2.0 Definition
2.1 Microzooplankton are defined, following Dussart (1963), as phagotrophic organisms that are < 200 mm in length. For simplicity’s sake, this encompasses the nanozooplankton (2-20 mm) of Sieburth et al. (1978).

2.2 Microzooplankton herbivory is defined as the rate of grazing of phytoplankton organic carbon by microzooplankton per unit volume of seawater. The units of this are mg C liter ^-1 day ^-1 .

3.0 Principle
Although several approaches for quantifying microzooplankton herbivory are possible and these have been summarized in the earlier JGOFS Report on Core Measurement Protocols (SCOR, 1989), one of these has been used routinely in JGOFS. This is the “dilution approach” of Landry & Hassett (1982). The dilution approach protocol is based on the experimental determination of phytoplankton growth in a dilution series. The dilution series is made up by combining the natural microbial community with seawater that has
been filtered free of microbial components. The theoretical and practical considerations of this technique are fully described in Landry and Hassett (1982) and updated in Landry (1993). Essentially, phytoplankton growth is assumed to be density independent with specific growth rates that are constant for all dilution conditions. In situations where this is an issue, controls should be run with amended nutrient concentrations. Per capita
clearance rates of microzooplankton are assumed to be constant among the dilution treatments, leading to proportionately higher phytoplankton mortality with greater concentrations of microzooplankton. Consequently there is a progressive uncoupling with dilution between phytoplankton growth and mortality due to grazing. It is further assumed that phytoplankton growth and grazing mortality are appropriately represented by
exponential rates.

The protocol is therefore based on quantifying the specific growth rates of phytoplankton in dilutions of different known concentrations. Phytoplankton growth rates are determined from time course incubations.
Microzooplankton biomass measurements (see Chapter 22) of the experimental water should be made in support of the experimental work.

4.0 Apparatus
4.1 Polycarbonate (or Teflon) experimental ware including incubation bottles. The latter should be 2 to 5 liter capacity scaled to the concentration of phytoplankton in the experimental water and the method used for its determination.

4.2 Free floating rigs for in-situ incubation or an incubator for simulated in-situ incubations.

4.3 Nitex 200 mm gauze and large volume filtration system for the production of seawater free from pigment-containing particles.

4.4 High sensitivity fluorometer, spectrophotometer or HPLC system plus ancillary equipment for quantification of phytoplankton chlorophyll (see chapters 13 and 14). Access to suitable room to minimize temperature changes to experimental water while setting up dilutions.

5.0 Reagents
Acetone (90%) and HCl (10%) for extraction and quantification of chlorophyll a and other pigments, if required.

6.0 Sampling
6.1 Experimental samples should be taken using clean acid-rinsed Go-Flo or Niskin water bottles from the depths of interest by CTD/rosette or conventional profiling. Experimental water should be obtained from a minimum of two depths; one of these should target depth of highest grazing—the depth of highest phytoplankton production is a good marker. Other sampling depths should be associated with any relevant
oceanographic markers (e.g. subsurface chlorophyll maximum, pycnocline, etc.).

6.2 On board ship, samples must be treated carefully as many protozoa are delicate. Samples to be siphoned from the water bottle directly into polycarbonate bottles prior to addition of particle-free dilution water.
6.3 Sampling for microzooplankton herbivory experiments should coincide with obtaining samples for primary production and, if possible, macrozooplankton herbivory and bacterial production. To coincide with primary production, water sampling would typically occur prior to local dawn.

7.0 Procedures
7.1 Before starting experiments, ensure experimental bottles are marked up for appropriate dilution (e.g. 40% concentration should be marked externally with water proof marker to hold 40% of its full volume) and that all experimental polycarbonate ware has been acid cleaned and then distilled water rinsed. Typical concentrations used in a dilution series should be 100%, 70%, 40% and 10% of ambient concentration with
triplicate bottles incubated at each concentration. A larger number of dilutions would be preferable for greater precision.

7.2 Water must be collected with appropriate clean water bottle techniques, as described above. Filter as much water as required (approximately half the overall water) free of phytoplankton using 0.2 mm porosity Gelman Suporcap filter capsules. If these capsules are unavailable, use Triton-free acid washed methyl cellulose filters. Filters must have been acid (10% HCl) washed and rinsed with Milli-Q water before use.
Discard the first few liters of filtered seawater and retain the remainder. Add filtered seawater to bottles as appropriate.

7.3 Carefully, but rapidly, siphon experimental water through 200 mm gauze into experimental bottles and fill to the top.

7.4 Store experimental bottles temporarily in dim light at close to ambient in situ temperature. Mix bottles gently by inverting them slowly. Take subsamples from each bottle or sacrifice duplicate bottle for phytoplankton pigments and filter onto 0.2 mm Nuclepore filter. Store filter deep frozen until required for analysis. Sub-samples should also be taken from each bottle for determination of microzooplankton at
beginning and end of experiment which should last 24 hours.

