A new chemical analyzer for in situ measurement of nitrate and total sulfide over hydrothermal vent biological communities

Marine Chemistry 72 Ž2000. 1–15 www.elsevier.nlrlocatermarchem

A new chemical analyzer for in situ measurement of nitrate and total sulfide over hydrothermal vent biological communities N. Le Bris a,) , P.-M. Sarradin a , D. Birot b, A.-M. Alayse-Danet c b

a DRO–EP, IFREMER, Brest, BP 70, 29280 Plouzane, ´ France TMSI–TSI–ME, IFREMER, Brest, BP 70, 29280 Plouzane, ´ France c DMON, IFREMER, Brest, BP 70, 29280 Plouzane, ´ France

Received 18 August 1999; received in revised form 2 May 2000; accepted 16 May 2000

Abstract A new submersible chemical analyzer, ALCHIMIST ŽAnaLyseur CHIMique In SiTu., based on colorimetric detection and flow injection analysis ŽFIA., was adapted to allow in situ measurements of nitrate q nitrite ŽN q N. and total dissolved sulfide ŽÝS. in the deep sea hydrothermal environment. Before in situ trials, the influence of hydrostatic pressure and temperature on the analytical responses was examined under simulated conditions Ž1–300 = 10 5 Pa, 5–258C.. First trials were performed during dives of the Remotely Operated Vehicle ŽROV., VICTOR 6000, over the Lucky Strike hydrothermal vent field ŽMid-Atlantic Ridge.. ALCHIMIST, installed on the ROV, enabled simultaneous N q N and ÝS calibration and measurements at 1650 m depth, at a rate of 22 analyses per hour. At depth, the precision of the in situ ÝS and N q N measurements is estimated to be, respectively, 1.1% and 0.8%. The detection limit is 0.8 mM for ÝS and 0.5 mM for N q N. At a vent site of the Lucky Strike area, ALCHIMIST enabled to resolve, at the decimeter scale, the chemical gradients which characterize the patchy distribution of hydrothermal fauna. Additionally, temperature–concentration relationships offered further information on the processes controlling the chemistry of the habitats. Like former in situ analyzers used in this field, this new instrument should be valuable to characterize the physico-chemical characteristics of vent fauna environment. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Hydrothermal; In situ analysis; Nitrate; Sulfide; FIA

1. Introduction In the absence of light, the deep sea hydrothermal ecosystem is based mainly on a chemolithoau-

) Corresponding author. Tel.: q33-2-98224672; fax: q33-298224757. E-mail address: [email protected] ŽN. Le Bris..

totrophic bacterial primary production. A large part of this production is accomplished in symbiotic systems relying on both reduced and oxygenated compounds ŽFisher, 1990; Tunnicliffe, 1991; Childress and Fisher, 1992.. These symbiotic organisms are present in the turbulent mixing zone, where the environmental conditions are constantly switching from hydrothermal fluid to seawater-dominated conditions, as shown by the extreme variability of tem-

0304-4203r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 0 3 Ž 0 0 . 0 0 0 5 7 - 8

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Table 1 Characteristics of the in situ chemical analyzers developed for deep sea hydrothermal environment studies Johnson et al. Ž1986a.; Sakamoto-Arnold et al. Ž1986.; Coale et al. Ž1991.

Gamo et al. Ž1994.

Gamo et al. Ž1997.

Massoth and Milburn Ž1997.

Radford-Knoery et al. Ž1997.

This study

Name Parameters Method Detection

SCANNER Ža. Si, ÝS; Žb. Fe, Mn CFA colorimetryr mono-wavelength in situ Si 0–800 mM, 3 mM, 2% ÝS a 0–75 mM, 0.12 mM, 1.9 % 0–200 mM, 1.2 mM, 3.7% Fe, Mn, np

MCA-2000 Si, ÝS CFA colorimetryr mono-wavelength in situ, 2 standards Si 0–120 mM, 3 mM, 2% ÝS 0–40 mM, 1 mM, 2%

