Daniel flint

A Viability study on the use of enriched sediment verses natural sediment to increase growth and survival rates of Zostera marina.

Daniel j. Flint FdSc Marine Science Falmouth Marine School May 2012

Abstract Zostera marina is a key habitat in Northern European coastal waters due to the nursery grounds that it provides for many juvenile species. Unfortunately due to the naturally occurring fungal disease found within Zostera marina whipping out over 90% of the naturally occurring eelgrass beds in northern European waters in the 1930’s. This naturally occurring disease which normally has no effect on the eelgrass due to its natural defence of phenoic acid was able to take such a devastating effect due to the very poor weather conditions in the 3 years prior to the epidemic resulted in the eelgrass not being able to build the nitrogen reserves and therefore the phenoic acid as a natural defence against pathogens. This study has looked into the viability of the most effective methods of cultivating eelgrass, with the long term goal of reintroduction in to the natural environment, either replenishing existing Zostera m. beds or possibly seeding new Zostera m. beds. This study looks at the effect of nutrient enriched sediment verses natural sediment of the effect of growth rate and therefore net nitrogen gain/reserves. By creating a system in which both populations of Zostera m. where exposed to the same environmental conditions, with only one being in nutrient enriched sediment the sole effect of the sediments can be reliably measured. Through statistical tests of the comparative growth rates of both populations of Zostera m. it is proven that enriched nutrient sediments increases

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growth and survival rates of Zostera m. and therefore the viability of reintroduction into the natural environment.

Introduction

waters (Borum & Greve 2004) Z.

The lower Fal and Helford intertidal

marina inhabit from the intertidal

areas are classified as SSSIs (Natural

sones down to 5 - 15 meters depth in

England, 2011). Within these areas

northern European waters (J. Borum et

there are habitats designated as SACs.

al. 2004) being able to do so has

Special Areas of Conservation (SACs)

resulted in Zostera marina or Eelgrass

are sites that have been adopted by the

is the most dominant species of

European Commission and formally

seagrass in northern temperate coastal

designated by the government of each

waters. Eelgrass (Zostera marina)

country in whose territory the site lies

inhabits these areas due to its

(Natural England, 2011). Zostera

preference to grow in sheltered

marina, Eelgrass is one of four closely

intertidal zones and down to 5-15

related species of seagrass in European

meters in northern European waters. (J.

water. Seagrasses have evolved from

Borum et al, 2004). With the fal having

different freshwater species, some

wide areas of shallow sheltered water

being more related than others. There

produced from the tin mining further

are other species of aquatic plants that

up stream producing fine sand, mud

inhabit the marine areas of low to

and silt deposits, producing anoxic

moderate salinity however the only

conditions under the sediment. This is

group that can be classified as seagrass

favourable for Zostera as typical

are found in high salinity oceanic

growth occurs in highly reducing

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sediments. Photosynthesis-mediated 02

primary production. (McMillan and

supplied to below-ground tissues

McRory. 1977) and (Zieman and

sustains aerobic respiration during

Witzel. 1980) Studies have shown that

photosynthetic periods. (R.D. Smith,

zosteras physiological responses of

1998). Zostera is able to survive and

temperate seagrass, Zostera marina,

thrive in these conditions through

ability to absorb light. These factors

being able to obtain O2. Strict

have been expanded upon by the fact

anaerobic conditions inhibit plant

that over the past 10 years 62% of all

growth in all but a few species of plant.

published journals on seagrass have

The ecological success of eelgrass in

purely focused on light intensity and

anoxic sediments must rely

epiphytes while only 18% have

predominantly on anatomical and

focused on hydrodynamics, now

physiological features that prevent

thought to be one of the key factors in

below ground tissues from becoming

eelgrass growth. 17% have focused on

permanently anaerobic. (Armstrong

sediment characteristics only major

1978, Crawford 1978, Davies 1980).

factor contributing to eelgrass growth

However the physiological adaptive

that previously was thought to be a less

features of this ecologically important

significant factor. Only 3 % of the

species are not well understood.

journals published have been focused

Extensive reviews by McRoy and

on seagrass geochemistry. (E. W. Koch

Mcmillan and Zieman and Witzel have

2001). Though the understanding of

stated that there is very little

eelgrass hydrodynamics it has become

experimental data available on

clear that there are far more

photosynthetic characteristics on these

understanding needed than purely the

species beyond the estimates of plant

light intensity required as even with

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high light intensity if the flow rate is

factor governing seagrass production

too high (above 150 cm/s there will be

in moderately nutrient enriched

reduced average growth rates due to

environments. (K. A. Moore & R. L.

