L C Hall
email: hall13@llnl.gov

Surflan TM and Oryzalin Impair Reproduction in the Teleost Medaka (Oryzias latipes)

Keywords: endocrine-disruptor, medaka, herbicides, ecotoxicology, toxicity, Surflan, oryzalin

We conducted studies to determine if the xenoestrogens Surflan TM and its active ingredient oryzalin, affect indices of reproductive fitness in medaka (Oryzias latipes). Oryzalin (0.5, 0.25 mg/l) or Surflan TM (2.0 µl/l) and oryzalin (0.5 mg/l) significantly increased the mean number of non-fertilized eggs produced by treated females paired with untreated males, or by untreated females paired with treated males. Oryzalin (1.0, 0.5, 0.25 mg/l) and Surflan TM (3.8, 2.0, 1.0 µl/l) significantly affected the time to hatch of eggs from treated females paired with untreated males, and from untreated females paired with treated males. Surflan TM (3.8, 2.0, 1.0 µl/l) induced intersex lesions in 80-100% of males. Oryzalinexposed males exhibited a significant increase in the incidence of necrotic spermatids and necrotic spermatogonia, while oryzalin-exposed females had significantly fewer immature oocytes and an increase in the occurrence of hyperplastic ovaries. Our results indicate that Surflan TM and oryzalin affect both reproduction and gonadal histology in male and female medaka.

Introduction

Surflan TM is a proprietary herbicide emulsion formulated and marketed by Dow Elanco that contains 40.4% oryzalin (3,5-dinitro-N4, N4-dipropylsulfanilamide). We have identified both Surflan TM and oryzalin as xenoestrogenscompounds that may be capable of eliciting many of the biological responses of endogenous estradiol [START_REF] Hall | Identification of the herbicide Surflan™ and its active ingredient oryzalin, a dinitrosulfonamide, as xenoestrogens. Accepted[END_REF].

In teleosts, endogenous estrogens (primarily 17β-estradiol) have a multitude of functions in normal physiology, and are involved in sexual differentiation of embryos [START_REF] Yamamoto | Sex differentiation[END_REF], sexual maturation ( [START_REF] Van Den Hurk | Steroidogenesis in the gonads of rainbow trout fry (Salmo gairdneri) before and after the onset of gonadal sex differentiation[END_REF][START_REF] Patino | Sexual dimorphism of plasma sex steroid levels in juvenile coho salmon, Oncorhynchus kisutch, during smoltification[END_REF], and reproductive behavior [START_REF] Hutchinson | Aromatase: neuromodulator in the control of behavior[END_REF]. Long-considered to be principally a female reproductive hormone due to its roles in vitellogenesis and choriogenesis (Chester-Jones, Ingleton & Phillips, 1987), the identification of estrogen receptors (ERs) in brain and reproductive tissues of male vertebrates [START_REF] Callard | Exclusive nuclear location of estrogen receptors in Squalus testis[END_REF][START_REF] Cooke | Estrogen receptor expression in developing epididymis, efferent ductules, and other male reproductive organs[END_REF] point to estrogens' importance in male reproduction as well.

Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

Estrogen homeostasis is critical to the normal function of male and female gonads [START_REF] Van Den Hurk | Steroidogenesis in the gonads of rainbow trout fry (Salmo gairdneri) before and after the onset of gonadal sex differentiation[END_REF], and to reproductive behavior [START_REF] Hutchinson | Aromatase: neuromodulator in the control of behavior[END_REF]. Exogenous estrogen and xenoestrogens appear capable of disrupting this homeostasis. Administration of exogenous estradiol, the pharmaceutical estrogen 17α-ethynylestradiol (17α-EE2), or various xenoestrogens has been correlated with adverse reproductive effects in fish including: decrements in fertility (Gray, Teather & Metcalfe, 1999a;[START_REF] Van Den Belt | Reproductive effects of ethinylestradiol and 4t-octylphenol on the zebrafish (Danio rerio)[END_REF]; testicular degeneration and atrophy [START_REF] Christiansen | Effects of nonylphenol and 17β-oestradiol on vitellogenin synthesis, testicular structure and cytology in male eelpout Zoarces viviparous[END_REF][START_REF] Miles-Richardson | Effects of waterborne exposure to 4-nonylphenol and nonylphenol ethoxylate on secondary sex characteristics and gonads of fathead minnows (Pimephales promelas)[END_REF]; inhibition of spermatogenesis [START_REF] Jobling | Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals[END_REF][START_REF] Kinnberg | Concentration-dependent effects of nonylphenol on testis structure in adult platyfish Xiphorus maculatus[END_REF][START_REF] Sohoni | Reproductive effects of long-term exposure to bisphenol A in the fathead minnow (Pimephales promelas)[END_REF]; inhibition of testicular development (Gimeno, Komen, Venderbosch & Bowmer, 1998;[START_REF] Panter | Adverse reproductive effects in male fathead minnows (Pimephales promelas) exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone[END_REF]; development of intersex testes (Metcalfe, Metcalfe, Kiparissis, Nii, Foran & Benson, 2000;[START_REF] Yokota | Life-cycle toxicity of 4nonylphenol to medaka (Oryzias latipes)[END_REF]; and ovarian degeneration or suppressed ovarian development [START_REF] Lange | Effects of the synthetic estrogen 17α-ethinylestradiol on the life-cycle of the fathead minnow (Pimephales promelas)[END_REF][START_REF] Van Den Belt | Reproductive effects of ethinylestradiol and 4t-octylphenol on the zebrafish (Danio rerio)[END_REF]. In general, the physiological alterations underlying these effects have not been identified. However, certain studies have implicated administration of exogenous estradiol or xenoestrogens in the suppression of gonadotropins [START_REF] Lessman | Estradiol-17B silastic implants suppress oocyte development in the brook trout, Salvelinus fontinalis[END_REF][START_REF] Harris | Nonylphenol affects gonadotropin levels in the pituitary gland and plasma of female rainbow trout[END_REF], adverse effects on reproductive behavior (Gray et al., 1999a;[START_REF] Hall | Serum vitellogenin levels and reproductive impairment of male Japanese medaka (Oryzias latipes) exposed to 4-tert-octylphenol[END_REF][START_REF] Bjerselius | Male goldfish reproductive behavior and physiology are severely affected by exogenous exposure to 17B-estradiol[END_REF][START_REF] Van Den Belt | Reproductive effects of ethinylestradiol and 4t-octylphenol on the zebrafish (Danio rerio)[END_REF]; and alterations in p450arom activity [START_REF] Melo | Sexual dimorphism of brain aromatase activity in medaka; induction of a female phenotype by estradiol[END_REF] or expression [START_REF] Scholz | 17-α-ethinylestradiol affects reproduction, sexual differentiation, and aromatase gene expression of the medaka (Oryzias latipes)[END_REF].

Medaka (Oryzias latipes) are commonly chosen as a model organism for use in reproductive studies due to their high and relatively consistent reproductive capacity. Under specific culture conditions of photoperiod, water temperature, and regular feeding, reproductively mature females produce eggs approximately daily on a year-round basis [START_REF] Koger | Variations of Temperature regimes and resulting effects on reproductive parameters in medaka (Oryzias latipes)[END_REF]. Medaka eggs, typically produced in clusters of 10-25, develop externally. Their transparent chorion allows simple visual determination of fertilization, detectable within 0.2 h by the presence of yolk and a perivitelline space. In our laboratories, medaka larvae hatch within 9-12 days.

As newly-identified xenoestrogens, it is important to characterize the effects of Surflan TM and oryzalin on indices of reproductive fitness such as fertility and fecundity. Measurement of these parameters can give the first indication of the likelihood that effects of exposure may extend beyond the individual and potentially impact a population. Accordingly, the present study was designed to determine whether exposure of reproductively-active, mature male and female medaka to Surflan TM and oryzalin would affect fertilization success, fecundity, viability of eggs, time-to-hatch, or number of Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

deformities in the offspring. Gonadal tissues of males and females were also evaluated in order to identify and characterize any exposure-related effects.

Materials and Methods

The medaka used in this study were the golden strain of O. latipes, originally purchased from Carolina Biological Supply and maintained at the University of California, Davis Aquatic Center for 17 y. The colony's brood stock has been out-crossed twice with wild-type Northern strain golden medaka to prevent inbreeding [START_REF] Davis | The influence of gender on hepatocarcinogenesis in Japanese medaka, Oryzias latipes[END_REF].

Reproductive effects study design.

Sexually mature, five month-old medaka (n = 168) were separated into six replicate groups for each treatment, with two female and two male fish per replicate. Only reproductively-active females were utilized, and were identified by the presence of an attached, fertilized clutch of eggs on the day of collection. Each group of four medaka was placed in an aerated 1-l glass beaker of U.S. EPA reconstituted water [START_REF] Horning | Short-term methods for estimating chronic toxicity of effluents and receiving waters to freshwater organisms[END_REF], and maintained at 25 + 1°C.

Glassware was cleaned before use by soaking for 24 h in a detergent solution, rinsed, soaked for an additional 24 h in a 1% nitric acid solution, and rinsed. A photoperiod of 16h light, 8h dark was maintained throughout the experiment, with light provided at 400-450 lux by Vita-Lite (Coralife Chromatic™) bulbs.

Final exposure concentrations of Surflan™ and oryzalin were selected from the results of a preliminary study in which 168 medaka were exposed for 21d to nominal concentrations of Surflan™ (8.5, 5.8, or 3.8 ul/l), or oryzalin (2.0, 1.3, or 1.0 mg/l). Final concentrations for the high-dose group of each chemical were selected from the preliminary study as the 21-day maximally-tolerated concentration (MTC) where tolerance was considered to be those concentrations associated with <15% lethality during the exposure period (3.8 ul/l and 1.0 mg/l for Surflan™ and oryzalin, respectively).

Final exposure concentrations for each of the mid-and low-dose groups were set at approximately half of the concentration of each higher dose.

