Drinking water source and reproductive outcomes in Sprague-Dawley rats

Pergamon

0890-6238(95)02005-8

Reproductive Toxicology, Vol. 9, No. 6. pp. 549-561, 1995 Coavrieht 0 1995 Elsevier Science Inc. Printed in the USA. All rights reserved

OWO-6238/95 $9.50 + .OO

DRINKING WATER SOURCE AND REPRODUCTIVE OUTCOMES IN SPRAGUE-DAWLEY RATS’

JANET Y. URIU-HARE,* SHANNA H. SWAN,? LINH M. Bur,* RAYMOND R. NEUTRA,~ and CARL L. KEEN**

*Department of Nutrition, *Department of Internal Medicine, University of California at Davis, Davis, California, tDivision of Environmental and Occupational Disease Control, California Department

of Health Services, Berkeley, California

Abstract - As part of our work on the influence of water source on reproductive outcome, Sprague-Dawley rats were randomized to tap water, bottled water, or deionized water treatment groups, utilizing 160 animals per treatment; animals received the water prior to and during pregnancy. Rats were shipped in four batches (A-D). Batch effects were seen for several reproductive parameters. Because the tap water supply was interrupted by an earthquake resulting in an unbalanced design, primary analyses utilized only batches C and D, which included most of the tap water-treated rats. A treatment effect with respect to resorption frequency was seen that was marginally significant using a fixed-effects analysis of variance (P = 0.053), but not when batch was entered as a random effect (I’ = 0.36). The data were modeled by logistic regression, controlling for batch, litter size, and batch-treatment interaction. The odds ratio comparing tap to bottled water was 1.8 (95% CI 1.0 to 3.3, P = 0.05), which was similar to the epidemiologic result that prompted this study. The magnitude of this association varied by batch, and the difference in resorption frequency was within the range of variation seen for control animals. Although these findings do not justify pubiic health action at this time, further investigation is warranted.

Key Words: reproduction; pregnancy outcome; resorption; water; bacteria; rats.

INTRODUCTION

Several recent epidemiologic studies conducted by the California Department of Health Services suggested that risk of spontaneous abortion may be related to source and amount of drinking water consumed during early pregnancy (l-4). In this group of studies, conducted primarily in one county in California, spontaneous abortion rates were higher among women who reported drinking tap water during the first trimester than among women who reported drinking bottled water. Although the magnitude of the association differed somewhat among studies, the results suggested a 25 to 50% greater risk of spontaneous abortion in women who drank tap (or mostly tap) water during the first trimester of their pregnancy compared to those who drank bottled (or mostly bottled) water (5). If causal, these results could be explained by a reproductive toxicant

‘Supported in part by the California Department of Health Services, and HD 072241 (J.Y.U-H).

Address correspondence to Dr. Carl L. Keen, Department of Nutrition, University of California at Davis, Davis, CA 95616- 8669.

Received 3 March 1995; Revision received 5 June 1995; Ac- cepted 10 June 1995.

549

in the tap water in (some or all of) the study areas, by something beneficial in the bottled water consumed in those areas, or both. These results prompted additional epidemiologic studies, exten- sive water analyses, and two animal studies. The first of these animal studies (6) examined resorption frequency in Fischer 344 rats treated between days 0 and 20 of gestation. The study included three treatment groups, each with 60 dams, to which rats were assigned after mating. Treatments included tap water collected from the homes of subjects in one of the epidemiologic studies, a commercial bottled water used widely in the study area, and HPLC laboratory- grade water (water in which all organics were below the limit of detection using -high-performance liquid chromatography). That study found a somewhat higher resorption frequency (average resorptions/ litter) in rats fed tap water compared to bottled water (5.5% vs. 3.4%; one-sided P-value = 0.05). This result of a 60% greater resorption frequency in tap than bottled water-fed rats was similar to results in the epidemiologic studies. In the first animal study it was necessary to examine 78 HPLC water-fed rats, compared to 69 bottled and 69 tap water-fed rats that showed sperm plugs to reach the desired

550 Reproductive Toxicology Volume 9. Number 6. 199.5

sample size of 60 “successful” pregnancies (i.e.. evidence of fetuses and/or resorptions). Although these differences were not statistically significant (P = 0.12 comparing HPLC-water vs. either tap OI bottled water), they suggested that HPLC-water had no advantage over distilled deionized water, the usual control water in this laboratory. Therefore, in this second experiment, distilled deionized water was selected as the treatment against which tap and bottled water were compared.

The study reported here differed from the first animal study in other ways. First, Sprague-Dawley rats were used instead of Fischer 344 rats to obtain data from a second strain, as well as to utilize a strain of animals commonly used in our laboratory. Secondly, the sample size was increased to 160 animals per treatment group so that a 50% increase in the proportion of dams with one or more resorptions could be reliably detected, even in this strain with a low resorption frequency. Resorption frequencies in “control” groups have typically averaged < SC/c over the past several years in our laboratory. Third, dams were treated prior to, as well as during, pregnancy in an attempt to amplify any differences among treatment groups.

