Journal of Plankton Research Vol.14 no.2 pp.201-221, 1992

A comparison of in situ and simulated in situ methods for estimatingoceanic primary production

Steven E.Lohrenz, Denis A.Wiesenburg1, Charles R.Rein2, RobertA.Arnone2, Craig D.Taylor3, George A.Knauer and Anthony H.Knap4

Center for Marine Science, University of Southern Mississippi, Stennis SpaceCenter, MS 39529, 1Geochemical and Environmental Research Group, TexasA&M University, College Station, TX 77843, 2Naval Oceanographic andAtmospheric Research Laboratory, Stennis Space Center, MS 39529, 3BiologyDepartment, Woods Hole Oceanographic Institution, Woods Hole, MA 02543,USA and 4Bermuda Biological Station for Research, Inc., 17 Biological Lane,Ferry Reach GE01, Bermuda

Abstract. Primary production data measured by in situ (IS) and 'simulated' in situ (SIS) incubationswere compared. To minimize differences between the two types of incubations, SIS experimentswere conducted in temperature-controlled incubators in which the spectral distribution andirradiance were adjusted to approximate IS conditions. IS available irradiance (/Is) was computedfrom vertical attenuation of integrated surface irradiance. Vertical attenuation was estimated using aspectral irradiance model, validated by measured profiles of the vertical attenuation coefficient. ISincubations were carried out using two methods. The first involved deployment of bottles on adrifting array for whole-day (dawn to dusk) incubations. The second method employed anautonomous submersible incubation device that performed short term (<1 h) incubations at multipledepths. Differences between whole-day IS and SIS incubation estimates were attributed partially todifferences between / ]S and SIS-available irradiance (/Sis)- Photosynthesis-irradiance (P-I)properties of IS and SIS populations from the whole-day incubations were not significantly different.P-I properties of the short-term IS and SIS populations were significantly different, althoughestimates of P 8 (mg C mg Chi"1 rT1) from contemporaneous IS and SIS incubations did not differ by>40%. Integrated water-column primary production (IPP) estimated using P-I models derived fromSIS data were within 15% of IS estimates of IPP.

Introduction

Estimates of primary production are fundamental to investigations of biologicaltransformations of carbon and nitrogen in the upper ocean. The most widelyused approach for estimating primary production involves the introduction of14C-inorganic carbon into an enclosed sample (Steeman Nielsen, 1952).Although various processes have been recognized as potential causes ofuncertainty in 14C-primary-production estimates (e.g. Eppley, 1980; Harris,1980; Peterson, 1980) the technique itself remains generally accepted as a highlysensitive assay of photosynthetic carbon fixation. An issue yet to be resolvedregarding 14C-primary-production incubations is the magnitude of differencesbetween results obtained using in situ incubations from those obtained withshipboard incubators.

Ideally, incubations should be conducted in situ with the minimum possibleperturbation of sample environmental conditions. However, this is not alwayspossible or practical. In situ incubations require remaining on or returning to agiven location, thus monopolizing valuable ship time and limiting the area that

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can be sampled. In addition, time course incubations, which are particularlyimportant for resolving short-term changes in rates, are especially difficult withcommonly employed in situ methods.

Shipboard incubators used to simulate in situ conditions provide a convenientalternative (e.g. Steeman Nielsen, 1958). A variety of investigations hascompared in situ (IS) and 'simulated' in situ (SIS) incubation measurements. Insome cases, results have compared favorably (e.g. Berge, 1958; Head, 1976;Gieskes et al., 1990). However, many studies have reported differences (Jitts,1963; Doty et al., 1965; Jitts et al., 1976; Cadee and Hegeman, 1979; Colijn etal., 1983; Grande et al., 1989).

An alternate approach to SIS methods uses measured solar irradiance,attenuation coefficients, and mathematical descriptions of photosynthesis-irradiance (P-I) relationships to estimate IS primary production (e.g. Fee,1973a,b; Colijn et al., 1983; Cote and Platt, 1984; Harrison et al, 1985; Hermanand Platt, 1986). Data for constructing P-I relationships are generally obtainedby incubating samples in artificial light gradients (e.g. Fee, 1973a,b; Jitts et al.,1976; Harrison et al., 1985). It is also possible to derive 'composite' P-Iparameters from measurements of available irradiance and primary productiondetermined by IS or SIS techniques (e.g. Gallegos and Schiebe, 1986; Hermanand Platt, 1986; Bower et al., 1987; Lohrenz et al., 1990). The P-I modelapproach has clear advantages over conventional IS and SIS methods whenincreased temporal and spatial resolution of primary production is desired.However, as with IS versus SIS comparisons, significant differences betweenmodeled estimates and IS measurements of primary production have beenreported (Jitts et al., 1976; Gargas et al., 1976; Fee, 1978; Fahnenstiel andScavia, 1987).

Lack of agreement of IS measurements with other techniques has beenattributed to errors in matching irradiances (Jitts, 1963), differences in spectraldistribution (Jitts, 1963; Kiefer and Strickland, 1970; Jitts etal., 1976; Fee, 1978;Harrison et al., 1985; Herman and Platt, 1986; Grande et al., 1989), anddifferences in incubation duration (Bower et al., 1987; Fahnenstiel and Scavia,1987). To date, no consensus exists about the magnitude of differences that canbe expected between IS and SIS measurements of primary production.