self shading from neighbouring

Wetzel). As is apparent there is still

eelgrass. (E. W. Koch 2001) however a

much debate between the effects and

flow rate lower than 18 cm/s will result

causes of Zostera standing stock

in a increased DBL thickness in turn

diminishment. These factors have to be

reducing the CO2 absorption resulting

further understood as to provide

in reduced photosynthesis, over

adequate conservation. The first step in

prolonged periods can be the cause of

conservation is understanding the

death in the species. (Jones et al.

biological makeup and physical

2000). Having said these studies still

identification of Zostera marina.

have shown that light intensity is a

Zostera marina has leaf bundles with

major contributing factor, as light

terminal shoots on horizontal

shading studies indicated a massive

rhizomes. Branching of the rhizomes

reduction in standing stock of Zostera

occurs during the growth season

marina due to light irradiance,

forming the new terminal shoots. (J.

(Dennison 1979). Though studies

Borum and T. M. Greve. 2004). Each

carried out on light intensity and

new leaf formed produces a new

nutrient enrichment it was clear that

rhizome segment (internode) and two

only in light saturated conditions were

bundles of roots develop from the

nutrients an additional factor in

nodes between the segments. The roots

increased seagrass production. The

themselves are thin (0.2-1mm),

results of the experiment suggested

covered in fine hairs that may be up to

that the available light is the principle

20cm long. The rhizomes segments on

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which the roots develop are generally

Nitrogen and phosphate. The Redfield

2-6mm in thickness and vary from 5-

ratio of C:N at 106:16 of carbon to

40mmin length. Begin as a white/green

nitrogen (Redfeild et al. 1963) being a

colour and change to dark brown in the

marine primary producer one might

older segments. (J. Borum and T. M.

expect that eelgrass would contain the

Greve. 2004). Male and female flowers

same ratio of carbon to nitrogen

are found on the same individual. Male

however it considerably lower than in

and female flowers of Zostera marina

other marine species such as

are small, greenish and hidden in

phytoplankton. (Atkinson & Smith.

pockets within leaf sheaths. Zostera

1984, Duarte 1990). Although this is

marina may produce thousands of

the case overall nitrogen requirement

seeds per square meter and flower

for maximum eelgrass production per

regularly under optimum conditions

m2 is still 3 time higher than that of

predominantly flowering from early

phytoplankton production. (M. F.

spring till autumn. During this period

Pedersen. J. Borum. 1992). Eelgrass

the Zostera begins to change

unlike most angiosperms not only

morphology to produce more leaf

absorb nutrients from the sediment

bundles separated by long thin stem

they also uptake nutrients from the

segments. (J. Borum and T. M. Greve.

water column. (Iizumi & Hattori, 1982,

2004). Being an angiosperm Zostera

Thursby & Harlin, 1982, Short &

marina has the same nutrient

McRoy, 1984). Sediments where

requirements as terrestrial plants, but

Zostera can be located have generally a

obviously with a few key differences.

much high concentration of nitrogen

The major nutrients required for

than in the water column. (Lizumi &

Zostera growth are Carbon dioxide,

Hattori, 1982, Boon 1986, Dennison et

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al. 1987) although this is the case

However due to a disease in the 1930’s

Zostera marina leaves have a much

sweeping across Europe depleting the

higher uptake affinity than root

population by 90% (Koch 2001) only

rhizomes (Short & McRoy 1984)

those in brackish waters survived.

suggesting that the sediment is not the

Zostera marina is under huge amounts

primary source of nutrients. however

of pressure still due to human activity,

later studies have shown that nutrient

sediment disturbance is a massive

uptake can be accredited to both the

influence to the growth and well being

leaves and roots-rhizomes as both

of Z.marina if the sediment is

contribute significantly to overall

disturbed, the turbidity increases, this

nitrogen uptake in eelgrass (Iizumi &

reduces light intensity to the Z.marina

Hattori, 1982, Short & McRoy 1984,

leaves this as we know from Zosteras

Zimmerman et al, 1987).

high light intensity requirements very detrimental to growth rates. For these reasons, this study was carried out to in a bid to asses the viability of culturing Zostera marina in the lab, with the idea to reintroduce Zostera m. back into the natural environment either

Fig 1. Depiction of natural geographical distribution of Zostera marina.

reseeding existing Zostera m. beds or potentially seeding new Zostera m. beds.

Method and materials

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A 130 litre tank was divided into two

sediment of Zostera m. beds in this that

sections by the use of a purpose built

area.