This experiment also differed from many toxicology protocols in several respects. The difference in resorption frequency of4 to 5.5% that was hypoth- esized is smaller than that usually sought in toxicologic studies. Moreover, no single “agent” was tested. Therefore. there was no attempt to obtain a dose response. Although this might have been possible by concentrating or diluting the drinking water. such procedures might also have altered the (hypothetical) biologic or chemical agent. For this reason. animals were treated with tap and bottled water as consumed by subjects in the epidemiologic study. Although this approach is nontraditional, the prior epidemiologic and toxicologic results suggested that a change in resorption frequency of the magnitude predicted should be detectable with the study design and sample size employed.

MATERIALS AND METHODS

Virgin female Sprague-Dawley rats weighing between 180 and 200 g were obtained from a commercial vendor (Bantin and Kingman, Fremont. CA) between September 1989 and June 1990. Rats were housed individually in stainless steel cages (7 x

9-l /4 x 7-l/4”) in a controlled environment (2 1 to 23 “C; 12 h light: dark cycle, lights on at 0700 h). Humidity was 40 to 70%; air changes were 10 to 15/h. The animals were fed a commercial rat chow

(Purina Rat Chow, Belmont, CA) ad lib throughout the study. Tap water samples were taken from five of the homes in which participants in the epidemiologic study resided. These households were served by a single water company and received unchlorinated and untreated ground water. Water samples were collected weekly in autoclaved glass carboys and samples from the five households were pooled before storage. The brand of bottled water used in this study was that consumed most often by study subjects in the area from which tap water was obtained. Distilled deionized water is the control water routinely employed by this laboratory. Rats were randomly assigned to one of three treatment groups: tap water (TAP); bottled water (BOTTLED); and deionized distilled water (DEIONIZED). In the Fischer 344 rat study, conducted previously, an increase in normally occurring saprophytic bacteria had been observed after refrigerated storage of the water for several weeks. Therefore, in the current study, all water samples were collected weekly and stored in glass bottles at 4 “C to minimize bacterial growth. Water was provided to the rats in glass bottles with stainless steel sippers. Drinking water was changed and water intake recorded every 2 d.

Females were provided their respective waters for a 2-week adaptation period. Subsequently, rats were mated overnight using chow-fed Sprague-Dawley males. Mating was confirmed by the presence of sperm plugs (designated day 0 of pregnancy). Males were rotated within each group every 4 nights. Females not showing evidence of mating after 3 weeks were removed from the study. Males were provided distilled deionized water ad lib except on mating nights, when they had access to the same water as the females. Subsequent to mating, dams were given their respective water source throughout gestation. Animals were bred until a sample size of 160 “successfully” pregnant rats/ group was reached (i.e., evidence of fetuses and/or resorptions). Although this protocol is somewhat unusual, it was selected to obtain a fixed sample size in each treatment group. The sample size chosen insured that the study had 80% power to detect a 50% increase in the number of dams with at least one resorption. Animals were examined daily for any overt signs of toxicity. Successfully mated females were monitored every 5 d for body weight. Research staff were blinded to the treatment group of each animal except for those individuals who only fed and cared for the animals.

Because of the large sample size and practical limitations of the animal facility, female rats arrived in four batches (A-D). Each batch was intended

Water source and rat reproduction l J. Y. URIU-HARE ET AL. 551

to constitute a replicate experiment. However, on October 17, 1989, a major earthquake occurred in the San Francisco Bay Area (the Loma Prieta earthquake), which interrupted the supply of fresh tap water from the study area. There was only enough tap water left from the previous week’s supply to maintain a small subset (n = 7) of the TAP animals in batch A; the remaining rats in this group were removed from the study. Because of the unavailabil- ity of fresh tap water, these seven animals received tap water (during days 6 through 13 of their pregnancy) that had been stored for up to 2 weeks. This was contrary to study protocol, which required that fresh water samples be obtained weekly. In addition, the rats in batch B that had been adapting to tap water at the time of the earthquake had to be removed from the study. As a result, there were no TAP rats in batch B. Therefore, the majority of animals in the TAP treatment group came from the last two batches (C and D). For this reason, the primary analyses presented here only utilize animals from batches C and D.

On day 20 of gestation, all animals that had shown a copulatory plug were weighed, anesthetized with methoxyflurane (Pitman-Moore, Inc., Wash- ington Crossing, NJ), and killed via open cardiac puncture. After removing and weighing the uterus, the number of corpora lutea, implantation sites, resorptions, and live fetuses were recorded. Resorp- tions were classified as early (no discernible embryo or placenta), mid (discernible embryo, placenta or fetus, including autolyzed fetus), or late (dead fetuses with no obvious autolysis).