Cote and Platt (1984) pointed out the utility of the light-saturation curve as anoperational model for quantifying effects of environmental conditions onphytoplankton photosynthesis. We speculated that systematic differencesbetween IS and SIS incubation conditions would be manifest as differences inP-I relationships. Herman and Platt (1986) reported differences between P-Irelationships derived from IS primary production data and SIS data obtainedwith an artificial light incubator. To our knowledge, there have been no otherreported attempts to compare P-I relationships derived from IS and SIS dataobtained in oceanic waters.

In this study, we systematically evaluated the statistical significance ofdifferences between IS and SIS measurements of primary production. Bothshort-term and whole-day incubations were compared. We attempted tominimize differences by controlling incubation temperatures and spectral

202

In situ and simulated in situ primary production

composition of irradiance. P-I relationships determined for data obtained by ISand SIS methods were compared. The utility of using 'composite' P-I modelsconstructed from SIS data to adjust for differences between IS and SISirradiance is illustrated.

Method

Description of shipboard incubators

Our primary design objectives were that the incubation system regulatetemperature, and both irradiance and spectral distribution. The system also hadto withstand exposure on the deck of a ship. Exposed metal surfaces inside theincubators were minimized. Each incubator (61 cm length x 48 width x 38depth) was constructed from 1.27 cm thick clear polycarbonate. Three in-cubators were used, each individually temperature-controlled and light adjustedusing a combination of colored polycarbonate (light blue Acrylite 625-5) andneutral density screening. Sample bottles (0.25 1) were held in 50 cm lengths of10 cm diameter clear polycarbonate tubing. The tubing was wrapped withneutral density screening to further adjust irradiance as required. Cool waterfrom a refrigerated circulator (Neslab Model HX-150 with CP-20 pump orNeslab CRT-75) was continuously circulated through polyethylene cooling coils(1.25 cm i.d., 30 m length) in each incubator. The cooling coils were connectedin series beginning with the coldest incubator. A circulating pump (March ModelLC-2CP-MD) provided temperature equilibration inside each incubator. Inaddition, for each incubator temperature was monitored by a thermistor probeand maintained at a set point using a temperature controller (Omega Model 49).The controllers regulated power to stainless-steel immersion heaters (Ogden750 W) mounted in flow lines with the circulating pumps. The circulating pumpswere run continuously and temperatures inside the incubators were monitoredusing the thermistor/controller readouts calibrated with a mercury thermometer.

Temperature dependence of primary production

During May 1986 aboard the USNS Lynch (cruise 705-86) in the westernMediterranean Sea, an experiment was conducted to evaluate potentialvariations in primary production rates due to deviations from in situ tempera-ture. Samples were collected at station 20 (37°46.94' N, T58.30' E) from 70 mand incubated at both surface (18.0°C) and in situ (13.5°C) temperatures undersimulated in situ irradiance. Procedures for sample collection, incubation andanalysis were described in Lohrenz et al. (1988). The production rate of thesample incubated at surface temperature was 50% higher than that of the sampleincubated at the in situ temperature (Figure 1). These results show thattemperature control can be a critical factor in simulating in situ conditions with adeck incubation system. Even the relatively small difference of 4.5°C was foundto affect measured production rates substantially. Incubator temperatures forother experiments described here were generally maintained to within 1CC of thein situ temperature.

203

S.E.Lohrenzefa/.

•3-

ubo

,2-

o0-

8

18° C A

13.5° C

GMT12 14

Fig. 1. Temperature dependence of photosynthesis measured in a sample collected from 70 m atwestern Mediterranean Sea station. In situ temperature was 13.5°C, while surface temperature was18.0°C. Measurements were corrected for dark uptake.

Irradiance measurements

Measurements of surface irradiance were obtained with a Biospherical In-struments QSR-240 solar reference sensor. Spectral composition of surfaceirradiance was approximated using the clear-sky irradiance model of Bird (1984)as described by Sathyendranath and Platt (1988). To obtain modeled surfaceirradiance of a magnitude comparable to that measured with the QSR-240, wecomputed the sum of direct normal (Eq. 1 in Bird, 1984) and diffuse horizontal(Eq. 11 in Bird, 1984) irradiance. The Bird (1984) model and a subsequentversion (Bird and Riordan, 1986) were designed for terrestrial applications.Differences between aerosol properties and surface-air multiple interactions inmarine and terrestrial environments may have resulted in differences betweenour modeled estimates and actual spectral quality (cf. Gregg and Carder, 1990).In addition, cloud variations would affect spectral composition of surfaceirradiance. Despite these problems, we were able to achieve good agreementbetween computed and measured vertical attenuation (see Modeling in situirradiance). Hence, we concluded that any errors in modeled surface spectraldistributions had a negligible effect on our computations of total IS-availableirradiance.

Modeled estimates of surface irradiance were scaled to measured irradiancewhen available. Surface irradiance measurements were not available for theFebruary, April and June whole-day incubations. In these experiments, surfaceirradiance was estimated by the Bird (1984) model; on cloudy days (Februaryand April) a mean albedo due to clouds of 0.5 was assumed (cf. Platt andSathyendranath, 1988).

204

In situ and simulated in situ primary production

The percentage of surface irradiance in the incubators was determined bymeasurements obtained with a Biospherical Instruments QSL-100. Irradiancemeasurements were obtained at various positions within the tanks and over arepresentative range of solar zenith angles. Mean coefficient of variation ofirradiance as a function of location within the tanks was 22% (range = 0.7-70%). Because of ship motion, it is unlikely that individual bottles within anincubator experienced consistent differences in irradiance. This view wassupported by the low standard errors for replicate bottles (see Table I).

