 1000 Vacuum Membrane Degasser and an AS 1000 SpectraSYSTEM autosampler (Thermo Separation Product, Inc., Riviera Beach, FL). Optimum separation was achieved using a reverse-phase Beckman Ultrasphere Octyl 5-mm C-18 column (4.6 mm 25 cm) purchased from Varian Analytical (Walnut Creek, CA). The system was operated at ambient temperature and the flow rate was 1.0 mL/min with degassed methanol as the mobile phase. The injection volume was 50 mL. Detection was performed by a SpectraSYSTEM UV 3000 Multiple-Wavelength Detector (Thermo Separation Product, Inc., Riviera Beach, FL) set at 224 nm. A ChromQuest Chromatography Workstation for Windows NT (ThermoQuest, Inc., San Jose, CA) was used for integration.
2.5. Chronic reproduction studies The effect of NP and ethanol on the reproduction of daphnids was investigated under conditions that stimulate male production. Neonates (o24 h old, Xthird brood) were exposed to the following conditions: NP concentrations of 12.5, 25, and 50 mg/L using ethanol as carrier solvent (p8 ml/L); NP concentrations of 25 and 50 mg/L without ethanol; ethanol concentrations of 2, 4, and 8 ml/L. Ten beakers per treatment group were used with one daphnid per beaker. All experiments were conducted in an environmental chamber with constant temperature (2072 C) and short photoperiod (8-h light:16-h dark). The medium was renewed and daphnids were fed a green algae species, A. falcatus (3.75 106 cells/beaker), three times weekly (i.e., Monday, Wednesday, and Friday). During the experiments, temperature, dissolved oxygen, pH, total hardness, total alkalinity, and conductivity were monitored weekly. Endpoints included adult survival, total number of molts, total number of live neonates (fecundity), total number of aborted eggs, and sex ratio. The sex and morphology of neonates were observed and counted using a dissecting microscope. Male daphnids were identified by the presence of large, prominent first antennules. The sex ratio was determined as the total number of males divided by the total number of neonates (Dodson et al., 1999).
2.7. Statistical analyses The EC50’s were calculated by probit analysis using SAS statistical software (SAS Institute, Inc., Cary, NC). All chronic data were tested for statistical significance using a single factor one-way analysis of variance (ANOVA). Supplementary tests included Fisher’s least significant difference and Newman–Keul’s tests. Homogeneity of variances and normality among replicates were determined by Bartlett’s test and Komogorov– Smirnov test, respectively. All tests were performed using standard software (JMP IN, SAS Institute, Inc., Cary, NC).
3. Results 3.1. Acute toxicity studies The summary of 48-h acute toxicity tests with NP is presented in Table 1. The 48-h acute EC50’s without ethanol as the carrier solvent ranged from 234 to 337 mg/L (mean ¼ 281). The 48-h EC50’s with ethanol as the carrier solvent ranged from 94 to 199 mg/L (mean ¼ 155). Although the means are statistically
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significant, the 95% confidence limits overlap and the solvent studies exhibited a poorly defined dose–response relationship (r2 ranging from 0.78 to 0.83). However, these results suggest that the use of ethanol as a carrier solvent increases the acute toxicity of NP to daphnid neonates. Recoveries of NP stock solution (3.6 mg/L) without ethanol immediately after preparation averaged 94% (Table 2; CV=2.8%). The concentrations of the NP stock measured at 0, 48, and 72 h ranged from 89% to 95% (Table 2). These results indicate that the NP stock solutions without ethanol were accurately prepared and soluble prior to testing.
(Table 1). Examples of arrested development in embryos and neonate deformities are presented in Fig. 1. The most sensitive effect was late-stage neonate deformities (curved or unextended shell spines and undeveloped second antennae) observed at 50 and 200 mg/L (10% and 90%, respectively). Arrested egg development was only observed at higher NP concentrations (800 and 1000 mg/L). Embryos exposed to 800 mg/L for 48 h exhibited arrested development (Figs. 1E and F), and severe deformities in neonates were observed at 96 h (Figs. 1H and I).
3.2. Ex vivo embryo development study
3.3. Chronic reproduction studies
The 96-h ex vivo embryo development EC50’s for embryo lethality (arrested egg development) and neonate deformities were 738 and 263 mg/L, respectively
The effect of NP (with and without ethanol as a carrier solvent) and ethanol on daphnid survival and reproduction was investigated in 35-day chronic studies. Adult survival in NP concentrations of 12.5, 25, and 50 mg/L using ethanol as a carrier solvent was 80%, 90%, and 60%, respectively (results not presented). Adult survival in NP concentrations of 25 and 50 mg/L without ethanol was 100% and 90%, respectively (results not presented). No adult mortality was observed in the ethanol concentrations (2, 4, and 8 mL). No effects were observed in the total number of juvenile molts in any treatment group. The effects of different exposures on fecundity are presented in Fig. 2. Ethanol significantly increased fecundity at 4 and 8 mL (Fig 2A, ethanol control, C). There was a statistically significant decrease in fecundity at 12.5 mg/L compared to the ethanol control (Fig. 2A), but this decrease was not considered biologically significant because of a lack of significance compared to the media control. Nonylphenol concentrations of 25 and 50 mg/L with ethanol as the solvent carrier significantly decreased sex ratios compared to the ethanol control but not the media control (Fig. 3A). However, sex ratios in 25 and 50 mg/L without ethanol were not significantly different from controls (Fig. 3B). The increase in sex ratios observed in the ethanol control (8 ml/L; Fig. 2A) was not repeated in the ethanol study (Fig. 3C). The results indicate that sex ratios are variable and can be influenced by solvent carriers such as ethanol. A low, but statistically significant, increase in neonate deformities (3.3%) was observed at 50 mg/L
Table 1 Summary of 48-h acute toxicity tests and 96-h embryo development test with NP Test
EC50a (mg/L)
48-h acute tests No solvent 234 No solvent 272 No solvent 337
r2
95% Equation confidence limits (lower–upper, mg/L) 157–245 167–481 193–1014
y ¼ 7:943 þ 0:2479x 0.93 y ¼ 15:21 þ 0:2396x 0.91 y ¼ 17:59 þ 0:2003x 0.91
33–450 NA NA
y ¼ 0:0971 þ 0:2511x y ¼ 33:383 þ 0:1777x y ¼ 12:732 þ 0:2172x
NA NA
y ¼ 8:527 þ 0:0793x 0.84 y ¼ 20:322 þ 0:113x 0.80
Mean (SEM) 281 (30) Solvent Solvent Solvent
199 94 172
0.83 0.78 0.83
Mean (SEM) 155 (32)b 96-h embryo test Solvent 738c 263d a
Immobility or death. Statistically significant from no solvent. c Based on arrested egg development. d Based on deformed neonates, i.e., curved or unextended shell spines and undeveloped first antennae. b
Table 2 Measured concentrations of NP stock solutions without ethanol Nominal concentrations 3.6 mg/L
1 2 3 4 Mean (SEM)
Measured concentrations (mg/L) 0h
48 h
72 h
3.49 3.33 3.28 3.43 3.3870.048
2.97 3.56 3.35 3.25 3.2870.122
3.70 3.35 2.95 3.19 3.3070.157
Mean (SEM) (%)
Mean (%) nominal
3.3970.22 3.4170.074 3.1970.12 3.2970.072 3.3270.051
94 95 89 91 9271.4
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(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
(I)
Fig. 1. Developmental abnormalities elicited by 4-NP. (A) Embryo ( 100) in control group at 24 h. (B, C) Embyros ( 100) exposed to NP (800 mg/L) at 24 h. (D) Embryo ( 40) in control group at 48 h. (E, F) Embyros ( 40) exposed to NP (800 mg/L) at 48 h. (G) Normally developed nenonates ( 40) in control group at 96 h. (H, I) Deformed neonates ( 40) exposed to NP (800 mg/L) at 96 h.