The variables “number of resorptions per litter” and “proportion of dams with one or more resorptions” were both calculated using the number of dams who showed evidence of pregnancy (presence of live fetuses or resorptions) at the time of sacrifice (gestation day 20) as the denominator. However, these variables do not reflect the loss of an entire litter before implantation or the resorption of all implanted embryos very early in pregnancy. Therefore, we examined maternal weight gain as a means of identifying litters that might have been totally resorbed. For this purpose, the weight gain in females that had shown a copulatory plug at mating, but who had no evidence of pregnancy on day 20, was examined. Weight gain in these dams was compared to the weight gain of the known pregnant dams in our study, as well as to the expected weight gain for nonpregnant female rats, as obtained from the supplier. Rats without evidence of pregnancy at day 20 could be divided into two subsets. One subset consisted of rats with a pattern of weight gain compa-

rable to that of nonpregnant female rats. We as- sumed these were truly nonpregnant females. The second subset consisted of rats whose pattern of weight gain for approximately the first 10 d after mating was similar to that of pregnant dams (those with evidence of pregnancy on day 20 of gestation). However, weight gain in dams in this second subset slowed after day 10 in marked contrast to the pattern in rats who were known to be pregnant. We called this latter subset “possibly totally resorbed.” This subset may have included dams in whom no fetusus implanted, or alternatively, dams who were pseudo- pregnant. We examined the proportion of such rats in each batch and treatment group.

All fetuses were examined for external maifor- mations and anomalies, sexed, the crown-rump length measured, and the fetuses weighed. In addition, placental weights were recorded. Alternate fetuses were placed in 95% ethanol for subsequent staining with Alizarin Red S and Alcian Blue (7). The remaining fetuses were placed in 10% formalin. An average of two fetuses per litter were taken for visceral exam using standard protocols (8). All of the fetuses prepared for skeletal and cartilage development assessment were examined.

In an attempt to identify possible etiologic agents, the three water sources were characterized using a number of analytic and microbiologic tests. Concentrations of 21 trace elements were detet- mined by inductively coupled plasma mass spec- trometry. Volatile organic chemicals (VOC) were analyzed by the EPA methods 502.1, 503.1, and 524.2 (9). Total organic carbon (TOC) was detet- mined by EPA method 9060, and total organic ha- lides (TOX) were analyzed by EPA method 9020 (10). Water samples from some of the wells supply- ing homes in the study area were also analyzed.

A 100 mL sample from the tap water composite was sent to the Sanitary Engineering and Environ- mental Health Research Laboratory of the Univet- sity of California, Berkeley, for bacteriologic analyses, which were performed within 24 h of collection. Bacterial counts were obtained by the same laboratory on samples taken from the cage water bottles after 2 d of use. Counts of organisms culturable on heterotrophic plate media (HPC) were obtained using the pour plate method (11). These plates were incubated in a high humidity incubator at 35 “C for 48 h. In addition, direct bacterial counts, using acridine orange stain (AODC), were obtained using the method of Hobbie and collegues (I 2). Randomly selected isolates from HPC medium were purified and identification was attempted using two biochemical identification systems (API and Biolog). Also, iden-

552 Reproductive Toxicology Volume 9. Number 6. 1995

tification was attempted on a number of the isolates by analysis of the fatty acid content of the cell walls (Microbiological Identification, Inc.).

Statistical analysis The epidemiologic results could not distinguish

between three alternative hypotheses: I) an increased risk of spontaneous abortion in tap water drinkers; 2) a “protective” effect of bottled water; or 3) an increased risk in both tap and bottled water drinkers relative to some “ideal” control water, with tap water drinkers at greater risk than bottled water drinkers. By comparing tap water to both deionized and bottled water we hoped to distinguish among these three alternatives.

Although all animals within a treatment group are often combined for the purpose of statistical analysis, the analyses reported here consider the variation of resorption frequencies across batches. This was necessary because the October, 1989, earthquake resulted in approximately equal numbers of animals in the BOTTLED and DEIONIZED groups in each of the four batches, but few rats in the TAP group other than in batches C and D. Secu- lar trends in several reproductive parameters over the course of the study suggested that pooling data across batches was inappropriate.

Multiple reproductive endpoints are usually given equal weight in traditional toxicology studies. However, this study, as well as in the prior study of Fischer 344 rats, was conducted to follow-up on epidemiologic studies that linked spontaneous abortion to drinking water. Therefore, fetal resorption was singled out a priori as the parameter of primary interest. Traditional reproductive parameters, including fetal length and weight, proportion of females vs. males, number of corpora lutea, and fetal anomalies (gross, visceral, and skeletal), were examined as well.

Because of the statistical dependence of implantation sites within litter, all models in these analyses used the litter (or dam) as the unit of analysis. Two analysis of variance (ANOVA) models were initially utilized to test for the presence of a batch effect, a treatment effect, and a treatment-by-batch interaction. The first used a fixed effects model. However, because batch is more appropriately viewed as a random than a fixed effect, we also used a mixed model in which batch was random while treatment remained fixed (I 3).

ANOVA models assume that observations follow a normal (Gaussian) distribution. However, because of the high proportion of litters with no resorptions, the number of resorptions per litter has a

distribution that is extremely skewed (see Figure I). Several transformations were considered, including the Freeman-Tukey transformation, which was de- rived for binomial data (14), but the transformed data failed to fit a normal curve. Because all ANOVA models fit poorly, alternative methods were utilized to analyze resorption frequency.