GAMOS-I and II Mn CFA chemiluminescence

SUAVE Fe, Mn, ÝS CFA colorimetryr mono-wavelength in situ, 3 standards np

AIS ÝS FIA colorimetry dual-wavelength in situ np

ALCHIMIST NqN, ÝS FIA colorimetryr

Calibration Linearity range, detection limit, precision

np Mn d.l. 0.1 nM

CFA: conventional flow analysis; FIA: flow injection analysis; np: not published. a High and low sensitivity manifolds. b The linear range can be extended to 450 mM using the same manifold using partial injection ŽSarradin et al., 1999b..

in situ, 3 standards NqN 0–40 mM, 0.5 mM, 1.5 % ÝS b 0–80 mM, 0.8 mM, 1.5%

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Reference

N. Le Bris et al.r Marine Chemistry 72 (2000) 1–15

perature at one point ŽJohnson et al., 1988a; Chevaldonne´ et al., 1992.. The distribution of vent biota is also highly discontinuous, along steep spatial gradients of environmental conditions ŽJohnson et al., 1986b; Sarradin et al., 1998, 1999a.. Our knowledge of the physical and chemical environment of hydrothermal organisms is thus deeply affected by the scale of measurement and sampling frequency. Furthermore, as the mixing zone is characterized by non-equilibrium and pressurertemperature dependant chemical systems, the sample characteristics may change between actual collection at depth and further analysis on board ship ŽMillero et al., 1987.. Considering this, in situ chemical analysis appears to be the most relevant way to describe and understand the chemical processes driving this unusual and highly fluctuating biotope and, thus, the interactions between biota and environment. Several in situ analyzers have been developed for applications in deep sea hydrothermal environments. Their basic instrumental and analytical characteristics are presented in Table 1. All of these are based on the flow measurement principle, which allows in situ calibration with standard solutions. The continuous flow analysis ŽCFA. method is predominantly used and involves continuous drawing of a sample or standard solution, on-line mixing with reagents and flowing through a measurement cell. Flow injection analysis ŽFIA., which is defined by sequential injection of the sample into a carrier–flow before continuous reagent mixing and detection ŽRuzicka and Hansen, 1988.. FIA is used by the AIS ŽRadfordKnoery et al., 1997. and the ALCHIMIST. Colorimetric detection is principally used, as it is suitable for the analysis of a large number of chemical parameters. For high sensitivity measurement, such as needed for hydrothermal plumes detection, GAMOS I and II first use chemiluminescence detection. ALCHIMIST is the deep sea adapted version of a versatile in situ analyzer described by Birot et al. Ž1997, 1998.. This paper presents the settings and trials performed to adapt this device for simultaneous in situ analysis of N q N and ÝS, both being relevant parameters for hydrothermal vent fauna ŽFisher, 1990; Hentschel et al., 1993; Hentschel and Felbeck, 1993.. Prior to sea trials, evaluation of the response to temperature and pressure effects was conducted at

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the laboratory. This work in simulated conditions was necessary to check that the flow measurement conditions were suitable for in situ operation. Since ALCHIMIST permits in situ calibration, a precise quantification of these effects was, however, not needed. Subsequent installation of the analyzer on the Remotely Operated Vehicle ŽROV. and sea trials enabled to estimate the precision, accuracy and time response at depth. We also evaluated the capacity of this instrument to describe the chemical characteristics of the biological habitats.

2. Materials and methods 2.1. Analytical methods The FIA method for N q N determination described by Floch Ž1998., which involves reduction to nitrite on a Cd–Cu column and azo–dye spectrophotometric detection, was used. The method used for ÝS determination was adapted to FIA by Sarradin et al. Ž1999b. from Fonselius Ž1983., Cline Ž1969. and Sakamoto-Arnold et al. Ž1986.. The manifold is presented in Fig. 1. In comparison to the laboratory method, flow rates for ÝS measurement were increased to compensate the expected decrease of sensitivity at low temperature ŽSarradin et al., 1999b.. Reagents and standards ŽTable 2. were prepared according to Daniel et al. Ž1995a. and Sarradin et al. Ž1999b., using analytical grade reagents, ultrapure water ŽMilli-Q, Millipore w . or natural seawater. As described in Daniel et al. Ž1995b., a pseudoabsorbance FIA signal based on a dual-wavelength measurement was determined to overcome the Schlieren effect, produced by refractive index variation at the samplercarrier interface. This correction was convenient for ÝS measurement. Better results were obtained for N q N measurement by defining the pseudo-absorbance FIA signal as: A Ž t . s log