Spectral transmittance comparisons

Spectral transmittance of incubator materials (Figure 2) was determined using aspectral radiometer referenced to surface spectral irradiance (Arnone et al.,1986). The radiometer provided measurements of transmittance at 2 nmintervals from 400 to 700 nm. By varying combinations of neutral-density blackfiberglass screening and Acrylite 625-5, it was possible to approximate ISspectral quantity and quality. To illustrate, the spectral transmittance of naturalsunlight through incubator materials was compared with optical conditions attwo oceanic stations in the western Mediterranean Sea (Figure 2). Measure-ments were made near local noon aboard the USNS Lynch (cruise 705-86)during May 1986. One station was characterized by relatively low chlorophyllconcentrations (<0.2 mg m~3) and high transmittance. The other was charac-terized by higher chlorophyll (0.25-0.4 mg m"3) and lower transmittance (cf.Lohrenz ef a/., 1988).

Modeling in situ irradiance

An important objective of this study was to relate variations in photosyntheticrates to available irradiance. Our approach for estimating IS-available irradiance(/JS) was to adjust integrated surface irradiance to IS levels based on depthprofiles of the vertical attenuation coefficient. Wind conditions and averagesolar zenith angles in our experiments were consistent with surface reflectancelosses of <10% (Kirk, 1983). Therefore, no correction was made for surfacereflectance. To provide information for estimating profiles of the verticalattenuation coefficient, midday profiles of irradiance (Biospherical InstrumentsQSP-200L) were performed on days preceding whole-day incubation com-parisons. The vertical attenuation coefficient, K, was calculated according to thefollowing equation:

K(z) = In [^]/(*2 - z,) (1)

where /(zi) and /(z2) are measured light at depths Z\ and z2 respectively and z isthe depth at the midpoint of the interval. Factors which contributed to variabilityin K(z) within an irradiance profile included ship shadow and surface waves.Additional variation resulted from cloud-related surface light fluctuations duringthe vertical casts. These sources of variability limited the precision of K(z)

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S.E.Lohrenz et al.

60-,

40-

CJGCO

£20-

co

STN 22 21 mIncubator

500 600Wavelength (nm)

700

ou

0)

CJ

GCO£20-enCCO

0400

B STN 41 30 mIncubator

500 600Wavelength (nm)

700

Fig. 2. A comparison of water column and incubator spectral transmittance (% of surface spectralirradiance) at two western Mediterranean Sea stations. (A) Spectral transmittance determined at30 m depth at station 22 (37°11.04' N, 2°0.49' E) in relationship to transmittance of a 0.318 cm layerof Acrylite 625-5 plus 50% reduction in light with neutral density black fiberglass screening. Theaverage percent deviation between IS and SIS spectral irradiance was 23%, substantially lower than82% if neutral density screening had been used alone. (B) Spectral transmittance determined at 22 mdepth at station 29 (37°2.78' N, 3°0.45' E) in relationship to transmittance of a 0.318 cm layer ofAcrylite 625-5 plus a 90% reduction in irradiance with neutral density black fiberglass screening. Theaverage percent deviation between IS and SIS spectral irradiance was 30%, compared with 94% ifneutral density screening had been used alone.

206

In situ and simulated in situ primary production

estimates derived from measured submarine irradiance (Figure 3). This lack ofprecision made it difficult to match IS and SIS irradiance.

To provide more precise estimates of water column vertical attenuation, weused a spectral irradiance attenuation model. The model served two purposes. Itgenerated a smoothed profile of K(z) and provided extrapolated estimateswhere measured data were either not available or in error (e.g. due to shipshadow). The model was similar to that described by Sathyendranath and Platt(1988) with the exceptions that we omitted angular distribution terms, and weused the expression of Prieur and Sathyendranath (1981) to calculate chlorophyllabsorption at 440 nm in place of the expression given by Sathyendranath andPlatt (1988). We found that these modifications improved the fit of the model to

- 1

Attenuation (m )0.1 0.2 0.0 0.1 0.2

50

Q 100

MAR - MAY -

0

25

50

75

100

JUN - JUL

Fig. 3. Measured and modeled vertical profiles of attenuation coefficient, K(z), for whole-dayincubation comparisons. Measured data (solid line) were calculated from vertical profiles ofirradiance using equation (1). Modeled data (open circles) were estimated using proceduresdescribed in the text. Extinction due to dissolved substances was taken as zero for January-March.For April-July, an extinction coefficient for dissolved substances of 0.01 nT1 at 440 nm was used.

207

S.E.Lohrenzrfo/.

our data. Further improvements were also made by adjustment of the term forextinction due to dissolved substances (Figure 3). We used the model, adjustedfor each vertical profile, to compute attenuation of integrated surface irradiance,thereby yielding estimates of 7IS during incubations. This approach was also usedfor computing 7IS for the short-term incubations (Figure 4). In the latter case,irradiance data obtained with a cosine-corrected sensor mounted on the ISinstrumentation package (Taylor and Doherty, 1990) were used to determineactual K(z) by equation (1).