We turned to methods traditionally employed in epidemiology, including the odds ratio, the Man- tel-Haenszel method for combining estimates across strata, and logistic regression (15). Each of these methods assumes that the outcome of interest is a binomial variable. In this analysis, each dam is coded as a “failure” (one or more resorptions) or a “success” (no resorptions). Because the Mantel-Haenszel and logistic regression methods yield odds ratios (OR) as the estimate of the rate ratio, these have been used throughout for consis- tency. The OR estimates the relative risk, the rate of failure in one treatment group divided by the rate in a comparison group. It is usual epidemiologic practice to include a confidence interval for either the odds ratio or the relative risk (traditionally at the 95% confidence level). These confidence intervals incorporate the variability of the estimate and can also be used to provide a traditional test of significance: obtaining a 95% confidence interval that ex- cludes I .O is equivalent to obtaining an association that is significant at the 5% level (15). The odds ratios were first calculated separately for batches C and D. A summary estimate across batches C and D was then obtained using the method of Mantel-Haenszel (15) with confidence intervals calculated by the exact method of Greenland and Robins (16). To examine the possible effect of litter size on the probability of failure, this variable was entered into a multivariate model. A logistic regression model was used to estimate the treatment effect, while controlling for the effects of batch, treatment-by-batch interaction, and litter size. Separate logistic regression models were car- ried out to compare TAP to DEIONIZED and TAP to BOTTLED treatment groups.

RESULTS

As discussed above, only data from batches C and D were modeled when comparing all three treatments. Significant differences were seen between batch C and D for some parameters, though not all. For this reason, a batch effect is included in all analyses. We present results using both the fixed effects model of ANOVA and the mixed model. These are included here because ANOVA is often

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554 Reproductive Toxicology Volume 9, Number 6. 1995

the method of choice for the analysis of toxicologic data. However, we think these models are inappropriate for the analysis of resorption frequencies, though useful for variables such as weight gain or placental weight, which are approximately normally distributed (or transformable to approximately normal distributions). For this reason, resorption data were analyzed using the alternative statistical methods described above. Reproductive outcomes other than resorption frequency have been analyzed using ANOVA models only. Treatment effects for outcomes other than resorptions were minimal, and it is unlikely that results would be changed substantially using logistic regression.

and 0.09 for fixed effect and mixed models). Mean water intake was slightly less for batch C rats in the BOTTLED and DEIONIZED groups and only marginally different for TAP rats (41.5 vs. 41.8 mL/ d). The batch effect for water intake was significant only in the fixed-effects model (P = 0.006 vs. P = 0.21). An effect of water source on the number of resorptionsilitter was seen using the fixed effect model (P = 0.05), but not the mixed model (P = 0.36). As noted above, the departure from normality in the original and transformed resorption data resulted in a poor fit to these ANOVA models and led us to consider other models.

Reproductive outcomes The mean (?SEM) of selected maternal and

fetal parameters by batch and treatment are shown in Table I. The (two-sided) significance probabilities for these comparisons, obtained using ANOVA models, are shown in Table 2. Batch C rats were slightly heavier on gestation day 0 in all treatment groups but somewhat lighter on gestation day 20, resulting in a mean weight gain that was 11 g greater in batch D than in batch C. This batch effect was highly significant in both the fixed-effect and the mixed models (P = 0.0001 and 0.002, respectively). Batch C rats also showed evidence of mating about one-half day later than those in batch D (P = 0.06

The percentage of dams in each batch with evidence of fetal loss (one or more postimplantation losses) was compared between batches and treatment groups. Fetal loss among DEIONIZED animals nearly doubled between batches C and D (21.9 and 38.3%). increased slightly among the BOT- TLED group, but decreased by 22% in TAP animals (Table 3).

After controlling for batch using the Man-

tel-Haenszel summary odds ratio, the proportion of dams with one or more resorptions was 80% greater in TAP than BOTTLED (OR = 1.8,95% CI I .O-3.3) and 120% greater in TAP than DEIONIZED (OR = 2.2. 95% CI 1.2-4. I ). The incidence of the percentage of resorptions across batches is shown in Figure 1.

Table 1. Reproductive parameters by treatment and batch (mean i SEM)

Parameter BATCH TAP

Treatment

BOTTLED DEIONIZED

Number of dams

Weight gain (g, day O-20)

Water intake tmL/day) during gestation

Number implantation sites/litter

Number live fetuses/litter

Fetal wt (g)

Fetal length (cm)

Placental wt (g)

Number of resorptionsilitter

Postimplantation loss index“ (percent resorption)

Number corpora lutes/litter

c D C D C D C D c D c D C D C D C D C D C D

85 68

I38 -t 2 147 + 2

31.5 + 0.9 41.8 + I.0 14.4 * 0.3 14.0 i- 0.3 13.6 ? 0.3 13.4 + 0.3 3.44 t 0.03 3.55 i 0.05 3.85 + 0.01 3.94 + 0.02 0.49 * 0.01 0.49 ? 0.01 0.79 t 0. IO 0.60 ? 0.10

5.7 + 0.7 4.2 t 0.7

14.7 t 0.2 14.3 % 0.3

42 35

134 f 3 I46 -+ 3

40.1 + I.6 42.8 ? I.2 13.8 + 0.2 13.9 t 0.4 13.4 r 0.2 13.3 + 0.4 3.46 lr 0.0.5 3.40 2 0.03 3.84 2 0.02 3.87 + 0.03 0.50 % 0.01 0.50 t- 0.01 0.45 2 0.1 I 0.60 ? 0.21