Ž alref i Ž t . q b . rlmes i Ž t .

where lref iŽ t . and lmes iŽ t . are the intensities at the reference and measurement wavelengths, respectively. The a and b coefficients are the slope and

N. Le Bris et al.r Marine Chemistry 72 (2000) 1–15

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Fig. 1. N q N and ÝS manifold Žflow rates in ml miny1 are indicated for each pump channel..

intercept of the linear relation, which is obtained by plotting lmes i vs. lref i for a blank injection. Table 2 Preparation of reagents and standards Name Reagents ÝS Amine

FeCl 3 SW NqN

NH 4 Cl

SAN NED

Standards ÝS Stock solutions Standard solutions NqN Stock solutions Standard solutions

For quantitative analysis, peak height was used rather than peak area, considering that the N q N peak is quite large and could partially lie out the acquisition window Žsee Section 2.2..

Composition

2.2. Instrumental set up N, N-dimethyl-p-phenylene diamine dihydrochloride 1.0 g ly1 in HCl 0.1 N FeCl 3 , 3 g ly1 in HCl 0.9 N Natural seawater supplied on board NH 4 Cl 20 g ly1 in H 2 O 18 M V q4 ml NaOH 1M ŽpH s 7.5. Sulfanilamide 4 g ly1 in HC1 1.2 M N-Žnaphtyl-ethylene diamine. dihydrochloride 2 g ly1 in H 2 O 18 M V Na 2 S 50 mM 0, 20 and 40 mM in natural seawater supplied on board KNO 3 25 mM 0, 20 and 40 mM in synthetic seawater ŽKhoo et al., 1977.

ALCHIMIST ŽFig. 2. is composed of three main modules: hydraulic Žlength 325 mm, width 180 mm, height 291 mm., spectrophotometric Žlength 661 mm, diameter 120 mm. and electronic Žlength 580 mm, diameter 192 mm.. All the modules withstand pressure up to 600 = 10 5 Pa Ži.e. about 6000 m depth. ŽBirot et al., 1997, 1998.. Its total weight is 54 Da N in air, 27 Da N in water. The electronic package manages the energy supply and data communication between a personal computer and the spectrophotometric and hydraulic modules. The spectrophotometric module includes two custom-made PMMA flow measurement cells Ž3 cm pathlength. associated with two dual-wavelength spectrophotometric detection units. The hydraulic module consists of a peristaltic pump ŽGilson Minipuls MP3., two injection valves ŽUpchurch Sci-

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ALCHIMIST is operated from the surface, via the 10 km fiber optic transmission cable. The analyzer is programmed from a personal computer using a custom-designed software ŽVisual Basic. which enables communication in real time with the master electronic unit via an RS 232 serial link. The software enables to program a time sequence during which the four valves are individually activated and the data acquisition started at selected times. The dual-wavelength light intensity acquisition is performed simultaneously on the N q N and ÝS channels, and lasts 60 s at a rate of 1 point per second. Absorbance and peak height are calculated, recorded and edited at the end of each acquisition. This sequence can be automatically repeated. Fig. 2. Schematic view of the ALCHIMIST showing the three modules Ž1 — electronic, 2 — spectrophotometric, 3 — hydraulic. and the connection of sampling tubes to the selection valves Žthe rest of the manifold tubing is not represented here..

