The Effects of a Marine Sewage Outfal
on the Natural Succession of
Invertebrate Larval Settlement
Victor C. Anderlini
Hopkins Marine Station
Pacific Grove, Ca.
June 6, 1970
Introduction
The rocky headland knoun as Point Pinos, Monterey Co. Cal., re¬
ceives approximately 1.5 million gallons of primarily treated sewage
daily. Adjacent areas to the sewage outfall mouth, located inter-
tidally, are visibly deficient in fauna and flora typical of the
rocky Monterey coast.
The purpose of this six week investigation was to determine uhether
the sewage effluept affects the bacterial and diatomaceous films
knoun to produce a substratum for the metamorphosis of attached
invertebrate larvae. The importance of this early film to invertebrate
larval attachment has been debated for many. years. Most of thepprevious
microscopic work done on early stages of invertebrate settlement has
been concerned with the role of bacterial mats in fouling organism
succession studies.
The Australian worker, Uood (1949), in his work on fouling organisms,
suggested that bacteria were not necessary forthe attachment of some
species of barnacles and other foulers to submerged surfaces.
Zoßell and Allen (1935), however, felt that bacteriall and diatomaceous
slimes are not only beneficial to invertebrate larval attachment,
but that for some species this slime was also mandatory. An algal
and bacterial film has been shoun by Hinegardner (1969), to be nec¬
essary for the metamorphosis of settled sea urchins. Miller, Rapean
and Uhedon (1943), in their extensive revieu of the importance
of settlement surface preparation by bacteria, conclude that slimes
are beneficial but not required for settlement of all diatom and
invertebrate species.
The recent preliminary investigation of Golubic(1970), indicates
the importance of bacteria, diatoms, cyanophytes as en indicators of
organic pollution. In his study of nineteen Vugoslavian harbors
he has found bacterial-algal mats to persist throughout the year only
in the most heavily polluted areas. These mats consisted of a
number of species, some of uhich are directly dependent on water
that is rich in organic pollutants.
The study described here attempts to correlate variations in
micro-film formation with proximity to the Pacific Grove seuage
outfall.
Methods and Materials
To determine uhether the seuage effluent inhibits or enhances
bacterial and diatomaceous films, six stations were established
seaward of the Pacific Grove intertidal outfall. The stations
(Fig. 1), were located approximately 10 meters apart for a total
distance of approximately 65 meters away from the outfall mouth.
Stations were chosen that were similar in terms of tidal height,
wave shock, light and spatial orientation.
Since the study was of short duration and microscopic organisns
were being investigated, I felt 2" x 2" glass lantern slides (Semon
Bache & Co., Spec. 175-356) would provide an adequate settling surface
for bacteria, diatoms and any chance maroinvertebrate larvae
settling out. Dr. Eugene Haderlie, of the U.S. Naval Post-Graduate
School, Monterey Ca., in his studies (Haderlie 1968, 1970), has
shoun the choice of settlement surface to be inconsequential.
Dak holding frames (Fig. 2), uere constructed to secure the slides in
the intertidal area. The 1/4" x 2' x 6" frames were secured to granite
boulders at the six stations, the first of which was located three
feet directly north of the outfall mouth. All frames dere secured
at the 0.O tide level and on the rock face opposite prevalent uave
direction. Holes were drilled by star drill and hammer and the frames
attached by expanding lead plugs and 2" x 1/4" lag bolts. Tuenty-
four slides for each station were boiled in nitric acid and autoclaved
at 250 F. for 15 minutes. The slides were placed in their respective
racks on April 30th. One slide from each station was recovered daily
for the follouing tuo days. These preliminary plates shoued a great
abundance of bacteria of various morphologic types. To better
characterize bacteria accumulating, the standard Gram stain test
was chosen.
After six days exposure, the slides,though appearing clean to the
unaided eye, contained a remarkable assemblage of microscopic organisns.
To follou the very early stages of microbial succession, five neu
plates were placed in the frames, and one plate removed successively
for the following five days. This phase of experiment, designated
Short Term Study 1 (5TS-1), was duplicated by Short Term Study 2
(STS-2). Plates originally placed in the frames were left for
Long Term analyses.
An overall one month picture of early settlement was obtained by
this method.
Collected plates were transported in individual containers kept
in fresh seawater in a styrofoam bucket. Plates were examined imm-
ediately uhen possible. Studies on STS-1 & 2 concentrated on enum¬
eration of bacteria. Studies on plates collected on day 4 & 5 con-
centrated on counting andidentification of diatoms, and any other
microscopic organisms found on the plates. The recovered slides used
for bacterial investigation were allowed to air dry, after which time
bacteria were heat fixed. The standard Gram stain was employed as
a means of seperating gram-negative and gram-positive species.
A Zeiss light microscope with phase objectives and occular magnifier
uas used throughout the investigation. The area of a standard cover¬
slip (484mm), was traced on the backs of collected plates and bac-
teria wera counted at 2000x, by observing 10 random fields within this
area. Diatoms were counted at 200x along the diagonals of quadrants
traced on the backs of slides as shn in the pattern below. (Fig. 3).
Fig. 3