3.2 ? 0.8 4.5 + I.4

14.0 f 0.2 14.4 + 0.4

32 47

I38 + 3 I49 + 3

37.4 ?z I.1 42.9 t I.2 13.8 k 0.3 14.3 ? 0.3 13.5 2 0.3 13.8 + 0.3 3.43 + 0.03 3.55 2 0.07 3.84 -’ 0.02 3.94 t 0.03 0.48 + 0.01 0.49 + 0.01 0.31 2 0.11 0.51 2 0.12

2.2 -t 0.8 3.8 2 I.0

14.2 2 0.3 14.7 + 0.2

aPostimplantation loss index = #implantation sites - # live births x loo

#implantation sites


Table 2. Significance probabilities for two analysis of variance models (fixed effect vs. mixed) comparing reproductive parameters by treatment and batch

Parameter

Wt gain (g. day O-20)

Water intake (mL/day) during gestation

Number implantation sites/litter

Number live fetuses/litter

Fetal wt (g)

Fetal length (cm)

Placental wt tgl

Number of resorptionsilitter

Postimplantation loss index” (percent resorption)

Number corpora lutes/litter

Treatment 0.38 0.16 Batch 0.0001 0.002 Treatment x Batch 0.83 0.83 Treatment 0.45 0.75 Batch 0.006 0.21 Treatment X Batch 0.09 0.09 Treatment 0.62 0.64 Batch 0.76 0.77 Treatment X Batch 0.42 0.42 Treatment 0.71 0.37 Batch 0.92 0.84 Treatment x Batch 0.82 0.82 Treatment 0.29 0.59 Batch 0.17 0.40 Treatment x Batch 0.16 0.16 Treatment 0.06 0.36 Batch 0.0001 0.07 Treatment x Batch 0.20 0.20 Treatment 0.29 0.07 Batch 0.41 0.09 Treatment x Batch 0.90 0.90 Treatment 0.05 0.36 Batch 0.61 0.72 Treatment x Batch 0.19 0.19 Treatment 0.08 0.46 Batch 0.56 0.73 Treatment x Batch 0.11 0.11 Treatment 0.62 0.75 Batch 0.41 0.60 Treatment x Batch 0.24 0.24

P-value

Fixed effect model Mixed model

aPostimplantation loss index = #implantation sites - # live births x loo

#implantation sites

We considered the possible effect of litter size on the probability of failure; a smaller litter might have a smaller probability of including one or more resorptions than a larger one. We also included a term to assess the interaction between batch and treatment. These factors were explored using logis-

Table 3. Odds ratio for risk of resorption” by treatment adjusted for batch

Treatment Batch C Batch D Adjusted Table 4. Logistic regression analysis of risk of

(9%) %‘c) odds ratioh 95% Cl P-value resorption” (TAP vs. DEIONIZED)

TAP 48/85 30/68 - - - (56.5) (44. I)

BOTTLED 14142 14135 1.8C I .o-3.3 0.05 (33.3) (40.0)

DEIONIZED 7132 18/47 2.2d I .2-4.1 0.01 (21.9) (38.3)

aDams with one or more resorptions. bAdjusted for batch using Mantel-Haenszel procedure, with Greenland/Robins 95% confidence intervals (Cl). CTAP vs. BOTTLED. dTAP vs. DEIONIZED.

tic regression (Table 4). Treatment effect was statistically significant (in this analysis), as was the treatment-by-batch interaction. Litter size appeared to have little effect on risk of failure after controlling for batch and treatment (RR = 1.08, P = 0.09).

Resorptions were classified as early, mid, or late. As expected, most resorptions were early in all three treatment groups. Numbers were small and

Treatment Batch Litter size

Treatment x batch

Coefficienth 1.50 -0.47 0.08 -1.24 P-value 0.001 0.08 0.09 0.03 Odds Ratioh I .08

Batch C 4.48 Batch D 1.30

aDams with one or more resorptions. bAdjusted for treatment, batch, litter size, and treatment x batch. (Odds ratio estimates the rate ratio).

556 Reproductive Toxicology Volume 9, Number 6, 1995

Table 5. Fetal skeletal ossification by treatment and batch (mean 5 SEM)

Treatment

Parameter BATCH TAP

Number fetuses examined C 534 (85) (number of litters) D 442 (68) Number of ossification centers”

Sternum C 4.61 t 0.04 D 4.73 r 0.06

Caudal vertebrae c 5.25 + 0.05 D 5.36 t 0.08

Metacarpals C 3.18 ? 0.03 D 3.17 f 0.03

Anterior phalanges C 0.02 ? 0.01 D 0.09 + 0.06

“Number of fetal ossification centers expressed as an average per litter.

BOTTLED DEIONIZED

270 (42) 206 (32) 226 (35) 313 (47)

4.68 2 0.08 4.S2 2 0.07 4.52 + 0.08 4.61 + 0.09 5.19 2 0.09 5.21 t 0.06 5.16 2 0.08 5.17 k 0.11 3.14 2 0.04 3.13 2 0.03 3.13 2 0.04 3.17 + 0.05 0.07 i 0.07 0.00 5 0.00 0.00 k 0.00 0.11 + 0.06

treatment effects were nonsignificant in each of these categories (data not shown). The number of corpora lutea did not differ among treatment groups or batches, suggesting that implantation rates were similar, at least in dams with a continuing pregnancy (Table 1).