without ethanol and were identical to those observed in the ex vivo study (Figs. 1H and I). These deformities were consistent with late-stage developmental toxicity, i.e., curved or unextended shell spines and undeveloped second antennae. No other statistically significant increases in deformities were observed in NP concentrations with ethanol or in the ethanol study alone.
4. Discussion Nonylphenol was acutely toxic to daphnid neonates (with or without ethanol as a carrier solvent) with mean EC50 values of 155 and 281 mg/L, respectively. The literature reports acute toxicity values in daphnids ranging from 104 to 440 mg/L (Monsanto, 1985; Huls, .
*
120 100 80 60 40 20 0
Sex Ratio (%)
Fecundity
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#
Control
(A)
Ethanol Control
12.5
25
35 30 25 20 15 10 5 0
*
Control
50
(A)
120
Sex Ratio (%)
Fecundity
100 80 60 40 20 0 25
120
Sex Ratio (%)
Fecundity
Control
(B)
*
100
*
80 60 40 20 0 Control
(C)
2
4
12.5
#
25
50
35 30 25 20 15 10 5 0
50
4-nonylphenol (ug/l)
(B)
Ethanol Control
#
4-nonylphenol (ug/l)
4-nonylphenol (ug/l)
Control
335
8
Ethanol (ul/l)
50
4-nonylphenol (ug/l) 35 30 25 20 15 10 5 0 Control
(C)
25
2
48
Ethanol (ul/l)
Fig. 2. The effect of NP and ethanol (mL/L) on fecundity of D. magna under reduced photoperiod and food levels. All values represent mean7SEM. (A) The effects of NP using ethanol carrier solvent. (B) The effects of NP without ethanol carrier solvent. (C) The effects of ethanol. *Statistically significant from control, Po0:05: #Statistically significant from ethanol control, Po0:05:
Fig. 3. The effect of NP and ethanol (mL/L) on the sex ratio D. magna under reduced photoperiod and food levels. All values represent mean7SEM. (A) The effects of NP using ethanol carrier solvent. (B) The effects of NP without ethanol carrier solvent. (C) The effects of ethanol. *Statistically significant from control, Po0:05: #Statistically significant from ethanol control, Po0:05:
1992; Brooke, 1993; Comber et al., 1993). It is interesting to note that in the present study, significantly higher acute toxicity values were observed in the presence of ethanol as a carrier solvent (p8 ml/L) compared to studies without ethanol. This result was repeated over time by different technicians using different batches of daphnids. Similarly in the chronic study, 50 mg/L with ethanol resulted in 40% adult mortality compared to 10% in the same concentration without ethanol. Solvents are routinely used to deliver and enhance the solubility of NP in aqueous solutions. However, rigorous attempts to solubilize NP in water for studies without the use of ethanol (repeated mixing and sonication) were successful. Analytical recoveries of NP stock solutions (3.6 mg/L) averaged 94% of nominal concentrations, and these solutions were relatively stable for 72 h. Differences in the acute toxicity of NP with and without solvent may still be due to differences in solubility since all EC50 values were based on nominal
concentrations and not on measurements of actual test solutions. A more probable explanation is that solvents such as ethanol may enhance absorption of organic chemicals across biological membranes. However, the biological significance of this apparent increase may not be that dramatic since acute toxicity endpoints for a single species often differ by a factor of 2 depending on experimental design, i.e., exposure conditions, concentrations employed, and so on. Nevertheless, the potential influence of solvents on toxicity endpoints should be considered when evaluating and comparing data. The effects of NP on embryo development demonstrated a dose–response increase in arrested eggs (EC50=738 mg/L) and deformed neonates (EC50= 263 mg/L), the latter being a more sensitive indicator of toxicity in this assay. At NP concentrations of 200 mg/L, developmental deformities such as curved or unextended shell spines and undeveloped second antennae were observed. These deformities are characteristic of
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late-stage toxicity (stages 4–6 as described by KastHutcheson et al., 2001). Early stage embryo toxicity characterized by arrested egg development was only observed at the two highest concentrations (800 and 1000 mg/L). Direct exposure of C. riparius larvae to 100 mg/L NP was shown to cause mouthpart deformities (Meregalli et al., 2001). However, in contrast to the present study, LeBlanc et al. (2000) observed no developmental effects following direct NP concentrations of 100 and 200 mg/L in D. magna. The reason for these apparent discrepancies is currently unknown. Chronic studies using conditions that stimulate male production resulted in different reproductive endpoints depending on whether NP was delivered using ethanol as a solvent carrier. A previous study in our laboratory demonstrated that acetone (100 ml/L) was effective at increasing fecundity in a variety of exposure conditions (combinations of food levels and photoperiods) except in a high food and reduced photoperiod group (Zhang and Baer, 2000). Other investigators have observed similar results in conditions that favor male production. For example, Shurin and Dodson (1997) observed an increase in female offspring production in D. galeata mendotae at acetone levels of 80 ml/L compared with controls. However, Comber et al. (1993) using 90 ml/L acetone and experimental conditions conducive to parthenogenetic reproduction observed no increase in fecundity. The present study demonstrated that exposure to ethanol at 4 or 8 ml/L increased fecundity during conditions that stimulate male production. One possible explanation may be a stimulatory effect of ethanol on algal growth since increasing food levels have been shown to increase fecundity in a concentration-dependent fashion (Zhang and Baer, 2000). Ethanol was shown to have a slight stimulatory effect on algal growth at 8 ml/liter (results not presented), but this increase was not sufficient to explain the increase in fecundity in the present study. Previous studies indicate that high concentrations of ethanol (100 ml/liter) increased fecundity independent of food levels and photoperiod (Zhang and Baer, 2000). These results seem to indicate a direct effect of ethanol on the endocrine system, i.e., possibly by enhancing vitellogenesis and increasing reproduction endpoints. The effects of NP at 25 and 50 mg/liter had no effect on fecundity either with or without the use of ethanol as a solvent carrier. This is in contrast to LeBlanc et al. (2000), who demonstrated a dosedependent stimulatory effect of NP on fecundity using concentrations ranging from 31 to 163 mg/liter. In this study, ethanol used as a carrier for these concentrations was kept constant at 10 ml/liter. In the present study, the effect of NP on daphnid neonate sex ratios was dependent on the use of ethanol as a carrier solvent. NP concentrations of 25 and 50 mg/ L with ethanol as a solvent (8 ml/L) caused a statistically significant decrease in sex ratios compared to the
