K P Krishnan

S O Fernandes

P A Loka Bharathi

L Krishna Kumari

Shanta Nair

Anil K Pratihari

B Ramalingeswara Rao

B Ramalingeswara Rao

Anoxia over the western continental shelf of India: Bacterial indications of intrinsic nitrification feeding denitrification

Keywords: Denitrifiers, Nitrifiers, Coastal anoxia, Arabian Sea

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Coastal anoxia -Intrinsic nitrification feeds denitrification 3 INTRODUCTION

Progressive and cyclic developing of oxygen deficient waters over the continental shelf of the western India has received much attention [START_REF] Naqvi | Increased marine production of N 2 O due to intensifying anoxia on the Indian continental shelf[END_REF]. West India Coastal Current (WICC) flows equatorward along the west coast of India during June-September in response to alongshore wind stress and consequent upwelling can be seen along the coast [START_REF] Shetye | Hydrography and circulation off west coast of India during the southwest monsoon[END_REF]). The biological activity over the continental shelf is enhanced during the summer due to intense coastal upwelling and Ekman pumping [START_REF] Brock | The phytoplankton bloom in the northwestern Arabian Sea during the southwest monsoon of 1979[END_REF]. The upwelled water over the shelf covers an area of 180,000 km 2 [START_REF] Naqvi | Increased marine production of N 2 O due to intensifying anoxia on the Indian continental shelf[END_REF] and is extremely oxygen depleted [START_REF] Banse | Hydrography of the Arabian Sea Shelf of India and Pakistan and effects on demersal fishes[END_REF]). [START_REF] Naqvi | Increased marine production of N 2 O due to intensifying anoxia on the Indian continental shelf[END_REF] reported that this coastal water body could be one of the largest low-oxygen zones, which begins its annual development in June, reaching peak intensity by September-October and dissipating by December. Further, they measured the highest accumulations of hydrogen sulphide (H 2 S) and nitrous oxide (N 2 O) in open coastal waters and attributed to the addition of anthropogenic nitrate and its subsequent denitrification, which is favored by hypoxic conditions. It is now well understood that coastal anoxia is more widespread geographically with periodic cyclicity. Hence, the cues that trigger such processes could be linked to large scale oceanic processes than hitherto understood. A 5-10 m thick cap of warm, relatively less saline waters over the upwelled water clearly indicate the magnitude of terrestrial runoff accompanied by precipitation [START_REF] Banse | On upwelling and bottom-trawling off the southwest coast of India[END_REF].

However, despite its origin from land runoff with sufficient nutrient load, low nitrate values observed in this warm-water cap is attributed to efficient algal uptake [START_REF] Naqvi | Increased marine production of N 2 O due to intensifying anoxia on the Indian continental shelf[END_REF]. If this were to happen, then the denitrification process would be constrained by the availability of nitrate.

The observed high N 2 O accumulation [START_REF] Naqvi | Increased marine production of N 2 O due to intensifying anoxia on the Indian continental shelf[END_REF] is indicative of availability of nitrate despite algal uptake. Consequently, there could be a more significant pathway dynamically linked to denitrification contributing significantly to NO 3 and N 2 O flux. The low O 2 conditions are also known to enhance the yield of N 2 O during nitrification, and so the possibility of contribution coming through this pathway cannot be excluded [START_REF] Goreau | Production of NO -2 and N 2 O by nitrifying bacteria at reduced concentrations of oxygen[END_REF]. [START_REF] Shailaja | Respiratory enzyme activities in the oxygen-deficient waters of the Arabian Sea[END_REF] Hence we hypothesize, that ammonification fed nitrification under hypoxic conditions could be more important than previously thought of in these systems. The alarming nitrous oxide input into the atmosphere could be due to a tighter nitrification-denitrification coupling, rather than denitrification driven by extraneous nitrate alone.

MATERIALS AND METHODS

Study area and sampling:

A cross shelf transect off Goa (15 o 30'N, 72 o 40'E to15 o 30'N, 72 o 59'E) was selected to study the spatial and temporal variation of nitrifiers and denitrifiers with respect to the water chemistry for two contrasting periods viz. April (oxic) and October (anoxic) during the cruises SaSu-40 (April, 2003), 49 (October, 2003) and 103A (October, 2005) onboard C.R.V. Sagar-Sukti. Though the cruises conducted in April and October 2003 are the best representative of the contrasting conditions, analysis of additional data from other cruises almost corresponding to the period were undertaken [SaSu-26 (September 2002), 29 (December, 2002)]. A similar transect off Mangalore (13 o 00'N, Coastal anoxia -Intrinsic nitrification feeds denitrification 5 included in this analysis. Such extended analyses were undertaken to get a better perspective of the accumulation/oxidation of dissolved ammonium and inorganic phosphate in the water column.

Portable CTD (conductivity, temperature, depth) was used to obtain temperature and salinity profiles at sampling locations. Seawater was collected using Niskin water samplers attached to the winch wire and triggered with a messenger. Seawater sub-samples for microbiological analyses were collected into sterile containers immediately after retrieval of the bottle. Sub-samples for nitrate and phosphate measurements were frozen for onshore analysis, whereas ammonium and nitrite were measured onboard.

Water column chemistry:

Dissolved oxygen (DO) and nutrients (ammonium, nitrite, nitrate and phosphate) in the seawater samples were measured by colorimetric and titrimetric methods respectively [START_REF] Grasshoff | Methods of Seawater Analysis[END_REF].

Abundance of nitrifiers and sulphide oxidizing denitrifiers:

Nitrifiers and Sulphide-oxidizing denitrifiers (Thiobacillus denitrificans like organisms, TDLO) were enumerated as Colony Forming Units (CFU) on different selective media. Nitrifiers were enumerated by spread plating onto solidified Winogradsky's medium [START_REF] Rodina | Methods in Aquatic Microbiology[END_REF]. After inoculation, the plates were incubated for 30-45 days at 15+2°C. The TDLO were enumerated by an agar shake method on modified Leiske's medium [START_REF] Bharathi | The occurrence of denitrifying colourless sulphur-oxidising bacteria in marine waters and sediments as shown by the agar shake technique[END_REF][START_REF] Bharathi | A preliminary study of anaerobic thiosulfateoxidising bacteria as denitrifiers in the Arabian Sea[END_REF]).

The culture tubes were incubated for ~30 days at 28+2°C.

Nitrification and denitrification studies:

Seawater for incubation experiments were collected at definite intervals (surface, 8, 13 and 18 m) from a 25m deep station off Goa on R.V. Sagar Sukti #103A in the month of October ( 2005

Contours, plots and statistical analysis:

Bacterial abundance contour map was generated using Surfer 8.0 (Golden Software, Golden, CO).

Gridding used a kriging algorithm with a linear variogram and no anisotropy, as employed in standard topographic applications. Scatter plots and line graphs were constructed using Grapher 4.0 (Golden Software, Golden, CO, USA). Statistical analyses were done using Analysis Tool Pack in Microsoft Excel.

RESULTS

Surface temperatures roughly followed the solar heating pattern with primary and secondary heating during the March-April and September-October. The salinity profile for October showed the influence of riverine input as evidenced by the development of a less saline, warm water cap. The SST was cooler during October than in April, possibly due to the upwelling and the imminent winter. However, the signatures were weak during the transition periods i.e. April and October.

Changes in water column chemistry (Figs.1 c-e and 2 c-e):

During October, the shelf was characterized by low-oxygenated bottom waters (<0.5 mlL -1 ) of ~40m overlying the shelf. Nitrate values increased gradually to reach >4 µM at around 90m depth. It was observed that the thickness of nitrate-poor surface waters had decreased considerably. It was also interesting to note that nitrite and nitrate values in the month of oxygen deficit (October) showed a similar distribution pattern as that of the oxic month (April) but with enhanced concentrations.

Patchiness in nitrite distribution seen during April was lost but replaced by a core of elevated nitrite at around 50 km from the coast (Fig. 1e). In April, the concentration of nitrate and nitrite was very low in the top 20 m of the oxic water and it gradually increased to >1.4µM at around 90m water depth. The distribution of nitrite was patchy and elevated concentrations were noticed at around 40 km from the coast (Fig. 2e).

Bacterial abundance (Table 1):

During April, the abundance of nitrifiers were in the order of 10 7-8 CFU L -1 and patchy. The TDLO numbers showed that there is a core of elevated abundance almost 40 km away from the coast. By October nitrifier counts fell sharply to even reach non-detectable levels. In October, the distribution of TDLO was uniform and generally increased away from the coast. There was not much difference in the abundance of TDLO (10 4-5 CFU L -1 ) in both the contrasting periods.

Nitrification and denitrification:

In October (2005), the temperature and salinity (Fig. 3a) ranged from 26.3-28. At the surface (Fig. 4a) the levels of nitrite did not vary much over an incubation period of 144 hours when denitrification was inhibited by chloramphenicol, but nitrate increased to almost 2 µM in 96 hours and plateaued at the end of incubation (144 hours). The ammonium levels increased at a slow rate over the incubation period to reach final values of around 5.5 µM. At 3m (Fig. 4b), the values of ammonium fluctuated with a decrease followed by an increase with very little variation in nitrite but with constant increase in nitrate, which reached 2.6µM in 144 hours. At 8m (Fig. 4c), the variation in nitrite values fairly resembled the surface and 3m incubations except for a sharp decrease in the first 24 hours. At 13m (Fig. 4d), nitrite and nitrate values evolved as in the 8m water depth though absolute values differed. The irregularities observed in the rise and fall of ammonium values were more pronounced at this depth. At bottom i.e., 18 m (Fig. 4e), it could be observed that both nitrite and ammonium values vary in phase at higher frequency, while nitrate values continuously increase over time, which was to all the sampled depths.

Nitrification blocked system (Fig. 5a-e):

At the entire sample depths nitrate fell below the levels of detection within 48-72 h. At the surface (Fig. 5a), when nitrification was inhibited by the addition of allyl thiourea, the levels of nitrite initially increased and followed an elliptical curve to reach values below the limits of detection. The nitrate values decreased to non-detectable levels within 48 hours of incubation due to nitrate reduction/denitrification, while change in concentrations of ammonium was sigmoidal though less pronounced. The trend in the variation of all the three nitrogen species at 3m (Fig. 3b) water depth

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Coastal anoxia -Intrinsic nitrification feeds denitrification 9 were similar to that observed at the surface waters. The concentrations in nitrite and ammonium varied inversely. Nitrite level increased at the beginning of incubation and then fell to below detection. The peak of nitrite is broader at 8m depth (Fig. 5c) and decreased with increasing depth.

Changes in ammonium at 8m resembled that of the surface; while at 13 (Fig. 5d) and 18m (Fig. 5e) it increased rapidly after an initial stationary phase and then flattened out. At 13m the peak of nitrite accumulation was observed at 48 hours while at 18m it was observed at 24 hours. It could also be observed that the fall in level of nitrite at 13m is more pronounced between 96-120 hours where as the depletion of nitrite was more gentle at 18m.