We also calculated the proportion of dams in each treatment group classified as “possibly totally resorbed.” Data are shown for DEIONIZED dams in batch C (Figure 2). In batches C and D, 13.6% (2.5 out of 184) of TAP rats and 8.6% (8 out of 93) of DEIONIZED rats were classified as “possibly

Data from Supplier - Non Pregnant (N=100)

I/ I

Fig. 2. Cumulative weight gain (grams) for batch C rats fed deionized water with and without evidence of implantation sites on gestation day 20 (Mean ;t SEM) compared to weight gain data of known nonpregnant rats (supplied by Bantin and Kingman, Fremont, CA). 0 *Pregnant-rats with evidence of one or more implantation sites on gestation day 20. + **Possibly pregnant-rats with no evidence of implantation sites on gestation day 20. A **Not pregnant-rats with no evidence of implantation sites on gestation day 20. W Nonpregnant-weight gain data of known non pregnant rats (supplied by Bantin and Kingman, Fre- mont, CA).

totally resorbed,” compared to only 2.5% of rats in the BOTTLED group (2 out of 80). In batches A and B, the percentages for TAP, DEIONIZED, and BOTTLED groups were 12.5%, 7.8%, and l.l%, respectively. Thus, the lower rate in the BOTTLED group is seen in all four batches. Although this obser- vation is intriguing, it needs to be confirmed using a study design in which rats are killed early in gestation rather than on day 20.

Fetal olrtcome Fetal length varied significantly by batch, while

fetal weight and placental weight were similar among batches and water sources (Tables 1 and 2). The proportions of males and females were similar in all batches and treatment groups (data not shown). The number of fetal skeletal ossification centers at any site were also similar in all treatment groups (Tables 5 and 6).

Table 6. Significance probabilities for two analysis of variance models (fixed effect vs. mixed) for comparing

fetal skeletal ossification by treatment and batch

P-value

Number of ossification centers Fixed effect Mixed

model model

Sternum Treatment 0.28 0.64 Batch 0.78 0.87 Treatment x Batch 0.1 I 0.11

Caudal vertebrae Treatment 0.17 0.26 Batch 0.81 0.78 Treatment x Batch 0.54 0.54

Metacarpals Treatment 0.49 0.20 Batch 0.78 0.58 Treatment x Batch 0.84 0.84

Anterior phalanges Treatment 0.93 0.95 Batch 0.36 0.54 Treatment x Batch 0.20 0.20


Table 7. Incidence of specific skeletal abnormalities by treatment (batches C and D)

Treatment

TAP BOTTLED DEIONIZED

No. fetuses examined 976 496 519 (No. of litters) (153) (77) (79) Hemiverfebrae 7 3 5 Unossified vertebral centra 14 5 Misaligned sternebrae 3 0 ; Supernumerary rib(s) 105 57 27 Missing 13th rib 0 0 I Very poorly ossified 13th rib 0 I 0

All fetuses in batches C and D were examined for gross external anomalies (Table 7). The only major malformation noted occurred in the tap water group with one fetus with both cranioschisis and gastroschisis. Supernumerary ribs comprised the majority of the skeletal anomalies. These were present in 5.2% of fetuses in the DEIONIZED group vs. 10.8% of fetuses in the TAP group and 11.5% in the BOTTLED group.

The proportion of litters in which one or more fetus had a skeletal anomaly was similar in the TAP and BOTTLED animals (44.4% and 45.5%, respectively) but elevated compared to those in the DE- IONIZED group (29.1%) (Table 8). Using a two- tailed test, TAP and BOTTLED animals had rates of skeletal anomalies that were significantly higher than those in the DEIONIZED group. As with resorption frequency, the difference between groups was more marked in batch C than batch D.

Water characterization None of the water samples had concentrations

of trace elements, VOC, TOC, TOX, or bacterial counts outside the range of typical levels found in the U.S. drinking water supplies (9,10), although there were some differences among the three types of water.

Fifteen elements (aluminum, barium, boron, calcium, chromium, copper, lead, lithium, magnesium, nickel, scandium, sodium, strontium, vanadium, and zinc) were present at higher concentrations in tap water than in either bottled or deionized water. Among these elements, the tap water samples contained, on average, 1320 Fug/L of zinc and 7.6 PgIL of lead, while bottled and deionized waters contained mean levels of zinc that were < 2 pg/L and undetectable amounts of lead. Although several elements were present in tap water at levels that exceeded typical US drinking water levels (17), they did not exceed California drinking water standards

(18). Bacterial counts (AODC and HPC) were consis-

tently higher in tap water than in bottled or deionized waters (Table 10). In tap water, there were approximately lo3 to lo4 organisms/ml by AODC. This measure represents a count of individual bacteria that are captured on a water filter and then stained with acridine orange. This count estimates the number of viable organisms in a given volume of water. The heterotrophic plate count (HPC) involves culturing a known aliquot of water on a medium designed to facilitate the growth of bacteria. Not all species can be induced to grow on this medium. Indeed, of the lo3 to lo4 bacteria recovered from tap water, only 10’ organisms/ml were culturable on HPC. In the bottled and deionized waters, there were approximately lo2 to lo3 organisms/ml by AODC, and very few culturable bacteria. After rats had drunk from the water bottles, the average HPC counts in cage water were about 10’ to lo6 organisms/ml, regardless of the type of water. However, the AODC on water samples from cages in the tap water group were consistently higher than the AODC on bottled and deionized cage water samples (lo5 vs. lo6 organisms/ml).