Whole-day incubations

Whole-day (dawn to dusk) IS and SIS incubations were conducted at the GlobalOcean Flux Study Bermuda Atlantic Time-series Study site (31°50' N,64°10' W). Experiments were performed using clean techniques (Fitzwater etal., 1982) aboard the R/V Weatherbird during January-July 1989. Three hoursbefore dawn, seawater samples were obtained using Glo-Flo bottles deployedon a Kevlar line. Depths were sampled to match incubator irradiance, generallyfrom 85 to 0.5% of surface irradiance. Acid-cleaned (0.5 N Baker Instra-Analyzed HC1) 0.25 1 polycarbonate bottles were filled directly from Glo-Flosampling bottles under dim incandescent light. An 80 u-Ci ml"1 working solutionof 14C was prepared by dilution of a 1 mCi ml"1 [14C]HCO3~ stock (ResearchProducts International) with 0.002 M Na2CO3 (Aldrich) prepared with Milli-Qwater. Under low light conditions, 0.25 ml of the 14C working solution (20 u.Ci)

0.0

Attenuation (m )0.1 0.2

100

Fig. 4. Measured and modeled vertical profiles of attenuation coefficient, K(z), for short-termincubation comparisons at station 34 (CH08-88). In situ irradiance measurements were used tocalculate water-column attenuation (solid squares) using equation (1). Modeled data (open circles)were estimated using procedures described in the text. An extinction coefficient for dissolvedsubstances of 0.005 m~* at 440 nm was used.

208

In situ and simulated in situ primary production

was added to each bottle using a cleaned polypropylene pipet tip. Measurementsat each depth were made in triplicate and dark uptake was subtracted; darkcorrections were generally <15% of near surface rates. Total added 14C activitywas assayed by placing a 0.25 ml aliquot in a 20 ml glass scintillation vialcontaining 0.25 ml ethanolamine (Aldrich). Ten milliliters of liquid scintillationcocktail (Aquasol, New England Nuclear) plus 2.5 ml Milli-Q water were thenadded to the vial before liquid scintillation counting.

Approximately 1 h before sunrise, IS incubation bottles were suspended atthe appropriate depths beneath a spar equipped with strobe flash and VHF radiobeacon. Simultaneously, shipboard incubation bottles were placed in incubatorsadjusted to appropriate temperature and light conditions. Approximately 0.5 hafter sunset, the productivity array was recovered and sample bottles wereremoved from the incubators. A 50 ml aliquot from each productivity bottle wasfiltered onto a 25 mm Whatman GF/F glass fiber filter at vacuum levels of^100 mm Hg. All sample handling was carried out in low light. The unrinsedfilter was placed in a 20 ml glass scintillation vial, covered with 0.25 ml 0.5 NHC1, and dried in a fume hood. Ten milliliters of liquid scintillation cocktail wereadded to the dried filters, and the filters allowed to clear (usually 24 h) beforeliquid scintillation counting. Samples were counted using a Packard Tri-Carb2000CA Liquid Scintillation Analyzer. An external gamma source was used toassess quenching. A dissolved inorganic carbon concentration of 25 mgC m~3

was assumed in calculations of 14C specific activity for inorganic carbon fixationrate estimates.

Samples (2.75 1) for chlorophyll a analysis were filtered on 25 mm GF/F filtersat vacuum levels of ^100 mm Hg and stored at — 20°C until analysis (usuallywithin 3 days). Filters were placed in 15 ml of 90% acetone and extracted indarkness at 4°C for 12 h. Samples were then brought to room temperature,centrifuged for 10 min at 1800 g and fluorescence in the extract was read on aPerkin Elmer 650-10S fluorescence spectrophotometer (excitation 443 nm,emission 669 nm). Concentrations were estimated based on calibrations usingpure chlorophyll a standards (Sigma).

Short-term incubations

A comparison of short-term IS and SIS primary production measurements wasmade at two stations during October 1988 aboard the R/V Cape Hatteras (cruise08-88). Station 33 (37°57.43' N, 7117.25' W) was in Slope Water immediatelynorth of the Gulf Stream and station 34 (37°58.25' N, 70°45.29' W) was locatedin the Stream. IS production was measured using an autonomous submersibleincubation device (SID; Taylor and Doherty, 1990). The instrument wassuspended on a line from floats that were tethered to the ship. The ship wasallowed to drift during the deployment. The device performed three consecutiveincubations. At station 33, the device was held at 15 m for the three incubations.At station 34, incubations were conducted at 15, 45 and 75 m. The depth of theSID was adjusted between incubations. Pressure data from sensors mounted onthe instrument were continuously recorded during deployment. For each

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incubation, a sample was introduced into a 0.4 1 glass (interior siliconized forinertness) chamber previously cleaned with 1.5 N HC1 (Baker Instra-analyzed)and Milli-Q water. During the aquisition of a sample, 0.6 mCi (final activity1.5 mCi I"1) [14C]HCO3~ was simultaneously introduced and mixed. Sub-samples (final volume 61 ml) were expelled from the chamber through a rotarysample distribution valve into plastic syringes each containing 2.5 ml 9 N H2SO4

as fixative. Subsamples were collected at time zero, 20 and 40 min for eachincubation. In addition, an unfixed sample was taken for determination of total14C activity and chlorophyll a. Total photosynthetic fixation of 14C in theacidified samples was estimated according to techniques of Cuhel et al. (1981).Briefly, the acidified sample was filtered through a 25 mm Whatman GF/F glassfiber filter and the filtrate sparged with air for 15 min to eliminate inorganic 14C.14C activity on the filter was counted as previously described. Replicate 5 mlaliquots of the filtrate, which contained acid-released low-molecular-weightmetabolites, were mixed with 10 ml scintillation cocktail (Aquasol-II) andcounted as previously described. Total 14C fixation was given as the sum ofparticulate + filtrate fractions.