Monitoring the changes in 'N' species in the controls (Fig. 6a-e):

At the surface (Fig. 6a) and 3m (Fig. 6b), when neither of the inhibitors were added, the levels of nitrite initially increased and then followed an elliptical curve to reach values below the limits of detection, while ammonium levels increased at a very slow rate. At all the depths nitrate values fell to non-detectable levels within 72 -120 hrs. The pattern of changes in ammonium and nitrite at 8m (Fig. 6c), 13m (Fig. 6d) and 18m (Fig. 6e) were very similar to that of the experiment where nitrification was inhibited by allyl thiourea (Figs 5c-e).

DISCUSSION

The presence of a 5-10 m thick cap of warm, less saline water over the upwelled water clearly indicates the magnitude of terrestrial runoff accompanied by precipitation [START_REF] Banse | On upwelling and bottom-trawling off the southwest coast of India[END_REF]. Low nitrate values observed in this warm water cap were solely attributed to efficient algal uptake [START_REF] Naqvi | Increased marine production of N 2 O due to intensifying anoxia on the Indian continental shelf[END_REF]. A more significant pathway dynamically linked to denitrification could contribute significantly to NO 3 and N 2 O flux and mediate N 2 O production. It was observed that the relationship between ammonium and total dissolved inorganic nitrogen (DIN) pool (r = 0.98, p <0.001, n =122; data obtained from various cruises as described in legend to Fig. 7) was stronger than the relationship between DIN to nitrite and nitrate. It is therefore inferred that ammonification is a predominant Coastal anoxia -Intrinsic nitrification feeds denitrification 10 process contributing to DIN. The oxygen-poor waters are conducive for reduction of iron and the subsequent phosphate release from ferric phosphate [START_REF] Caraco | Evidence for sulphate-controlled phosphorus release from sediments of aquatic systems[END_REF]1991). Enhanced dissolved inorganic phosphate in the water column is used as an indicator for the degree of anoxia in the region. The reduced compounds such as NH 3 , H 2 S, and CH 4 tend to accumulate under anaerobic conditions. Hence low oxygen regimes result in the accumulation of phosphate and ammonia.

However, our study showed that under low oxygen regimes with high phosphate concentrations, though ammonium is a significant fraction of DIN, accumulation of ammonium was low. This strengthens our observation that ammonium does not appear to be locked always under low oxygen regimes. Besides, the higher diffusion coefficient of ammonium as compared to other reduced compounds like sulphide [START_REF] Li | Diffusion of ions in seawater and in deep-sea sediments[END_REF] facilitates its mobility to oxic shallower layers. This could probably improve the spatial coupling between the substrate (ammonium) and the utilizer (nitrifier), which could get decoupled due to the development of acute anoxia. Unlimited substrate availability by degradation of organic matter could also eliminate the dependence of culturable bacterial abundance on the distribution of ammonia.

Studies generally reveal that 99% or more of the bacterial diversity remains uncultured and unexplored. However, estimates on data compiled for culturability indicate that in seawater and unpolluted estuarine water, percentage culturability can reach up to 0.1 and 3% of direct microscopic counts respectively [START_REF] Fuhrman | Microbial diversity[END_REF]. Table 1 presents contrasting distribution of TDLO and nitrifiers. While nitrifiers are mostly detectable only during the oxic period (April), TDLO could be retrieved during both the seasons (April and October) as TDLO is more metabolically versatile to respire either oxygen or nitrate. During April (2003), the culturable fraction of nitrifier population (10 7 CFU/ L -1 ) decreased towards the open sea. It was also observed that the nitrifier counts vary with water depth at around 40 km from the coast. A zone of high nitrite, high TDLO and low nitrifiers was observed in the same area. Increase of nitrate concentration below 20m depth indicates that nitrification is more pronounced in the deeper depths than in the surface. These observations provide a glimpse of the distribution pattern of bacteria well before the onset of chemical signatures

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Coastal anoxia -Intrinsic nitrification feeds denitrification 11 of anoxia. In the post monsoon season (October, 2003) the nitrifier population declined to non detectable levels from ~ 10 8 CFU L -1 . During this period, very low nitrifier counts in conjunction with a more heterogeneous and regular abundance pattern of TDLO showed that the water column is at the peak of anaerobic conditions. The depth of nitrate-poor surface layers has decreased considerably but showed inputs of nitrate by nitrification from subsurface levels. Though the nitrifier counts have dropped by almost two orders, the elevated nitrate observed could be attributed to higher specific activity of nitrifying bacteria or suboxic nitrification, perhaps at the expense of iron and manganese. The nitrite and nitrate values in the anoxic period showed a similar distribution pattern as that of the oxic period, but the enhanced magnitude due to net nitrate production being more than nitrate reduction suggests that the nucleus of microbial activity spreads during the period of anoxia to cover the entire water column, changing the chemistry. Hence it is proposed that the microbial indicators of oxygen deficit appear as a well-defined nucleus almost 40 km from the coast even during the oxic period (April).

Net changes of inorganic nitrogen species in nitrification blocked and unblocked experimental systems during the period of anoxia (October) show that denitrification is the predominant process in the water column consuming available nitrate (~ 0.5 µM) to near zero levels within 48 -96 hours of incubation. Similar experiments conducted by [START_REF] Naqvi | Increased marine production of N 2 O due to intensifying anoxia on the Indian continental shelf[END_REF] showed that majority of the available nitrate (21.8µM) is converted to nitrite in the first 48 hours of incubation. Initial levels of nitrite varied with depth and could range between 0.44-3.87µM. The levels of nitrite encountered in the incubation experiments were comparatively higher (~7-65 times) than that reported by [START_REF] Naqvi | Increased marine production of N 2 O due to intensifying anoxia on the Indian continental shelf[END_REF]. When denitrification was blocked, the absence of a welldefined nitrite peak coupled to a steady increase in nitrate clearly indicates the efficiency with which ammonium is oxidized to nitrate. It was found that nitrification could contribute almost 4.5 µM to the total nitrate pool, suggesting that ammonium is actively produced by detrital breakdown, but efficiently consumed through the nitrification process. Moreover, the highest rate of ammonia Coastal anoxia -Intrinsic nitrification feeds denitrification 17

  has shown that both denitrification and nitrification occur in several closely spaced layers in the Arabian Sea and the Coastal anoxia -Intrinsic nitrification feeds denitrification 4 contribution of nitrification to nitrite enrichment in the perennial oxygen minimum zone is significant. Despite the growing interest in mechanisms involved in the development and regression of coastal anoxia and emission of potential green house gases into the atmosphere, the role of microbial processes in driving this ecosystem has received little attention. Ammonia-oxidizing nitrifiers and sulphide-oxidizing denitrifiers (Example: Thiobacillus denitrificans like organisms; TDLO) are physiologically diverse bacterial groups, which respond in tandem to environmental chemistry. Recording the spatial and temporal distribution of these groups and their synchronization with the water column chemistry gives us a better understanding of the dominant microbial processes and their potential contribution to gaseous inputs to the atmosphere.

  ). Sub-Coastal anoxia -Intrinsic nitrification feeds denitrification 6 samples in triplicates were then transferred into sterile, plugged, 250 ml, glass conical flasks, which were incubated in the dark onboard under ambient temperature. The temperature in the incubation flasks varied between 26-29 o C. Three sets of incubation experiments were carried out viz., -(1) Nitrification was inhibited by adding Allyl thio urea to a final concentration of 86 µM (Ginestet et al. 1998), (2) Denitrification was inhibited by adding Chloramphenicol to a final concentration of 300 mg L -1 (Brooks et al. 1992), and (3) Sub-samples of third set were monitored without adding any inhibitors (controls). Aliquots were analyzed at 24 hrs intervals up to 144 hrs (6 days) for nitrate, nitrite and ammonium.

  Variations in temperature and salinity during October (Figs.1a and 1b respectively) showed an upwelling signature, possibly during the waning phase of the WICC. The WICC flows northward during January-April, which results in downwelling along the coast. The temperature/salinity profiles for April (Figs.-2a and 2b respectively) also showed downwelling signatures, though weak.

  Figs. 4-6.

Fig. 1 .

 1 Fig.1. Hydrography along the transect Off Goa transect in October a-temperature ( o C); b -salinity (psu); c -oxygen (mlL -1 ); d -nitrate (µM); e-nitrite (µM). Station # 1 was sampled at 1, 5 and

Fig. 2 .

 2 Fig.2. Hydrography along the transect Off Goa transect in April. temperature ( o C); b -salinity (psu); c -oxygen (mlL -1 ); d -nitrate (µM); e-nitrite (µM). Details of sampling depths same as Fig 1.

Fig

  Fig.3a-b. 3a represents T-S profiles for station #2 during the cruise SaSu 103A (October, 2005). 3b

Fig. 7 .

 7 Fig.7. Plot of dissolved inorganic phosphate (DIP) versus ammonium under different oxygen levels

Fig

  Fig.3 a and b

Fig.7.

Acknowledgement: This work was carried out as a part of the institute project "Marine Biodiversity and Ecosystem Functioning". Authors wish to express their gratitude to Dr. Satish R. Shetye, Director, NIO and Dr. Rasik Ravidra, Director, NCAOR for their interest in this work. Drs. S. Raghukumar and C.T. Achuthankutty are duly acknowledged for their inputs and all round support. Dr M.V. Ramana, Project Leader, Gas Hydrates Group, is highly acknowledged for his support and logistics during Sagar Sukti 103A cruise. Scientific team and crew onboard C.R.V. Sagar Sukti was extremely cooperative. Fruitful discussions with Nuncio M. from the Physical Oceanography Division at NIO have been extremely useful. KPK and SOF record their gratefulness to UGC and CSIR (Government of India) for the award of Senior Research Fellowship. Inputs from an anonymous reviewer considerably improved the earlier version of the manuscript. This is NIO contribution number xxxx.

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Coastal anoxia -Intrinsic nitrification feeds denitrification 12 oxidation (0-144 hours) was found to be at the surface (2.34 ng at NH 4 + -N l -1 h -1 ) and the lowest rate of 0.72 ng at NH 4 + -N l -1 h -1 was observed at 13 m depth. But contrary to the observed oxidation maxima, nitrite and nitrate production maxima of 5.85 and 2.34 ng at NO 3 --N l -1 h -1 were observed at 18m depth. Since the incubation systems were isolated and denitrification was blocked it could be assumed that a major fraction of nitrate was produced by the oxidation of ammonium with an intermediate production of nitrite. The production of nitrate occurred at a much higher rate than ammonium oxidation. This highlights the predominance of ammonification and its contribution to denitrification via nitrification. In addition, the highest rate of nitrate reduction in nitrification blocked systems (0-144 hours) was found to be at 13 m depth (0.05 ng at NO 3 --N l -1 h -1 ) and the lowest rate of 0.03 ng at NO 3 --N l -1 h -1 at the surface. High ammonium oxidation and low nitrate reduction in surface waters suggest that the physiologically forced synthetic pathway as electron sink is much more dominant in these systems than hitherto accepted. Oxidation of NH + 4 to NO - 2 in the euphotic zone has been observed in many regions [START_REF] Mccarthy | Sources and sinks of nitrite in the water column of the York River and the Chesapeake Bay[END_REF][START_REF] Ward | Biological nitrogen cycling in the nitracline[END_REF][START_REF] Dore | Nitrification in the euphotic zone as a source for nitrite, nitrate and nitrous oxide at station ALOHA[END_REF]. Studies by [START_REF] Sambrotto | Nitrogen production in the northern Arabian Sea during the Spring Intermonsoon and Southwest Monsoon seasons[END_REF] have also shown that ammonium regeneration rates and concentrations are higher along the coastal waters of Oman during the southwest monsoon season.