Exposure to nominal concentrations of oryzalin (1.0, 0.50, or 0.25 mg/l) or Surflan™ (3.8, 2.0, or 1.0 µl/l) or reconstituted water was initiated after fish had been allowed to acclimate for five days, and it had been established that each replicate group was actively producing eggs. Exposure solutions were made fresh daily, and the entire solution of each beaker was replaced every day for the 21-d exposure period. On a daily basis, fish were fed a purified casein (PC)based diet [START_REF] Dekoven | Evaluation of conventional and purified rations for the Japanese medaka, Oryzias latipes: Refining a fish model for toxicological research[END_REF] at 2% of their body weight prior to solution change, then received brine shrimp at approximately 3.5% of body weight after solution replacement.

At the end of the 21-d exposure period, fish from each treatment replicate were separated by dose group and gender. Two untreated males were placed with each pair of treated females, and two untreated (reproductively active) Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

females placed with each pair of treated males. Newly-introduced fish were of the same broodstock and hatch date as the treated animals. Each new group of four medaka was placed in a fresh 1-l beaker, and was maintained in reconstituted water for the duration of the experiment. The entire 1-l volume of water in each beaker was replaced daily throughout the remainder of the experiment. All fish received the same diet as described above.

Following the placement of untreated medaka with treated medaka, fish were allowed to acclimate for two days.

Egg collection began on day three post-exposure, and continued for five days thereafter. Eggs were siphoned twice daily from each beaker for five days, counted, and evaluated visually under a microscope to determine fertilization status. Egg masses were separated with forceps to obtain individual eggs, and unfertilized eggs were counted, recorded and discarded.

For the first three days of egg collection, fertilized eggs from each replicate were counted and transferred to a 600-mL aerated beaker and observed daily for mortality and hatch. To minimize fungal growth, embryos that died were counted and removed from beakers daily. Approximately 75% of the water was exchanged daily from each beaker. Beakers were observed on a daily basis to detect the presence of newly-hatched larvae. Larvae were counted and the date of hatch noted.

Larvae were transferred to an aerated 40-l aerated tank for grow-out (one tank per treatment group). The water temperature and photoperiod were maintained as previously described. Larvae were fed a PC diet, and were raised until approximately 30 days of age. At that time, a sample of 50 juvenile fish from each tank were sacrificed and examined visually for the presence of any gross abnormalities.

Histologic effects study design

Eighty four, seven month-old sexually mature medaka were separated into three replicate groups for each treatment, with two male and two female fish per replicate. Each group of four medaka were placed in an aerated 1-l beaker of reconstituted water, and maintained as described above. Following a two-day acclimation period, exposures to Surflan™ and oryzalin were initiated. Exposure concentrations, solution preparation, solution exchange procedures, and feeding regimens were the same as those used in the reproductive effects study.

At the end of the 21-d exposure period, medaka were euthanized by immersion in tricaine methane sulfonate (MS222, Sigma). Abdominal cavities were surgically opened, and fish were immediately immersed in 10% neutral buffered formalin. Formalin-preserved fish were processed with standard methods for paraffin embedding. Two initial sections were obtained from each paraffin block, mounted on glass slides, and stained with hematoxylin and eosin (HE). If representative sections of gonads were not obtained during the initial processing, paraffin blocks of fish were stepsectioned (at 50 µm intervals) for an additional four to six levels as needed to obtain adequate gonadal views.

Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

Glass HE slides were initially sorted by gender and then randomized prior to reading and scoring. All slides were read and scored semi-quantitatively without knowledge of exposure history. Table 1 identifies lesion types and lesion scoring criteria for male and female medaka.

Statistical analyses

Fertility and fecundity data and both testicular and ovarian lesion scores were evaluated by applying the nonparametric Kruskal-Wallis test for one-way ANOVA using NCCS ® or JMP ® statistical software. When comparisons were made on the basis of dose group, α values were initially adjusted by a Bonferroni correction to account for multiple comparisons, such that p < 0.008 represents a result that is significantly different from controls. The Bonferroni correction is a multiple-comparison correction used to preserve the desired α value when several independent statistical tests are performed simultaneously. Adjustments of α values were not made for testicular and ovarian lesion-scores evaluated by treatment category, where an outcome of p < 0.05 was identified as statistically significant. Given that the Bonferroni approach examines only the p-values of each test without accounting for the number of tests within a series that are significant, we also applied the Bernouli equation [START_REF] Moran | Arguments for rejecting the sequential Bonferroni in ecological studies[END_REF] to determine the probability of obtaining 'n' number of significant results in a series by chance alone. If both the individual ANOVA result and the Bernouli equation applied to a series of tests yielded a p value < 0.05, we concluded that the result was statistically significant. Time-to-hatch data were tested for statistical significance by application of the Wald-Wolfowitz Runs Test, which tests the hypothesis that any two samples (i.e., distributions) came from populations with identical distribution functions. A result of p < 0.05 in the Wald-Wolfowitz Runs Test represents statistical significance.

Results

Exposure to Surflan TM or oryzalin for 21d adversely affected the reproductive outcomes of medaka, significantly affected the hatching time of eggs, and increased the incidence and/or severity of testicular and ovarian lesions.

The daily mean production of non-fertilized (NF) eggs by treated females paired with untreated males, and by untreated females paired with treated males was consistently greater than controls in all dose groups (Table 2). These differences in the mean number of NF eggs were statistically significant in low-and mid-dose oryzalin females, and in mid-dose Surflan TM and mid-dose oryzalin males. Considering treated females and males together (F+M), the daily total production of NF eggs was significantly greater than controls in the high-and mid-dose Surflan™ groups, as well as in the mid-and low-dose oryzalin groups.

The mean daily total production of eggs was not uniformly affected by exposure to Surflan TM or oryzalin. Although several treatment groups produced lower total numbers of eggs than controls, none of these apparent Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

effects reached statistically significant levels (Table 3). Viability of fertilized eggs was also not affected by treatment, with a mean hatch rate in all groups typically > 90% (data not shown). Additionally, there were no treatment-related effects on the incidence of gross deformities in juveniles sampled at approximately 30-d post-hatch (data not shown).

The results of the Wald-Wolfowitz Runs Test (Table 4) demonstrate that the time-to-hatch in all Surflan TM and oryzalin treatment groups was significantly different from controls. In eggs produced by untreated females paired with treated males, hatching time was significantly increased relative to controls. The opposite trend was apparent in eggs produced by treated females paired with untreated males, where time-to-hatch occurred significantly earlier than in eggs produced by controls.

A 21-d exposure to Surflan TM or oryzalin induced intersex lesions with stage one oocytes (S1-Oo) and/or stage 2/3 oocytes (S2/3-Oo's) in male medaka in all treatment groups (Table 5). Pre-intersex lesions (PIL) were also observed in all treatment groups, and there was a low but measurable occurrence of PIL in controls. One control male exhibited a S1-Oo lesion (see discussion). Mean intersex lesion scores were significantly different than controls for S1-Oo (high-and low-dose Surflan TM groups); and for S1-Oo and S2/3-Oo's combined (all Surflan TM groups as well as the middose Oryzalin group). There was a marked difference in prevalence of S1-Oo's, S2/3-Oo's, or total intersex lesions (S1-Oo or S2/3-Oo) between treated and control groups, where prevalence refers to the number of animals in a treatment group to manifest a specific intersex lesion(s). Intersex prevalence data by treatment category (control, oryzalin, or Surflan TM ) are presented in Figure 1, and an example of a male with intersex testis is provided as Figure 2. Some necrosis of spermatids (NST) and necrosis of spermatogonia (NSG) were observed in control animals (Table 5), although the mean severity scores for these lesions were generally higher in animals exposed to Surflan TM or oryzalin. The mean lesion severity score for NST approached statistical significance in mid and low dose oryzalin animals. Mean lesion severity scores for NSG were significantly different from controls in the mid-dose Surflan TM and low-and mid-dose oryzalin groups. Evaluation of NST and NSG by treatment category (n = 18) demonstrated that oryzalin (p < 0.05) but not Surflan TM caused statistically significant increases in the mean lesion severity score for these categories of testicular lesion (Table 6).

Of the five categories of ovarian lesions; ovarian macrophage aggregates (OMA), oocyte atresia (OA), ovarian follicular necrosis (OFN), immature oocytes (IO), and ovarian hyperplasia or hypertrophy (OHH), three showed little to no effect of exposure to either Surflan TM or oryzalin (OMA, OA, OFN). All oryzalin dose groups exhibited mean lesion scores for IO's that were approximately half those determined for controls. None of these results was statistically significant when evaluated on the basis of dose (data not shown); however, when evaluated by treatment category, the Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J. mean lesion severity scores for IO's in oryzalin-exposed animals was significantly lower than in controls (Table 7). The severity of OHH also seemed to be affected by exposure to Surflan TM and oryzalin, with mean lesion severity scores in all dose groups greater than those in controls (data not shown). Evaluation of OHH by treatment category (Table 7) demonstrated that exposure to oryzalin was associated with a statistically significant higher mean lesion score for OHH relative to controls.

Discussion

Male medaka undergo sexual differentiation of the gonad at approximately 2 weeks post-hatch [START_REF] Okada | A further note on testis-ova in the teleost Oryzias latipes[END_REF].

Induction of intersex lesions in genotypic adult male medaka with differentiated testes following exposure to estradiol or xenoestrogens has been reported by [START_REF] Egami | Production of testis-ova in adult males of Oryzias latipes I. Testis-ova in the fish receiving estrogens[END_REF], [START_REF] Shibata | Evidence for the sexual bipotentiality of spermatogonia in the fish, Oryzias latipes[END_REF], and Gray, Niimi & Metcalfe (1999b) among others. The gonadal plasticity exhibited by male medaka has been attributed to the existence of bipotential spermatogonia, whereby type B (secondary) spermatogonia retain the ability to form ovarian tissue under estrogenic influences [START_REF] Shibata | Evidence for the sexual bipotentiality of spermatogonia in the fish, Oryzias latipes[END_REF].