Water samples for short-term SIS measurements were collected from sixdepths (including those sampled for IS measurements) using acid-cleanedNiskin bottles mounted on a Rosette (General Oceanics). Silicon rubber wassubstituted for all rubber parts of the Niskin bottles that came into contact withthe water sample. For SIS incubations, 1 1 polycarbonate bottles were filled fromthe Niskin bottles and inoculated with 0.2 mCi (final activity 0.2 mCi I"1)[14C]HCO3~ (Research Products International). The contents of each 1 1 bottlewere dispensed into four 0.25 1 polycarbonate bottles, one of which wasanalyzed immediately for a zero-time sample and the remaining bottles placed inthe incubator adjusted to approximate IS conditions. One hydrocast was madeat each station preceding deployment of the SID. SIS incubation bottles weresampled at intervals roughly corresponding to each consecutive IS incubation.The entire contents of the 0.25 1 bottles were filtered onto 25 mm Whatman GF/F filters at =£100 mm Hg vacuum. The filters were placed in 20 ml glassscintillation vials and covered with 0.5 ml 1 M acetic acid. Filters were dried in afume hood, moistened with 0.5 ml Milli-Q water, and 10 ml scintillation cocktail(Scintiverse II) added. Total 14C activity was determined by placing a 50 u.1aliquot in a vial containing 50 u.1 phenethylamine (Aldrich), 50 |il 1 M Tris-HC1, 100 u,l Milli-Q water, and 4 ml scintillation cocktail. Liquid scintillationanalysis was performed as previously described.

Samples for dissolved inorganic carbon concentrations were collected in 50 mlglass serum bottles, sealed with butyl rubber serum stoppers and preserved byaddition of sodium azide to a final concentration of 0.001 M. For inorganiccarbon specific activity estimates, dissolved inorganic carbon concentrationswere determined by acid-volatilization and analysis with a Horiba model PIR-2000 IR carbon analyzer.

Standard fluorometric assays of photosynthetic pigments were performed(Holm-Hansen et al., 1965). A problem of unknown origin with chlorophyllmeasurements developed midway through cruise 08-88 leading to unusually low

210

In situ and simulated in situ primary production

acid ratios and low chlorophyll a readings. Comparisons with concurrent HPLCanalyses of chlorophyll a (Mantoura and Llewellyn, 1983) led us to conclude thatthe offset was systematic and internally consistent (S.E.Lohrenz, unpublished).The low values were corrected based on HPLC analyses to be consistent with theinitial fluorometer calibration. All chlorophyll measurements used for short-term incubation comparisons in this study were treated in an identical fashion sothat relative differences were unaffected.

Results

Comparison of IS and SIS primary production estimates

Although whole-day IS and SIS experiments often yielded significantly differentresults (Table I), there was no consistent trend. We speculated that differences

Table I. Comparison of whole-day (dawn to dusk) IS and SIS measurements of primary production

Date(1989)

Jan. 27

Feb. 19

Mar. 26

Apr. 17

May 15

June 22

Depth(m)

3.217.741.161.781.1

102.1

2.019.146.6

4.025.053.0

5.032.064.0

3.314.324.839.067.397.0

3.016.024.039.053.066.0

IS(mg C m"3 day"1)

lost3.78 (0.20)3.50 (0.11)2.03 (0.08)0.76 (0.03)0.49 (0.05)

28.3 (0.9)b

8.10 (1.78)1.85 (0.08)

4.02 (0.14)3.81 (0.05)2.96 (0.11)

3.50 (0.06)2.49 (0.12)2.14 (0.04)

2.42 (0.84)b

3.39 (0.26)0.86 (0.10)2.17 (0.38)1.82 (0.01)0.68 (0.15)

4.11 (0.17)b

4.39 (0.30)4.27 (0.29)1.85 (0.48)4.45 (0.22)3.92 (0.03)

SIS

4.21 (0.26)4.53(0.16)4.57(0.16)3.62 (0.12)2.04 (0.10)1.20 (0.07)

24.0 (0.6)11.4 (0.2)4.49 (0.23)

2.32 (0.10)2.06 (0.07)1.02 (0.05)

2.17 (0.17)1.85 (0.05)0.95 (0.08)

3.98 (0.34)2.97 (0.33)1.90 (0.28)1.69 (0.06)1.06 (0.16)0.25 (0.08)

5.13 (0.20)4.00 (0.27)3.54 (0.13)1.27 (0.06)3.00(0.13)2.12(0.15)

Student's t

_-2.92-5.51

-11.0-12.3-8.25

4.20-1.84

-10.8

9.8820.316.0

7.374.92

13.3

-2.041.00

-3.491.254.742.52

-3.550.972.301.205.67

11.8

_0.0430.0050.0000.0000.001

0.0250.140°0.000

0.0010.0000.000

0.0020.0080.000

0.134c

0.374c

0.0250.279c

0.0090.065°

0.0380.387c

0.083c

0.296c

0.0050.000

Measurements are means of triplicate samples (except where indicated) corrected for dark uptake.Values in parentheses are standard errors."P = probability of a numerically larger value of |r|.bOnly duplicate samples were available.cDifference between treatments was not significant at the 95% confidence level.

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between IS and SIS treatments partially resulted from unintentional differencesbetween 7IS and SIS-available irradiance (/sis)- Indeed, differences wereapparent when /IS for whole-day incubations was compared to IS1S (Table II).Estimates of IS and SIS water-column-integrated primary production, calculatedby trapezoidal integration of measurements from six depths, were alsosignificantly different in two out of three experiments (Table III). Again, nosystematic trend was apparent as SIS measurements both over- and under-estimated IS results.