Perspex divider see fig. 2. This allowed the separation of the two sediment types, natural sediment. Both sections where then filed with a base layer of coral sand this was placed on the bottom of both sections to enable water flow to prevent excess anoxic conditions. This can be seen on the left of Fig. 2.

Fig. 3 Google maps, location for source of natural sediment.

The second section was filled with a nutrient enriched sediment, (Aquatic fertiliser). On top of these sediments a second layer of fine coral gravel and sand was placed this was to prevent the water movement causing the sediments to be stirred into the water column causing a increase in turbidity and therefore effecting light intensity and

Fig. 2 Cultivation tank set up. (D.Flint 2011)

Once the base layer was in place and levelled out, the sediments where then put into place one side containing the natural sediment a mud/slit mixture that was taken from Deveron mudflats, see fig. 3 for details of specific area.

The sediment was taken from this area as this is consistent with the natural 7|Page

therefore growth rates. Also the use of coral gravel provides a natural buffering of the water to help maintain the natural pH of the water maintaining a level between 7.4 and 8.0 recreating the natural conditions (J. Borum and T. M. Greve. 2004). The lighting required to recreate the natural light conditions, a Halide lighting system with a 150

watt 5000k bulb alongside two 24watt

by (J. Borum. 2004) Zostera m.

T5 bulbs. These were put on a

requires a wave period of 0.4-0.7

electronic timer which was set for 16

seconds. Once set up the Zostera m.

hours a day for recreate the natural

may be planted placed into the

light duration at the time of the start of

sediment only up to the crown under

the study. The filtration system used

low light intensity to establish the

was a canister filter to provide good

growth of the root systems. Once

water quality and decent water flow

established and any non viable

without reducing aquarium space or

specimens removed form the tank and

causing shading. To increase water

the study may begin.

flow as recommended by (J. Borum

Measurements were taken on a weekly

and T. M. Greve. 2004) two 1600lph

basis measurement to be taken from

power heads were placed on opposite

the crown to the tip of the tall frond.

ends of the tank on a waver maker

Each specimen was numbered by a

system this allowed not only increased

small cable tie being numbered and

water flow but intermittent water flow

placed around the base of the

causing a wave motion in the tank

specimen. The number of fronds is to

preventing the build up of macroalgaes

be recorded this together with the

and filamentous algae which would

labelling system will allow reliable

reduced growth of Zostera m. as stated

record to be kept.

Results.

From the data collected from the

the graph in Fig. 4 taking into account

cultivation tanks, the overall growth

the loss of the samples in the natural

has been established and placed on to

sediment as opposed to no losses in the

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enriched sediment once the trial had

sediment type was not the sole

begun. As can been seen form the 2.

contributor to this as the time that the

Growth (mm) Nutrient Enriched Natural 9 5 10 9 10 XXX 3 12 14 XXX 10 9 10 12 13 10 11 9 8 XXX 7 12 10 11 10 10 Averages Nutrient Natural

9.875 O 9.875 4.125

Critical value Statistical value

4.125 E 7 7

O-E 2.875 -2.875

(O-E)2 8.265625 8.265625

(O-E)2/E 1.180803571 1.180803571 2.361607143

3.841 2.362

Table 1. Chi Squared Statistical test of overall growth rates

study was carried out coincided with

of the two populations of Zostera marina

the time of year Zostera m begins graph there is not the same amount of

to stop growth, as well as the fact that

data for each sediment type, this is due

Zostera is a protected species could

to in the settlement period there was

not be harvested for this study due to

considerably more loss of Zostera m

Zostera m being a Protected species

samples within the natural sediment

within the Fal under SSSI and SAC

that in the enriched sediment. However

legislation. The Zostera m. had to be

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collected form specimens that had been

significantly different. Due to the

washed ashore is rough weather

limited amount of data that could be

therefore prior damage to the Zostera

collect from this study it would appear

may have occurred that may not have

that enriched sediment does have a

been apparent, degradation of the

huge bearing on the growth and

Zostera may have already begun. This

survival rates of Zostera which is

being the case the Zostera samples in

reinforces the hypothesis of (J. Bourm

the nitrogen rich, nutrient enriched

2004) who stated that the only effect of

sediment did show a marked

higher nitrogen levels within the

improvement in the survival of the

sediment will only replace the nitrogen

specimens, as show in table 1, this may

lost from nitrogen recycling by the

be due to the nitrogen deficit normally

Zostera as the species is very effective

occurred in nitrogen recycling by the

at recapturing any nitrogen lost from

Zostera (J. Borum. 2004). Carrying out

decaying leaves and roots. To back up

a chi squared statistical test on the

these results a much more

samples as shown in table 2. shows a

comprehensive study on a larger

significant difference in the growth

scales would be needed to qualify

rates of the two populations favouring

these results.

the enriched sediment see table 2 for details. The statistical value is lower than that of the Critical value therefore

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Overall Growth measurements.