ethanol control (8 ml/L). However, there was a statistically significant increase in sex ratios between the ethanol controls and media controls. This increase could not be repeated in the ethanol study using concentrations of 2, 4, and 8 ml/L. When daphnids were exposed to NP directly without solvent carrier, a slight but nonstatistically significant decrease in sex ratios was observed. Shurin and Dodson (1997) observed no effects on sex ratios of D. galeata mendotae exposed to NP at similar concentrations or in acetone controls. Sex ratios were also not affected by NP exposure in juvenile amphipods (Corophium volutator) (Brown et al., 1999). There are several possibilities for these apparent differences in sex ratios between studies. Ethanol may sensitize daphnids to conditions that stimulate male production and exaggerate the suppressive effect of NP when compared to solvent control groups. There might be an interaction between ethanol and NP, in which NP may disrupt the pathway ethanol uses to exert its effect on male offspring production. Results may also suggest that sex ratio in D. magna is too variable to be used as a reliable endpoint for evaluation of reproductive toxicity. Mean sex ratios in media controls could not be repeated between studies. Apparent differences may be due to annual cycles in the production of daphnids or variation in food quality. Based on the results, in order to see a statistically significant difference (Po0:05), a power test indicates a minimum number of replicates equaling 34. This large number of replicates needed makes the use of sex ratios as a general endpoint in reproductive studies impractical. It appears that the most sensitive effect to NP exposure during the chronic study was developmental abnormalities. Maternal exposure of NP at concentrations of 25 and 50 mg/L increased developmental deformities when fecundity was not affected. These deformities were similar to those following direct exposure, although at higher concentrations mentioned earlier, and are characteristic of late-stage developmental toxicity. The consistency in deformities in the chronic study and ex vivo study indicate that NP may affect specific ontogenetic processes directly or indirectly (Shurin and Dodson, 1997). In contrast, LeBlanc et al. (2000) showed that late-stage developmental toxicity was caused by maternal exposure to NP concentrations of 100 and 200 mg/L, but not by direct exposure. These investigators concluded that NP is not directly embryotoxic to daphnid embryos but requires exposure of gravid females during embryo development. Furthermore, this effect does not appear to be elicited by increasing endogenous testosterone levels but by interfering with maternal precursors of endogenous constituents critical to normal embryo development. In the present study, NP concentrations of 50 mg/L caused similar deformities following maternal exposure and direct embryo exposure. Further research is warranted
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to understand the mechanisms of developmental toxicity of NP in daphnids.
5. Conclusion When all results of the present studies are taken into consideration, it does not appear that NP presents a significant environmental hazard to D. magna. Although neonate deformities were observed at 25 and 50 mg/L, the percentages of total reproductive output were low (0.75% and 3.3%, respectively). These small increases in neonate deformities would not be expected to significantly affect the populations since total fecundity was not significantly decreased. Furthermore, no significant changes were observed in sex ratios following NP exposure (without solvent). However, the use of carrier solvents commonly used in ecotoxicity testing needs to be carefully considered. The present study indicates a possible interaction of ethanol with NP in modulating acute toxicity endpoints as well as reproductive endpoints such as fecundity and sex ratios. A greater understanding of these effects will greatly avoid confounding the interpretations of toxicity endpoints for industrial chemicals.
Acknowledgments The authors thank Nancy Baer for her valuable editorial comments on the article.
References Ahel, M., Giger, W., 1985. Determination of alkylphenols and alkylphenol mono- and diethoxylates in environmental samples by high-performance liquid chromatography. Anal. Chem. 57, 1577–1583. Baer, K.N., Goulden, C.E., 1998. Evaluation of a high-hardness COMBO medium and frozen algae for Daphnia magna. Ecotoxicol. Environ. Saf. 39, 201–206. Baer, K.N., Owens, K.D., 1999. Evaluation of selected endocrine disrupting compounds on sex determination in Daphnia magna using reduced photoperiod and different feeding rates. Bull. Environ. Contamin. Toxicol. 63 (3), 214–221. Baldwin, W.S., Graham, S.E., Shea, D., LeBlanc, G.A., 1997. Metabolic androgenization of female Daphnia magna by the xenoestrogen 4-nonylphenol. Environ. Toxicol. Chem. 16 (9), 1905–1911. Bennie, D.T., 1999. Review of the environmental occurrence of alkylphenols and alkylphenol ethoxylates. Water Qual. Res. J. Can. 34 (1), 79–122. Blackburn, M.A., Waldock, M.J., 1995. Concentrations of alkylphenols in rivers and estuaries in England and Wales. Water Res. 29, 1623–1629. Brooke, L.T., 1993. Acute and chronic toxicity of nonylphenol to ten species of aquatic organisms. Report to the US Environmental Protection Agency, Duluth, MN (contract no. 68-C1-0034). Lake
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Superior Research Institute, University of Wisconsin-Superior, Superior, WI. Brown, R.J., Conradi, M., Depledge, M.H., 1999. Long-term exposure to 4-nonylphenol affects sexual differentiation and growth of the amphipod Corophium volutator (Pallas, 1766). Sci. Tot. Environ. 233, 77–88. Christiansen, T., Korsgaard, B., Jespersen, A., 1998. Effects of nonylphenol and 17b-oestradiol on vitellogenin synthesis and testicular structure and cytology in male eelpout Zoarces viviparus. J. Exp. Biol. 201, 179–192. Comber, M.H.I., Williams, T.D., Stewart, K.M., 1993. The effects of nonylphenol on Daphnia magna. Water Res. 27, 273–276. Dodson, S.I., Frey, D.G., 1991. Cladocera and other Branchiopoda. In: Thorp, J.H., Covich, A.P. (Eds.), Ecology and Classification of North American Freshwater Invertebrates. Academic Press, San Diego, CA, pp. 723. Dodson, S.I., Merritt, C.M., Shannahan, J.-P., Shults, C.M., 1999. Low exposure concentrations of atrazine increase male production in Daphnia pulicaria. Environ. Toxicol. Chem. 18 (7), 1568–1573. Hob!ck, A., Larsson, P., 1990. Sex determination in Daphnia magna. Ecology 71, 2255–2268. Huls, . A.G., 1992. Acute and chronic toxicity of nonylphenol to Daphnia magna. Final Study Report Numbers DK-522, DL-143, Marl, Germany. Jobling, S., Sheahan, D., Osborne, J.A., Matthiessen, P., Sumpter, J.P., 1996. Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals. Environ. Toxicol. Chem. 15 (2), 194–202. Kast-Hutcheson, K., Rider, C.V., LeBlanc, G.A., 2001. The fungicide propiconazole interferes with embryonic development of the crustacean Daphnia magna. Environ. Toxicol. Chem. 20 (3), 502–509. Kinnberg, K., Korsgaard, B., Bjerregaard, P., Jespersen, A., 2000. Effects of nonylphenol and 17b-estradiol on vitellogenin synthesis and testis morphology in male platyfish Xiphophorus maculates. J. Exp. Biol. 203, 171–181. LeBlanc, G.A., Mu, X., Rider, C.V., 2000. Embryotoxicity of the alkylphenol degradation product of 4-nonylphenol to the crustacean Daphnia magna. Environ. Health Perspect. 108 (12), 1133–1138. Marcomini, A., Giger, W., 1987. Simultaneous determination of linear alkylbenzenesulfonates, alkylphenol polyethoxylates, and nonylphenol by high-performance liquid chromatography. Anal. Chem. 59, 1709–1715. Meregalli, G., Pluymers, L., Ollevier, F., 2001. Induction of mouthpart deformities in Chironomus riparius larvae exposed to 4-n-nonylphenol. Environ. Pollut. 111, 241–246. Monsanto Industrial Chemicals Co., 1985.Chemical hazard information profile: nonylphenol. Report to the Office of Toxic Substances, US Environmental Protection Agency, Washington, DC. Naylor, C.G., Mieure, J.P., Adams, W.J., Weeks, J.A., Castaldi, F.J., Ogle, L.G., Romano, R.R., 1992. Alkylphenol ethoxylates in the environment. J. Am. Oil Chem. Soc. 69, 695–703. Ohta, T., Tokishita, S., Shiga, Y., Hanazato, T., Yamagata, H., 1998. An assay system for detecting environmental toxicants with cultured Cladoceran eggs in vitro: malformation induced by ethylenethiourea. Environ. Res. 77, 43–48. Shurin, J.B., Dodson, S.I., 1997. Sublethal toxic effects of cyanobacteria and nonylphenol on environmental sex determination and development in Daphnia. Environ. Toxicol. Chem. 16 (6), 1269–1276. Zhang, L., Baer, K.N., 2000. The influence of feeding, photoperiod and selected solvents on the reproductive strategies of the water flea, Daphnia magna. Environ. Pollut. 110 (3), 425–430.