We succeeded in culturing only a small proportion of the organisms counted by AODC. Of these

Table 8. Skeletal abnormalities by treatment (batches C and D)

TAPa

Treatment Significance probability

BOTTLEDa DEIONIZEDa TAP vs. BOTTLED TAP vs. DEIONIZED

Fetuses with ~1 122 (12.5) Jl_ (12.3) 36 (6.9) 0.91 0.001 skeletal abnormality 976 496 519

Fetuses with multiple 6 (0.6) 3 (0.6) 0 (0.0) 0.98 0.07 skeletal abnormalities 976 496 519

Litters with 21 68 (44.4) 35 (45.5) 23 (29. I) 0.88 0.02 skeletal abnormality 153 77 79

“# Abnormal (%) # Examined

558 Reproductive Toxicology Volume 9. Number 6. 1995

Table 9. Concentrations of trace elements by water source, in comparison to California drinking water standards and typical levels in United States drinking water supplies

(pg/L) (average of two measurements)

Element Tap Bottled Deionized

California Drinking

Water Standard (17)

Typical U.S.

drinking water

level (Range) (18)

Barium

Boron

Calcium”

Chromium

Copper

Lead

Lithium

Magnesium”

Nickel

Scandium

Sodium”

Strontium

Vanadium

Zinc

24.5 4.0 2.5 1,000 74 (ND-2.760) I 06 I.5 0.8s I .ooo 29 (ND-172)

123.5 <3 <3 NA NA

S5,SOO 1214.5 555 NA 26.000 (to 145,000)

Il.4 CO.2 10.3 SO 8.S (ND-I 12)

156.5 O.SS co.3 I .oooh 100 (ND-450)

1.6 co.2 co.2 SO 3.7 (ND-621

9.6 10.2 SO.2 NA NA

32.350 817.S 44.5 NA 6.250 (to 120.000)

9.9 0,s <0.4 NA S (0.4-75)

41.1s 10.2 10.2 NA NA

24.050 4150 <20 NA 28,000 (to 220,000)

449.5 3.0 <0.2 NA 10,000 (2,200-12,000)

3.8.5 10.2 co.2 NA 4 (ND-70)

I320 1.35 0.7s 5,oooh 200 (ND-1,500)

“Analyzed in well water only

bAesthetic standard only.

NA = Not available.

ND = Nondetectable.

colonies cultured, most could not be identified. Or- ganisms identified from tap water included opportu- nistic pathogens such as Pusterrrella haemolytica. Pasteurella anatipesti$er, Vihrio damselrr. and Pseudomonas spp. However. these identifications are suspect because the identification systems employed are primarily used in the identification of clinical isolates. On matched isolates, identifications

by these three systems seldom agreed to the genus level.

DISCUSSION

This study was designed with a sample size large enough to have a high probability of detecting a 50% increase in fetal loss (the end point of concern in

Table 10. Bacterial counts by treatment

Type of water II Percentile AODC” HPCh HPCiAODC (%)

Fresh

TAP

BOTTLED

DEIONIZED

Cage water TAP

BOTTLED

DEIONIZED

64

66

32

25th 4.200 SOth 7.037 75th I0.000 2Sth 94 50th 235 75th I.135 25th II7 50th 274 75th I.500

33

39

41

25th 690,000 125,000 8.70 50th I ,600.OOO 330,000 22.00 75th 2,300,ooo 520,000 72.50 2Sth 250,000 350,000 66.70 SOth 430,000 670,000 103.30 75th 670,000 990,000 254.60 25th 60.200 78,000 55.80 50th 140.000 I70,OOO 106.90 7Sth 235.250 300.000 171.90

S6

83

I03 <I

4

<I

<I I

0.50 1.04

2.74

0.04 0.22

1.92

0.00 0.05

0.30

“AODC in counts/ml. ‘HPC in colony forming units/ml.


humans). This is an increase similar to that seen in several epidemiologic studies (5). After controlling for batch using the mixed model ANOVA, the effect of treatment on number of resorptions per litter was not significant (P = 0.36). Because the number of resorptions per litter followed a distribution that was highly skewed, we chose to compare the number of dams with one or more resorptions (“failures”) in the tap water group to that proportion in the other treatment groups. These data are summarized as odds ratios that compare the risk of failure between two treatment groups. For the TAP and BOTTLED water comparison, we observed an odds ratio of 1.8 (95% CI l.O-3.3), after controlling for batch. When TAP was compared to DEIONIZED, this association appeared slightly stronger (OR = 2.2, 95% CI I .2-4. l), and both associations were significant (P < 0.05). However, logistic regression identified a significant batch-treatment interaction; the eleva- tion in resorption frequency was seen primarily in batch C.