In the present study, intersex was induced in 10/18 (55.6%) of medaka exposed to oryzalin and in 15/17 (88%) of medaka exposed to Surflan™ for 21 d (Figure 1). These are comparatively high values relative to the 17% incidence reported by Gray et al. (1999b) for adult medaka exposed to 200 or 300 µg/l octylphenol for 18 or 36 d, and are generally comparable to the ~ 70% incidence reported by [START_REF] Egami | Production of testis-ova in adult males of Oryzias latipes I. Testis-ova in the fish receiving estrogens[END_REF] in medaka exposed to 80 or 160 µg/l estradiol for 31 to 37 d.

The biological significance of intersex lesions to an individual fish is unclear, in part because intersex lesions tend to regress once the estrogenic stimulus is withdrawn [START_REF] Okada | A further note on testis-ova in the teleost Oryzias latipes[END_REF][START_REF] Shibata | Evidence for the sexual bipotentiality of spermatogonia in the fish, Oryzias latipes[END_REF]. Furthermore, it is not known whether intersex lesions within a testis affect fertility (Gray et al., 1999a).

The spontaneous development of intersex testes in naïve medaka has not been formally reported in the open literature. However, the experience in our laboratory has been that intersex testes do occur -albeit infrequently-in naïve fish (Koger, Teh & Hinton, 2000;[START_REF] Davis | Personal communication with L.C.H. concerning observations of intersex lesions in male medaka raised at the Institute of Ecology[END_REF]. One possible explanation for the occurrence of intersex testes in one of the control males is that these lesions can be induced by a potentially estrogenic compound present in the PC diet used by our laboratory. The PC diet formulated and prepared in our laboratory contains soy lecithin at approximately 6% of the total dry weight. Soy food products are estrogenic in humans (e.g., see [START_REF] Kurzer | Hormonal effects of soy in premenopausal women and men[END_REF], and it is likely that they would have similar activity in teleosts. Alternatively, estrogens released by females into aquaria water may be able to induce intersexeither singly, or in combination with the soy-derived lecithin. That biologically-active levels of estrogen may occur in aquaria water is indicated by reports of vitellogenin (Vg) in unexposed male fish (Tyler, van der Eerde, Jobling, Panter & Sumpter, 1996;[START_REF] Panter | Adverse reproductive effects in male fathead minnows (Pimephales promelas) exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone[END_REF][START_REF] Tyler | An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin[END_REF]. Vitellogenin expression is Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J. regulated by estradiol, and typically, Vg is not produced at measurable levels in males in the absence of estrogenic stimulus. Whether biologically active levels of estrogen are present in aquaria water in our facilities has not been established, nor is it known whether levels of estrogen, if present, are sufficient to induce intersex testes. Although the occurrence of the S1-Oo lesion in a control male cannot be definitively explained, it is clear that both Surflan™ and oryzalin dramatically affect the occurrence and severity of intersex lesions in medaka.

In this study, Surflan™ and oryzalin adversely affected spermatogenesis, increasing the incidence and severity of NST and NSG in treated animals above that observed in controls. In mammals, germ cell degeneration is a common phenomenon in adult males [START_REF] Huchkins | The morphology and kinetics of spermatogonial degeneration in normal adult rats: an anlysis using a simplified classification of germinal epithelium[END_REF][START_REF] Kerr | Spontaneous degeneration of germ cells in normal rat testis: assessment of cell types and frequency during the spermatogenic cycle[END_REF], and Nonclerq, Reverse, Toubeau, Beckers, Sulon, Laurent et al.

(1996) documented a low but measurable incidence of apoptosis in the seminiferous epithelium of untreated male hamsters. Although analogous data specific to teleosts does not exist, it is likely that a similar phenomenon occurs and is the underlying basis for the occurrence of NST and NSG in controls.

The effects of Surflan™ and oryzalin on NST and NSG are consistent with previously-reported effects of estrogens or xenoestrogens on spermatogenesis, in which necrotic spermatids [START_REF] Miles-Richardson | Effects of waterborne exposure to 4-nonylphenol and nonylphenol ethoxylate on secondary sex characteristics and gonads of fathead minnows (Pimephales promelas)[END_REF], inhibition of spermatogenesis [START_REF] Jobling | Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals[END_REF]Kinnberg et al., 2001[START_REF] Sohoni | Reproductive effects of long-term exposure to bisphenol A in the fathead minnow (Pimephales promelas)[END_REF], mild degeneration of testes [START_REF] Lange | Effects of the synthetic estrogen 17α-ethinylestradiol on the life-cycle of the fathead minnow (Pimephales promelas)[END_REF], and testicular atrophy [START_REF] Jobling | Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals[END_REF][START_REF] Panter | Adverse reproductive effects in male fathead minnows (Pimephales promelas) exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone[END_REF] have been reported. These adverse effects on gonad structure and gametogenesis encompass a range of diagnostic criteria and a spectrum of severity of effect. Underlying these differences are the use of separate species, distinct chemicals, different exposure durations, and different dosages.

Notwithstanding the existence of these variables, there is considerable consistency in the nature of effects on the male gonad associated with exposure to estrogenic substances. How a xenoestrogen elicits these effects is not clear, but Surflan™ and oryzalin may act directly at the gonad by either suppressing testosterone synthesis, altering testosterones' rate of conversion to estradiol by affecting p450aromatase, or indirectly via feedback effects at the pituitary that impact the synthesis and secretion of gonadotropins and in turn, affect steroid levels within the gonad.

The development of ovarian lesions after estrogenic challenge is both more poorly defined and less frequently identified than effects in males. We identified treatment-related effects on two categories of ovarian lesion; IO and OHH.

When analyzed by treatment type, oryzalin but not Surflan™ was associated with a significant decrease in the mean severity score for IO's. The scoring citeria for IO (Table 1) were established such that the lower the score, the fewer the number of immature oocytes present in an ovary. Oocytes are derived from mitotically-active oogonia, stem cells that develop from primordial germ cells, which migrate into the ovary during embryogenesis. Oogonial proliferation and oocyte formation are followed by development of the oocyte within a follicle. Under the influence of gonadotropins, Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

follicles synthesize testosterone and estradiol, where estradiol is produced via the p450 aromatase-mediated conversion of testosterone [START_REF] Hall | Gonadotropin action on gametogenesis and steroidogenesis in teleost gonads[END_REF]. The decrease in the number of immature oocytes in ovaries of exposed females may be a consequence of oryzalin acting to inhibit the formation or maturation of oocytes -either by some direct action on oogonia, or by interference with estrogen or gonadotropin homeostasis. [START_REF] Van Den Belt | Reproductive effects of ethinylestradiol and 4t-octylphenol on the zebrafish (Danio rerio)[END_REF] reported a qualitatively similar effect in zebrafish exposed to 17α-EE2 i.e., highly reduced ovarian somatic indices and an absence of "yolky oocytes", indicating that 17α-EE2 interferes with oocyte development in this species.

Many of the same treatment groups that displayed a decrease in the number of IO's, also exhibited OHH. The lesion score data for individual animals were reviewed, and in most cases, both lower IO scores and higher OHH scores were documented in the same ovary. Considering the scores for these two criteria, our data indicates that there was both a relative decrease in the number of IO's, and an increase in the number of developing oocytes and/or an overall enlargement of the ovary, primarily in oryzalin-treated animals. From estrogen receptor knockout mice, it has been established that estradiol is not required for the development of primary and secondary oocytes, but is critical to the ability of an oocyte to progress beyond the secondary stage to an antral state (for review, see [START_REF] Drummond | The role of estrogen in folliculogenesis[END_REF].

Subsequent oocyte maturation and ovulation then depend on estradiol and FSH-mediated expression of LH receptors, stimulation of an LH surge, and LH-mediated terminal differentiation of granulosa cells (reviewed in [START_REF] Robker | Hormonal control of the cell cycle in ovarian cells: proliferation versus differentiation[END_REF]. Therefore, interference with estradiol, FSH (GtH-I), or LH (GtH-II) synthesis, or xenoestrogen-induced suppression of GtH-I or GtH-II could impair normal ovulation and manifest as an increase in the number or size of maturing oocytes within an ovary as well as a greater ovarian size. However, if oocyte development or ovulation is impaired by treatment with oryzalin, the effect appears to be relatively subtle, as oryzalin did not measurably affect the fecundity of treated females.

Exposure to Surflan™ or oryzalin increased the production of nonfertilized eggs (and thus decreased the production of fertilized eggs) in all treatment groups of both genders. The production of nonfertilized eggs was a highly variable event, and was typically manifested as most or all of a given clutch of eggs failing to be fertilized on the day of collection. This tended to alternate with successful fertilization for a period of days, followed by another failed fertilization. This pattern of fertilization failure was observed in all of the treated groups, and yielded fertilization data characterized by substantial variability. This variability in the response of individuals within all treatment groups made it exceptionally difficult to demonstrate statistical significance.

Exposure to both Surflan™ and oryzalin was associated with reduced fertilization capacity of treated male medaka paired with untreated females. This phenomenon has been observed in zebrafish, where an exposure to 17α-EE2 Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J. caused a dose-related reduction in the number of unexposed females that spawned when paired with exposed males [START_REF] Van Den Belt | Reproductive effects of ethinylestradiol and 4t-octylphenol on the zebrafish (Danio rerio)[END_REF]. Male medaka exposed to the xenoestrogen octylphenol (OP) for 6 months were also found to be significantly less likely to fertilize eggs than untreated controls. These latter data, reported within the context of OPinduced effects on behavior, also documented a significant correlation between impaired courtship behavior in treated males and a lower egg fertilization success rate (Gray et al., 1999a). [START_REF] Bjerselius | Male goldfish reproductive behavior and physiology are severely affected by exogenous exposure to 17B-estradiol[END_REF] showed that a relatively brief exposure to estradiol (24-28 d) negatively impacted a number of courtship parameters in treated male goldfish paired with an unexposed, artificially-induced spawning female.Therefore, for males in particular, but also potentially for females, decreased fertility may be due to effects of treatment with estrogenic substances on reproductive behavior. Although certain data indicate that exogenous estrogen may impact brain p450arom activity, a causal link between altered p450aromatase activity and behavior have not been established [START_REF] Callard | The goldfish as a model for studying neuroestrogen synthesis, localization, and action in the brain and visual system[END_REF][START_REF] Melo | Sexual dimorphism of brain aromatase activity in medaka; induction of a female phenotype by estradiol[END_REF].