The effects of 4-nonylphenol and ethanol on acute toxicity, embryo development, and reproduction in Daphnia magna L. Zhang,a R. Gibble,b and K.N. Baerb,* a
The McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-8591, USA b Department of Toxicology, College of Pharmacy, The University of Louisiana at Monroe, Monroe, LA 71209-0470, USA Received 27 February 2002; accepted 8 July 2002
Abstract The mean 48-h EC50 (n ¼ 3) of 4-nonylphenol (NP) using ethanol as the carrier solvent was 155 mg/L, compared to a mean 48-h EC50 (n ¼ 3) of 281 mg/L without ethanol. The 96-h EC50’s for embryo lethality (arrested egg development) and deformities (curved or unextended shell spines and undeveloped second antennae) were 738 and 263 mg/L, respectively. Reproduction studies were conducted using conditions that stimulate male production (i.e., reduced photoperiod and food levels). An increase in neonate deformities was observed at 50 mg/L (without ethanol), but no changes were observed in fecundity or sex ratios. A decrease in sex ratios was observed at 25 and 50 mg/L (with ethanol) compared to the ethanol control. However, an increase in sex ratios was observed in the ethanol control compared to media controls. The use of ethanol as a solvent carrier confounds the effects of 4-NP on acute toxicity and male production in daphnids. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Daphnia; Reproduction; Sex ratio; 4-nonylphenol; Ethanol
1. Introduction Daphnia magna is one of the most important freshwater species employed in ecotoxicity testing throughout the world. Adequate survival, growth, and reproduction of daphnids are crucial for success in the environment, but more important is the ability of the species to survive adverse environmental conditions during winter. Most daphnids are cyclic parthenogenetic species capable of both asexual and sexual reproduction (Dodson and Frey, 1991). The survival strategy in sexual reproduction is to produce sufficient numbers of resting or winter eggs known as ephippia. Resting eggs require the presence of males for fertilization and development. These eggs then enter a diapause stage and hatch in spring as parthenogenetic females. The switch to male offspring production and subsequent sexual reproduction appears to be triggered naturally by environmental cues such as photoperiod, temperature, density, and food availability (Hobc! k and Larsson, 1990). The timing *Corresponding author. Fax: +1-318-342-1901. E-mail address: [email protected] (K.N. Baer).
of sexual reproduction and the optimal ratio of male to female offsprings appear critical for adequate ephippia production. Exposure to endocrine disrupting chemicals may disrupt this process. Nonylphenol (NP), a major degradative product of NP ethoxylates, has recently drawn attention because of its intrinsic estrogenic potential. Nonylphenol concentrations of 54.3 mg/L have been demonstrated to induce the formation of vitellogenin and inhibit testicular growth in male trout (Jobling et al., 1996). Similar effects attributed to NP exposure have been reported in other fish species (Christiansen et al., 1998; Kinnberg et al., 2000). Nonylphenol has been measured in sewage effluent and river water in the UK at concentrations of 330 and 180 mg/L, respectively (Blackburn and Waldock, 1995). However, concentrations in US rivers generally range from o0.11 to 0.64 mg/L (Naylor et al., 1992; reviewed in Bennie, 1999). Nonylphenol has been demonstrated to be acutely toxic to daphnids; however, at concentrations substantially higher than those normally found in the environment. For example, acute EC50 values in Daphnia
0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(02)00081-7
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neonates range from 104 to 440 mg/L (Monsanto, 1985; Huls, . 1992; Brooke, 1993; Comber et al., 1993). Recent studies have focused on the toxicity of NP to developing embryos and larvae, perhaps a more sensitive invertebrate life stage to these environmental contaminants. Nonylphenol concentrations of 100 mg/L caused mouthpart deformities in chironomid larvae, Chironomus riparius (Meregalli et al., 2001). However, in D. magna, LeBlanc et al. (2000) observed developmental retardation via maternal exposure to 100 and 200 mg/L NP, but not during direct embryo exposure. Chronic studies with NP have also been conducted to assess survival, growth, and asexual reproduction in daphnids. The no-observed-effect concentration (NOEC) for reproduction ranges from 10 to 116 mg/L, still higher than most environmental concentrations (Huls, . 1992; Comber et al., 1993; Brooke, 1993; Baldwin et al., 1997; Shurin and Dodson, 1997). However, studies have demonstrated that sexual reproduction may be more sensitive to certain toxicants than asexual reproduction. For example, changes in Daphnia sex ratio were observed to be one to two orders of magnitude more sensitive to atrazine than survivorship or fecundity (Dodson et al., 1999). The influence of NP on sex ratios in D. magna is not clear. Several studies have not observed changes in sex ratios following NP exposure (Shurin and Dodson, 1997), whereas previous studies in our laboratory indicate a possible interaction between carrier solvents and NP on sex ratios (Baer and Owens, 1999; Zhang and Baer, 2000). These interactions need to be clarified before interpretations can be made concerning NP exposure on sex ratios in daphnids. In recent studies, carrier solvents routinely employed in ecotoxicity testing have produced some surprising effects. Dodson et al. (1999) observed a 40% increase in the sex ratio of D. pulicaria in acetone controls (45 ml/L) compared to water controls. However, under different conditions, acetone (80 ml/L) increased female offspring in D. galeata mendotae while having no effect on male production (Shurin and Dodson, 1997). The increase in fecundity was not observed in D. magna at an acetone concentration of 90 ml/L (Comber et al., 1993). Studies in our laboratory indicate that acetone (100 ml/L) and ethanol (10 ml/L) caused a significant increase of fecundity at low food levels while the effects on sex ratio varied depending on experimental conditions (Zhang and Baer, 2000). The possibility that carrier solvents may be modulating reproductive endpoints under certain experimental conditions may have farreaching implications for ecotoxicity testing protocols. In view of these findings, the objective of the present study was to investigate the effects of 4-NP and ethanol on acute toxicity, embryo development, and reproduction in D. magna.