Table II. Comparison of available irradiance during whole-day (dawn to dusk) IS and SISincubations

Date Depth IS SIS(1989) (m) (E m"2 day"1)

Jan. 27 surface 34.0 34.027.313.35.542.961.390.653

Feb. 19 surface" 27.2 27.211.75.171.16

Mar. 26 surface 67.0 67.026.311.02.76

Apr. 17 surface" 36.0 36.015.56.841.54

May 15 surface 35.9 35.922.612.07.433.490.7720.170

June 22 surface" 85.8 85.860.328.617.18.063.791.78

"Measured surface irradiance not available. Cloud albedo of 0.5 used in determining modeledsurface irradiance."Measured surface irradiance not available. Clear sky conditions used in determining modeledsurface irradiance.

212

surface3.2

17.741.161.781.1

102.1

surface"2.0

19.146.6

surface4.0

25.053.0

surface"5.0

32.064.0

surface3.3

14.324.839.067.397.0

surface"3.0

16.024.039.053.066.0

34.020.07.492.761.290.6520.318

27.218.84.951.02

67.035.811.84.31

36.018.45.021.53

35.921.710.57.763.991.420.328

85.848.723.417.210.66.283.86

In situ and simulated in situ primary production

Short-term SIS estimates of primary production were generally higher thanthe instrument-derived IS estimates (Table IV). However, there was substantialtime-course variability and differences between incubation time and durationmay have contributed to differences between IS and SIS rates. Whencontemporaneous IS and SIS rates, expressed as chlorophyll a-specific primaryproduction (P8, mg C mg Chi"1 h"1), were compared, differences were <40%.

Photosynthesis-irradiance P-I relationships

We compared P-I relationships determined from IS and SIS data to evaluatewhether differences in photoadaptive physiology contributed to observeddifferences between IS and SIS treatments. Values of PB were determined forwhole-day SIS (Pfis) a nd IS (Pfs) incubations. A regression of ln(P^) versusln(/IS) was not significantly different from that of ln(P|IS) versus ln(/SiS) for allwhole-day incubation data (Figure 5A). Hence, there was no evidence ofconsistent differences between photosynthetic properties of IS and SIS popu-lations as a result of differences in incubation conditions. In contrast to whole-day incubations, there was evidence of differences between photosyntheticproperties of the short-term IS and SIS populations (Figure 5B). The interceptsof the ln(Pfs) versus ln(/IS) and ln(Pfis) versus ln(/SIS) regressions weresignificantly different (P < 0.05). This was a reflection of higher SIS ratesmeasured in near-surface waters (Table IV).

To compare P-I relationships of IS and SIS data from specific seasons, we fitSIS data to the following expression (cf. Webb et ai, 1974; Platt et al., 1980):

- exp(- - ^ ) \ (2)' m a x

Table in . Comparison of IPP (mg C m~2 day"1) derived from IS and SIS incubations

Date Treatment IPP Student's t" Pb

Jan. 89

May 89

June 89

IPP was calculated by trapezoidal integration of estimates at discrete depths given in Table I. Valuesin parentheses are propagated standard errors.'Represents value for comparison of the IS estimate with either the SIS or SIS model estimate ofintegrated production. Total degrees of freedom for statistical comparison of IS versus SIS estimateswas 4. Total degrees of freedom for comparison of IS versus SIS model estimates was 12 in Januaryand 10 in May and June.bP = probability of a numerically larger value of \t\.'Difference between treatments was not significant at the 95% confidence level.

213

ISSISSIS Model

ISSISSIS Model

ISSISSIS Model

249 (5)356 (4)275 (4)

176 (9)137 (5)169 (6)

246(6)204(3)209(6)

-16.7-2.87

3.790.54

6.262.95

0.0000.012

0.0000.598

0.0030.012

S.E.Lohrenze/a/.

Table IV. Comparison of short-term IS and SIS measurements of primary production (PP, mg C m 'h"1) and chlorophyll a-specific production (/*, mg C mg Chi"1 h"1)

Depth(m)

Station 3315

15

15

Station 3414

42

71

GMT

13.05-13.3013.30-13.55

13.00-14.00

14.20-14.4514.45-15.10

14.00-15.00

15.25-15.5015.50-16.15

15.00-16.00

19.30-19.4519.45-20.00

19.30-20.1020.10-20.5020.50-21.30

20.20-20.4020.40-21.00

19.30-20.1020.10-20.5020.50-21.30

21.10-21.3021.30-21.50

19.30-20.1020.10-20.5020.50-21.30

ISPP

1.41.4

2.32.2

1.81.4

0.730.82

0.350.28

0.170.17

Chi"

0.36

0.36

0.33

0.20

0.25

0.25

P°

3.93.9

6.56.1

5.44.1

3.64.1

1.41.1

0.680.70

SISPP

2.1

2.7

2.4

0.960.791.0

0.620.420.18

0.430.280.23

Chi"

0.43

0.20

0.22

0.24

pB

4.9

6.3

5.9

4.84.05.0

2.81.90.77

1.81.20.93

Measurements were corrected for zero time activities." Chlorophyll a concentrations (mg Chi irT3) were determined in unfixed subsamples collectedduring each IS incubation series.b Chlorophyll a concentrations were determined in hydrocast subsamples. Constant values wereassumed for a given depth throughout an SIS incubation series.

where PB (z) is the chlorophyll a-normalized production rate at depth z, P^M isthe production rate at optimal irradiance, aB is the initial slope of the P-I curve,and /(z) is irradiance. This equation can be thought of as a Poisson model of therelationship between the instantaneous light-limited photosynthetic rate andirradiance (Dubinsky et al., 1986; Peterson et al., 1987; Sakshaug et al., 1989;Cullen, 1990). Our data did not justify inclusion of terms for photo-inhibition,although the expression can be adapted to account for this (Platt et al., 1980).