There were statistically significant differences in the time-to-hatch of eggs from all groups of males and females treated with Surflan™ and oryzalin (Table 4). In all Surflan™ and oryzalin dose groups, eggs produced by untreated females paired with treated males took significantly longer to hatch than eggs from controls, and eggs produced by treated females paired with untreated males hatched significantly earlier than controls. For exposed medaka of both genders, the effects on time-to-hatch were remarkably consistent, occurring in all dose groups. The biological significance of these effects is not clear, given that survival of progeny was not affected by exposure, nor were there any effects on the incidence of deformities in juveniles grown to approximately 30 d post-hatch.

Although we have documented gonadal lesions in both males and females and altered fertilization success in both genders of medaka exposed to Surflan™ or oryzalin, there is not a complete correspondence between the groups that manifested the most severe lesions and the groups that exhibited the greatest effects on fertility. However, in general, treatment with oryzalin at 0.5 mg/l or 0.25 mg/l, and treatment with Surflan™ at 2.0 or 1.0 µg/l had the greatest adverse effects on male and female fertility as well as on the severity of non-intersex gonadal lesions.

The maximum dose of oryzalin and maximum concentration of Surflan™ used to assess reproductive toxicity were selected on the basis of range-finding studies that identified an apparent MTC for each compound. Mid-and lowdose amounts of Surflan™ and oryzalin were set at approximately 50% decrements of the MTC or of the mid-dose quantity, respectively. Surflan™ is an emulsion that contains 40.4% oryzalin, and at the concentrations used, gave nominal concentrations of oryzalin of 1.5 mg/l, 0.8 mg/l, and 0.4 mg/l, levels that are approximately 50-60% higher than were administered for oryzalin given alone. Despite the administration of greater amounts of oryzalin in the Surflan™ groups, in general, Surflan™ did not elicit more severe effects than the lower doses of oryzalin administered alone. The Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

one clear exception to this is seen in the prevalence and severity data for induction of intersex lesions, where Surflan™ consistently caused more severe lesions as well as a greater prevalence of these lesions than oryzalin alone. It is also noteworthy that the high-dose oryzalin group (1 mg/l) had no statistically significant or marginally significant effects on non-intersex testicular lesions or on any measure of male or female fertility, and in only one category of ovarian lesion, OHH, did effects approach statistical significance. In fact, the most severe lesions often occurred in the low-dose oryzalin group, or the effects in that group were similar in magnitude to those observed from treatment with the next higher dose.

Similarly, the highest concentration of Surflan™ was not associated with significant effects on non-intersex testicular lesions or on any ovarian lesion. These results suggest that oryzalin administered alone, or administered within the mixture Surflan™, has a distinctly non-linear dose response for the reproductive parameters assessed here. This phenomenon has been documented for certain other xenoestrogens, but is not a universal characteristic of these compounds [START_REF] Andersen | Quantitative mechanistically-based dose-response modeling with endocrine-active compounds[END_REF][START_REF] Melnick | Summary of the National Toxicology Program' s report of the endocrine disruptors low-dose peer review[END_REF]. The National Toxicology Program's Panel on low-dose effects and nonmonotonic dose-response relationships for endocrinedisrupting chemicals (EDCs) focused on mammalian testing of EDCs, and did not extend their review to fish.

Nonetheless, the panel concluded that current testing 'paradigms' for the assessment of reproductive and developmental toxicity may require reassessment to account for this fact [START_REF] Melnick | Summary of the National Toxicology Program' s report of the endocrine disruptors low-dose peer review[END_REF]. It is clear from these data that a lowestobserved-adverse-effect-level for reproductive effects of oryzalin and Surflan™ has not been defined by our work.

Further testing to evaluate the consequences of exposure to environmental levels of oryzalin must consider the doseresponse phenomenon suggested by our data and structure studies accordingly.

Conclusion

A relatively brief (21 d) exposure of mature, reproductively-active medaka to Surflan™ and oryzalin impaired fertilization success of both males and females, and affected the hatching time of eggs. Fecundity, viability of eggs, and incidence of deformities of offspring were not affected by exposure. Exposure to Surflan™ or oryzalin increased the prevalence and severity of intersex lesions, and exposure to oryzalin increased the severity of NST and NSG. Whether the testicular lesions caused by exposure led directly to the observed decrements in fertility in males, or whether there are indirect effects of treatment that adversely affect fertility -such as impairment of reproductive behavior-are not known.

Oryzalin affected the number of immature oocytes present in the ovaries of exposed females, potentially via inhibition of the mitotic division of oogonia. Alternatively, oryzalin may inhibit oocyte development and/or impair ovulation, leading to the observed paired phenomena of decreased IO and increased OHH scores. Oryzalin also adversely affected spermatogonia, and thus it appears that oryzalin may be capable of affecting the viability or function of germ cells Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

of both males and females. Although the mechanism(s) of these effects remains to be identified, it appears plausible that a phenomenon experienced by both genders -such as alteration in estrogen homeostatis-may underlie the toxicity elicited by these compounds.

Table 1. Histologic lesion definitions and criteria. A criterion score of 0 was assigned if no lesion was present.

Gender

Lesion (criterion score) a Definition Males Pre-intersex lesion (PIL) (1: < 2 PIL cells; 2: 2-5 PIL cells; 3: >5 PIL cells).

Hypertrophied spermatogonia; karyomegaly; prominent single nucleoli; increased euchromatin; numerous provitelline nucleoli. Intersex: stage 1 oocytes (S1-Oo) (1: < 1 S1-Oo; 2: 1-2 S1-Oo clusters; 3: > 2 S1-Oo clusters).

Clusters of small cells as discrete acini distinct from normal seminiferous tubules. Pale eosinophilic to amphophilic cytoplasm; higher than normal nucleus to cytoplasm ratio; prominent oval nuclei with more euchromatin and an absence of distinct nucleoli. Intersex: stage 2/3 oocytes (S2/3-Oo) (1: 1-2 stage 2 oocytes; 2: >2 stage 3 single oocytes or 1 oocyte cluster; 3: >2 clusters of stage 3 oocytes).

Stage 2 oocytes: 5-15 µm, usually solitary; large central nuclei with diffuse euchromatin and small amounts of intensely basophilic cytoplasm; Stage 3 oocytes: clusters of 15-45 cells, to 30 µm; cells with abundant intensely basophilic cytoplasm, marked karyomegaly (nuclei 4-30 µm). Translucent euchromatin and provitelline nucleoli. Necrosis, spermatids (NST) (1: < 2 dead spermatids; 2: 2-20 dead spermatids; 3: > 20 dead spermatids. Numbers are per 40X field).

Spermatids with irregular hyperchromatic heads (2-5 times normal size).

Necrosis, spermatogonia (NSG) (1: 2-10 dead spermatogonia; 2:11-20 dead spermatogonia; 2:>20 dead spermatogonia. Numbers are per 40X field).

Rounding of individual spermatogonia, hyper-eosinophilic cytoplasm, and nuclear pyknosis.

Females Ovarian macrophage aggregates (OMA) (1: < 3 OMA; 2: 3-10 OMA; 3: > 10 OMA Numbers are per 20X field).

Large macrophages (> 50 µm) with foamy pale eosinophilic debris concentrated in the medullary portion of the ovary.

Ovarian follicular necrosis (OFN) (1: < 4 necrotic follicles); 2: 4-5 necrotic follicles; 3: > 6 NF). Numbers are NF per 4, 10X fields.

Presence of large necrotic follicles (several hundred µm diameter), collapsed, distorted, and empty of oocyte. Follicles lined by necrotic columnar epithelium.

Oocyte atresia (OA) (1: < 3 atretic oocytes (AO per ovary); 2: 3-5 AO; 3: > 5 AO).

Atretic oocytes or follicles with distinct chorions and abundant yolk protein. Chorion distorted and collapsed, yolk disorganized and fragmented; variable influx of macrophages. Ovarian hypertrophy/hyperplasia (OHH) (1: ovary 20-30% of abdominal cavity (AC); 2: ovary 31-40% of AC; 3: ovary >40% of AC).

Overall enlargement of ovary, with an increased number and density of developing ooctyes.

Immature oocytes (IO) (1:IO 5-10% of total oocytes (TO); 2: IO 11-50% TO; 3: IO>50% TO).

Characterized by scoring the percentage of immature oocytes within an ovary by small size, absence of definitive chorion, absence of distinct yolk protein or oil droplets, abundant basophilic cytoplasm, distinct nuclei. ψ Probability of obtaining 4 of 6 tests with p < 0.05 by chance = 0.00008 [START_REF] Moran | Arguments for rejecting the sequential Bonferroni in ecological studies[END_REF].

τ Probability of obtaining 2 of 6 tests with p < 0.05 by chance = 0.03 [START_REF] Moran | Arguments for rejecting the sequential Bonferroni in ecological studies[END_REF]. Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J. Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

Figure 2. A. Intersex testis in a mature, sexually active male medaka exposed to 0.5 mg/l oryzalin. Arrows point to primary oocytes (100X). Higher magnification view (600X white box) reveals B. a significant number of primary oocytes (>10) seen within a testicular tubule (arrows), and C. several degenerated primary oocytes. All views from hematoxylin and eosin stained tissue.