entific, V451, six ports. and two selection valves ŽUpchurch Scientific, V421, six ports.. Each selection valve has six inlet ports. These ports were connected to three bags containing standard solutions and to three sampling tubes. We thus had the possibility to draw the sample through one of the three sample inlets and to change the inlet if some problems of clogging were encountered after a while. Each sampling tube ŽPEEK 0.8 mm i.d., 7 m length. was divided just before the selection valves using a T junction to be connected to the two selection valves ŽFig. 2.. The sample intake probes were modified to adjust 40 cm titanium tubing Ž0.8 mm i.d.. fitted with PEEK frits Ž10 mm. at the PEEK and titanium tube junction. The three intake probes and a temperature probe were tied together in order to be manipulated by the remotely operated arm of the submersible. As shown in Fig. 1, the injection valves are positioned in the manifold, in such a way that the sample is drawn through the injection loops instead of being pushed, to minimize the dead volume. Standards and reagents are contained in flexible bags ŽTransfer pack, Baxter Healthcare., which ensures that the entire flow system is maintained at the ambient hydrostatic pressure.

2.3. Laboratory temperature and pressure tests Prior to sea trials, we examined the influence of temperature and pressure on the peak shape, peak delay and its consequence on the position of the peak in the acquisition window and the sensitivity of the method. Temperature influence on the analyzer response for ÝS and N q N was tested over the range 5–208C. The ÝS and N q N tests were done separately, as the N q N acquisition channel failed for the first test. The three modules were placed in a thermostated oven, together with the plastic bags containing reagents and standards. The temperature inside the oven was regulated to within 0.18C. Calibrations were performed at least 2 h after the temperature was stabilized in the oven allowing the solutions and electronics to reach the thermal equilibrium. It should be noted that the pump speed was higher for these tests than for pressure test and in situ operation Ž6 rpm instead of 5 rpm., due to a change of the configuration of the pump electronic board. Measurements were performed at a hydrostatic pressure up to 300 = 10 5 Pa in a pressure test tank. The temperature of the tank water, measured with a Pt 100 probe, was stabilized at 12 " 18C during the experiment. The pump speed was periodically controlled using a video camera placed in the pressure tank for this experiment. Automatic measurements of N q N and ÝS concentrations in standard solutions were made while the hydrostatic pressure was cycled

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Ž8 = 10 5 Pa miny1 . between 3 = 10 5 and 300 = 10 5 Pa. Calibrations with three standards were performed at constant pressure Ž3 and 300 = 10 5 Pa.. 2.4. In situ operation ALCHIMIST was tested at sea during the operational trials of the deep sea ROV, VICTOR 6000 ŽVictor Premiere ` cruise, Mid-Atlantic Ridge.. The

analyzer, placed at the back of the ROV, was deployed at depths ranging between 1600 and 2100 m and operated for 20 h at depth. Standard injections were performed during the descent and on the bottom, before operation in the hydrothermal area. At 1650 " 50 m depth and 4.5 " 0.58C, calibration lines were determined with three standards Ž0, 20 and 40 mM. for N q N and two standards Ž0 and 40 mM. for ÝS. Repeatability of

Fig. 3. Modification of the peak shape induced by temperature variation. Ža. ÝS: 40 mM; Žb. N q N: 50 mM.

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measurements was also established on ambient bottom water for N q N and on the 40 mM standard for ÝS. On the AElisabethB site of the Lucky Strike hydrothermal area ŽFouquet et al., 1995; Van Dover et al., 1996; Desbruyeres ` et al., 2000., in situ measurements were made around the dominant organisms, in an area limited by a 50 = 50 cm quadrat. The sample intake probe was placed successively at four different points of the quadrat and maintained for about 15 min on each of these points, which were characterized by different temperature ranges. Diffuse fluids of higher temperature were analyzed 1 or 2 m away from the quadrat.

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3. Results and discussion

3.1. Influence of temperature and pressure on the response

3.1.1. Temperature Fig. 3a shows how the ÝS peak is delayed Žq2 s., broadened Ž w 1r2 from 14 to 16 s. and its height decreased Žy0.1 absorbance unit. between 158C and 58C. The variation of the calibration slope vs. temperature reaches 2% 8Cy1 . The effect is similar for the N q N peak Žq2 s delay, q2.5 s w1r2 , y0.1

Fig. 4. Influence of pressure on the FIA peak shape. Ža. ÝS: 50 mM, Žb. N q N: 15 mM.