Slime scrapings were taken from the wooden slide holders and
from rocks adjacent to Stations 4 & 5. (see Fig. 1-x).
A cursory one day chemical analysis was carried out on May 22
using standard seawater analysis methods, (Strickland and Parsons, 1968).
Station 5 was "lost at sea" on May 25, consequently, there are
minor gaps in some graphs and tables.
Results
The results of this study will be described in three parts, the
first presenting bacterial trends and the second presenting diatom
and other microscopic form data. The third phase of this study provides
physical data for the Point Pinos area.
Bacteria-
After many hours of preliminary observation of various morphologic
types eight definite morphologic forms were chosen as test 'species".
The forms and pertinent information are given below in Fig. 4.
Table I presents the results of direct counts in the tuo short
term studies and Table II presents the combined counts. The tun
counts were combined to reduce variations due to external factors
such as chlorine content fluctuations, exposure time, light intensity
and seasonal variation.
Graphs 1-8 present the combined bacterial counts for single spedies
in graph form shouing the grouth trends over three days and at each
station. Graph 9 shows the total counts of all bacterial "species'
and the percent increase of cells for three days. Tables I & II shou
the number of cells at Station l to be higher than at other stations
on day one. Subsequent days shou the number of cells at Stations 2-
6 to shou a higher percent increase than at Station 1.
The Long Term Studv (LIS) slides showed the bacterial populations
to remain approximately at the levels reached on the fourth day of
exposure.
Diatoms-
After four days exposurs the number of bacteria became impractical
to count.. Noticeable numbers of diatoms uere observed at the end of
four days exposure and days 4 8 5 of the short term studies uere
devoted to counting and identification of these forms.
Table III presents the results of direct diatom counts for both
Short Term and Long Term Studies.
Graphs 10-13 present the species felt to represent overall trends
in diatom populations for the various exposure times. Stations 3 & 4
appear to be enriched by the ingreased nutrient levels shoun to be
highest in adjacent areas. Graph 14 express the results of the one-
day chemical analysis carried out on May 20th.
The cryptomonad Cryptomonas sp. and the phytomonad Ochromonas sp
were noted on all slides at various times, except those collected at
Station 1. Counts of these organisms proved to be inconclusive.
A marine fungi and the cyanophyte Oscillatoria sp. Uere also numerous
but direct counts provided no observable correlations. Fig. 5 depicts
Cryptomonas sp. and Ochromonas sp. Photo 1 shous a mycelial filament
of the observed fungi. A unicellular organism tanatively identified
as a unicellular blue-green alga is shoun in Photo 2.
The sequence of succession during the course of this study was
recorded and appears to occur in the follouing order:
lst Day- Detritus heavily adsorbed on Station 1 plates with almost
none at other stations. Numerous bacteria of many marph¬
ologic types arrive with the detritus at Station 1 with
lesser numbers and variety occurring at other stations.
2nd Day- Diatoms, including some pelagic types, begin settling and
some specimens of the unicellular blue-green algae.
Cryptomonas sp., Ochromonas sp., Uscillatoria sp. along with
3rd Day.
the filamentous fungi are found at the end of this period.
4th Day- All the above organisms uere numerous and specimens of the
red alga Erythrocladia subintegaa were found at Stations
3 and 5.
Scrapings collected from the surface of rocks adjacent to Stations
4 & 5 (Fig. 1-x), uere composed of numerous Navicula sp., Fragiillaria
sp., Nitzschia sp., Licmophora sp. and naviculoid-type diatoms.
Numerous bacteria of the 3u rod size raige tere also seen. Scrapings
collected from the wooden slide racks at Stations 1 & 3 shoued sim-
ilar numbers and species composition to those adsorbed to the glass
plates at these stations.
Discussion and Conclusions
The results of the bacterial study shou the general trand of bac-
terial populations during this month long study. Most of the species
studied are apparently introduced by the sewage effluent as evidenced
by their three day grouth patterns (Graphs 1-9). The first day of the
short term studies is characterized by higher numbers and greater
variety of morphologic types at Station 1 as seen in Table II. Day
tuo shous a slight decrease in the number of cells at almost all
stations folloued on day three by a tremendous percent rise in cell
numbers at stations auay from the outfall area (Graph 9).
Detrital adsorption showed a definite decreasing gradation away
from the outfall as was expected. The amount of detritus present at
each station over the three study days could not be correlated with
bacterial grouth.
The study plates uere subjected to fluctuations in natural en¬
vironmental parameters such as tides, sunlight etc., as well as
any fluctuations in the quality of the effluent. Replication of this
phase of the report combined with differential plating techniques
to identify bacterial species, would allou a more complete picture
of early bacterial succession. This information could possibly then
be more closely correlated to subsequent larval settlement.
Slides collected on days 4 & 5 of the short term studies ad
those collected for long term effects uere concerned with counting
and identification of diatoms.
Stations 3 & 4, 20 and 30 meters distant from the outfall mouth,
generally shou the greatest number of species and individuals adsorbed
to plates, (Graphs 10-13). Comparing nutrient data (Graph 14); with the
diatom counts shous that the stations with the most diatoms also appear
to be most enriched by PO or NO». These stations are also charac¬
terized by the least tidal fluctuation. Station 6 showed what is
believed to be a "normal" grouth pattern.
The genera Navicula, Licmophora, Fragillaria and Nitzschia are
frequent inhabitants of intertidal waters and dense populations are
often indicative of organic pollution, (Golubic, 1970). Their
occurrence andhigh numbers at Stations 3 & 4 and on rocks in an
adjacent tide ponl (Fig. 1-x), suggests the possibility of utilizing
them as quantatative as well as qualitative indicators of pallution
levels. The genus Navicula has been reported to live near outfallsiand
Byldo (1970), has reported grouth in various concantrations of sewage.
Houever, Baldo also found that the genera Nitzschia and Fragillaria
appear to be more susceptible to seuage than is Navicula. Thus the
ratios of these genera in the outfall area could aid in determining
the concentrations of seuage in specific areas.
The appearance of diatomaceous films on test slides also coincided
with the appearance of flagellates, ciliates, cyanophytes and an as
yet unidentified filamentous fungi.
I feel the natural succession of early substrate colonizers (as
seen on page 5), is perhapa being aborted at this stage in the immed¬
iate outfall area. The significantly high, possiblyttoxic, detrital
cover and the lou numbers of diatoms at Station l suggest this area
to be inhospitable for invertebrate larval settlement.
From the results, I feel Station 2 to be a marginal area in regards
to natural succession. It is influenced by fluctuating sewage concen¬
trations and may also be inhospitable for settling larvae. All
other stations appear to be follouing a natural sequence of settlement
(Zoßell & Allen, 1935), with Stations 3 & 4 showing a possible acceler-
ation and enhancement dde to increased, but non-toxic nutrient levels.
Natural variation in diatom populations could explain the observed
results, but replication of data makes this explanation unlikely.
Our limited one-day chemical analysis of the outfall area shous
the significant daily fluctuations of physical parameters prevalent
at Stations 1 & 2. These fluctuations, no doubt, influence the numbers
and types of bacteria and diatoms that can tolerate this environment.
The stations established at Pt. Pinos will be left throughout the
summer months in an attempt to study the long term effects of the out¬
fall on invertebrate larvae.
Acknouledgements
I would like to thank Doug Muchmore and Craig Blencoue for their
kind help and muscle power in establishing my stations at Pt. Pinos.
I would also like to exprass my appreciation to Dr. Epel of Hopkins
Marine Station for his help and counsel throughout this project.
Bibliography
Baldo, Angela
1970. Personal communication.
Cupp, Easter E.
Marine Plankton Diatoms of the West Coast of North America.
1943.
University of California Press, Berkeley and Los Angeles.
237pp.
Golubic
Stjepko
Effect of Organic Pollution on Benthic Communities.
1970.
Marine Pollution Bulletin, 1(4): 56-57.
Hinegardner, Ralph
Grouth and Development of the Laboratory Cultured Sea
1969.
Urchin. Bio. Bull., 137(3): 465-475.
Rapean and Uhedon
Miller
The Role of Slime Film in the Attachment of Fouling Or
1943.
ganisms. Bio. Bull., 94: 143-157.
Strickland and Parsons
A Practical Handhook of Seawater Analysis. Fisheries
1968.
Research Board of Lanada. 311pp.
Uood, E
The Role of Bacteria in the Early Stages of Fouling.
1949.
ZoBell and Allen
The Significance of Marine Bacteria in the Fouling of
1932.
Submerged Surfaces. Journal of Bacteriology, 29(3): 239-
251
metets
610
50