The unaccounted (~20%) portion of total ammonium sink in the study was attributed to nitrification.

Moreover, [START_REF] Mantoura | Nitrogen biogeochemical cycling in the northwestern Indian Ocean[END_REF] have attributed elevated N 2 O production to nitrification from the Omani coastal region. Hence, the sequential processes viz. ammonification, nitrification and denitrification operate in more spatial and temporal proximity than hitherto believed suggesting their significant role in the dynamics of nitrogen species. Further, the study suggests the potential role of anaerobic/suboxic nitrification that could intrinsically feed the denitrification process.

CONCLUSION

The microbial indicators of ensuing oxygen deficit appear as a well-defined nucleus almost 40 km from the coast even during the oxygenated period and spreads during the period of anoxia to cover the entire zone governing the water column chemistry. Nitrification operates at the base of this

Coastal anoxia -Intrinsic nitrification feeds denitrification 13 suboxic system, very efficiently feeding denitrification. The alarming nitrous oxide input into the atmosphere could be relatively more due to a tighter intrinsic coupling of nitrification-denitrification, rather than denitrification driven by extraneous nitrate alone.


M G Ikonomou
email: ikonomoum@dfo-mpo.gc.ca

R F Addison

Polybrominated diphenyl ethers (PBDEs) in seal populations from eastern and western Canada: an assessment of the processes and factors controlling PBDE distribution in seals

Polybrominated diphenyl ether (PBDE) concentrations were measured in the blubber of five mother-pup pairs of grey seals (Halichoerus grypus) from Sable Island, Nova Scotia (NS) sampled in 1995 and in 20 British Columbia (BC) sampled in 1991-92. Concentrations in maternal grey seals from Sable Is. averaged 113 ± 55.2 ng/g lipid (mean ± s.d.) and were over twice the concentrations measured in their pups; lower brominated PBDEs showed a distinct decline in PBDEs transfer efficiency in motherpup pairs as a function of increasing K o/w . Hepta-BDEs and higher molecular weight (MW) congeners appear not to be transferred efficiently from the grey seals mother to pup during lactation on the basis of lipid/water partitioning (as indicated by ln K o/w ) and this may be a consequence of molecular size (MW >600 Daltons) which may limit diffusion and partitioning. PBDE concentrations in blubber from harbour seals from the Strait of Georgia averaged 319 ± 132 ng/g lipid (mean ± s.d., n = 13), while those from the more remote and less contaminated Quatsino Sd. averaged 27.8 ± 11.8 ng/g (mean ± s.d., n = 7). Tri-to hexa-BDEs were appreciably enriched in Strait of Georgia harbour seals relative to those from Quatsino Sd, suggesting that the former were closer to PBDE sources. The main congeners identified in all samples were BDEs 47, 99, 100, 153 and 154. Data were compared to those in ringed seals sampled from Holman, North West Territories (NWT), in 1996. The Holman samples had a higher proportion of lower bromination congeners, and a lower proportion of higher bromination congeners, than did the Sable Island and BC seal samples, consistent with selection in favour of more volatile compounds during atmospheric transport to the Arctic.

Introduction

For approximately the last decade polybrominated diphenyl ethers have been identified as pollutants of growing concern. These chemicals have been used since the 1980's, and continue to be used though under increasingly restricted conditions [START_REF] Hites | Polybrominated diphenyl ethers in the environment and in people: a metaanalysis of concentrations[END_REF][START_REF] De Wit | Levels and trends of brominated flame retardants in the Arctic[END_REF] mainly as flame retardants in fabrics and other materials. Like other organic pollutants, PBDEs accumulate in higher trophic level-feeding marine biota, and have been described in marine mammals from eastern and western Canada, and in populations far removed from local PBDE sources [START_REF] Ikonomou | Exponential increases of the brominated flame retardants, polybrominated diphenyl ethers, in the Canadian Arctic from 1981 to 2000[END_REF][START_REF] Rayne | PBDEs, PBBs and PCNs in three communities of free-Ranging killer whales (Orcinus orca) from the Northeastern Pacific Ocean[END_REF][START_REF] Lebeuf | Levels and temporal trends (1988-1999) of polybrominated diphenyl ethers in beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Canada[END_REF][START_REF] De Wit | Levels and trends of brominated flame retardants in the Arctic[END_REF].

In this paper, we describe PBDE distribution in mother-pup pairs of grey seals (Halichoerus grypus) from Sable Island NS, sampled in 1995 and in two populations of harbour seals (Phoca vitulina) sampled from the BC coast in 1991-92; both populations were sampled previously to provide information on contamination by polychlorinated biphenyls (PCBs) and polychlorinated dibenzop-dioxins and furans, but no data have been published describing their PBDE contamination.

We compare the distribution of PBDEs with that of legacy POPs measured in these samples, and with PBDEs and other organic pollutants in other seal populations from various sites in Canada and elsewhere. Since the production and use of PBDEs changed rapidly between 1980 and 2000 (e.g., [START_REF] Ikonomou | Exponential increases of the brominated flame retardants, polybrominated diphenyl ethers, in the Canadian Arctic from 1981 to 2000[END_REF] our comparisons have focussed deliberately on samples taken only during the fairly narrow interval of 1991-92 (harbour seals) and 1995 (Sable Island grey seals). We have also compared these data to PBDE concentrations in Holman ringed seals sampled in 1996 and published elsewhere [START_REF] Ikonomou | Exponential increases of the brominated flame retardants, polybrominated diphenyl ethers, in the Canadian Arctic from 1981 to 2000[END_REF]. These comparisons allow us to assess the processes and factors which control the exposure of seals to these chemicals and their distribution in seals. The data sets compared in this paper captures most of the Canadian seals PBDE data published so far, and since all the samples were analyzed by the same laboratory following the same analytical protocols the analytical data are internally consistent.

Materials and Methods

Blubber from five isolated mother-pup pairs of grey seals were sampled on Sable Island in February 1995. Detailed sample information can be found in [START_REF] Addison | Polychlorinated dibenzo-p-dioxins and furans and non-ortho and mono-ortho chlorine substituted polychlorinated biphenyls in grey seals (Halichoerus grypus) from Sable Island, Nova Scotia, in 1995[END_REF].

(Mother-pup pair 149204 was omitted from PBDE analyses.) The samples had previously been analysed for residues of polychlorinated biphenyls (PCBs) and for polychlorinated dibenzo-dioxins and furans (PCDD/F) [START_REF] Addison | Polychlorinated dibenzo-p-dioxins and furans and non-ortho and mono-ortho chlorine substituted polychlorinated biphenyls in grey seals (Halichoerus grypus) from Sable Island, Nova Scotia, in 1995[END_REF] and the archived extracts from these analyses were re-analysed for PBDEs. Blubber was sampled from harbour seals in 1991-92 from the Strait of Georgia, and from Quatsino Sd., Vancouver Island. These animals were shot as predators on fish farms, and so variables such as sex and age could not be controlled. The blubber samples were analysed for PCBs and PCDD/F [START_REF] Addison | PCDD/F and PCB in harbour seals (Phoca vitulina) from British Columbia: response to exposure to pulp mill effluents[END_REF] and 20 archived extracts from these analyses were re-analysed for PBDEs. Figure 1 shows the location of the sampling sites, and of Holman, NWT. The archived extracts for both the Sable Island and the west coast samples (Strait of Georgia, and Quatsino Sd) were stored in amber vials in the dark at -20 o C.

As noted above, both the grey seal and the harbour seal samples were analyzed for PCBs and PCDD/Fs as part of other studies. The sample extraction and clean-up procedures used to process the samples for PCBs and PCDD/Fs have been described in detail elsewhere [START_REF] Ikonomou | A Comprehensive Multiresidue Ultra-Trace Analytical Method , Based on HRGC/HRMS, for the Determination of PCDDs, PCDFs, PCBs, PBDEs, PCDEs, and Organohalogen Pesticides in Six Different Environmental Matrices[END_REF]. Four fractions were collected from each of the samples and those were analyzed for target PCBs and PCDD/Fs by high-resolution gas chromatography highresolution mass spectrometry (HRGC-HRMS). Following these analyses the four fractions were combined, spiked with a suite of 13 C-labeled PBDE standards (method internal and performance) and analyzed for mono-to hepta-BDEs by HRGC-HRMS. The extracts from the Sable Island samples were analyzed for octa-to deca-BDEs as well but the concentrations measured were close to the levels measured in the procedural blanks and thus not reported.

The composition of the 13 C-labelled PBDE standards used for quantification, the instrumental analysis conditions used, the quantification protocols, the criteria used for congener identification, and the quality assurance/quality control (QA/QC) measures undertaken for the HRGC-HRMS analysis of PBDE target analytes are described in detail elsewhere [START_REF] Ikonomou | A Comprehensive Multiresidue Ultra-Trace Analytical Method , Based on HRGC/HRMS, for the Determination of PCDDs, PCDFs, PCBs, PBDEs, PCDEs, and Organohalogen Pesticides in Six Different Environmental Matrices[END_REF]2002). Since the samples were not spiked at the extraction step with labelled PBDE internal standards, percent recoveries of the PBDE method internal standards were unknown. However, extensive method validation experiments with this matrix have enabled us to confirm that the extraction efficiency of the PCBs and PBDEs are similar under the experimental conditions used. Therefore, we used percent recoveries of the labelled PCB internal standards, obtained from the corresponding analyses described above, to establish the percent recoveries of the labelled PBDEs internal standards and to quantify the native PBDE concentrations accordingly. The mono-to hepta-BDE levels measured in the procedural blanks associated with the analysis of these samples totalled 3500 pg/g and were accounted for mainly by BDE 138/166 (2000 pg/g) and BDE 140 (1000 pg/g). Detection limits for the Sable Is. grey seal analyses were in the range 1.6 pg/g (mono-and hepta-BDEs) to 13.7 pg/g (penta-BDEs) and for the BC harbour seal analyses were in the range 0.6 pg/g (mono-and penta-BDEs) to 21.5 pg/g (hexa-BDEs). The concentrations of the mono-to hepta-BDE congeners measured in all the samples were orders of magnitude higher than those measured in the blanks and the data were not blank corrected.

To provide information about the importance of atmospheric transport in controlling the distribution of PBDEs in seal populations, data from these analyses were compared in detail with data from ringed seals (Phoca hispida) from Holman, NWT, sampled in 1996 details of which can be found in [START_REF] Ikonomou | Exponential increases of the brominated flame retardants, polybrominated diphenyl ethers, in the Canadian Arctic from 1981 to 2000[END_REF]. Data were analysed using software from Statistica (Statsoft, OK).

Results

PBDE residues in Sable Island grey seals.