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absorbance unit for peak height; Fig. 3b.. The temperature dependence of the calibration slope is 1.2% 8Cy1 .

Such a modification of the peak shape with decreasing temperature was described in Sarradin et al. Ž1999b. and compared to the evolution observed as

Fig. 5. Evolution of peak height during pressure cycling Žfilled circles: 3–300 = 10 5 Pa; open circles: 300–3 = 10 5 Pa.. Ža. ÝS: 50 mM, Žb. N q N: 15 mM.

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Table 3 Calibration slopes for ÝS and N q N analysis in the pressure test tank and in situ Ž n is the number of calibration points. Experiment

Temperature Ž8C.

Pressure Ž10 5 Pa.

N q N slope Žabsorbance unit mMy1 .

ÝS slope Žabsorbance unit mMy1 .

Pressure tests Pressure tests In situ

12.1 12.1 4.5

3 300 160

0.0062 Ž r 2 s 0.996, n s 7. 0.0066 Ž r 2 s 0.990, n s 7. 0.0064 Ž r 2 s 0.993, n s 11.

0.0037 Ž r 2 s 0.990, n s 7. 0.0039 Ž r 2 s 0.989, n s 7. 0.0040 Ž r 2 s 0.9995, n s 16.

the flow rate was decreased by changing the pump tube diameters. The peak broadening indicates a larger axial dispersion of the sample in the carrier stream. A change of the viscosity of fluids due to temperature variation can increase the dispersion of the sample as displayed by Kolev and Pungor Ž1988.. According to these authors, an increase of fluid viscosity also leads to a decrease of peak height and of Aretention timeB. Then, the larger delay between injection and beginning of the peak at low temperature cannot be related to a modification of fluid viscosity. This rather suggests that flow rates are reduced at low temperature. As the pump speed did not vary during these trials, modification of the properties of pump tubes Želasticity and inner diameter. with temperature is a possible cause for this effect. Though temperature effects in air or water may not be quantitatively equivalent, these results lead to

expect a slight decrease of the sensitivity of the methods, and a shift in the appearance of the peaks from ambient to in situ temperature. For an in situ temperature of about 58C, these variations remain limited and modification of the flow measurement parameters were not considered as necessary.

3.1.2. Pressure The influence of pressure on the ÝS peak shape is shown in Fig. 4a. The peak is delayed Žq3 s., the peak width increases Ž w1r2 from 18 to 22 s. and its height increases Žq0.015 absorbance unit. between 3 and 300 = 10 5 Pa. The peak broadening indicates a larger dispersion of the sample in the carrier possibly due to an increase of fluid viscosity with pressure. Enhanced dispersion should also lead to a decrease in peak height. Contrary to this, the ÝS peak height increases linearly with pressure ŽFig. 5a.. The rea-

Fig. 6. Decrease of the ÝS standard peak height with time at 1650 m depth and 4.58C Žinitial concentration 40 mM..

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sons explaining this apparently complex phenomenon remain to be clarified. A similar peak broadening Ž w 1r2 : q2.5 s. and delaying Ž1–2 s. is observed for N q N ŽFig. 4b.. In this case, the evolution of the peak height is not uniform and seems to be minimum around 150 = 10 5 Pa. However, considering the anomalous peaks and the apparent hysteresis between pressure cycling, this conclusion remains ambiguous ŽFig. 5b.. We experienced that the reduction column is quite sensitive to mechanical perturbation and could be a major source of instability in the N q N measurement method. It is thus possible that mechanical constraints induced by pressure are the cause of the anomalies and hysteresis recorded here. An increase of the peak delay is shown for both ÝS and N q N. The video control enabled to check that the pump speed remained constant during pressure rise. Thus, if a flow rate decrease is assumed to be responsible for this shift, it would be due to a modification of the pump tube properties. The linearity of the response was verified at 300 = 10 5 Pa. The calibration slopes obtained for the