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2. Materials and methods 2.1. Chemicals 4-Nonylphenol (B85% based on p-isomers) was purchased from Fluka Chemika (Milwaukee, WI). Ethanol (Absolute-200 Proof) was purchased from AAPER Alcohol and Chemical Company (Shelbyville, KY). High-performance liquid chromatography(HPLC) grade acetonitrile and methanol used in the HPLC system were purchased from Fisher Scientific (Houston, TX). 2.2. Animal cultures Daphnia magna Straus cultures (clone 5 obtained from the Academy of Natural Sciences of Philadelphia) were held in 1-L glass beakers containing B900 mL of H-H COMBO medium (Baer and Goulden, 1998). The temperature was maintained at 2072 C and a 16-h light:8-h dark photoperiod (illumination ranged between 300 and 450 lux) was employed. The medium was renewed and daphnids were fed a green algae species, Ankistrodesmus falcatus (15 106 cells/beaker), three times weekly (i.e., Monday, Wednesday, and Friday). These conditions allowed for continuous parthenogenetic reproduction in laboratory cultures. 2.3. Acute toxicity studies To evaluate the effects of ethanol as a carrier solvent on the 48-h acute toxicity of NP, randomly selected neonates from laboratory cultures (o24 h old, Xthirdbrood) were used. Nominal test concentrations of NP with and without ethanol as a carrier solvent were 39, 65, 108, 180, 300, and 500 mg/L. At each concentration, two replicates with five neonates per zeplicate were employed. Stock solutions of NP using a carrier solvent were prepared by dissolving the appropriate amount of NP into ethanol. The solvent limit in these test concentrations was p8 ml/L (ethanol control was 8 ml/ L). For studies without ethanol as the carrier solvent, a stock solution of NP (3.6 mg/L) was prepared by dissolving 2 mL NP into distilled water followed by ultrasonication for 1 h. A 100-mL syringe was loaded with NP from behind by a 1-mL syringe, then 2 mL NP was injected from the 100-mL syringe into a 1-L beaker containing 500 mL distilled water. The solubility and stability of the NP stock solution (3.6 mg/L) were measured by HPLC (see below). All studies were conducted in a Cryo-Fridge environmental chamber (Revco Scientific, Asheville, NC) with a constant temperature (20 72 C) and normal photoperiod (16-h light:8-h dark). Temperature, dissolved oxygen, and pH were monitored at 0, 24, and 48 h. Immobility (synonymous with death) was observed daily.
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2.4. Ex vivo embryo development study
2.6. Analytical methodology
Daphnid embryos are capable of normal development outside of the brood pouch and are therefore suitable for ex vivo studies. Adult females from normal laboratory cultures bearing more than 10 parthenogenetic eggs less than 6 h old (stage 1 as described by Kast-Hutcheson et al., 2001) were selected and placed in petri dishes containing H-H COMBO medium. The daphnids were placed under a dissecting microscope and the abdominal side of the carapace was held with the aid of fine forceps. Eggs were removed carefully from the brood chamber by gently swirling the body of the animal in the medium (Ohta et al., 1998). The isolated eggs were pooled and placed into 10 plastic cups containing 25 mL of NP test solutions (10 eggs/ cup). Nominal test concentrations using ethanol as the carrier solvent (8 ml/L) were 50, 200, 800, and 1000 mg/L. All test containers were covered and placed in the environmental chamber with a constant temperature (2072 C) and normal photoperiod (16-h light:8-h dark). Embryo development was observed at 24, 48, and 96 h. The live and/or deformed neonates were observed at 96-h.
The solubility and stability of NP stock solution in distilled water (3.6 mg/L; without ethanol) was measured at 0, 48, and 72 h according to methods previously reported (Ahel and Giger, 1985; Marcomini and Giger, 1987). Nominal NP concentrations used in the study were below the limit of detection for this method. Prior to HPLC analysis, samples were subjected to solid-phase extraction (SPE) using C-18 Bond Elut LRC extraction cartridges that were preconditioned with methanol. Nonylphenol stock solutions (n ¼ 4) were eluted with methanol, ultrasonicated, and analyzed by HPLC. Samples and standards (prepared in methanol) were analyzed on a P2000 SpectraSYSTEM gradient pump equipped with a SCM 1000 Vacuum Membrane Degasser and an AS 1000 SpectraSYSTEM autosampler (Thermo Separation Product, Inc., Riviera Beach, FL). Optimum separation was achieved using a reverse-phase Beckman Ultrasphere Octyl 5-mm C-18 column (4.6 mm 25 cm) purchased from Varian Analytical (Walnut Creek, CA). The system was operated at ambient temperature and the flow rate was 1.0 mL/min with degassed methanol as the mobile phase. The injection volume was 50 mL. Detection was performed by a SpectraSYSTEM UV 3000 Multiple-Wavelength Detector (Thermo Separation Product, Inc., Riviera Beach, FL) set at 224 nm. A ChromQuest Chromatography Workstation for Windows NT (ThermoQuest, Inc., San Jose, CA) was used for integration.
2.5. Chronic reproduction studies The effect of NP and ethanol on the reproduction of daphnids was investigated under conditions that stimulate male production. Neonates (o24 h old, Xthird brood) were exposed to the following conditions: NP concentrations of 12.5, 25, and 50 mg/L using ethanol as carrier solvent (p8 ml/L); NP concentrations of 25 and 50 mg/L without ethanol; ethanol concentrations of 2, 4, and 8 ml/L. Ten beakers per treatment group were used with one daphnid per beaker. All experiments were conducted in an environmental chamber with constant temperature (2072 C) and short photoperiod (8-h light:16-h dark). The medium was renewed and daphnids were fed a green algae species, A. falcatus (3.75 106 cells/beaker), three times weekly (i.e., Monday, Wednesday, and Friday). During the experiments, temperature, dissolved oxygen, pH, total hardness, total alkalinity, and conductivity were monitored weekly. Endpoints included adult survival, total number of molts, total number of live neonates (fecundity), total number of aborted eggs, and sex ratio. The sex and morphology of neonates were observed and counted using a dissecting microscope. Male daphnids were identified by the presence of large, prominent first antennules. The sex ratio was determined as the total number of males divided by the total number of neonates (Dodson et al., 1999).