Figure 1 .

 1 Figure 1. Prevalence of intersex lesions in testes of control medaka and those exposed to oryzalin and Surflan™. S1-Oo = stage 1 oocytes; S2/3-Oo = stage 2 or 3 oocytes; Total Intersex = combined stage 1 and stage 2 or 3 intersex lesion severity scores.

Table 2 .

 2 Daily mean production of non-fertilized eggs in medaka after exposure to either Surflan™ or oryzalin. Data are from 12 replicates; 6 containing treated males (paired with untreated females) or 6 treated females (paired with untreated males). Each replicate contained 2 males and 2 females. b Data are from 6 replicates containing treated females paired with untreated males. Each replicate contained 2 males and 2 females. c Data are from 6 replicates containing treated males paired with untreated females. Each replicate contained 2 males and 2 females. n.s. = not significant. *p < 0.05 determined by ANOVA. **p < 0.005 determined by ANOVA. ***p < 0.001 determined by ANOVA.

	Exposure concentration	Treated medaka
		Females and Males a	Treated Females b	Treated Males c
	Control Surflan TM 3.8 µl/l Surflan TM 2.0 µl/l	1.7 3.7* ψ 3.7 ** ψ	1.7 3.1 n.s. 2.9 n.s.	1.8 4.4 n.s. 4.6* τ
	Surflan TM 1.0 µl/l	2.7 n.s.	2.3 n.s.	3.2 n.s.
	Oryzalin 1 mg/l Oryzalin 0.5 mg/l Oryzalin 0.25 mg/l	5.1 n.s. 4.8 *** ψ 4.7 ** ψ	5.3 n.s. 4.5 * τ 5.2 ** τ	4.9 n.s. 4.9 * τ 4.2 n.s.

a

Table 3 .

 3 Mean daily total egg production of medaka after exposure to either Surflan™ or oryzalin. The tests of significance are Kruskall-Wallace one-way ANOVA nonparametric tests of differences in the medians between the individual treatment and control groups. All α values for determining statistical significance were adjusted by a Bonferroni correction to account for multiple comparisons. Data are from 12 replicates; 6 containing treated males (paired with untreated females) or 6 treated females (paired with untreated males). Each replicate contained 2 males and 2 females. Kruskall-Wallace ANOVA results for Treated medaka, Females and Males: F ratio, 1.81; p = 0.095; DF = 6 b Data are from 6 replicates containing treated females paired with untreated males. Each replicate contained 2 males and 2 females. Kruskall-Wallace ANOVA results for Treated Females: F ratio, 2.44; p = 0.0269; DF = 6

	Exposure concentration	Treated medaka
		Females and Males a	Treated Females b	Treated Males c
	Control	28.0	30.8	25.1
	Surflan TM 2.5 µl/l	23.3 *	23.6 *	23.0 n.s.
	Surflan TM 1.3 µl/l	26.1 n.s.	24.8 n.s.	27.4 n.s.
	Surflan TM 0.67 µl/l	28.8 n.s.	29.7 n.s.	27.8 n.s.
	Oryzalin 1 mg/l	28.4 n.s.	35.6 n.s.	21.1 n.s.
	Oryzalin 0.5 mg/l	28.2 n.s.	31.7 n.s.	24.7 n.s.
	Oryzalin 0.25 mg/l	22.8 *	26.9 n.s.	18.6 *

a

Table 7 .

 7 Mean ovarian lesion scores in medaka compared by category of treatment. OMA, ovarian macrophage aggregates; OA, oocyte atresia; OFN, ovarian follicular necrosis; IO, immature oocytes; OHH, ovarian hyperplasia or hypertrophy. b Scores are the mean of observations on 6 females per treatment group. c Scores are the mean of observations on 18 females per treatment group. d Result is statistically significant (p = 0.007) by one way ANOVA.

	Treatment category	OMA a	OA a	OFN a	IO a	OHH a
	Control b	1.83	0.17	1.83	1.2	2.2
	Oryzalin c	2.10 n.s.	0.17 n.s.	2.01 n.s.	0.67 d	2.80 d
	Surflan TM c	2.30 n.s.	0.17 n.s.	1.67 n.s.	1.28 n.s.	2.30 n.s.

a

Acknowledgements Funding provided by the California Department of Transportation.

Hall, L.C., Okihiro, M., Johnson, M.L, and Teh, S.J.

c Data are from 6 replicates containing treated males paired with untreated females. Each replicate contained 2 males and 2 females. Kruskall-Wallace ANOVA results for Treated Males: F ratio, 1.72; p = 0.1164; DF = 6 n.s = not significant. * p < 0.05. Not significant by the criterion for statistical significance (p < 0.008) established by use of the Bonferroni correction for multiple comparisons; data are included to indicate that a value approached statistical significance. ψ Probability of obtaining 4 of 6 tests with p < 0.05 by chance = 0.00008 [START_REF] Moran | Arguments for rejecting the sequential Bonferroni in ecological studies[END_REF].

φ Probability of obtaining 3 of 6 tests with p < 0.05 by chance = 0.04 [START_REF] Moran | Arguments for rejecting the sequential Bonferroni in ecological studies[END_REF].

τ Probability of obtaining 2 of 6 tests with p < 0.05 by chance = 0.03 [START_REF] Moran | Arguments for rejecting the sequential Bonferroni in ecological studies[END_REF].


Produced water undergoes changes in its physical chemistry including precipitation of heavy metals after being discharged and mixed with ambient seawater. Potential impacts of the precipitation of heavy metals on their transport and toxicity were studied using samples from offshore oil production sites on the Scotian Shelf off eastern Canada. Concentrations of aluminum, cadmium, chromium, cobalt, copper, iron, lead, manganese, nickel and zinc were measured in total, particulate and dissolved fractions together with Microtox ® tests for assessment of toxicity.

Heavy metals in produced water were transformed from dissolved to particulate phase in a period of hours under oxygenated conditions, and aggregated to larger particles that settle rapidly (>100m/day) over a few days. In addition, there was production of buoyant particles comprised of heavy metal precipitates sequestered onto oil droplets that were transported to the surface.

The particulate fraction was generally more toxic than the dissolved fraction. This was evident at the mixing interface between produced water and seawater where elevated particulate and toxicity levels were observed. Laboratory studies suggest an increase in the toxicity of discharged produced water over time. Time-series experiments showed a sustained toxic response for more than a week following the oxidation of freshly discharged produced water that initially elicited little or no toxic response in the Microtox ® Test.

Chemical processes identified in this study, namely precipitation of heavy metals and consequent settling and rising fluxes of particles, will influence the toxicity, the fate and the transport of potential contaminants in the produced water. Therefore, these processes need to be considered in assessment of the environmental impact associated with offshore oil and gas operations.

INTRODUCTION

Produced water comprising naturally occurring formation water in the oil and/or gas reservoir and seawater injected to maintain reservoir pressure, has been classified as the largest contributor to the offshore waste stream [START_REF] Krause | Spatial and Temporal Variability in Receiving Water Toxicity near an Oil Effluent Discharge Site[END_REF], constituting approximately 80% of the wastes and residuals produced from natural gas production operations [START_REF] Mccormack | Analysis of Oilfield Produced Water and Production Chemicals by Electrospray Ionization Multistage Mass Spectrometry [ESI-MSn[END_REF]. It is usually a hot, saline reduced water that contains high concentrations of heavy metals, radionuclides, hydrocarbons and ammonia compared to the receiving environment. It may also contain process chemicals such as demulsifers and corrosion inhibitors added during the operation of the well [START_REF] Somerville | Environmental Effect of Produced Water from North Sea Oil Operations[END_REF]. Despite the identification of a multitude of potential contaminants in produced water, regulatory guidelines for its disposal in Canada are solely based on the concentration of residual hydrocarbons (National Energy Board et al., 2002).

The produced water flow from an oil/gas well and its trace chemical content can be quite variable. Data on chemical composition from the PanCanadian Cohasset/Panuke production facility on the Scotian Shelf (SOEP, 1996) indicate that trace metal concentrations in the produced water are 10 2 to 10 5 times those in seawater. Maximum volume discharge for offshore installations on the Scotian Shelf has been estimated to be of the order of hundreds of cubic metres per day. In consideration of the concentration of potential contaminants, future anticipated discharge volumes and number of production sites, this may represent a substantial input of contaminants to the Scotian Shelf.

Environmental impact of produced water has been assessed by measuring chemical composition of freshly discharged produced water [START_REF] Tibbets | A comprehensive determination of produced water composition[END_REF][START_REF] Jacobs | The composition of produced water from shell operated oil and gas production in the North Sea[END_REF]Flynn et al., 1992), by plume dispersion-transport models (Brandsma and[START_REF] Brandsma | Dispersion modeling perspectives on the environmental fate of produced water discharges[END_REF][START_REF] Yeats | Dispersion of produced water in a coastal environment and its biological implications[END_REF] and by ecotoxicological measurements [START_REF] Brendehaug | Toxicity testing and chemical characterization of produced water -a preliminary study[END_REF][START_REF] Neff | Aromatic hydrocarbons in produced water: bioaccumulation and trophic transfer in marine food webs[END_REF]. Because of high variability in its chemical composition, toxicity of produced water shows a wide range of results (summarized by [START_REF] Patin | Environmental impact of the offshore oil and gas industry[END_REF]. A response of vulnerable zooplankton, especially in embryonic and larval stages, has been reported and the accumulation of lipophilic hydrocarbons is suspected to cause a reduced survival rate [START_REF] Gamble | Mesocosm experiments on the effect of produced waters from offshore oil platforms in the northern North Sea[END_REF][START_REF] Davies | Sources of environmental disturbances associated with offshore oil and gas development In North Sea oil and the environment. Developing oil and gas resources, environmental impoacts and responses[END_REF]. In California, [START_REF] Osenberg | Spatial scale of ecological effects associated with an open coast discharge of produced water[END_REF] described the effects of produced water on infauna populations near a discharge. Purple sea urchin (Strongylocentrotus purpuratus) fertilization tests at the same site showed toxicity for dilution rates as high as 1,000,000:1 [START_REF] Krause | Effects of produced water on early life stages of a sea urchin: stage-specific responses and delayed expression[END_REF]. Ecosystem models by Rivkin et al. (2002) and [START_REF] Khelifa | Numerical Investigation of Impact of Nutrient Inputs from Produced Water on the Marine Planktonic Community[END_REF] predicted that the discharge of inorganic nutrients (e.g. ammonia and phosphorus) and dissolved organic carbon fractions (e.g. volatile fatty acids) within the produced water stream may alter planktonic community structure.