The PBDE concentrations of the all the congener groups for the five mother-pup pairs examined in this study are summarized in Table 1. Total PBDEs were distributed normally (Kolmogorov-Smirnov test) in both mothers and pups. Total PBDE concentrations were around 110 ng/g wet weight in maternal blubber, and about 46 ng/g wet weight in pups. No mono-BDEs were detected, and only BDE 15 was detected among the di-BDEs. The main tri-BDE was the pair BDE 28/33 which accounted for about 80% of this group. BDE 47 comprised over 90% of the tetra-BDEs, and BDE 99 and BDE 100 represented (respectively) about 50% and 40% of the penta-BDEs. Similarly, BDE 153 and 154 represented about 50% and 40% respectively of the hexa-BDEs. Tetra-, penta-, and hexa-BDEs represented (respectively) about 62%, 18% and 18% of total BDEs in mothers; because of differences in the "transfer efficiency" of these compounds from mother to pup via milk, discussed in detail below, these three congener groups in the pup represented about 79%, 14% and 5% of the total. Only traces of hepta-and higher PBDEs were found in either mother or pup. Figure 2 summarises the major individual BDE congener concentrations in mothers and pups.

Total PBDE concentrations in adult female grey seals showed no statistically significant variation with age, but sample size was small (n=5).

The transfer of PBDE from mother to pup during lactation declined consistently with increasing Br substitution from di-to hexa-BDE, and then levelled off (Table 1). However, variance in the concentrations of higher BDE groups (above hexa-BDE) was high (s.d. usually about equal to the mean, whereas in di-to hexa-BDEs, s.d. was about 60% of the mean) perhaps due partly to the fact that concentrations were approaching the detection limit. This increasing variance in higher BDE congener groups may have skewed the relationship with increasing bromination. Figure 3 shows a plot of mean % transfer of di-to hepta-BDE from mother to pup versus ln K o/w ( calculated from data of [START_REF] Braekevelt | Direct measurement of octanol-water partition coefficients of some environmentally relevant brominated diphenyl ether congeners[END_REF].

2. PBDE residues in British Columbia harbour seals.

The total PBDE concentrations of the major PBDE congener groups measured in two populations of harbour seals are summarized in Table 2. Again, total PBDEs were distributed normally in both Strait of Georgia and Quatsino Sd. sample sets. PBDE concentrations differed widely in the blubber of harbour seals from these two populations, with tetra-and penta-BDEs in samples from the Strait of Georgia being more than an order of magnitude higher than those in Quatsino Sd. samples. Differences in tri-, hexa-and hepta-BDEs, although large, are more probably related to general "background" contamination. As noted previously [START_REF] Addison | PCDD/F and PCB in harbour seals (Phoca vitulina) from British Columbia: response to exposure to pulp mill effluents[END_REF] the Strait of Georgia samples had about half the blubber thickness of the Quatsino Sd. samples, so the PBDE burden (as opposed to concentration) of the former would probably be only about five times as great as in the latter. There was little difference between the sexes in total PBDE concentrations at either site: in Strait of Georgia samples, females contained 306 ± 99 ng/g lipid (mean ± s.d., n=9) and males 348 ± 205 ng/g lipid (n=4) in Quatsino Sd., females contained 26 ± 19 ng/g lipid (n=3) and males 29 ± 6 ng/g lipid (n=4). Sample sizes were too small, and the range of ages covered too large, for the possible effect of age on sex differences to be detected. The distribution of the six major congeners in the two sample sets are presented in Fig. 4; BDE 47 (representing >98% of tetra-BDEs), BDE 99 and 100 (65-75% and 23-33% of penta-BDEs), BDE 153 and 154 (63-86% and 9-23% of hexa-BDEs) and BDE 183 (>90% of hepta-BDEs) predominated. Table 2 also shows that there was a considerable enrichment of tetra-and penta-BDEs, and possibly of tri-and hexa-BDEs, in the Strait of Georgia samples.

In Strait of Georgia female harbour seals (n=9) there was no statistically significant variation with age. Sample sizes of male seals in the two BC harbour seal populations were too small (n=4) to undertake regression analyses.

Discussion

Of the three populations sampled, the harbour seals from the Strait of Georgia had the highest total blubber PBDE concentrations (> 300 ng/g lipid). These total concentrations are in the same range as those reported in harbour seals from the San Francisco area in 1989 and 1993 [START_REF] She | PBDEs in the San Francisco Bay Area: measurements in harbor seal blubber and human breast adipose tissue[END_REF] but slightly lower than those found in North Sea harbour seals [START_REF] Boon | Levels of polybrominated diphenyl ether (PBDE) flame retardants in animals representing different trophic levels of the North Sea food Web[END_REF], and generally lower than killer whales and porpoise from the same general area [START_REF] Ikonomou | Exponential increases of the brominated flame retardants, polybrominated diphenyl ethers, in the Canadian Arctic from 1981 to 2000[END_REF][START_REF] Rayne | PBDEs, PBBs and PCNs in three communities of free-Ranging killer whales (Orcinus orca) from the Northeastern Pacific Ocean[END_REF]. Sable Island grey seals had appreciably lower total PBDE concentrations (>100 ng/g lipid in adult females); these concentrations are similar to those reported for female northern fur seals collected in the mid-1990's from northern Japan [START_REF] Kajiwara | Polybrominated diphenyl ethers and organochlorines in archived Northern Fur Seal samples from the Pacific Coast of Japan, 1972-1998[END_REF] but slightly lower than those reported for European gray seals in the early to mid-1990's [START_REF] Law | Levels and trends of polybrominated diphenylethers and other brominated flame retardants in wildlife[END_REF]. Harbour seals from Quatsino Sd. had the lowest total PBDE concentrations (< 30 ng/g lipid); these concentrations are below most of those reported in any marine mammals from northern hemisphere mid-latitudes, but more than an order of magnitude higher than concentrations in western Arctic ringed seals sampled in the mid-1990's [START_REF] Ikonomou | Exponential increases of the brominated flame retardants, polybrominated diphenyl ethers, in the Canadian Arctic from 1981 to 2000[END_REF]. The distribution of the major PBDE congeners, with BDE 47 predominant followed by BDEs 99 and 100 (Fig. 1) is similar to that reported for other north American marine mammals and probably reflects (mainly) the use of the "penta" PBDE mixture in North America.

The distribution of POPs (exemplified by PCBs and the DDT group) in seals is controlled by external factors such as exposure (usually through diet) and endogenous factors such as the age, sex and condition of the animals (Addison, 1989). In the case of the Sable Island grey seal pups, for practical purposes the only exposure route is ingestion of maternal milk. There may be some deposition of residues during foetal development, but this is very small in comparison to exposure through lactation. "Transfer efficiency" of PBDEs from mother to pup via lactation showed a clear decline with increasing K o/w (Fig. 3) but the relationship failed beyond the hexa-BDEs, as noted above. [START_REF] Braekevelt | Direct measurement of octanol-water partition coefficients of some environmentally relevant brominated diphenyl ether congeners[END_REF] have noted that the generally linear relationship between Br substitution and ln K o/w also tends to become asymptotic beyond hepta-BDEs. The factors which govern the transfer from mother to pup of hepta-BDEs and higher are not clear: it may be significant that hexa-and hepta-BDEs have MW of 698 and 786 Daltons, respectively, and there is evidence the transport of lipophilic compounds of MW above about 600 Daltons across biological membranes may be difficult (e.g., [START_REF] Niimi | Influence of molecular weight and molecular volume on dietary absorption efficiency of chemicals by fishes[END_REF]. The process of transfer may be further complicated by the fact that some re-synthesis of lipid in mammary tissue may take place [START_REF] Iverson | Lipoprotein lipase actvity and its relationship to high milk fat transfer during lactation in seals[END_REF]. The transfer of PBDEs, particularly the higher congeners, from mother to pup via milk is therefore not simply a process of diffusion and partition.. A similar reduction in transfer from mother to pup in higher congener groups was also observed with mono-ortho-PCBs and [START_REF] Addison | Polychlorinated dibenzo-p-dioxins and furans and non-ortho and mono-ortho chlorine substituted polychlorinated biphenyls in grey seals (Halichoerus grypus) from Sable Island, Nova Scotia, in 1995[END_REF] and was attributed to increasing octanol-water partition coefficient (K o/w ) with increasing Cl substitution (or increasing MW or molecular size, all of which co-vary); this implied increase in lipid solubility with increasing Cl substitution would probably reduce partitioning into blood, which is the first step in transfer of POPs residues from blubber into milk. Similar factors appear to govern the transfer of at least the lower PBDE congener groups from mother to pup.

Total PBDE transfer from mother to pup was 44.4 ± 9.9% (n=5). This is fairly similar to the transfer of PCBs which were estimated to be in the 30-40% range but probably less than DDT-group transfer (40-60%: [START_REF] Addison | Organochlorine residues in maternal blubber, milk and pup blubber from grey seals (Halichoerus grypus) from Sable Island, Nova Scotia[END_REF][START_REF] Smith | The ringed seal, Phoca hispida, of the Canadian western Arctic[END_REF].

In adult seals, the main route of exposure to POPs must be through diet, whose contamination in turn depends on various inputs which would include at a minimum local (perhaps "point source") discharges, and/or long range atmospheric or advective transport.

Comparison of the PBDE congener distribution patterns in the seal population discussed here, and with others, allows us to infer something about the relative importance of these sources.

Consider first the BC harbour seals: the Strait of Georgia represents the "receiving waters" for industrial and municipal discharges from the greater Vancouver and SE Vancouver Island area (approximate population 3M). Quatsino Sd. is a relatively remote inlet in western Vancouver Island, receiving wastes only from the small community of Port Alice and a local pulp mill. In previous analyses [START_REF] Addison | PCDD/F and PCB in harbour seals (Phoca vitulina) from British Columbia: response to exposure to pulp mill effluents[END_REF] it was shown that the polychlorinated dibenzo-dioxin and furan (PCDD/F) distribution in these samples reflected both the expected qualitative and quantitative differences in discharges of industrial wastes in these two areas.

Thus, total PCDD and PCDF burdens in the Strait of Georgia samples were estimated to be (respectively) about 8-fold and 3-fold higher than in Quatsino Sd.; furthermore, enrichment of 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran (both of which were characteristic of bleached kraft mill discharges during the 1990's) in the Strait of Georgia samples identified such effluents as a significant source of these chemicals. In other words, the PCDD/F data show that these two seal populations reliably reflect quantitative and qualitative differences in local contaminant sources. The relative distribution of PBDEs in the two harbour seal populations follows the pattern of PCDD/F; i.e., total PBDE concentrations were about 11-fold higher in the Strait of Georgia samples than in Quatsino Sd., corresponding to a difference in residue burdens (because of differences in condition of the two samples: see [START_REF] Addison | PCDD/F and PCB in harbour seals (Phoca vitulina) from British Columbia: response to exposure to pulp mill effluents[END_REF] for a discussion) of about 5-fold. Furthermore, the relative enrichment of tri-to hepta-BDEs in the Strait of Georgia suggests that these samples may have been closer to the putative source of PBDEs in the Strait of Georgia. However, when congener group distribution is expressed as a percentage of total PBDEs, there is little evidence in favour of the skewing of PBDEs in Quatsino Sd. samples in favour of lower MW (and hence more volatile) components (Fig. 4). This suggests that atmospheric transport may not be a significant route of transport of PBDEs into Quatsino Sd.