2.7. Statistical analyses The EC50’s were calculated by probit analysis using SAS statistical software (SAS Institute, Inc., Cary, NC). All chronic data were tested for statistical significance using a single factor one-way analysis of variance (ANOVA). Supplementary tests included Fisher’s least significant difference and Newman–Keul’s tests. Homogeneity of variances and normality among replicates were determined by Bartlett’s test and Komogorov– Smirnov test, respectively. All tests were performed using standard software (JMP IN, SAS Institute, Inc., Cary, NC).
3. Results 3.1. Acute toxicity studies The summary of 48-h acute toxicity tests with NP is presented in Table 1. The 48-h acute EC50’s without ethanol as the carrier solvent ranged from 234 to 337 mg/L (mean ¼ 281). The 48-h EC50’s with ethanol as the carrier solvent ranged from 94 to 199 mg/L (mean ¼ 155). Although the means are statistically
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significant, the 95% confidence limits overlap and the solvent studies exhibited a poorly defined dose–response relationship (r2 ranging from 0.78 to 0.83). However, these results suggest that the use of ethanol as a carrier solvent increases the acute toxicity of NP to daphnid neonates. Recoveries of NP stock solution (3.6 mg/L) without ethanol immediately after preparation averaged 94% (Table 2; CV=2.8%). The concentrations of the NP stock measured at 0, 48, and 72 h ranged from 89% to 95% (Table 2). These results indicate that the NP stock solutions without ethanol were accurately prepared and soluble prior to testing.
(Table 1). Examples of arrested development in embryos and neonate deformities are presented in Fig. 1. The most sensitive effect was late-stage neonate deformities (curved or unextended shell spines and undeveloped second antennae) observed at 50 and 200 mg/L (10% and 90%, respectively). Arrested egg development was only observed at higher NP concentrations (800 and 1000 mg/L). Embryos exposed to 800 mg/L for 48 h exhibited arrested development (Figs. 1E and F), and severe deformities in neonates were observed at 96 h (Figs. 1H and I).
3.2. Ex vivo embryo development study
3.3. Chronic reproduction studies
The 96-h ex vivo embryo development EC50’s for embryo lethality (arrested egg development) and neonate deformities were 738 and 263 mg/L, respectively
The effect of NP (with and without ethanol as a carrier solvent) and ethanol on daphnid survival and reproduction was investigated in 35-day chronic studies. Adult survival in NP concentrations of 12.5, 25, and 50 mg/L using ethanol as a carrier solvent was 80%, 90%, and 60%, respectively (results not presented). Adult survival in NP concentrations of 25 and 50 mg/L without ethanol was 100% and 90%, respectively (results not presented). No adult mortality was observed in the ethanol concentrations (2, 4, and 8 mL). No effects were observed in the total number of juvenile molts in any treatment group. The effects of different exposures on fecundity are presented in Fig. 2. Ethanol significantly increased fecundity at 4 and 8 mL (Fig 2A, ethanol control, C). There was a statistically significant decrease in fecundity at 12.5 mg/L compared to the ethanol control (Fig. 2A), but this decrease was not considered biologically significant because of a lack of significance compared to the media control. Nonylphenol concentrations of 25 and 50 mg/L with ethanol as the solvent carrier significantly decreased sex ratios compared to the ethanol control but not the media control (Fig. 3A). However, sex ratios in 25 and 50 mg/L without ethanol were not significantly different from controls (Fig. 3B). The increase in sex ratios observed in the ethanol control (8 ml/L; Fig. 2A) was not repeated in the ethanol study (Fig. 3C). The results indicate that sex ratios are variable and can be influenced by solvent carriers such as ethanol. A low, but statistically significant, increase in neonate deformities (3.3%) was observed at 50 mg/L
Table 1 Summary of 48-h acute toxicity tests and 96-h embryo development test with NP Test
EC50a (mg/L)
48-h acute tests No solvent 234 No solvent 272 No solvent 337
r2
95% Equation confidence limits (lower–upper, mg/L) 157–245 167–481 193–1014
y ¼ 7:943 þ 0:2479x 0.93 y ¼ 15:21 þ 0:2396x 0.91 y ¼ 17:59 þ 0:2003x 0.91
33–450 NA NA
y ¼ 0:0971 þ 0:2511x y ¼ 33:383 þ 0:1777x y ¼ 12:732 þ 0:2172x
NA NA
y ¼ 8:527 þ 0:0793x 0.84 y ¼ 20:322 þ 0:113x 0.80
Mean (SEM) 281 (30) Solvent Solvent Solvent
199 94 172
0.83 0.78 0.83
Mean (SEM) 155 (32)b 96-h embryo test Solvent 738c 263d a
Immobility or death. Statistically significant from no solvent. c Based on arrested egg development. d Based on deformed neonates, i.e., curved or unextended shell spines and undeveloped first antennae. b
Table 2 Measured concentrations of NP stock solutions without ethanol Nominal concentrations 3.6 mg/L
1 2 3 4 Mean (SEM)
Measured concentrations (mg/L) 0h
48 h
72 h
3.49 3.33 3.28 3.43 3.3870.048
2.97 3.56 3.35 3.25 3.2870.122
3.70 3.35 2.95 3.19 3.3070.157
Mean (SEM) (%)
Mean (%) nominal
3.3970.22 3.4170.074 3.1970.12 3.2970.072 3.3270.051
94 95 89 91 9271.4
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(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
(I)
Fig. 1. Developmental abnormalities elicited by 4-NP. (A) Embryo ( 100) in control group at 24 h. (B, C) Embyros ( 100) exposed to NP (800 mg/L) at 24 h. (D) Embryo ( 40) in control group at 48 h. (E, F) Embyros ( 40) exposed to NP (800 mg/L) at 48 h. (G) Normally developed nenonates ( 40) in control group at 96 h. (H, I) Deformed neonates ( 40) exposed to NP (800 mg/L) at 96 h.
without ethanol and were identical to those observed in the ex vivo study (Figs. 1H and I). These deformities were consistent with late-stage developmental toxicity, i.e., curved or unextended shell spines and undeveloped second antennae. No other statistically significant increases in deformities were observed in NP concentrations with ethanol or in the ethanol study alone.
4. Discussion Nonylphenol was acutely toxic to daphnid neonates (with or without ethanol as a carrier solvent) with mean EC50 values of 155 and 281 mg/L, respectively. The literature reports acute toxicity values in daphnids ranging from 104 to 440 mg/L (Monsanto, 1985; Huls, .