214

In situ and simulated in situ primary production

7 100

u00

1 0 •

• A

/0

1

1 ' .••• V *

<*> •

0

0

o in situ 1• Incubator r

i i i

0.1 10 100-2 -1

I r radiance ( E m d )

0.01

Irradiance (Em

Fig. 5. Comparison of ln(PB) versus ln(/) regressions of IS and SIS data. Variables were transformedto their natural logarithms to normalize variances over the range of the regressions. (A) Data werefrom whole-day (dawn to dusk) IS and SIS experiments conducted from January to July 1989 at theBermuda Atlantic Time Series Site. The dotted line represents a model II linear regression(Bartlett's three group method; Sokal and Rohlf, 1969) described by the equation: ln(Pre) = 0.77(95% confidence limits 0.64-0.92) x ln(/IS) + 2.3 (2.0-2.4), r2 = 0.793, n = 41. The solid linerepresents the equation: ln(Pf,s) = 0.61 (0.48-0.74) x ln(/SIS) + 2.4 (2.2-2.6), r2 = 0.784, n = 35.(B) Data were from short-term IS and SIS experiments conducted at stations 33 and 34 (R/V CapeHatteras cruise 08-88). The dotted line represents a model II linear regression described by theequation: ln(Pfs) = 0.62 (95% confidence limits 0.50-0.86) x ln(/,s) + 1.20 (1.16-1.28), r2 =0.914, n = 12. The solid line represents the equation: l n ^ s ) = 0.68 (0.57-0.78) x ln(/SIS) + 153(1.43-1.64), i2 = 0.921, n = 19.

Photosynthetic parameters, P^ax and aB, were derived for whole-day SIS datacollected in January and May-June (Figure 6). PB and irradiance data werefitted to the model using non-linear regression techniques (Systat). A P-I curvederived for the whole water column in this manner was assumed to represent a'composite' of adapted rates (e.g. Cullen, 1990). The values of aB and PB

ax

showed distinct seasonal differences (Figure 6). This apparently reflectedphysiological differences between winter and summer photosynthetic popu-

215

S.E.Lohrenzefo/.

60

40

20

n

-f'1

-

January

10 20 30 40

—2 ~ 1I r r a d i a n c e ( E m d )

Fig. 6. P-I relationships for the whole-day SIS experiments conducted in January and May-June atthe Bermuda Atlantic Time Series Site. Curves represent nonlinear fit of equation (2) to SISincubation data (closed circles). Data obtained from IS experiments (open circles) are shown forcomparison. P-I parameters derived for January were: / * „ = 48.9 mg C mg Chi"1 d"1 (95%confidence limits 45.0-52.9), aB = 21.1 mg C mg ChP1 (E m"2)"1 (16.7-25.4). For May-Juneparameters were: /* a x = 196 (138-254), a* = 6.67 (4.93-8.40).

lations. The IS incubation data were generally consistent with the P-Irelationships derived for SIS incubation data (Figure 6). No attempt was madeto derive P-I parameters for the IS incubation data because of the small samplesize (n = 5) in January and scatter in May-June.

We used the P-I relationships derived from the whole-day SIS incubationdata to estimate water-column-integrated primary production (IPP) correspond-ing to IS irradiance. Error analysis revealed that, in two out of threecomparisons, differences between the SIS model and IS estimates of IPP were

216

In situ and simulated in situ primary production

statistically significant (Table III). Despite statistical significance, the differ-ences between these estimates did not exceed 15%.

1 Discussion

Earliest efforts to substitute SIS incubations for IS methods of estimatingprimary production recognized incubator irradiance and spectral distribution ascritical factors (Steeman Nielsen, 1958). In addition to these factors, ourmethods emphasized control of temperature in SIS incubations (e.g. Figure 1).The potential for short-term temperature-dependent responses of productionrates has been previously shown in laboratory cultures (Morris and Glover,1974), and in tropical, arctic and antarctic phytoplankton populations (Neoriand Holm-Hansen, 1982; Smith and Platt, 1985; Tilzer et al., 1986). Thus it is notsurprising that we found our deck incubation measurements of primaryproduction to be temperature dependent (Figure 1). Our results highlight theinadequacy of using surface seawater for temperature control of deep sampleswhen temperature varies significantly within the photic zone.

In our study, we determined that there were frequently differences inavailable irradiance between IS and SIS incubations (Table II). Thesedifferences were apparently a significant factor in differences between IS andSIS incubation measurements of primary production (Tables I and IV). Some ofthe differences between /IS and / S j S could be attributed to difficulties inobtaining a priori accurate assessments of /IS (e.g. Figure 3). Comparison ofwhole-day IS and SIS P-I properties (Figures 5A and 6) revealed no significantdifferences. These results support the view that the spectral composition of /IS

was adequately approximated by our blue acrylic light filters. Such findings areconsistent with those of Gieskes et al. (1990) who reported good agreementbetween IS and SIS production estimates when appropriate blue-light filterswere used for SIS incubations. Herman and Platt (1986) found that the initialslope of the P-I curve, aB, was consistently higher for IS data compared withSIS data. Their results may have been a consequence of lower photosyntheticefficiency associated with the artificial light source used for SIS incubations (cf.Harrison et al., 1985).