Sable Island is considered relatively remote and free from local sources of contamination; nevertheless, it is both downstream and downwind from the St. Lawrence basin which represents a large urbanised and industrialised area. [START_REF] Bidleman | Organic contaminants in the Northwest Atlantic atmosphere at Sable Island, Nova Scoitia, 1988-89[END_REF] have documented atmospheric transport of contaminants into Sable Island via the prevailing westerly winds, and either atmospheric or water advective transport (or both) probably contributed to contamination of the gray seal population by DDT and its degradation products [START_REF] Addison | Trends in organochlorine residue concentrations and burdens in grey seals (Halichoerus grypus) from Sable Island, NS, Canada between 1974 and 1 994[END_REF]. Mean total PBDE concentrations were lower than in the Strait of Georgia harbour seals, which would be expected given Sable Island's relative remoteness; however a potentially confounding factor is the fact that grey seals and harbour seals may feed at slightly different trophic levels, or may differ in their capacity for metabolic degradation of compounds such as PBDEs. The relative distribution of the main PBDE congener groups (Fig. 5) did not differ appreciably between the Sable Island grey seals and the BC harbour seals, which suggests that "sorting" of PBDE congeners on the basis of their vapour pressure or other physico-chemical properties is relatively unimportant.

In contrast, comparison of the Sable Island grey seal data and the BC harbour seal data with data from western Arctic ringed seals illustrates the probable importance of atmospheric transport in introducing PBDEs to the Arctic. Figure 5 shows the main congener group distribution in ringed seals compared to those in the Sable Island and BC seals: it is clear that lower brominated PBDEs represent a higher fraction of total PBDEs, and higher brominated groups a lower fraction, in the Arctic ringed seals. This suggests that there has probably been some "sorting" of the supply of PBDEs to the western Arctic which is based on congener volatility; this is not seen over the shorter distribution routes to Quatsino Sd. or to Sable Island.

Finally, the role of metabolic degradation and the significance of trophic level are factors which must be considered in making inter-species comparisons, as we have done here.

At first sight, grey seals may appear to function at a higher trophic level than the other two species, as they face no direct predators, unlike the harbour and ringed seas which are preyed on by (respectively) killer whales and polar bears. In practice, however, all three seal species are, to a varying extent, fairly opportunistic feeders, and feed on small-to medium-sized fish and large invertebrates (e.g., [START_REF] Smith | The ringed seal, Phoca hispida, of the Canadian western Arctic[END_REF][START_REF] Addison | Trends in organochlorine residue concentrations and burdens in grey seals (Halichoerus grypus) from Sable Island, NS, Canada between 1974 and 1 994[END_REF]) and have been assigned to similar trophic levels based on dietary analyses [START_REF] Pauly | Diet composition and trophic levels of marine mammals[END_REF]. In this sense, our inter-species comparisons are justified. The role of metabolic degradation in controlling PBDE congener distribution is more difficult to assess. Experimental studies on BDE congener metabolism by aquatic species has been limited to studies on fish, from which it has been inferred from comparative congener distribution and from the occurrence of hydroxylated and methoxylated metabolites that some degradative capacity exists (e.g., Stapleton et al., 2004a, b;[START_REF] Isosaari | Dietary accumulation efficiencies and biotransformation of polybrominated diphenyl ethers in farmed Atlantic salmon (Salmo salar)[END_REF] though it is recognised that some of the methoxy-derivatives may have a natural source (e.g., [START_REF] Marsh | Identification of hydroxylated and methoxylated polybrominated diphenyl ethers in Baltic Sea salmon (Salmo salar) blood[END_REF]. Observational studies on natural marine food chains [START_REF] Wolkers | Congener-specific accumulation and food chain transfer of polybrominated diphenyl ethers in two Arctic food chains[END_REF] have also suggested that some degradative capacity for some BDE congeners may exist in ringed seals, and components of a cytochrome P-450 system (which could contribute to BDE degradation) exist in both grey and harbour seals [START_REF] Addison | Characterization of ethoxyresorufin-O-de-ethylase in grey seal, Halichoerus grypus[END_REF][START_REF] Addison | Mixed function oxidase activity in the harbour seal (Phoca vitulina) from Sable Island, NS[END_REF]. However, the significance of possible differences in degradative capacity among the species discussed here is impossible to assess.

Figure 3 .Figure 4 .

 34 Figure 3. Mean transfer of major brominated diphenyl ether (BDE) congeners from

Figure 5 .

 5 Figure 5. Percentage of total polybrominated diphenyl ether (PBDE) residues

Table 1 :

 1 Major PBDE congeners and congener groups (pg/g lipid) in blubber of mother and pup grey seals (Halichoerus grypus) and calculated transfer (pup conc. as % maternal conc.) from Sable Island, NS, in 1995. Data quoted to 3 significant figures as mean ± s.d. (n=5 unless otherwise stated) or as range (pup maturity). ND, not determined.

	Group:	Mothers	Pups	Pup blubber conc.
				as % maternal
	PBDE congener or			blubber conc.
	group
	Age (y)	13.8 ± 3.70	Stage III-V
	Blubber thickness	2.66 ± 0.300	4.82 ± 0.680
	(cm)
	Total mono-BDE	ND	ND
	Total di-BDE	15.3 ± 9.76	14.1 ± 7.48	100 ± 25.1
	BDE 17	119 ± 65.6	114 ± 74.5
	BDE 28/33	634 ± 383	437 ± 276
	Total tri-BDE	877 ± 623	555 ± 336	66.1 ± 17.4
	BDE 47	69700 ± 30400	39500 ± 21100
	BDE 66	177 ± 100	51.8 ± 16.9 (n=4)
	BDE 75	40.3 ± 1.21 (n=3)	11.1, 5.30 (n=2)
	Total tetra-BDE	70200 ± 30800	39600 ± 21100	55.1 ± 11.9
	BDE 99	10700 ± 7250	3920 ± 2520

Table 2 :

 2 PBDE congener and congener group concentrations (pg/g lipid) in harbour seals (Phoca vitulina) from Strait of Georgia (SoG) and Quatsino Sd. (QS), British Columbia in 1991-92. Sexes have been combined at each site. Data are quoted to 3 Figure 1. Map of Canada showing seal sampling sites discussed in the study. QS, Quatsino Sd., BC; SoG, Strait of Georgia, BC.Figure 2. Concentrations of major brominated diphenyl ether (BDE) congeners in mother and pup grey seals (Halichoerus grypus) from Sable Island, NS in 1995. Note that BDE concentrations (y axis) are on a log scale to illustrate mother pup differences in congeners present at concentrations over 4 orders of magnitude.

	significant figures and presented as mean ± s.d. (no of samples). P is probability (by
	t-test) of spatial differences arising by chance. SoG/QS is ratio of mean
	concentrations at the two sites.
	BDE group	Harbour seal, SoG	Harbour seal, QS	P	SoG/QS
	Sampling period:	1991-92	1991-92
	No. of samples taken:	9F, 4M	3F, 4M
	Age (y)	4.90 ± 5.10 (13)	8.40 ± 4.90 (7)
	Blubber thickness (mm)	18.5 ± 6.1 (13)	38.1 ± 13.0 (7)
	total di-BDE	26.0 ± 10.8 (8)	20.5 ± 18.1 (6)	NS	1.3
	BDE 17	325 ± 132 (12)	53.7 ± 31.8 (7)
	BDE 28/33	1420 ± 486 (12)	350 ± 148 (7)
	total tri-BDE	1,770 ± 529 (12)	417 ± 184 (5)	<0.01	4.2
	BDE 47	225,000 ± 94,300 (13)	18,700 ± 7730 (7)
	BDE 66	568 ± 280 (12)	127 ± 58.4 (7)
	BDE 75	60.4 ± 38.5 (4)	60.2 ± 29.4 (5)
	total tetra-BDE	229,000 ± 98,000 (13)	19,000 ± 7,790 (7)	<0.001	12.0
	BDE 99	53,800 ± 27,100 (13)	3,540 ± 1,970 (7)
	BDE 100	16,600 ± 6,300 (13)	1,840± 844 (7)
	total penta-BDE	70,400 ± 31,800 (13)	5,410 ± 2,680 (7)	<0.001	13.0

Acknowledgments

The authors thank DFO's Regional Contaminants Laboratory chemists and laboratory assistants involved in the processing and analyses of these samples. The financial support of the Environmental Sciences Strategic Research Fund (Fisheries and Oceans Canada), the Health Canada Toxic Substances Research Initiative Program (TSRI), and the regional Fisheries and Oceans funding available to the Regional Dioxin Laboratory at IOS made this work possible and is appreciated.

Addison R.F. 1989. Organochlorines and marine mammal reproduction. Can. J. Fish. Aquat. Sci. 46,[360][361][362][363][364][365][366][367][368]


Peter Thomas

Jennifer Sweatman

Interference by atrazine and bisphenol A with progestin binding to the ovarian progestin membrane receptor and induction of oocyte maturation in Atlantic croaker

Keywords: Atrazine, Bisphenol-A, Oocyte maturation, Nongenomic endocrine disruption

Recent studies have shown that several environmental chemicals which disrupt classical genomic steroid actions can also interfere with nongenomic steroid actions initiated at the cell surface. The effects of bisphenol-A and atrazine on the nongenomic actions of a progestin, 17,20 ,21-trihydroxy-4-pregnen-3-one (20 -S), on oocyte maturation (OM) were investigated an in vitro Atlantic croaker (Micropogonias undulatus) oocyte bioassay. Treatment of croaker follicle-enclosed oocytes with either bisphenol-A or atrazine blocked OM in response to 20 -S in a concentration-dependent manner at 10-25 M (2.2-5.7 ppm) and higher concentrations. These compounds were also effective competitors at concentrations of 10 -6 to 10 -5 M of [ 3 H]-20 -S binding to the ovarian progestin membrane receptor that mediates the OM response to 20 -S. The results suggest that diverse classes of environmental chemicals can disrupt nongenomic progestin actions via receptor-mediated mechanisms.

Progestin membrane receptor; Atlantic croaker *Corresponding author: Tel: +1-361-749-6788; fax: +1-361-749-6777 E-mail address: thomas@utmsi.utexas.edu (P. Thomas) In addition to the classic genomic mechanism of steroid action via binding and activation of nuclear steroid receptors, steroids can also act at the cell surface of many target tissues including the brain, pituitary, bone, heart, gonads and gametes to initiate rapid, nongenomic responses mediated by binding to steroid membrane receptors. Recent studies have shown that nongenomic steroid actions, like genomic ones, are susceptible to interference by environmental estrogens and nonestrogenic endocrine disrupting chemicals (EDCs, Ghosh and Thomas, 1999;Nadal et al., 2000;Thomas and Doughty, 2005). In the present study the effects of an estrogenic EDC, bisphenol-A and a nonestrogenic one, atrazine, on progestin induction of oocyte maturation (OM) and binding to the ovarian membrane progestin receptor were investigated in Atlantic croaker. Currently, there is considerable interest and controversy over the mechanisms of action of these two EDCs. Interference with a nongenomic progestin action was investigated in an in vitro oocyte maturation bioassay which determines the percentage of oocytes that undergo germinal vesicle breakdown (GVBD), an easily observable endpoint during oocyte maturation (Ghosh and Thomas, 1995). Fragments of Atlantic croaker ovaries containing inducing steroid, 17,20 ,21-trihydroxy-4-pregnen-3-one (20 -S, 87 nM) in the presence of the test chemicals (1 -50 M ). Atrazine ( ChemService, Inc.,Westchester, PA,) and bisphenol-A (City Chemical (Westhaven, CT) were added dissolved in methanol and ethanol, respectively (final conc. <1%). The carrier solvents were also added to the controls treated with 20 -S alone.