7.5 Under extreme conditions of oligotrophy, when phytoplankton growth may be nutrient limited, supplementary nutrients should be added to minimize this effect. This is discussed by Landry (1993).

7.6 Experimental dilution bottles should be incubated in situ on a free-floating rig in parallel with the conventional primary production measurements. This ensures direct intercomparisons are valid. If this is impossible, incubation under simulated in situ conditions onboard ship may be carried out either using an illuminated incubator or deck incubation equipped with appropriate light attenuation filters.

7.7 Phytoplankton should be quantified through measurement of chlorophyll or other photopigments as soon after sampling as possible, since pigments degrade rapidly. Photopigments may be analysed fluorometrically (Yentsch & Menzel, 1963), by spectrophotometry (Jeffrey & Humphrey, 1975) or by HPLC (Mantoura & Llewel-lyn, 1983) with appropriate modifications recommended in the JGOFS Protocol. HPLC analysis is the preferable approach since it allows the quantification of taxon-specific photopigments. The coupling of HPLC analysis of photopigments to the dilution technique allows considerably greater interpretation of microbial dynamics (Burkill et al., 1986: Verity et al., 1993). However, HPLC is much more specialized and resource-demanding than conventional fluorometry.

7.8 An alternative and perfectly acceptable approach for the quantification of phytoplankton is via conventional microscopical analysis of phytoplankton cells in the experimental bottles. This approach will yield information on the dynamics of individual phytoplankton taxa.

8.0 Calculation and expression of results.
An example of the complete computation is shown below:
8.1 Dilution experiment No 1 results: 190 JGOFS Protocols—June 1994

8.1.1 Calculate turn-over rate of phytoplankton by microzooplankton (days ^-1 ) from slope of regression equation (= 1-e ^-slope ).

8.1.2 Calculate rate of grazing of chlorophyll (mg chl liter ^-1 day ^-1 ) from turnover rate by multiplying by ambient chlorophyll concentration (= chlorophyll concentration* turnover rate).

8.1.3 Calculate rate of grazing of phytoplankton carbon by microzooplankton from chlorophyll rate * carbon to chlorophyll ratio. This ratio varies between 10 and 200. An average for the N Atlantic in 1989 was 32. It should be determined independently.

8.1.4 For further details on these see Landry and Hassett (1982) and Burkill et al. (1986).

9.0 Quality control and assessment
There is no standard for this assay and the accuracy cannot be determined. A minimum of two experiments should be performed during the occupancy of each station. Several estimates made on one station a few days apart will allow interpretation of the temporal pattern of grazing.

10.0 Notes
It should be remembered that many microzooplankton organisms are fragile; water samples should be treated with care prior to fixation. Experiments should be carried out as soon as possible after collection.

11.0 Intercomparison
No intercomparisons have been carried out in JGOFS, although this is clearly desirable for the future.

12.0 References & JGOFS papers published using these techniques
Burkill, P.H., Edwards, E.S., John, A.W.G, & Sleigh, M.A. 1993. Microzooplankton and their herbivorous activity in the north-east Atlantic Ocean. Deep-Sea Res. II. 40: 479- 494. 1993
Burkill, P.H, Mantoura, R.F.C., Llewellyn, C.A. and Owens, N.J.P. 1986.Microzooplankton grazing and selectivity of phytoplankton in coastal waters. Mar.Biol. 93: 581-590.
Dussart, B.M. 1963. Les differentes categories de plancton. Hydrobiol. 26: 72-74.JGOFS Protocols—June 1994 191
Jeffrey, S.W. and Humphrey, G.C. 1975. New spectrophotometric equations for the determination of chlorophylls-a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Plantz. 167: 191-194.
Landry, M.R 1993. Estimating rates of growth and grazing mortality of phytoplankton by the dilution method. In: Handbook of Methods in Aquatic Microbial Ecology (Kemp, P.F., Sherr, B.F. & Cole, J.J. Eds) pp 715-722 Lewis, Boca Raton.
Landry, M.R. and Hassett, R.P. 1982. Estimating the grazing impact of marine
microzooplankton. Mar. Biol. 67: 283-288.
Scor 1990. Grazing by microzooplankton. In: SCOR/JGOFS Report No 6: Core Measurement Protocols. Reports of the Core Measurement Working Groups. pp 31-37.
Sieburth, J.McN., Smetacek, V. and Lenz, J. 1978. Pelagic ecosystem structure: Heterotrophic compartments of the plankton and their relationships to planktonic size fractions. Limnol. Oceanogr. 23: 1256-1263.
Verity, P.G., Stoecker, D.K., Sieracki, M.E. & Nelson, J.R. 1993. Grazing, growth and mortality of microzooplankton during the 1989 North Atlantic spring bloom at 47°N, 18°W. Deep-Sea Res. 40: 1793-1814.