*
120 100 80 60 40 20 0
Sex Ratio (%)
Fecundity
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#
Control
(A)
Ethanol Control
12.5
25
35 30 25 20 15 10 5 0
*
Control
50
(A)
120
Sex Ratio (%)
Fecundity
100 80 60 40 20 0 25
120
Sex Ratio (%)
Fecundity
Control
(B)
*
100
*
80 60 40 20 0 Control
(C)
2
4
12.5
#
25
50
35 30 25 20 15 10 5 0
50
4-nonylphenol (ug/l)
(B)
Ethanol Control
#
4-nonylphenol (ug/l)
4-nonylphenol (ug/l)
Control
335
8
Ethanol (ul/l)
50
4-nonylphenol (ug/l) 35 30 25 20 15 10 5 0 Control
(C)
25
2
48
Ethanol (ul/l)
Fig. 2. The effect of NP and ethanol (mL/L) on fecundity of D. magna under reduced photoperiod and food levels. All values represent mean7SEM. (A) The effects of NP using ethanol carrier solvent. (B) The effects of NP without ethanol carrier solvent. (C) The effects of ethanol. *Statistically significant from control, Po0:05: #Statistically significant from ethanol control, Po0:05:
Fig. 3. The effect of NP and ethanol (mL/L) on the sex ratio D. magna under reduced photoperiod and food levels. All values represent mean7SEM. (A) The effects of NP using ethanol carrier solvent. (B) The effects of NP without ethanol carrier solvent. (C) The effects of ethanol. *Statistically significant from control, Po0:05: #Statistically significant from ethanol control, Po0:05:
1992; Brooke, 1993; Comber et al., 1993). It is interesting to note that in the present study, significantly higher acute toxicity values were observed in the presence of ethanol as a carrier solvent (p8 ml/L) compared to studies without ethanol. This result was repeated over time by different technicians using different batches of daphnids. Similarly in the chronic study, 50 mg/L with ethanol resulted in 40% adult mortality compared to 10% in the same concentration without ethanol. Solvents are routinely used to deliver and enhance the solubility of NP in aqueous solutions. However, rigorous attempts to solubilize NP in water for studies without the use of ethanol (repeated mixing and sonication) were successful. Analytical recoveries of NP stock solutions (3.6 mg/L) averaged 94% of nominal concentrations, and these solutions were relatively stable for 72 h. Differences in the acute toxicity of NP with and without solvent may still be due to differences in solubility since all EC50 values were based on nominal
concentrations and not on measurements of actual test solutions. A more probable explanation is that solvents such as ethanol may enhance absorption of organic chemicals across biological membranes. However, the biological significance of this apparent increase may not be that dramatic since acute toxicity endpoints for a single species often differ by a factor of 2 depending on experimental design, i.e., exposure conditions, concentrations employed, and so on. Nevertheless, the potential influence of solvents on toxicity endpoints should be considered when evaluating and comparing data. The effects of NP on embryo development demonstrated a dose–response increase in arrested eggs (EC50=738 mg/L) and deformed neonates (EC50= 263 mg/L), the latter being a more sensitive indicator of toxicity in this assay. At NP concentrations of 200 mg/L, developmental deformities such as curved or unextended shell spines and undeveloped second antennae were observed. These deformities are characteristic of
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late-stage toxicity (stages 4–6 as described by KastHutcheson et al., 2001). Early stage embryo toxicity characterized by arrested egg development was only observed at the two highest concentrations (800 and 1000 mg/L). Direct exposure of C. riparius larvae to 100 mg/L NP was shown to cause mouthpart deformities (Meregalli et al., 2001). However, in contrast to the present study, LeBlanc et al. (2000) observed no developmental effects following direct NP concentrations of 100 and 200 mg/L in D. magna. The reason for these apparent discrepancies is currently unknown. Chronic studies using conditions that stimulate male production resulted in different reproductive endpoints depending on whether NP was delivered using ethanol as a solvent carrier. A previous study in our laboratory demonstrated that acetone (100 ml/L) was effective at increasing fecundity in a variety of exposure conditions (combinations of food levels and photoperiods) except in a high food and reduced photoperiod group (Zhang and Baer, 2000). Other investigators have observed similar results in conditions that favor male production. For example, Shurin and Dodson (1997) observed an increase in female offspring production in D. galeata mendotae at acetone levels of 80 ml/L compared with controls. However, Comber et al. (1993) using 90 ml/L acetone and experimental conditions conducive to parthenogenetic reproduction observed no increase in fecundity. The present study demonstrated that exposure to ethanol at 4 or 8 ml/L increased fecundity during conditions that stimulate male production. One possible explanation may be a stimulatory effect of ethanol on algal growth since increasing food levels have been shown to increase fecundity in a concentration-dependent fashion (Zhang and Baer, 2000). Ethanol was shown to have a slight stimulatory effect on algal growth at 8 ml/liter (results not presented), but this increase was not sufficient to explain the increase in fecundity in the present study. Previous studies indicate that high concentrations of ethanol (100 ml/liter) increased fecundity independent of food levels and photoperiod (Zhang and Baer, 2000). These results seem to indicate a direct effect of ethanol on the endocrine system, i.e., possibly by enhancing vitellogenesis and increasing reproduction endpoints. The effects of NP at 25 and 50 mg/liter had no effect on fecundity either with or without the use of ethanol as a solvent carrier. This is in contrast to LeBlanc et al. (2000), who demonstrated a dosedependent stimulatory effect of NP on fecundity using concentrations ranging from 31 to 163 mg/liter. In this study, ethanol used as a carrier for these concentrations was kept constant at 10 ml/liter. In the present study, the effect of NP on daphnid neonate sex ratios was dependent on the use of ethanol as a carrier solvent. NP concentrations of 25 and 50 mg/ L with ethanol as a solvent (8 ml/L) caused a statistically significant decrease in sex ratios compared to the
ethanol control (8 ml/L). However, there was a statistically significant increase in sex ratios between the ethanol controls and media controls. This increase could not be repeated in the ethanol study using concentrations of 2, 4, and 8 ml/L. When daphnids were exposed to NP directly without solvent carrier, a slight but nonstatistically significant decrease in sex ratios was observed. Shurin and Dodson (1997) observed no effects on sex ratios of D. galeata mendotae exposed to NP at similar concentrations or in acetone controls. Sex ratios were also not affected by NP exposure in juvenile amphipods (Corophium volutator) (Brown et al., 1999). There are several possibilities for these apparent differences in sex ratios between studies. Ethanol may sensitize daphnids to conditions that stimulate male production and exaggerate the suppressive effect of NP when compared to solvent control groups. There might be an interaction between ethanol and NP, in which NP may disrupt the pathway ethanol uses to exert its effect on male offspring production. Results may also suggest that sex ratio in D. magna is too variable to be used as a reliable endpoint for evaluation of reproductive toxicity. Mean sex ratios in media controls could not be repeated between studies. Apparent differences may be due to annual cycles in the production of daphnids or variation in food quality. Based on the results, in order to see a statistically significant difference (Po0:05), a power test indicates a minimum number of replicates equaling 34. This large number of replicates needed makes the use of sex ratios as a general endpoint in reproductive studies impractical. It appears that the most sensitive effect to NP exposure during the chronic study was developmental abnormalities. Maternal exposure of NP at concentrations of 25 and 50 mg/L increased developmental deformities when fecundity was not affected. These deformities were similar to those following direct exposure, although at higher concentrations mentioned earlier, and are characteristic of late-stage developmental toxicity. The consistency in deformities in the chronic study and ex vivo study indicate that NP may affect specific ontogenetic processes directly or indirectly (Shurin and Dodson, 1997). In contrast, LeBlanc et al. (2000) showed that late-stage developmental toxicity was caused by maternal exposure to NP concentrations of 100 and 200 mg/L, but not by direct exposure. These investigators concluded that NP is not directly embryotoxic to daphnid embryos but requires exposure of gravid females during embryo development. Furthermore, this effect does not appear to be elicited by increasing endogenous testosterone levels but by interfering with maternal precursors of endogenous constituents critical to normal embryo development. In the present study, NP concentrations of 50 mg/L caused similar deformities following maternal exposure and direct embryo exposure. Further research is warranted
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to understand the mechanisms of developmental toxicity of NP in daphnids.