Atrazine significantly impaired 20 -S-induced OM at concentrations of 10 M and above in a dose-dependent manner, with fewer than 10% of the oocytes maturing at the highest concentration, 50 M (16.4ppm, Fig. 1A). The herbicide also significantly impaired maturation of spotted seatrout oocytes in a parallel in vitro bioassay at concentrations of 0.1 M and 1 M (results not shown). Bisphenol-A treatment also decreased OM in response to 20 -S, although it appeared to be less potent than atrazine, since maturation of croaker oocytes (Fig. 1B) and spotted seatrout oocytes (results not shown) was not significantly decreased by concentrations below 25 M.

Possible interactions of these chemicals with the membrane progestin receptor mediating progestin-induced OM was investigated in competitive binding radioreceptor assays (Das and Thomas, 1999). Plasma membrane fractions of ovarian tissues containing full grown oocytes (>450 m) and radiolabelled 20 -S ([ 3 H]-20 -S,41.9 Ci/mmol) were prepared as described previously (Patino and Thomas, 1990). Competition assays were conducted with 5 nM [ 3 H]-20 -S in the presence of different concentrations of the test chemicals (range 1 nM-10 M, dissolved in 1-5 l ethanol, which does not affect ligand binding in the receptor assays). After a 30-min incubation at 4˚C with the membrane fractions, the reaction was stopped by filtration (Whatman GF/B filters, presoaked in assay buffer), washed, and bound radioactivity was measured by ACCEPTED MANUSCRIPT scintillation counting. The displacement of the radiolabelled steroid binding by the steroid competitors was expressed as a percentage of the maximum specific binding of the steroid for its receptor. Both atrazine and bisphenol-A were effective competitors of [ 3 H]-20 -S binding to the croaker ovarian membrane progestin receptor, causing significant displacement at a concentration of 10 -6 M and approximately 50% displacement at 10 -5 M (Fig. 2). Scatchard binding analyses will be required to determine whether the binding of these compounds to the receptor is competitive (i.e. same binding site as 20 -S). Previous studies suggest Kepone binds competitively to the seatrout ovarian 20 -S receptor (Das and Thomas, 1999), whereas another estrogenic EDC, diethylstilbestrol, binds to a different site on the goldfish ovarian progestin receptor (Tokumoto et al., 2007).

The ovarian 20 -S receptor mediating OM has been identified as a novel seventransmembrane receptor, called membrane progestin receptor alpha (mPR , Zhu et al., 2003).

Recent studies suggest mPR also mediates 20 -S induction of sperm hypermotility in this species (Thomas et al., 2005). It is not surprising, therefore, that atrazine and bisphenol -A are also potent inhibitors of sperm hypermotility in response to 20 -S in croaker (Thomas and Doughty, 2005). Overall, progestin stimulation of sperm motility appears to be much more susceptible to interference by chemicals than OM and is significantly impaired by less then onetenth of the minimum effective concentrations observed in the present study (Thomas and Doughty, 2005). These differences may be due to lower effective concentrations of the test compounds at the oocyte surface than at the sperm membrane because the oocytes are surrounded by follicle cells. Bisphenol-A also binds to GPR30, the novel seven-transmembrane membrane estrogen receptor that has been characterized in breast cancer cells (Thomas et al., 2005), where it exerts an estrogen action (Thomas and Dong, 2006). GPR30 has also been identified in croaker ovaries (Pang and Thomas, unpubl.), raising the possibility that this environmental estrogen could influence ovarian and oocyte functions by interfering with estrogen as well as progestin nongenomic actions through interactions with both mPR and GPR30.

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Figure captions

Fig. 1 .

 1 Fig. 1. Concentration -dependent inhibition by (A) atrazine, and (B) bisphenol-A of 20 -S-

Fig. 2 .

 2 Fig. 2. Competition by atrazine and bisphenol-A for [ 3 H]-20 -S binding to Atlantic croaker

  Figure 1

Acknowledgements

Supported by NIH grant ESO12961 to P.T. and a McNare Scholarship to J.S.


Jared V Goldstone

John J Stegeman
email: jstegeman@whoi.edu

Gene structure of the novel cytochrome P4501D1 genes in stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes)

Keywords: Fish, isozymes, microsomal oxidases, CYP1A, bioinformatics, ortholog

The cytochrome P450 1 (CYP1) family has expanded with the addition of the CYP1B and CYP1C subfamilies. We recently identified a new CYP1 subfamily in zebrafish, CYP1D, with a single gene, CYP1D1. Here we examined sequences found in other fish genomes, i.e., stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes), for similarities among fish CYP1D1 genes. The full-length deduced amino acid sequences for CPY1D1 in these two species averaged about 43% identity to the CYP1As, but nearly 50% when sequence alignment ambiguities were masked. CYP1D1 has seven exons, similar in size and position to the exons in CYP1D1 and CYP1A in zebrafish. However, the intronic distances were substantially smaller in the medaka and stickleback. There also were differing numbers of xenobiotic response elements in the CYP1D1 of the various species. Whether the stickleback or medaka genes are inducible by aryl hydrocarbon receptor (AHR) agonists is yet to be determined.

Members of the CYP1 of monooxygenase family catalyze the oxidation and often bioactivation of a wide variety of common environmental carcinogens and promutagens.

CYP1 substrates include aromatic hydrocarbons, especially polycyclic aromatic hydrocarbons such as benzo[a]pyrene, as well as aromatic amines and heme degradation products (Nebert et al., 2004). Planar PCB congeners such as 3,3'4,4'-tetrachlorobiphenyl are also substrates, although metabolized very slowly (White et al., 1997). In mammals, three CYP1 genes have been described: CYP1A1, CYP1A2 and CYP1B1. Homologous CYP1A and CYP1B genes also have been described in fish. In addition, however, fish also express two paralogous genes in the CYP1C subfamily, CYP1C1 and CYP1C2, not found in mammals (Godard et al., 2005).

Recently we identified a fourth CYP1 subfamily in fish, CYP1D, with a single gene CYP1D1. CYP1D1 transcript is expressed in many organs of adult zebrafish but appears to be most highly expressed in early embryos. CYP1D1 transcript is not induced by the potent AHR agonist PCB 126 (3,3',4,4',, implying a possible endogenous role during development (Goldstone et al., submitted). The amino acid sequence of CYP1D1 in zebrafish inferred from cDNA and from genomic sequence is more like that of CYP1A than of CYP1B1 or the CYP1Cs. We showed that the differences in sequence between CYP1D1 and CYP1A are not evenly distributed along the protein, but that there are regions of higher or lower amino acid similarity, as expected. We also have found CYP1D1 sequences in other fish species. Here, we compare the sequence identities and gene structure of CYP1D1 in medaka (Oryzias latipes) and three-spined stickleback (Gasterosteus aculeatus), showing that CYP1D1 resembles CYP1A also in these species.

The sequences for stickleback and medaka genomes and gene predictions were obtained from the Ensembl database (Hubbard et al., 2007) and searched using BLAST. Gene predictions for CYP1A, CYP1B, CYP1C1, CYP1C2, and CYP1D1 were refined using Genscan and FGeneSH. Exon mapping was performed using GCG (v. 10.3; Accelrys, San Diego, CA).

The percent identity of amino acid sequences inferred for CYP1D1 and CYP1A genes in stickleback, medaka and zebrafish are shown in Table 1. In these species the unambiguously aligned CYP1D1 sequences are 47 to 50% identical to the CYP1As. In comparison, fish CYP1B and CYP1C sequences average only 43 and 41% amino acid identity to CYP1As, respectively. Similar to zebrafish, medaka and stickleback CYP1D1 deduced amino acid sequences display regions of higher and lower sequence similarity to CYP1A (data not shown). Some of these regions of high similarity correspond to structural elements common to many CYPs, including the A, B, E, G, I, K, and L α -helices and β-sheets 3 and 4.

Regions showing higher sequence similarity between CYP1D1 and CYP1A include putative substrate recognition sites (SRS; Gotoh, 1992) 4, 5 and 6. In contrast, the sequences of SRS 2 and 3 are quite different between CYP1D1 and CYP1A, and exhibit lower sequence similarity than immediately surrounding regions of the protein (Goldstone et al., submitted).

The differences in these two SRSs imply differences in substrate preferences between CYP1A and CYP1D1, assuming CYP1D1 is translated into protein.

Examination of the CYP1D1 gene structure revealed seven exons in both medaka and stickleback (Figure 1), with a relative arrangement highly similar to the exon-intron structure of the CYP1As in these species, quite like the similarity detected in zebrafish. Furthermore, the exon-intron boundaries are the same in the CYP1D1s and the CYP1As, with the exception of the first part of the coding region in CYP1D1 being split over the first two exons, while the first exon in CYP1A is non-coding. The CYP1D1 genes in medaka and stickleback are compressed on the chromosome relative to the zebrafish CYP1D1 (Figure 1). This may be a consequence of the smaller genome sizes in medaka (816 Mb) and stickleback (714 Mb) relative to zebrafish (1.7 Gb) (Ishikawa, 2000).

Mapping of consensus AHR binding sites in the proximal promoter regions (i.e., xenobiotic response elements or XREs; KNGCGTG), shows that medaka CYP1D1 has zero and stickleback CYP1D1 has four putative XREs in the upstream regulatory region (Figure 1); zebrafish CYP1D1 has two putative XREs (Goldstone et al., submitted). In contrast, the AHR2-regulated zebrafish genes CYP1A, CYP1B1, CYP1C1, and CYP1C2 have 12, 6, 4, and 6 XREs, respectively (Jönsson et al., 2007a(Jönsson et al., , 2007b)). Whether XREs in CYP1D1 are functional is not known; not all XREs in zebrafish CYP1A are functional (ZeRuth & Pollenz, 2005, 2007).

The CYP1D and CYP1A subfamilies apparently diverged from one another subsequent to the divergence leading to the CYP1B and CYP1C subfamilies. Thus, within the vertebrate CYP1 gene family, the CYP1As and CYP1Ds together constitute one clade, while the CYP1Bs and CYP1Cs constitute another. As we have noted (Goldstone et al., in press, submitted), establishing the timing of the divergences leading to the four subfamilies will require further taxonomic sampling.

Fig. 1 .

 1 Figure Caption:

Table 1 .