Chernoff and co-workers reported significant month-to-month variation in maternal and fetal parameters in mice given either distilled water or a municipal drinking water source over an &month study (19). In our study, the number of resorptions per litter increased in successive batches in both the deionized and bottled water groups, but resorptions in the tap water group decreased in successive batches. Although interbatch variation for resorption frequencies was substantial, logistic regression analysis showed a significant treatment effect for resorptions, even after controlling for this batch effect. This is also true when the full data set (including batches A and B) is analyzed.

Because of small numbers, we chose not to present data from batches A and B. However, results from these batches are of interest. Among the seven TAP rats in batch A, five had evidence of fetal loss (71.4%), compared to a rate of 28.3% in the 46 BOTTLED rats and 18.4% in the 49 DEIONIZED rats. Unlike TAP rats in batches C and D, these seven rats received water stored for up to 2 weeks during days 6 through 13 of their pregnancy. This was due to the interruption of the tap water supply as a result of the Loma Prieta earthquake. It is possible that longer storage amplified some component in tap water that resulted in a higher resorption frequency in these animals. This difference might be the result of chance, although the resorption frequency in the seven TAP rats was significantly higher than corresponding frequencies for the BOTTLED (P < 0.01) and DEIONIZED (P < 0.001) groups.

We also compared the proportion of “failures” (dams with one or more resorption) between the BOTTLED and DEIONIZED animals across all four batches. Although the failure rate was somewhat higher in BOTTLED rats in each batch, the magnitude of the excess was inconsistent. The Man- tel-Haenszel summary odds ratio for BOTTLED compared to DEIONIZED was similar in magnitude to that seen when comparing TAP and BOTTLED rats in batches C and D, though the former did not achieve statistical significance (OR = 1.41, 95% CI 0.81-2.46).

The four manifestations of developmental toxicity typically considered in standard toxicology studies are fetal growth, functional deficits, embryolethality, and structural changes. No effect of water treatment on fetal growth was seen, and functional deficits were not assessed in this study. However, our data suggest a difference between treatment groups with respect to embryolethality. In addition, both TAP and BOTTLED groups showed somewhat greater frequencies of fetal skeletal anomalies than the DEIONIZED group, and this was seen in all batches. Chernoff and co-workers (19) also reported an increase in the incidence of supernumerary ribs in fetuses from mice drinking municipal drinking water vs. purified distilled water. The significance of these anomalies, which are considered to be minor variations by some investigators, is uncertain. How- ever, it is clear that they reflect a subtle alteration in normal development.

Characterization of the three waters did not identify an etiologic agent. None of the chemical constituents nor bacteria were present at levels unusual for U.S. drinking water (10). Could bacterial differences in the source waters, at the time of collection, or after laboratory handling, explain the differences in the resorption rates seen in this study? Several studies have dealt with the bacteriologic quality of water and reproductive outcomes. It has been suggested that disinfection of laboratory water can affect bacteriologic quality (20) and that water treatment may influence reproduction (21). The tap water did contain higher bacterial counts than either bottled or deionized waters. However, less than 2% of the bacteria in tap water based on AODC were culturable on HPC medium and most culturable organisms could not be identified. Of those bacteria that were identified, none are known to cause reproductive toxicity.

Unlike standard toxicology studies, no single agent was tested in this experiment. Rather, both tap and bottled water contained an unknown and complex mixture of possible agents. Moreover, ani-

560 Reproductive Toxicology Volume 9, Number 6, 1995

mals were exposed to a single “dose level” of water as consumed by humans. Dilution would have provided a range of “doses,” but would have required a prohibitively large sample size. We chose not to concentrate samples by boiling, lyophilization, or evaporation, because such treatment might have altered the (hypothetical) unknown biologic or chemical agent. Instead, we conducted a large study with sufficient power to detect the small effect in which we were interested. In the typical toxicologic protocol the expected treatment effect is large and significantly exceeds the range of background variability. In this study, however, the effect of interest (a 50% increase in a resorption frequency that is typically very low) was similar in magnitude to the fluctuation in the resorption frequency in the DEIONIZED group over the course of the study. Although an effect at this level is unusual for toxicology studies, it is frequently reported for epidemiologic studies in which exposures are at low levels and background rates of disease fluctuate appreciably.

somewhat Iower than rates previously seen by this laboratory, using this strain of animals. For these reasons, we believe that these results do not justify regulatory action. However, the results of the current animal study lend support to results from recent epidemiologic studies (5); the consumption of tap water from the test area was associated with reproductive risk of the type and magnitude seen in humans. Thus, while the biologic significance of the increased resorption phenomenon in tap water-fed rats can be debated, the results from these studies merit additional experimental investigation.

Ackno~~‘/e&~?zanf.s - This work was supported in part by the California Denartment of Health Services. and HD 072241 (J.Y.U-H). The authors acknowledge the valuable assistance of Karen Kan. Kirsten Wailer, and Jack Gerson in data analysis. John Andrew in water collection, Rick Danielson in bacterial analysis. and Raimund Roehl in analysis of metals and organics.

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Drinking water source and reproductive outcomes in Sprague-Dawley rats

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