5. Conclusion When all results of the present studies are taken into consideration, it does not appear that NP presents a significant environmental hazard to D. magna. Although neonate deformities were observed at 25 and 50 mg/L, the percentages of total reproductive output were low (0.75% and 3.3%, respectively). These small increases in neonate deformities would not be expected to significantly affect the populations since total fecundity was not significantly decreased. Furthermore, no significant changes were observed in sex ratios following NP exposure (without solvent). However, the use of carrier solvents commonly used in ecotoxicity testing needs to be carefully considered. The present study indicates a possible interaction of ethanol with NP in modulating acute toxicity endpoints as well as reproductive endpoints such as fecundity and sex ratios. A greater understanding of these effects will greatly avoid confounding the interpretations of toxicity endpoints for industrial chemicals.
Acknowledgments The authors thank Nancy Baer for her valuable editorial comments on the article.
References Ahel, M., Giger, W., 1985. Determination of alkylphenols and alkylphenol mono- and diethoxylates in environmental samples by high-performance liquid chromatography. Anal. Chem. 57, 1577–1583. Baer, K.N., Goulden, C.E., 1998. Evaluation of a high-hardness COMBO medium and frozen algae for Daphnia magna. Ecotoxicol. Environ. Saf. 39, 201–206. Baer, K.N., Owens, K.D., 1999. Evaluation of selected endocrine disrupting compounds on sex determination in Daphnia magna using reduced photoperiod and different feeding rates. Bull. Environ. Contamin. Toxicol. 63 (3), 214–221. Baldwin, W.S., Graham, S.E., Shea, D., LeBlanc, G.A., 1997. Metabolic androgenization of female Daphnia magna by the xenoestrogen 4-nonylphenol. Environ. Toxicol. Chem. 16 (9), 1905–1911. Bennie, D.T., 1999. Review of the environmental occurrence of alkylphenols and alkylphenol ethoxylates. Water Qual. Res. J. Can. 34 (1), 79–122. Blackburn, M.A., Waldock, M.J., 1995. Concentrations of alkylphenols in rivers and estuaries in England and Wales. Water Res. 29, 1623–1629. Brooke, L.T., 1993. Acute and chronic toxicity of nonylphenol to ten species of aquatic organisms. Report to the US Environmental Protection Agency, Duluth, MN (contract no. 68-C1-0034). Lake
337
Superior Research Institute, University of Wisconsin-Superior, Superior, WI. Brown, R.J., Conradi, M., Depledge, M.H., 1999. Long-term exposure to 4-nonylphenol affects sexual differentiation and growth of the amphipod Corophium volutator (Pallas, 1766). Sci. Tot. Environ. 233, 77–88. Christiansen, T., Korsgaard, B., Jespersen, A., 1998. Effects of nonylphenol and 17b-oestradiol on vitellogenin synthesis and testicular structure and cytology in male eelpout Zoarces viviparus. J. Exp. Biol. 201, 179–192. Comber, M.H.I., Williams, T.D., Stewart, K.M., 1993. The effects of nonylphenol on Daphnia magna. Water Res. 27, 273–276. Dodson, S.I., Frey, D.G., 1991. Cladocera and other Branchiopoda. In: Thorp, J.H., Covich, A.P. (Eds.), Ecology and Classification of North American Freshwater Invertebrates. Academic Press, San Diego, CA, pp. 723. Dodson, S.I., Merritt, C.M., Shannahan, J.-P., Shults, C.M., 1999. Low exposure concentrations of atrazine increase male production in Daphnia pulicaria. Environ. Toxicol. Chem. 18 (7), 1568–1573. Hob!ck, A., Larsson, P., 1990. Sex determination in Daphnia magna. Ecology 71, 2255–2268. Huls, . A.G., 1992. Acute and chronic toxicity of nonylphenol to Daphnia magna. Final Study Report Numbers DK-522, DL-143, Marl, Germany. Jobling, S., Sheahan, D., Osborne, J.A., Matthiessen, P., Sumpter, J.P., 1996. Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals. Environ. Toxicol. Chem. 15 (2), 194–202. Kast-Hutcheson, K., Rider, C.V., LeBlanc, G.A., 2001. The fungicide propiconazole interferes with embryonic development of the crustacean Daphnia magna. Environ. Toxicol. Chem. 20 (3), 502–509. Kinnberg, K., Korsgaard, B., Bjerregaard, P., Jespersen, A., 2000. Effects of nonylphenol and 17b-estradiol on vitellogenin synthesis and testis morphology in male platyfish Xiphophorus maculates. J. Exp. Biol. 203, 171–181. LeBlanc, G.A., Mu, X., Rider, C.V., 2000. Embryotoxicity of the alkylphenol degradation product of 4-nonylphenol to the crustacean Daphnia magna. Environ. Health Perspect. 108 (12), 1133–1138. Marcomini, A., Giger, W., 1987. Simultaneous determination of linear alkylbenzenesulfonates, alkylphenol polyethoxylates, and nonylphenol by high-performance liquid chromatography. Anal. Chem. 59, 1709–1715. Meregalli, G., Pluymers, L., Ollevier, F., 2001. Induction of mouthpart deformities in Chironomus riparius larvae exposed to 4-n-nonylphenol. Environ. Pollut. 111, 241–246. Monsanto Industrial Chemicals Co., 1985.Chemical hazard information profile: nonylphenol. Report to the Office of Toxic Substances, US Environmental Protection Agency, Washington, DC. Naylor, C.G., Mieure, J.P., Adams, W.J., Weeks, J.A., Castaldi, F.J., Ogle, L.G., Romano, R.R., 1992. Alkylphenol ethoxylates in the environment. J. Am. Oil Chem. Soc. 69, 695–703. Ohta, T., Tokishita, S., Shiga, Y., Hanazato, T., Yamagata, H., 1998. An assay system for detecting environmental toxicants with cultured Cladoceran eggs in vitro: malformation induced by ethylenethiourea. Environ. Res. 77, 43–48. Shurin, J.B., Dodson, S.I., 1997. Sublethal toxic effects of cyanobacteria and nonylphenol on environmental sex determination and development in Daphnia. Environ. Toxicol. Chem. 16 (6), 1269–1276. Zhang, L., Baer, K.N., 2000. The influence of feeding, photoperiod and selected solvents on the reproductive strategies of the water flea, Daphnia magna. Environ. Pollut. 110 (3), 425–430.