 1 Percent predicted amino acid identity between zebrafish (DANRE), stickleback (GASAC) and medaka (ORYLA) CYP1D1 and CYP1A sequences (lower left) and with regions of ambiguous alignment excluded by masking (upper right).

		DANRE_1D GASAC_1D ORYLA_1D DANIO_1A ORYLA_1A GASAC_1A
	DANRE_1D		76.6%	73.3%	49.0%	48.3%	48.1%
	GASAC_1D	67.1%		83.7%	47.7%	48.3%	47.0%
	ORYLA_1D	63.6%	74.1%		49.7%	50.2%	47.4%
	DANRE_1A	44.4%	40.8%	42.0%		73.3%	75.4%
	ORYLA_1A	42.9%	42.2%	42.3%	69.6%		77.7%
	GASAC_1A	42.7%	40.3%	40.4%	72.5%	75.2%

The similarity of CYP1D1 to CYP1A has intriguing functional implications. However, at this time the expression of a CYP1D1 protein and any functions thereof has yet to be established. If the relatively greater levels of CYP1D1 transcript expression in zebrafish embryos are accompanied by protein expression, it would suggest that there may be endogenous functions accomplished by CYP1D1, although CYP1D1 could have catalytic functions involving the transformation of xenobiotics. Interestingly, CYP1D1 has not been found in either of the two pufferfish genomes [Takifugu rubripes or Tetraodon nigroviridis;(Nelson, 2003;Goldstone et al., submitted)]. Clearly, determining expression and catalytic functions of CYP1D1 protein is an important objective; such studies are underway.

Figure 1

Acknowledgements

We thank two anonymous reviewers for comments. This research was supported in part by the Superfund Basic Research Program, NIH grant 5P42ES007381 (JS) and by NIH grant 1R01ES015912 (JS).


Irvin R Schultz

Ann Skillman

Dana Woodruff

Domoic acid excretion in dungeness crabs, razor clams and mussels

Keywords: Domoic acid, Bivalves, Crabs, Toxicokinetics

Domoic acid (DA) is a neurotoxic amino acid produced by several marine algal species of the Pseudo-nitzschia (PN) genus. We studied the elimination of DA from hemolymph after intravascular (IV) injection in razor clams (Siliqua patula), mussels (Mytilus edulis) and Dungeness crabs (Cancer magister). Crabs were also injected with two other organic acids, dichloroacetic acid (DCAA) and kainic acid (KA). For IV dosing, hemolymph was repetitively sampled and DA concentrations measured by HPLC-UV. Toxicokinetic analysis of DA in crabs suggested most of the injected dose remained within hemolymph compartment with little extravascular distribution. This observation is in sharp contrast to results obtained from clams and mussels which exhibited similarly large apparent volumes of distribution despite large differences in overall clearance. These findings suggest fundamentally different storage and elimination processes are occurring for DA between bivalves and crabs.

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E-mail address: ir_Schultz@pnl.gov (I. Schultz) The marine algal toxin domoic acid (DA), produced primarily by several species of pennate marine diatoms of the Pseudo-nitzschia (PN) genus is the causative agent of amnesic shellfish poisoning. High levels of DA have been periodically found in razor clams Siliqua patula and Dungeness crabs Cancer magister along the Pacific Coast of Washington (Wekell et al., 1994). A few studies have examined the disposition of DA in crabs. An early biomonitoring effort, measured DA in viscera (unspecified tissues) ranging from 0.8 to 30 mg/kg (Wekell et al., 1994). Similar levels of DA were still observed 3-4 months later in crabs captured from the same estuaries. The assimilation efficiency of DA in crabs after four consecutive daily feedings of razor clam meat was observed to be 68 % based on hepatopancreas levels (Lund et al., 1997). In this study, 16 % of the first dose was detectable in the hepatopancreas after four hours, which still had increasing concentrations of DA 24 hours after the last feeding (Lund et al., 1997).

These results suggest that DA is rapidly and extensively absorbed from the digestive system in crabs.

Profound differences in the toxicokinetics of DA have been identified in a wide variety of shellfish including several species of mussels (Mytilus sp.; Wohlgeschaffen et al., 1992, Blanco et al., 2002) and razor clams (Drum et al., 1993). Depuration of DA in field-exposed mussels has been reported to be both mono-exponential (Novaczek et al., 1992) and bi-exponential (Blanco et al., 2002), although in both studies >90 % of the toxin was eliminated within 96 hours. Mussels retain most of the toxin in the digestive gland and the kidney with low levels found in the hemolymph (Novaczek et al., 1991).

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In this study, we applied an experimental approach involving direct intravascular injection of DA in mussels, clams and crabs and subsequent characterization of the toxicokinetics using a similar compartmental model. Additional experiments with crabs studied the elimination of two other organic acids, dichloroacetic (DCAA) and kainic (KA) acids to provide a comparison to the DA results.

All crabs and mussels were collected from Sequim Bay WA, USA. Razor clams were collected from various sites along the WA coastline. Water quality values measured during the study were: temperature: 9.0-10.2 o C, pH: 7.9-8.1; salinity: 31.0 ; dissolved oxygen 8.2-8.9 mg/L. After acclimation for at least two weeks, individuals were transferred into indoor holding tanks under otherwise similar conditions.

All shellfish were injected intravascularly with DA (1.0 mg/Kg; dissolved in Cortlands saline, pH 7.4). Separate crabs were also dosed dichloroacetic acid (DCAA; 9 mg/Kg) and kainic acid (KA; 2 mg/kg). Subsequent hemolymph samples were removed from different legs by inserting the sampling syringe into the pereiopod arteries and removing approximately 0.1-0.2 ml of hemolymph. Bivalves were similarly injected at a point posterior from the gills. Domoic acid was measured by HPLC using UV detection at 242 nm (Quilliam et al., 1995). KA was measured by fluorescent detection using methods described by James et al. (2000). DCAA was measured by GC-ECD as described by Schultz et al. (1999). After administration of the DA to all shellfish and DCAA or KA to crabs, the decline in hemolymph concentration-time profiles appeared bi-exponential and we chose to fit these data to a 2compartment clearance volume compartmental model using WinNONLIN. The toxicokinetic parameters are the apparent volumes of distribution (V1, V2), total body clearance (Clb) and the elimination half-life (t½).

A summary of the hemolymph concentration-time profiles for clams and mussels is presented in Figure 1a and 1b and for crabs in Figure 2A-C. In mussels, DA declines in a log-linear manner with a elimination half-life of 55 hrs and a steady-state volume of distribution of 4746 ml/kg (Table 1). In clams, DA levels initially declined and then appeared to stop or even increase slightly.

Kinetic analysis is thus limited to estimation of distributive volume which appears to be similar to that observed for mussels (Table 1). In contrast to the results from bivalves, the volume of distribution of DA in crabs was quite small (approximately 280 ml/kg). This value is very similar to the hemolymph volume in crabs, suggesting very little of the injected DA dose distributed outside the vascular fluid compartment. Comparison with the results from the DCAA and KA experiments indicated these organic acids have larger distributive volumes than DA and greater clearance, although both DCAA and KA also primarily distributed into a large central pool or compartment (see V1 estimates, Table 1). The DA results in crabs are surprising given the established ability of the hepatopancreas to retain DA after oral dosing. Instead, most of the injected DA is retained in the hemolymph compartment suggesting a diffusional barrier exists, which prevents DA in the hemolymph from distributing into the hepatopancreas.

Fig. 1 .

 1 Figure captions:

  Figure1

Acknowledgement

Financial support was provided by the US EPA-STAR grants program.


Irv Schultz
email: ir_schultz@pnl.gov

Kim H Brown

James J Nagler

Effect of parental exposure to trenbolone and the brominated flame retardant BDE-47 on fertility in rainbow trout (Oncorhynchus mykiss)

Keywords: Trenbolone, Flame retardants, BDE-47, Rainbow trout, Reproduction

We exposed sexually maturing male rainbow trout (Oncorhynchus mykiss) to BDE-47 (a polybrominated diphenyl ether) and female rainbow trout to trenbolone (an anabolic steroid).

Male trout were orally exposed for 17 days to 55 g/Kg/day BDE-47 and female trout continuously exposed for 60-77 days to a measured trenbolone water concentration of 35 ng/L.

After the exposure, eggs and semen were collected and in vitro fertilization trials performed using a sperm:egg ratio of 300,000:1. In the BDE-47 study, eggs from control females were fertilized with semen from exposed males, while in the trenbolone study, eggs from exposed females were fertilized with semen from control males. All treatments were evaluated at two-three early developmental time-points representing first cleavage (0.5 day), embryonic keel (9 days), and eyed stages (19 days), respectively. The results indicated that BDE-47 exposure did not alter

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fertility as embryonic survival was similar between control and exposed groups. Trenbolone exposure also did not alter embryo survival. However, in the embryos fertilized with eggs from trenbolone exposed females, a noticeable delay in developmental progress was observed. On day 19 when eye development is normally complete, the majority of the embryos either lacked eyes or displayed under-developed eyes, in contrast to control embryos. This finding suggests steroidal androgen exposure in sexually maturing female rainbow trout can impact developmental timing of F1 offspring.

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In previous studies, we demonstrated that the synthetic estrogen ethynylestradiol (EE2) reduces the fertility of sexually maturing male rainbow trout (Oncorhynchus mykiss) by decreasing progeny survival (Schultz et al., 2003;Brown et al., 2007). At EE2 exposure rates comparable to environmental levels (e.g. < 15 ng/L), embryo mortality between developmental days 9 (embryonic keel formation) and 19 (eyed stage) approached 50%. This suggests some form of genomic instability rather than an effect on sperm morphology and/or motility is responsible (Brown et al., 2007). This raises important ecotoxicological questions in regards to whether this effect is limited to estrogens or can other classes of endocrine disruptors known to impact testicular development produce similar effects. Alternatively, can exposure to steroidal androgens impact female rainbow trout in a similar manner? In this study, we pursued these questions by exposing sexually maturing male rainbow trout to 2,2,4,4-tetrabromodiphenyl ether (BDE-47; commercially used as a flame retardant) and female rainbow trout to the anabolic steroid trenbolone. We chose to expose male trout to BDE-47 based on the findings from a previous study, using pair breeding fathead minnows (Pimephales promelas), which documented a reduction in spawning due to decreased production of mature spermatozoa in males (Muirhead et al., 2006). Trenbolone is a widely used growth promoter in livestock production that is known to cause masculinization of female fish (Ankley et al., 2003).

Sexually mature, male isogenic rainbow trout of the OSU x Arlee strain (Young et al., 1996) (22 months in age;body weight: 733 ± 166 g (mean ±SD); n=5), raised at MSL holding facilities were orally exposed for 17 days to BDE-47 at a dose rate of 55 g/Kg/day. The BDE-47 was incorporated into the pelleted trout feed and assayed by GC-ECD using methods described in Muirhead et al. (2006) to verify the concentration and stored at -20 o C until day of feeding. Each morning of the exposure, a known mass of BDE-47 feed was provided to individual trout. Complete consumption of the dose within 5 minutes was verified visually each day. Control fish body weight: 727 ± 93 g; n=4; identical in age and genetics) were fed uncontaminated trout feed.