Rapid dilution of produced water with ambient water is often argued as sufficient to mitigate any impacts on the marine environment. [START_REF] Somerville | Environmental Effect of Produced Water from North Sea Oil Operations[END_REF] modelled the dilution of produced water discharged from the Brent platform in the North Sea. For a 10,000 m 3 /day discharge rate, they estimated a 100-fold dilution at 50 m and a 2,800-fold dilution at 1,000 m from the platform. [START_REF] Furuholt | Environmental Effects of Discharge and Reinjection of Produced Water[END_REF] modelled a 2,000 m 3 /day discharge and found a 1,000:1 dilution approximately 50 m downstream of the discharge point. For a 20,000 m 3 /day discharge he found a 1,000:1 dilution approximately 500 m from the discharge point. However, Washburn et al. (1999) modeled the dispersion of produced water at the Carpinteria production site in California using a buoyant plume model, and found that concentrations can exceed lethal levels for sea urchin fertilization bioassay >1000 m from the discharge site.

Physical dispersion models of projected discharges from the SOEP wells (SOEP, 1996) indicate that rapid dilution of the discharge will dilute contaminant concentrations to non-acutely toxic levels within very short distances from the discharge points. These models, however, treat chemicals as passive tracers. Produced waters can contain tens to hundreds of milligrams per litre of elements such as aluminum, iron and manganese that will precipitate on contact with oxygenated seawater. Based on produced water concentrations reported for the Venture well in the SOEP project (SOEP, 1996), there could be enough dissolved aluminum and iron to generate >1 g/l of suspended particulate matter (SPM). How important will the transformation of these dissolved metals to precipitates be in sequestering other more toxic contaminants, altering their toxicity, and transporting them by routes other than passive dilution in the receiving waters? The experiments being reported in this paper were designed to address these questions based on 1) measurements of major and trace element concentrations in produced water, 2) experiments to determine the chemical phase transitions that occur when produced water reacts with oxygen and seawater, and 3) toxicity tests with a regulatory assay on the produced water samples and the various chemical fractions generated in the experiments.

MATERIALS AND METHODS

Twenty produced water samples were collected from Aug. 5, 1998 to June 21, 1999 at the Cohasset and Panuke oil production sites on Sable Island Bank, Nova Scotia, Canada (Fig. 1).

Water samples were collected by the rig staff using high density, polyethylene bottles (2-4 l) that were pre-cleaned with 6 N HCl, then rinsed with milli-Q water. Most samples were collected in the morning, immediately air-lifted to the Halifax airport and transported to the laboratory.

Using this protocol, we could start our experiments within a few hours of sample collection at the oil production sites. Sample bottles were modified with a conical plastic insert in the cap that facilitated the collection of a sample without trapping air in the bottle. Most samples arrived in the lab without showing any outward signs of oxidation, however, some did contain trapped air and indications that oxidation had occurred. Flow rates at time of sampling as well as produced water temperatures were recorded by the rig staff, as were hydrocarbon concentrations for many of the samples. Oxygen saturation levels and salinity were measured in our laboratory.

Manipulation of samples including sub-sampling for various experiments and filtration for various analyses was conducted in a glove box purged with nitrogen. Concentrations of Al, Cd, Cr, Co, Cu, Fe, Pb, Mn, Ni and Zn were measured in filtered or unfiltered water samples and in the particulates collected during filtration. Thirty ml water samples were acidified with 30 µl of high purity HNO 3 and analyzed by ICP-MS (PE/Sciex ELAN 6000) without any preconcentration or separation. Because of the high salt content of the samples, all water samples were diluted with milliQ water (50 to 150 fold dilutions) before analysis. The particulate fractions were collected by filtering aliquots of water through cleaned, tared 0.2µm Nuclepore filters. After rinsing with Milli-Q water to remove salt and drying, each filter was weighed on a microbalance to determine SPM concentration, then digested with 2.0 ml of HNO 3 and 1.0 ml of HF in a Loran teflon bomb [START_REF] Loring | Manual for the geochemical analyses of marine sediments and suspended particulate matter[END_REF]. We used minimum amount of Milli-Q water to rinse the salt, however, the loss of some metals is possible. The bomb was heated inside a plastic pressure chamber in a microwave oven for 45 seconds. The digested solution was heated in a teflon beaker on a hot plate (170-200 ºC) in a clean bench to evaporate the solution to dryness. The inside walls of the beaker were washed down with two 10 ml aliquots of 2% HNO 3 , and left to sit for 30 min to allow re-solution of digested metals. Concentrations were measured by ICP-MS. Oxygen was measured using an oxygen meter (Orion, model 840) and salinity by refractometer. Toxicity was assessed with the Microtox ® Test based on the reduction of bioluminescence by the marine bacterium Vibrio fischeri on exposure to toxicants [START_REF] Johnson | Microtox® toxicity test system -New developments and applications[END_REF][START_REF] Qureshi | Microtox toxicity test systems -Where they stand today[END_REF]Environment Canada, 1992).

In addition to measurements of chemistry and toxicity of untreated produced water, three separate experiments were conducted to study the changes in composition and toxicity after mixing with seawater, and on exposure to oxygen.

First, a preliminary time series study of settling particulate matter from different mixtures of produced water and seawater was conducted using the produced water sample collected on Sep. 16, 1998. Four sets of dilutions included raw produced water and three mixtures of produced water and seawater at dilution rates of 1:2, 1:10 and 1:100. Filtered seawater (0.2 µm Nuclepore filter) from the Scotian Shelf was used for dilutions. Four dilutions containing different ratios of produced water and seawater were placed in Inhoff glass cones for 148 hours. Sub-samples (50ml) were collected from the bottom of cones at time intervals: 0, 24, 95, 119 and 148 hours and particulate matter concentrations were measured gravimetrically.

Secondly, produced water/seawater interface experiments were conducted to study precipitation, flocculation and settling behaviour of heavy metals. Produced water samples used in these experiments were collected on Sep. 28, Oct. 14 and Oct. 19, 1998. For each experiment, 200 ml of produced water were layered under 200 ml of seawater in Inhoff cones (Fig. 2). In this experiment, seawater was measured into the Inhoff cones and produced water, which has a higher density than seawater, was slowly introduced from the bottom of the Inhoff cones under the seawater layer using a peristaltic pump. The density interface made by this method is stable and mixing is limited by the diffusion at the interface (Azetsu-Scott and [START_REF] Azetsu-Scott | Measuring physical characteristics of particles: A new method of simultaneous measurement for size, settling velocity and density of constituent matter of particles[END_REF]. This stratification allowed us to observe chemical processes occurring at the interface between produced water and ambient seawater under a controlled environment. Two Inhoff cones were set using the Sep. 28 produced water sample to take time series samples at t=3.9 and t=25 hours and three Inhoff cones were set for Oct. 14 and 19 samples for time series samples at t=1.5, 71.5 and 144 hours. Sub-samples were taken from the bottom of the each Inhoff cone (Fig. 2) from two produced water layers (70 ml for L-1 and 90 ml for L-2), one produced water/seawater interface layer (80 ml for L-3) and two seawater layers (90 ml for L-4 and 70 ml for L-5). At the end of the Oct. 14 and 19 experiments, particles adhering to the walls of the Inhoff cones were recovered by washing with 50 ml of distilled water, although some particles could not be removed and still adhered to the walls. Heavy metal concentrations were measured for both particulate and dissolved fractions in each layer during the experiment using the Oct. 14 sample.

Microtox ® Tests were conducted on unfiltered and filtered samples from each layer.

Thirdly, two experiments were conducted to investigate the effect of oxygen on produced water. Aliquots of 100 ml of produced water were placed in 500 ml high density polyethylene bottles and stirred on a rotating table. Time series samples were taken at time 0 and 1 hour, and 1, 2, 3, 8 and 15 days for produced water collected on Dec. 14, 1998. For produced water collected on Feb. 8, 1999, samples were taken at 0 and 3 hours, and 1, 2, 3, 4, 7, 10, 14, 16 and 21 days. Heavy metal concentrations in the particulate fraction were measured together with particulate matter concentration, oxygen saturation and microtoxicity tests.

Metal precipitates from several of these experiments were observed using Scanning Electron microscopy with Energy Dispersive X-ray analysis and fluorescent microscopy.

RESULTS AND DISCUSSION

(1) Temporal variability of heavy metal concentrations and toxicity of source produced water Metal concentrations measured in unfiltered produced water samples (Table 1) clearly show that the concentrations can be very high (as much as 50,000 times ambient seawater) and very variable. High concentrations were especially evident for Cu, Fe and Zn. Enrichment of metal concentration in produced water compared to that in the ambient seawater is Zn>Fe>Cu>Mn>Pb,Ni>Al,Cd>Co. Although the composition of produced water varied widely, some affinity among elements was observed. These include Fe, Mn and Al, which varied in the same manner and showed highest concentrations on Oct. 14, while Cu and Zn co-varied and showed high concentrations for samples collected on Aug. 13 and Oct. 5. Observed concentrations of Al and Fe, elements that are responsible for the formation of precipitates, are lower than anticipated based on previously published data for these fields. Approximately 25-50 mg/l of SPM could be generated from the observed levels of Al, Fe and Mn. The samples were analyzed for metals by ICP-MS after diluting the samples with milli-Q water. As a result the detection limits are not as low as normally expected for analysis of aqueous samples. None of the samples had Cr or Co concentrations above their detection limits (80 and 2 µg/l, respectively).