In contrast to our findings for the whole-day incubation comparisons, we didfind differences between photosynthetic properties of short-term IS and SISpopulations. Although there was general agreement between the short-term P-Irelationships under low light conditions (Figure 5B), the relationships divergedat higher irradiance. This may have occurred partially as a result of dielvariations in P-I properties combined with the fact that IS and SIS samplingperiods were not identical (Table IV; cf. Bower et al., 1987). Jitts et al. (1976)attributed differences between IS and SIS treatments to differences in UVradiation exposure. Suppression of radiocarbon estimates of primary pro-ductivity by UV radiation of wavelengths 280-320 nm (UV-B) has beendemonstrated (e.g. Smith et al., 1980). Significant penetration of UV-B to adepth of 15 m may occur in clear ocean waters such as that of our station 34,although the glass IS incubation chamber would further reduce UV exposure (cf.

217

S.E.Lohrenzeto/.

Smith and Baker, 1981). In comparison, UV radiation of wavelengths <345 nmwould have been absorbed by the acrylic SIS incubators. That we did not seesimilar differences between whole-day IS and SIS treatments suggests theproblem was specific to the short-term incubations. Perhaps our short-termincubations were conducted at times of the day when sensitivity to UVsuppression of photosynthesis was high. This requires further investigation.

An alternative explanation for differences between near-surface short-term ISand SIS measurements was that SIS-available irradiance was utilized moreefficiently than IS irradiance. This situation would have been the inverse of onesuggested by Harrison et al. (1985). Their analysis indicated that the higherproportion of red light used for an artificial light P-I incubator would have led tolower estimates of production at depths where the IS irradiance field waspredominately blue. Selected comparisons of near-surface samples incubatedunder similar irradiance conditions using either neutral density or blue acryliclight filters revealed no consistent differences (S.E.Lohrenz, unpublished).Therefore we do not believe spectral dependency was an explanation for thelower near-surface rates we observed in the short-term IS incubations.

By fitting our whole-day SIS incubation data to a P-I model (Figure 6), wewere able to adjust rates to correspond to IS irradiance. Resulting estimates ofIPP were within 15% of the IS values (Table III). Stochastic variation in IPPassociated with independent profiles can often be larger than the analytical errorterms in Table III. Thus an error margin of 15% may be acceptable in manycases. These results illustrate a possible means of compensating for oftenunavoidable differences between /IS and 7SIS, provided that relative differencesare comparable to those we encountered (Table II). Jitts (1963) dealt with theproblem of matching 7iS and 7SIS using a 'balance-by-depth' method, by whichsampling depths were chosen by matching a submerged photometer with anidentical sensor placed in the incubator. However, in situ irradiance attenuationproperties may vary over the course of an incubation period. In addition, it is notalways feasible nor desirable to choose depths limited to those predetermined byincubator conditions. We suggest that 'composite' water column P-I relation-ships derived from SIS data can be used to estimate rates of IPP which arerepresentative of IS data. Our approach showed how P-I parameterization ofwater column production data can highlight changes in adapted photosyntheticresponses throughout the photic zone (Figure 6). Furthermore, the P-I modelscan be used for spatial and temporal extrapolations of primary production on thebasis of additional measurements of irradiance and chlorophyll profiles (cf.Harrison etal., 1985; Herman and Platt, 1986). We acknowledge, however, thatresults from this approach must be interpreted with caution. The 'composite' P-I model does not provide information about depth-dependent or diel variationsin P-I properties. If such variations are substantial, then large differencesbetween 7IS and 7SIS could result in errors in SIS estimates of IPP. An idealapproach for estimating IPP would probably involve a combination of IS, SISand P-I techniques, as well as alternative methods not discussed here.

Our results provide evidence that estimates of IPP representative of IS datacan be obtained from SIS incubations. Further evaluation of factors such as

218

In situ and simulated in situ primary production

spectral dependency and UV suppression of photosynthesis may lead to betteragreement between IS and SIS methods. Because of reduced complexity, ISincubations may be preferable given adequate availability of shiptime, and instudies where broad areal coverage is not an objective. However, in view ofincreased interest in interdisciplinary research programs requiring large-scalesurveys of primary production (e.g. US Joint Global Ocean Flux Study), it islikely that SIS techniques will continue to be important.

Acknowledgements

This work is dedicated to the memory of Charles R.Rein, whose ingenuity andenthusiasm were essential ingredients. We are grateful for the expert technicalassistance of M.Tuel, R.L.Dow, R.J.Johnson and K.Gundersen. We also thankthe captains and crews of the USNS Lynch, the R/V Cape Hatter as and the R/VWeatherbird I. We acknowledge the assistance of D.C.Young in the fabricationof the incubators and J.Montalvo for design input. An anonymous reviewerprovided useful comments. This work was supported by the Office of NavalResearch (Program Element 61153N through the Naval Ocean Research andDevelopment Activity Defense Research Sciences Program and OceanicBiology contract N00014-88-K-0155) and by the National Science Foundation(OCE-8801089, subcontract). The SID component of this work was supportedby funds from the University of Southern Mississippi ONR subcontract N00014-85-K-0155, National Science Foundation grant OCE87-08958 and the WoodsHole Oceanographic Institution (contribution no. 7320). This is contribution no.USM/CMS 118 from the University of Southern Mississippi Center for MarineScience and no. 1295 from the Bermuda Biological Station for Research, Inc.

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Received on October 22, 1990; accepted on July 8, 1991

221