For the trenbolone exposures, sexually maturing female rainbow trout, 2 + years in age were obtained from a local hatchery (Nisqually farms, Lacey WA, USA) and exposed to a nominal waterborne concentration of 50 ng/ L. The trenbolone was continuously added to the exposure tanks dissolved in a methanol carrier (<0.004 % methanol in tanks), control fish were only exposed to the methanol carrier. Actual trenbolone exposure levels were determined by GC-MS using the method described by Marchand et al. (2000). The mean value of four separate determinations made throughout the exposure was 35 ng/L. After 60 d of exposure, all fish were checked for ovulation twice weekly until five control and treated fish had ovulated. (BW = 1450 ± 220 g exposed; 1375 ± 301 g control fish). Thus, the duration of exposure lasted between 60 -77 days. At completion of the exposures, sperm or eggs were obtained by manual expression of the gametes from anesthetized fish and used in controlled fertility trials. For semen from the BDE-47 exposed males, the number of sperm/ml semen was quantified and fertilization trials performed using eggs from a single unexposed female donor at a sperm:egg ratio of 300,000:1.

Eggs collected from trenbolone exposed trout were immediately used in fertilization trials at a similar sperm:egg ratio using sperm collected from unexposed isogenic male trout. All treatments were evaluated at two or three developmental time-points: 0.5 days post fertilization (dpf), 9 dpf, and 19 dpf, representing first cleavage, embryonic keel, and eyed stages, respectively. An additional measure of eye pigmentation in the day 19 embryos from the trenbolone exposure was performed to assess the overall state of embryonic development. This was done subjectively by categorizing the level of pigmentation as: 0-no eye development, 1-eye development with no retinal pigmentation, 2-initial retinal pigmentation, 3-moderate retinal pigmentation, 4-fully eyed. Statistical analysis of the categorical data was done with SAS vs. 8.1

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using a chi-square test of independence and a general linear model that specifically compared within treatment effects on pigmentation.

The results of the BDE-47 fertilization trials are shown in Fig. 1. Fertilization rates are consistently high and no apparent difference was observed between control and the BDE-47 exposure when embryo survival was assessed at 1 st cleavage or at the eye stage of development. This result occurred despite the presence of BDE-47 in all semen samples (436 ± 109 ng/ml) from exposed trout used in the fertilization trials. Trenbolone exposure to female trout did not affect subsequent embryo survival, which was consistently above 80% in all treatment groups assessed at the three different developmental stages (Fig. 2A). During evaluation of embryo survival from the trenbolone experiment, it became apparent that the rate of embryonic development was delayed compared to control embryos. We chose to quantify this effect by subjectively categorizing the state of eye development at 19 days post-fertilization. These results are presented in Fig. 2B. The majority of embryos in the trenbolone exposed treatment group can be characterized as either having no eye pigmentation or a complete lack of eye development. This finding is in contrast to control embryos, the majority of which possessed fully developed embryonic eyes. Thus, exposure to an androgen in sexually maturing female trout does appear capable of impacting normal embryonic development.

Our results combined with past studies of EE2 in male trout would indicate that exposure to steroids during sexual maturation impacts genomic stability and subsequent viability of the F1 offspring. This raises serious concerns about the consequences of municipal sewage release (primary source for xenoestrogens) and run-off from cattle feed-lots (primary source for anabolic androgens) on fish populations.

Figure captions

Fig. 1 .

 1 Fig. 1. Embryonic survival assessed at 0.5 days of development (1 st cleavage) and at day 19 (eyed

Fig. 2 .

 2 Fig. 2. (A) Embryonic survival assessed at 0.5 days (1 st cleavage), 9 days (keel formation) and 19

Acknowledgement

Financial support was provided in part by the U. S. Department of Energy under contract DE-AC06-76RLO 1830 with Battelle PNNL, NIEHS grant 5R01ES012446-03 and NSF grant 0540693.


J C Leão

L A Geracitano

J M Monserrat

L L Amado

J S Yunes
email: jsyunes@furg.br

Microcystin-induced oxidative stress in (Polychaeta, Nereididae)

Keywords: Microcystin, Microcystis, Laeonereis, Oxidative stress, Lipid peroxidation, DNA-Protein Cross-link

Oxidative stress induced by microcystins was evaluated in an estuarine worm, Laeonereis acuta (Nereididae). Ten organisms were exposed to lyophilized cells of a toxic Microcystis aeruginosa strain RST9501 (≈2 mg/L microcystins, MC); ten were exposed to lyophilized cells of a nontoxic Aphanotece sp. strain RSMan92 and ten were maintained without cyanobacterial cells. Exposure time was 48 h. The enzymatic antioxidant defenses, as well as the oxidative damage, were analyzed. Toxic and nontoxic cyanobacteria lowered catalase activity with no changes in glutathione reductase and glutathione-S-transferase activities. This may have led to toxin intracellular accumulation, which should favor oxidative stress generation, observed by the high lipid peroxide and DNA-protein crosslink levels in the group exposed to MC.

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Microcystins (MC), common cyanobacterial hepatotoxins, are known to inhibit PP1 and 2A enzymes and to induce oxidative stress in different organisms leading to cell damage (Pflugmacher, 2004;Pietro et al., 2006).

Anthropogenic pollution has promoted cyanobacterial toxic blooms in water bodies.

Several blooms containing toxic Microcystis aeruginosa have occurred in the Patos Lagoon (Southern Brazil) over the last two decades with effects on the aquatic life. Physiological and behavioral changes with the death of some species have been observed; this fact may decrease the biodiversity (Yunes et al., 1998;Dewes et al., 2006).

The worm Laeonereis acuta has been regarded as an important bioindicator in the Patos Lagoon. Its environmental abundance and physiological characteristics, such as the ability to modulate its antioxidant defense system to several pollutants, make this worm an important tool for ecotoxicological studies (Geracitano et al., 2004).

The present study aimed to evaluate some antioxidant enzymatic parameters in L.

acuta exposed to acute MC levels, as well as oxidative damage.

Cells of a toxic M. aeruginosa strain (RST 9501) were cultured and employed as a source of MC. Cells of a nontoxic cianobacterium, Aphanotece sp. (RSMan92), were cultured in parallel with M. aeruginosa cultures.

Specimens of L. acuta were collected in an unpolluted area of the Patos Lagoon and immediately transferred to the laboratory in an ice-cold box. After 4 days of acclimation in plastic boxes with sand and water (10‰; pH 8.00), the worms were transferred to individual recipients (10 cm diameter) containing 50 mL water (10‰; pH 8.00) and 10 g sterilized sand at a fixed temperature (20 o C) and photoperiod (12L:12D).

Three groups (n=10) were tested; the first one was exposed to lyophilized cells of toxic M. aeruginosa (2 mg/mL) reaching 2 µg MC/mL, the second one to lyophilized cells of the nontoxic cyanobacterium, Aphanotece sp. (2 mg/mL), and the third group was the control. All tests lasted 48 hours.

According to Matthiensen et al. (2000), [D-Leu 1 ]MC-LR is the major MC variant produced by this Microcystis strain, and it presents an LD 50 value of 71 µg/kg in mouse bioassay. In virtue of the MC concentrations used, the assays were considered acute.

For protein concentration and enzyme activity analysis, every organism was homogenized (20% w/v) in a buffer [Tris base (20mM), EDTA (1mM), dithiothreitol (1mM, Sigma), sucrose (0.5 mM), KCl (150 mM); pH 7.6]. Samples were centrifuged at 9,000 x g (4 o C) for 30 min and the supernatants employed as the enzyme source.

The following parameters were measured: activity of glutathione-S-transferase (GST) and catalase (CAT) (Geracitano et al., 2004); glutathione reductase (GR) (Nagalakshmi and Prasad, 2001); lipid peroxides levels (LPO) (Geracitano et al., 2004);

and DNA-protein crosslink levels (Barker, 2005). Data were subjected to ANOVA (α =0.05).

After 48-h exposure, CAT activity was reduced (p<0.05) whereas GST and GR activities had no difference when compared to the control group (p>0.05; Fig. 1). Only the Microcystis-exposed group presented higher lipid peroxides and DNA-protein crosslink levels, when compared to the control group (p<0.05; Fig. 2).

Reactive oxygen species (ROS) are produced during several biochemical processes, thus an antioxidant system is necessary to protect the organism. CAT is an enzyme that catalyzes H 2 O 2 (precursor of HO , the most toxic ROS) degradation. GST catalyzes the conjugation of oxidized products with reduced glutathione (GSH). GST also catalyzes the detoxification process through the conjugation of MC with GSH, whereas GR catalyzes the regeneration of GSH from GSSG (oxidized glutathione) (Pflugmacher, 2004).

In this work, MC did not activate the antioxidant system; in fact, CAT was diminished. Besides, non-altered GST activity may induce MC intracellular accumulation, leading to an oxidative stress condition. Moreover, high levels of ROS such as superoxide anion may inhibit CAT activity (Kono and Fridovich, 1982). Dewes et al. (2006) have reported the rise of oxidative stress in an estuarine crab orally exposed to MC. Pflugmacher Oxidative stress condition is induced by an unbalance of pro-oxidants and the antioxidant capacity in favor of the pro-oxidant. In this work, high DNA protein crosslinks and LPO levels were observed. Although there is no evidence of MC-induced DNA-protein crosslink damage in the literature, Zegura et al. (2004) have observed that the genotoxicity of MC is mediated by the induction of ROS production. High LPO levels caused by MC have been observed in crabs and fish by Dewes et al. (2006) and Pietro et al. (2006). The fact that those variables were altered during exposure to MC indicates that there is a common response triggered by aquatic organisms, and also suggests that not only the accumulation of MC in tissues, but also the biochemical variables should be analyzed in order to estimate the toxicological risk to populations exposed to these toxins.

Figure Captions

  Microcystin-induced oxidative stress in Laeonereis acuta (Polychaeta, Nereididae) J.C. Leão, L.A. Geracitano, J.M. Monserrat, L.L. Amado, J.SPlease cite this article as: Leão, J.C., Geracitano, L.A., Monserrat, J.M., Amado, L.L., Yunes, J.S., Microcystininduced oxidative stress in Laeonereis acuta (Polychaeta, Nereididae), Marine Environmental Research (2008), doi: 10.1016/j.marenvres.2008.02.033

(

  2004) andPietro et al. (2006) have observed MC-induced oxidative stress in the aquatic macrophyte Ceratophyllum demersum and in the tilapia fish (Oreochromis sp.). The absence of antioxidant responses in L. acuta led the organism to an oxidative stress condition after acute MC exposure, as evidenced by LPO and DNA-PC damage.

Fig. 1 .

 1 Fig. 1. Enzymatic activity after 48-h exposure to 2 mg/mL lyophilized cells of toxic

Fig. 2 .

 2 Fig.2. Induced-oxidative stress damage after 48-h exposure to 2 mg/mL lyophilized cells

Acknowledgements

We thank the Brazilian agencies CAPES and CNPq for their support during the tenure of the PhD students J.C. Leão and L.L. Amado and for research grants. L.A.

Geracitano is recipient of a post-doctoral grant PRODOC (CAPES program) and J.S. Yunes and J.M. Monserrat are research fellows from CNPq.