The large number of not detected results would suggest that a more sensitive technique for determination of heavy metals would be preferred. Nevertheless, it is evident that heavy metal concentrations can exceed levels at which toxic effects would be expected. Concentrations of 4.2 mg/l Cu and 26 mg/l Zn are clearly in the potentially toxic range. Copper toxicity to marine plankton is seen at tenths of µg/l [START_REF] Fitzwater | Metal contamination and its effect on primary production measurements[END_REF][START_REF] Coale | Effects of iron, manganese, copper and zinc enrichments on productivity and biomass in the subarctic Pacific[END_REF] and at even lower levels in laboratory cultures [START_REF] Gavis | Toxic binding of cupric ion by marine phytoplankton[END_REF]. Zinc is not as toxic, but toxicity to plankton is seen at approximately 10 µg/l [START_REF] Davies | The effects of heavy metals upon natural marine phytoplankton populations[END_REF].

The Microtox Assay is a regulatory biotest for testing the toxicity of environmental samples.

It is based on the reduction of light produced by a photo-luminiscent marine bacterium that is used in the test system on exposure to toxicants [START_REF] Johnson | Microtox® toxicity test system -New developments and applications[END_REF]. Results from the Microtox ® Test also showed large variation, from 18.7% to >99.0% for unfiltered samples and 16.1% to >99.0 % for filtered samples (Table 2). This can be attributed to the fact that samples were recovered from different sectors of the formation as they were exploited by production operations during the experimental period. Toxicity (EC 50 ) derived from dose response curves is expressed as the percentage of produced water in a test solution that will generate a 50% decrease in bioluminescence. Thus, lower numbers indicate greater toxicity. Some of the non-toxic samples (EC 50 >99%; no toxicity at the EC 50 level observed in undiluted samples) did show evidence of a toxic response at the EC 20 level (>20% decrease in bioluminescence) and these samples were expressed as >99*. EC 50 values were not achieved in 66% of total and 83 % of dissolved produced water samples. When toxicity was observed, unfiltered samples showed higher toxicity than filtered samples, with the exception of the sample collected on Feb. 8, 1999. The difference between filtered and unfiltered samples inferred that the particulate phase had an influence on toxicity of discharged produced waters. No clear correlation was observed between toxicity results and salinity, temperature, flow rate, hydrocarbon or oxygen concentrations. Despite metal concentrations being much higher in produced water than those in the natural seawater, no significant correlation between toxicity and metal concentrations was observed, indicating that metal concentrations alone may not be responsible for the observed toxicity.

(2) Formation of particulate matter from produced water Although there is no indication in the results described above of a strong link between heavy metal concentration and toxicity, there are substantial quantities of metals such as Al, Fe and Mn that will precipitate on contact with seawater, absorb heavy metals and other chemicals, and settle to the bottom, thus altering the transport of the contaminants in the produced water plume and potentially focusing their accumulation onto the sediments. The actual location for this accumulation will depend on the oxidation/precipitation rates of the Al, Fe and Mn and current speeds. A series of experiments was conducted to investigate these chemical reactions and how they affect toxicity. In the first of these experiments, produced water was simply exposed to atmospheric oxygen in 500 ml settling bottles and the production and settling of precipitates was observed. Transformation of heavy metals from dissolved to particulate phase was observed at a time scale of hours in these experiments with raw produced water, and three mixtures at dilution rates of 1:2, 1:10 and 1:100 (Fig. 3). For all dilutions, more than 50 % of the SPM settled within 24 hours. Settling velocity of a particle (≈5 mm in diameter) observed in the undiluted produced water sample was measured directly and it exceeded 100 m/day. Formation and settling of particles would increase the vertical transport of heavy metals by one to two orders of magnitude over that from simple eddy diffusion in the ocean, indicating that chemical processes that alter the physical characteristics (i.e. the settling velocity) of produced water can have an impact on the transport of potentially toxic produced water chemicals.

(3) Heavy metal dynamics and toxicity at the seawater-produced water interface To investigate precipitation, aggregation and settling of metals in produced water during the interaction with ambient seawater in a more controlled manner, produced water and seawater were layered in Inhoff cones (Fig. 2). In these experiments a sharp interface between produced water and seawater layers was clearly visible because of their different refractive indices, and production and removal of particles at and from this interface was evident to the naked eye.

Figure 4 shows SPM concentrations and salinity in the five layers for three experiments. Rapid removal of particles from the interface layer, where particle production was active, was observed as occasional downward plumes, and resulted in high concentration of particulate matter in L1.

The net increase of SPM concentration in the interface and produced water layers during the experiment for the Oct. 14 sample was 50%, while the increase was 71% and 105% for the Sep. 28 and Oct. 19 samples, respectively. Increased concentrations of SPM in the very surface layer (L5) were observed with time (Fig. 4). Seawater was pre-filtered, therefore, no particles were in L5 at t=0. Salinity variation in this layer was minimal, so the increase in SPM concentrations in L5 indicates an upward transport of particles from the produced water layers rather than mixing of produced water with seawater.

Produced water collected on Oct. 14 and 19 contained high concentrations of hydrocarbons, 40 mg/l and 30mg/l, respectively, and white emulsion was evident in the Oct. 14 sample. At the end of the Oct. 14 and 19 experiments, the walls of the Inhoff cones were greasy due to the adhesion of residual particles from the L5 layer. These observations would suggest that buoyant oil droplets with attached particles are responsible for the upward transport to the surface layer.

Microscopic observation of the particles in the surface slick confirmed that these particles were oil droplets (Fig. 5-a) with inorganic particles adhering to their surface (Fig. 5-b).

Particulate and dissolved metal concentrations were measured during the experiment using produced water collected on Oct. 14. The initial produced water sample contained visible black particles and concentrations of total Fe, Mn, Ni and Al were high (Table 1). Over the course of the experiment, the concentrations of dissolved Cu, Fe and Zn decreased markedly (~10 fold), while those of Mn decreased only slightly (Figure 6). Much more rapid precipitation is seen for Fe than for Mn consistent with the slower oxidation kinetics of Mn 2+ .

Particulate Cr and Ni concentrations were positively correlated with particulate Mn concentration (Fig. 7-a), while particulate Al concentration was positively correlated with particulate Fe concentration (Fig. 7-b) for both produced water and seawater layers. These results suggest co-precipitation/adsorption of these metals with Fe and Mn oxides. Particulate Pb and Fe concentrations were correlated differently in the produced water and seawater layers, showing enrichment of Pb on particles in the seawater layer that are buoyant oil droplet aggregates transported from the produced water layer (Fig. 8). Similar enrichment of the surface layer particles was observed for Cu and Zn, but not for Al, Fe, Mn, Cr or Ni. It would appear that this mechanism actively transports some heavy metals (Pb, Cu and Zn) to the sea surface, separating them from the subsurface produced water layer and settling metal oxide particles enriched in Cr and Ni.

Coefficients for the pseudo-equilibrium distribution of metals between particulate and dissolved phases (K d = [Me p ] /[Me d ]) are frequently used in descriptions of metal distributions and dissolved-particle interactions in marine systems. In this experiment K d s for all of the metals increased from low values in the produced water to high values in the seawater layer at the end of the experiment. For Cu, Ni and Zn the final K d s for the seawater layer were approaching consensus values for pelagic sediment deep water K d s (as compiled for example by IAEA, 2004). This would imply that concentrations in the experiment are approaching equilibrium with the ambient particles and that dissolved concentrations that are still in excess of normal seawater concentrations would not be decreased further unless particles are removed from the system by settling. For Fe and Pb, dissolved concentrations at the end of the experiment were less than the detection limits employed in this project. If equilibrium implied by the consensus K d s were achieved, the actual dissolved Fe and Pb concentrations should be ~ 1 mg/l and 0.01 µg/l, reasonable values for shelf waters. These Kds are consistent with the greater particle reactivity and shorter residence times in seawater for Fe and Pb as compared to Cu, Ni or Zn, and similar trends between residence times and metal concentration factors for marine plankton observed by [START_REF] Fisher | On the Reactivity of Metals for Marine Phytoplankton[END_REF]. For Mn, the final observed K d s were much lower than the consensus values consistent with the fact that Mn concentrations over these time scales are governed by kinetic factors not K d s.

Microtox ® Tests show high toxicity at the produced water/seawater interface (Table 3). The interface layer (L-3) at t=1.5 h and 71.5 h was more toxic than the freshly discharged produced water (t=0), and even at t=144 h, the interface layer retained significant levels of toxicity (EC 50 =88.8%). Unfiltered samples were more toxic than filtered samples. Particles that adhere to the Inhoff cone wall also showed high toxicity (EC 50 =67.1%), which further implies the toxicity of particulate phase of metals. The surface layer (L-5) did not show significant toxicity, probably as a result of removal of oil-rich particulates to the surface film or vessel walls. The surface slick, when carefully sampled using a pipette, was found to be highly toxic (EC 50 =38.0%). There were no clear correlations between EC 50 and metal concentrations.

(4) Influence of aging produced water on formation and growth of metal precipitates and toxicity under oxygenated conditions

Observed changes in SPM concentration, oxygen saturation, Fe and Mn concentrations for produced water samples that were aged in contact with atmospheric oxygen are shown in Fig. 9.