16

Growth in mm

14 12 Nutrient enriched sediment

10 8

Natural sediment

6 4 2 0 1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16

Specimen number

Fig. 4 A graph to show the Overall growth rates of the Two populations of Zostera marina.

The three troughs in the graph of the

and resulted in several specimens

natural sediment show the number of

being uprooted. These specimens had

species that did not survive in the

to be replanted this would have set

cultivation tank. This may be due to

back growths rate and may very well

the bridging of the nitrogen deficit of

have resulted in the loss for specimens

nitrogen recycling (J. Bourm 2004).

in this section. Problems were also

However this may also be due to the

experienced with the lighting system, a

several contributing factor that affected

malfunction in the electronic timers

the results that were collected, firstly

resulted in the constant output of high

one of the power heads designed to

intensity light. This resulted in excess

created irregular water flow preventing

algae within the tank, lots of macro

shelf shading (Koch, 2001) fell of its

and filamentous algae was produced

placement into the sediment on the

which has a hugely negative effect on

natural sediment section. This caused

the growth of Zostera marina. (J.

massive disturbance of the sediment

Bourm, 2004).

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Discussion Through this study it is apparent that

each sediment sample. Also it would

the theory proposed by (J. Bourm,

be advisable to collect Zoster m. in

2004) that increased levels of nitrogen

early spring to summer as this is in

in the sediment will provide the small

Zostera m primary growth period

percentage of nitrogen that is lost to

therefore being healthier, stronger and

the environment from nitrogen

have larger nitrogen and phenoic acid

recycling by Zostera marina. Enabling

reserves (Koch 2001). It would also be

Zostera not only to survive stressful

advisable to use larger power heads to

conditions i.e. uprooting and replanting

create a larger intermittent flow

but to also show a growth pattern

reducing shelf shading and a more

larger than that of natural sediment.

adequate wave period of 0.4-0.7

Having said that it may also the case

seconds (J. Bourm, 2004). This would

that the contributing factors in this

result in more accurate and reliable

study such. The contributing factors in

results to more accurately identify the

this study being the power head

importance of using a nutrient enrich

disturbance and the faulty lighting

sediment to increase growth and

timer may have had adverse effects on

survival rates. As (Borum et al, 1989)

the natural sediment populations

suggested that nitrogen conservation

causing a less pronounced growth and

i.e. reclamation could potentially be an

survival rate seen in the enriched

important mechanism for eelgrass

sediment population. If this study were

nitrogen nutrition, as studies have

to be carried out again, suggestions

shown that this is the case, even with

would be to carry out the study on a

deciduous terrestrial, evergreen plants

larger scale using more specimens in

living in nutrient poor environment

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therefore providing the nitrogen to the

Zostera marina in the nutrient enriched

eelgrass that would normally have

sediment to be healthier than the

been lost though this almost

population in the natural sediment.

completely effective process can explain the reason for the population of

Acknowledgements I gratefully acknowledge the help and support provided by Craig Baldwin, Falmouth Marine School for help, advice and concerns with aquaculture as well as use of lab and equipment with funding from Southampton University, Falmouth Harbour commission and Falmouth Marine School. I would also like to thank Luke Marsh, Falmouth Marine School, for his help taking measurements and helping with maintenance of the study tank.

References Borum, J., Murray, L., Kemp,W.M., 1989. Aspects of nitrogen acquisition and conservation in eelgrass plants.

Aquat. Bot. 35, 289–300.

Giesen, W.B.J.T., van Katwijk, M.M. and den Hartog, C., 1990b. Eelgrass condition and turbidity in the Dutch Wadden Sea. Aquat. Bot., 37: 71-85.

Harborne and Williams, 1976 J.B. Harborne, C.A. Williams Occurrence of sulphated flavones and caffeic acid esters in members of the Fluviales Biochem. Syst. Ecol., 4 (1976), pp. 37–41 Harlin, M. M., Thorne-Miller. B. (1981). Nutrient enrichment of seagrass beds In a Rhode Island coastal lagoon. Mar Biol. 65: 221- Harrison, P.G. and Chan, A.T., 1980. Inhibition of the growth of micro-algae and bacteria by extracts of eelgrass (Zostera marina) leaves. Mar. Biol., 61: 21-26.

lizumi, H., Hattori, A. (1982). Growth and organic production of eelgrass (Zostera marina L.) in temperate waters of the Pacific coast of Japan. 111. The kinetics of nitrogen uptake. Aquat. Bot. 12: 245-256 Jens Borum, CM Duarte, D Krause-Jensen and TM Greve, 2004, European seagrasses: an introduction to monitoring and management, the M&MS project.