3 and 300 = 10 5 Pa pressure steps Ž12.0 " 0.18C. are presented in Table 3. Both N q N and ÝS slopes exhibit an increase of, respectively, 6.4% and 5.4% between 3 and 300 = 10 5 Pa. These laboratory experiments demonstrated that both temperature and pressure exert a significant influence on the response of the analyzer, which makes in situ calibration imperative. The modification of these two parameters from ambient to in situ conditions was expected to lead to a peak broadening and delaying. Nevertheless, we could reasonably consider that the flow measurement conditions Žpump tube diameters, pump rotation speed, acquisition windows. were compatible with the in situ temperature and pressure. 3.2. In situ analytical performances As expected from laboratory tests, the peak shape evolution during the travel to the bottom is mainly characterized by a broadening and delaying Ž4 s for ÝS, 10 s for N q N.. After reaching the bottom Ž1650 " 50 m depth., the signal were shown to be

Fig. 7. Data series of ÝS and N q N and temperature recorded with ALCHIMIST. The intake probe was placed at four different points within the 0.25 m2 quadrat Ž0:30–1:30. and in a shimmering diffusion zone few meters away Ž1:45–2:15..

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stable within 5 min. The standard deviations Ž s . of peak height obtained from eight successive injections are 0.8% for N q N and 1.1% for ÝS. Detection limit is defined as 3 s blank divided by the calibration slope where s blank is the peak height standard deviation for eight blanks. Detection limits of 0.8 mM for ÝS and 0.5 mM for N q N are obtained. ÝS calibrations performed along 7 h on the bottom exhibited a significant decrease of the peak height with time, whereas the peak width and delay did not change. This suggests a loss of ÝS in the standard due to oxidation by oxygen migrating from ambient seawater through the bag walls. The bag walls are not impermeable to oxygen: in one of these bags, a reduced solution of indigo carmin Ža redox indicator for dissolved oxygen. develops a blue coloration in few minutes. The exponential decrease of peak height ŽFig. 6. is consistent with a pseudo first order kinetic oxidation rate. The corresponding half

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time of about 10 h is much weaker than expected from Millero et al. Ž1987. for ÝS oxidation by oxygen in air saturated seawater free of metals at 58C at pH 8 Žabout 120 h.. Iron contamination from the pipes, which supply seawater on board may be the cause of this instability Ž100 nM of this metal in seawater were shown to enhance the sulfide oxidation rate by a factor of 10; Zhang and Millero, 1994.. Low metal seawater collected from the open ocean surface in clean conditions should prevent this problem. Standard injections performed within 15 min after arrival at the bottom were used for calibration, assuming a concentration of 40 mM for the ÝS standard. No significant variation of the standard peak heights for N q N calibration was observed over the whole dive duration. The slopes of in situ calibration lines are similar to those obtained in the laboratory ŽTable 3.. It can be noticed that the in situ ÝS slope

Fig. 8. Variation of ÝS and N q N concentrations vs. temperature Žerror bars represent the maximum temperature variation over "30 s around the sampling times..

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is slightly higher than that obtained in the pressure tank at 128C, 300 = 10 5 Pa, although an opposite tendency could have been expected from temperature and pressure tests. However, the reproducibility of flow conditions is probably not good enough to precisely relate the in situ slopes with the laboratory results Žin particular, ageing of pump tubes is known to be a main cause of discrepancies.. Nevertheless, this suggests that the error that could be due to ÝS loss before calibration at depth remains weak. The measurement sequence Ždraining–injection– acquisition. lasts 2 min 40 s for simultaneous N q N and ÝS measurement. The maximum measurement

rate is then 22 measurements per hour. As shown in Fig. 7, the evolution of the measured concentrations is well correlated to the temperature variations, provided that a 2-min time shift correction is made. This shift probably corresponds to the time transfer of the sample from the intake probe to the measurement cell. Fig. 7 shows that a steep variation of the ÝS concentration, induced by a displacement of the sample inlet, can be fully discriminated with two Žexceptionally three. successive measurements. Considering the analytical processes involved, ÝS corresponds, in fact, to the concentration of all labile sulfide species that will form H 2 S in the pH 1

Fig. 9. Comparison of discrete sampling data ŽDiva2, Marvel and Pico cruises. and ALCHIMIST results ŽVictor Premiere ` cruise. for the Lucky Strike hydrothermal area: Ža. ÝS concentrations vs. temperature at the Elisabeth site, Žb. N q N concentration vs. temperature at Elisabeth ŽALCHIMIST., Eiffel Tower and Bairro Alto sites Ždiscrete samples, no discrete data available at Elizabeth..