Oxygen saturation levels increased more than 80% within several hours and remained constant through the rest of the experiments. Particulate matter concentrations reached steady state in the first day of the experiment for the Dec. 14 sample, while a second phase of increase was observed after 10 days for the Feb. 8 sample. Particulate metal measurements demonstrated that particulate Fe was the major component of particulate matter in both experiments (Fe oxides making up as much as 70-75% of the SPM). Particulate Fe concentration increased with time but the Fe content of the SPM was virtually constant at 30-40% for the two experiments except for a decrease to only 20-25% at the end of the Feb. 8 experiment. As expected, particulate Mn concentrations increased more gradually than Fe in these experiments. Qualitatively, the increases in particulate Mn concentrations toward the end of the Feb. 8 experiment mirror the increases in SPM concentration, however, there is insufficient Mn to quantitatively account for the SPM. Particulate Cu and Zn concentrations also increase throughout the experiments, while concentrations of Al, Cr, and Ni decrease.

Physical changes of the SPM were observed using Scanning Electron Microscopy. While the concentrations during the Dec. 14 experiment remained essentially constant after 1 day, morphology changed dramatically. Particles at time 0 were mainly single grains (Fig. 10-a). As time progressed, dissolved metals precipitated (Fig. 10-b), and the particles grew to large compact aggregates by t= 4 days (Fig. 10-c). The majority of these particles were confirmed to be Fe-oxides by energy dispersive X-ray analysis (Fig. 11-b). Particles formed by the attachment of metal oxide particles to oil droplets were also frequently found in the samples (Fig. 11-a).

Toxicity of unfiltered samples increased with time and the peak toxicity occurred one to two days after the experiments were started (Table 4). Reduced but significant toxicity was retained until 8 days later for the Dec. 14 sample and until 14 days later for the Feb. 8 sample. Unfiltered produced water showed higher toxicity than filtered samples in more than 85% of samples. This agrees with results from the measurements and experiments described previously -toxicity is greater for unfiltered than filtered samples and maximum toxicity is observed hours to a few days after the experiments were started. Positive correlation of toxicity and particulate Zn concentration (p<0.02) (Fig. 12) indicates that particulate Zn may have some influence on toxicity. Marine bacteria are known to have affinity to large aggregates (marine snow) and bacterial concentration is one to three orders of magnitude higher in aggregates than in the surrounding water [START_REF] Alldredge | Characteristics, dynamics and significance of marine snow[END_REF]. Luminescent bacteria, used in this study to measure toxicity, may adhere to suspended metal precipitates produced from the oxidation of produced water and become exposed to the enhanced concentration of metals such as Zn, resulting in development of the observed toxicity.

CONCLUSIONS

Our experiments demonstrated that produced water undergoes chemical and physical transformations upon exposure to seawater and oxygen. The principal process is the phase transition of metals from dissolved to particulate form. This transformation occurs rapidly under oxygenated conditions and influences the transport and the fate of metals in produced water.

Because the produced water also contains oil droplets, two types of particles are produced, purely inorganic particles that are denser than seawater and settle quickly, and buoyant particles that are aggregates of oil droplets and inorganic particles and rise to the surface.

Toxicity assessment using the Microtox ® Test showed that unfiltered samples containing metal precipitates generally had higher toxicity levels than filtered samples. This indicates that the particulate fraction of produced water contains toxic substances that were inhibitory to the primary metabolic function of the bioluminescent bacteria. Zn toxicity seems to be indicated by some of our experiments, but most of the observed toxicity appears to have been caused by an unidentified, presumably non-metallic, toxicant. Our experiments demonstrated that the toxic response elicited by the produced water may increase following exposure to aerobic marine waters, even in cases where the freshly discharged produced water has little or no toxicity. The laboratory studies showed that toxicity peaked after the sample was oxidized for one to two days in association with the production of metal precipitates and could last for more than a week. Chemical processes that were identified in this study are important for the understanding of transport and fate of heavy metals in produced water, and the toxicity of produced water discharges to biological communities. Fig. 13 identifies the major physical and chemical processes that occur when produced water is discharged at an offshore production facility. Initial dilution of the produced water with seawater is accompanied by precipitation of metals and association of some of these inorganic precipitates with oil droplets. These particles will separate from the produced water plume and either rise to the surface or settle to the bottom depending on their aggregation and buoyancy, potentially causing biological effects at the sea surface or in the benthic boundary layer. Experiments included raw produced water and produced water diluted with seawater at 1:100, 1:10 and 1:2 ratios with a total volume of 400 ml. Cumulative particulate matter amount (mg) was plotted against time. Particles at t=148 hours (last points) indicate those adhered to the wall of settling bottles washed off with 50 ml super Q water after all samples were drained. Concentrations of particles attached to the walls of Inhoff cones were calculated from differences between total metal concentration at t=0 and those at t=1.5h, 71.5 and 144 h. .14, 1998 andFeb. 8, 1999.

Fig. 1

 1 Fig. 1 Map showing the produced water sampling location.

Fig. 2

 2 Fig. 2 Schematic diagram for produced water/seawater layering experiment using an Inhoff cone.

Fig. 4

 4 Fig. 4 Time series study of particulate matter concentrations and salinity in 5 layers in the Inhoff cone experiments using produced water collected on September 28 (A), October 14 (B) and October 19 (C).

Fig. 5

 5 Fig. 5 Verification of oil droplets with metal precipitates within the surface slick of the seawater layer using light microscopy with UV epi-fluorescence illumination (A) and phase contrast microscopy (B).

Fig. 6

 6 Fig.6Concentrations of Fe, Mn, Cu and Zn in dissolved and particulate fractions in each layer during the time series experiment using the produced water collected October 14. Concentrations of particles attached to the walls of Inhoff cones were calculated from differences between total metal concentration at t=0 and those at t=1.5h, 71.5 and 144 h.

Fig. 7

 7 Fig. 7 (a) Co-precipitation of Cr and Ni with Mn. For Ni, Y= 0.89X+208.89, r 2 =0.84. For Cr, Y= 0.61X+103.02 , r 2 = 0.66, (b) co-precipitation of Al and Fe. Y=0.15X-6.03, r 2 =0.53

Fig. 10

 10 Fig. 10 Scanning Electron Microscope observations of changes in the size of metal precipitate aggregates formed during the experiment using the produced water collected on December 14 at t=0 (a), t=1d (b) and t=4d (c).

Fig. 11

 11 Fig. 11 Environmental Scanning Electron Microscopy (ESEM) coupled with Energy Dispersive X-ray analysis to illustrate and characterize the (a) precipitation of Fe around oil droplets and (b) Fe oxide. Sample was collected during the aging experiments.

Fig. 12

 12 Fig. 12 Relationship between toxicity and particulate Zn concentrations.

Table 2 .

 2 Microtoxicity test results for the source produced water. Expressed as EC 50 values (percentage of produced water in test solution that produces a 50% reduction in the bioluminescence of the test organism). Toxic response at the EC 20 level (>20% decrease in bioluminescence at 100% produced water test concentration).

	Tables
	Table 1. Chemistry data for produced water samples

Table 3 .

 3 Microtox results for produced water/seawater layering experiment -Oct 14, 1998. Toxic response at the EC 20 level (only >20% decrease in bioluminescence attained in undiluted produced water samples).

	Time (h)	Layer	Total -EC 50 (%)	Filtered -EC 50 (%)
	0	PW	83.5	>99
	1.5	L-1	89.7	>99
		L-2	88.8	>99
		L-3	69.6	>99*
		L-4	>99	>99
		L-5	>99	>99
	71.5	L-1	51.6	94.0
		L-2	>99*	>99
		L-3	80.4	>99
		L-4	>99*	>99
		L-5	>99*	>99*
	144	L-1	>99*	>99
		L-2	>99*	>99
		L-3	88.8	>99
		L-4	>99	>99
		L-5	>99	>99
		wall	67.1	>99*
	*

Table 4 .

 4 Metal in particulates and toxicity results for the seawater aging experiments.

	time Al mg/g µg/g µg/g µg/g µg/g % Cd Cr Co Cu Fe	Pb µg/g mg/g µg/g µg/g total Mn Ni Zn EC 50	EC 50 filtered
	Dec. 14
	0	1.7	0.28 210	0.91 21	38	120	0.23 12	73	>99* >99*
	1h	1.1	0.18 180	34	23	39	170	0.29 38	210	84.2	80.5
	1d	0.63 0.23 130	4.8	28	36	76	0.68 17	410	29.4	57.6
	2d	0.63 0.52 130	2.1	32	36	84	0.71 36	410	58.3	84.3
	3d	0.67 0.15 100	3.2	51	36	90	0.71 11	450	72.8	98.0
	8d	0.54 0.19 91	2.0	36	36	89	0.63 19	330	71.1	80.2
	15d 0.48 0.16 94	2.9	46	35	84	0.54 8.3	230	>99* >99
	Feb. 8
	0	2.9	0.21 250	1.2	14	29	28	0.29 130	26	>99* 16.1
	3h	1.3	0.14 150	1.0	5.6	36	18	0.54 62	150	90.4	98.8
	1d	1.3	0.35 180	1.6	8.8	33	19	0.69 25	250	76.6	>99*
	2d	1.3	0.45 220	1.1	9.4	34	20	1.1	59	270	64.0	65.4
	3d	1.1	0.30 150	0.85 8.9	34	18	1.4	48	230	68.7	>99*
	4d	1.3	0.41 140	1.0	9.9	37	22	1.5	59	300	65.6	74.3
	7d	1.2	0.29 150	2.4	23	33	24	1.7	38	270	79.5	>99
	10d 1.2	0.08 130	1.7	11	32	22	2.5	35	260	70.6	63.7
	14d	0.78 0.07 110	0.78 9.9	21	17	2.7	16	190	75.7	>99*
	16d 0.99 0.10 130	1.1	13	24	27	2.3	96	200	>99* >99*
	21d 1.1	0.94 150	0.66 62	23	20	1.6	21	200	>99* >99*
	*>20% decrease in bioluminescence at 100% PW test concentration.

Acknowledgement

The authors thank Dr. Peter Strain for comments on the earlier version of the manuscript. This work was supported by Panel of Energy Research and Development (PERD), Natural Resources of Canada.