Jacobs, R. P. W. M. (1979). Distribution and aspects of the production and biomass of eelgrass, Zostera manna L., at Roscoff, France. Aquat. Bot. 7: 151-172 Koch, E.W., 1994. Hydrodynamics, diffusion-boundary layers and photosynthesis of the seagrasses Thalassia testudinum and Cymodocea nodosa. Mar. Biol. 118, 767-776.

13 | P a g e

Koch, E.W., 1996. Hydrodynamics of shallow Thalassia testudinum beds in Florida, USA. In: Kuo, J., Phillips, R.C., Walker, D.I., Kirkman, H. (Eds.), Seagrass Biology: Proceedings of an International Workshop, Rottnest Island, Western Australia, 25±29 January 1996. Sciences UW, Nedlands, Western Australia, pp. 105±110.

Krause-Jensen D, Middelboe AL, Christensen PB,Rasmussen MB, Hollebeek P (2001) Benthic Vegetation Zostera marina, Ruppia spp., and Laminaria saccharina. TheAuthorities’ Control and Mmonitoring Programme for the fixed link across Øresund. Benthic vegetation. Status report 2000. 115 pp Krause-Jensen D, Pedersen MF, Jensen C (2003) Regulation of eelgrass (Zostera marina) cover along depth gradients in Danish coastal waters. Estuaries 26:866-877 McRoy, C. P., McMillan, C. (1977). Production ecology and physiology of seagrasses. In McRoy, C. P., Helfferich. C. (eds.) Seagrass ecosystems, a scientific perspective. Marcel Dekker, New York, p. 53-88 Muehlstein, L.K., Porter, D. and Short, F.T., 1991. Labyrinthula zosterae sp. nov., the causative agent of wasting disease of eelgrass, Zostera marina. Mycologia, 83: 180-191.

Patriquin, D. G. (1972). The origin of nitrogen and phosphorus for growth of the marine angiosperm Thalassia testudinum. Mar. Biol. 15: 35-46 Pedersen, M. F., Borum, J. (1992). Nitrogen dynamics of eelgrass Zostera marina during a late summer period of high growth and low nutrient availability. Mar. Ecol. Prog. Ser. 80: 6573 Phillips RC, McRoy CP (1990) Seagrass research methods. Monographs on oceanographic methodology. UNESCO, Paris. 210 pp Rasmussen, E., 1977. The wasting disease of eelgrass (Zostera marina) and its effects on environmental factors and fauna. In: C.P. McRoy and C. Helfferich (Editors), Seagrass Ecosystems. A Scientific Perspective. Marcel Dekker, New York/Basel, pp. 1-52 Renn, C.E., 1936. The wasting disease of Zostera marina: I. A phytological investigation of the diseased plant.

Biol. Bull., 70: 148-158.

Short, F.T., Mathieson, A.C. and Nelson, J.I., 1986. Recurrence of the eelgrass wasting disease at the border of New Hampshire and Maine, U.S.A. Mar. Ecol. Prog. Set. 29: 88--92.ng, E.L., 1938. Labyrinthula on Pacific coast eelgrass. Can. J. Res., 16:115-117.

Thayer, G.W., Engel, D.W., LaCroix, M.W., 1977. Seasonal distribution and changes in the nutritional quality of living, dead, and detrital fractions of Zostera marina L. J. Exp. Mar. Biol. Ecol. 30, 109–127 Tutin, T.G., 1938. The autecology of Zostera marina in relation to its wasting disease. New Phytol., 37: 50-71.

Vergeer, L.H.T. and den Hartog, C., 1991. Occurrence of wasting disease in Zostera noltii.

Aquat. Bot., 40: 155163.

Vergeer, L.H.T. and den Hartog, C., 1994. Omnipresence of Labyrinthulaceae in seagrasses.

Aquat. Bot., 48: 120.

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Fig. 1. Natural Geographical Distribution of Zostera marina, D. Flint 2012, map sourced from google maps.

Fig. 2 Cultivation tank set up sediment set up through to filling. D. Flint 2011.

Fig 3. Google maps Deveron mudflats [Online] available at: http://maps.google.co.uk/maps?hl=en&tab=wl (Accessed on 29th April).

Fig. 4. A graph to show the Overall growth rates of the Two populations of Zostera marina

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