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reagent Ži.e. dissolved species and dissolvable particles not retained on the 10 mM porosity filter.. On the basis of previous discrete sampling data ŽSarradin et al., 1999a., N q N is presumed to be predomi. nantly nitrate in the medium studied ŽNOy 2 - 1 mM .

3.3. Application At first, these trials demonstrated that the ALCHIMIST is able to quantify variation of ÝS and N q N concentrations within a few decimeters ŽFig. 7.. These results enabled to discriminate the ÝS and N q N ranges over the different biological populations within the different parts of the 0.25 m2 quadrat, which could not be done previously using discrete sampling ŽDesbruyeres ` et al., 2000.. This advantage of in situ analysis was already highlighted by Johnson et al. Ž1986a. and Johnson et al. Ž1988b.. It is a satisfying first result in view of the heavy technical and analytical constraints imposed when investigating deep sea vent environment. Compared to discrete sampling, in situ analysis provides spatially and temporally consistent data sets, from which empirical concentration–temperature relationships can be established. The linear relationship between N q N and temperature ŽFig. 8. suggests that temperature is a conservative tracer of the seawaterrfluid mixing for the studied zone and that dilution is the dominant process controlling the concentration of this species ŽJohnson et al., 1988b.. On the contrary, the relationship between ÝS and T is clearly curved ŽFig. 8., exhibiting an exponential-like tendency. If a constant composition of the diffuse fluid source is assumed for the studied area, this suggests that, in addition to fluid dilution, ÝS consumption occurred. Similar ÝS tendencies were displayed by Johnson et al. Ž1986b., Johnson et al. Ž1988b. and Johnson et al. Ž1994. in the environment of hydrothermal organisms of Pacific vents. ÝS concentrations previously obtained by discrete sampling at the same vent site during the Diva2 94 ŽSarradin et al., 1999a., Marvel 97 and Pico 98 cruises ŽDesbruyeres ` et al., 2000. are consistent with the concentration–temperature trends established with the analyzer ŽFig. 9a.. This seems to indicate that the temperature-to-sulfide ratios of diffuse fluids

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flowing over the biological habitats were similar in 1994 and 1998. N q N data from the AElizabethB site were not available but, since N q N is expected to come from seawater, a comparison with data from other Lucky Strike sites does make sense. Despite a larger dispersion, discrete N q N concentrations obtained on the Lucky Strike area are quite close to the linear profile established with the ALCHIMIST ŽFig. 9b..

4. Conclusion The new deep sea chemical analyzer, ALCHIMIST, was successfully deployed in a deep sea hydrothermal environment. The ALCHIMIST provided simultaneous ÝS and N q N calibration and measurement at 1650 m depth, at a rate of 22 analyses per hour, with precision and detection limits which seems convenient for the characterization of microhabitats. Our results confirm the potentialities of in situ analysis for hydrothermal ecosystem studies previously highlighted by Johnson et al. In particular, the ability to characterize spatial gradients and to establish concentration–temperature dependence is of major interest.

Acknowledgements We particularly acknowledge B. Leilde, A. Le Noac’h, E. Menut, R. Merceur, J.Y. Coail, J. Crozon and N. Lanteri for their involvement in the development of the ALCHIMIST and their assistance for the laboratory test and at-sea operation. We are grateful to the NrO Thalassa captain and crew and the ROV Victor team for their helpful collaboration. A.-M. Alayse-Danet was the chief scientist of Victor Premiere ` cruise. We thank D. Dixon, Bruce Shillito and D. Desbruyeres ` for their useful comments and corrections. This work was supported by IFREMER and the EU MAST III AMORES project ŽMAS3-CT950040..

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