30
210
PT. PINOS
Fig. 1

2

ee
tidepoo




sand
20


L
—





I
LYPE
Fig. 4
2


gram-negative Tod



2.54
gen rod
S

44
2

5
gen coccl
S
54
gram-positive cocci
3
g-nrod
g-p rod
G
g-n rod
C
1.5
H

O
gen rod
X g-n forms make up 15% of observed specles.
Da
May
May
May 11
May 22
May 23
May 25
May 9
May 10
May 11
May 22
May
May 24
May 9
May 10
May 11
May 22
May 23
May 24
May 9
May 10
May 1
May 22
May 23
May 24
May s
May 10
May 11
Tune
24
20
19
293
100
34
79
102
18
83
96
163
86
84
60
14
626
124
14
27
12
28
24
42
40
46
TABLE
Stations
516
24
10
17
22
23
31
24
38
14
121
54
15
24
24
10
18
16
28
11
385
24
24
48
10
12
14
16
473
—*
54
-*
16
42
24
3
TABLE ICon't
Stations
Da
Type
May 22
May 23
May 24
May
16
May 10
38
May 11
34
May 22
May
38
32
May 24
May
May 10
Maylll
Ma
22
10
May 23
May 24
16
156
34
May
313
May 10
86
571
493
May 11
47
May 22
72
224
234
May 23
May 24
178
212
Rough weather, no plate recovered.
14
63
574
30
100
110
12
120
30
122
22
16
355
24
17
61
-*
12
-*
26
618
V
TABLE I:
tatior
ther
132
239
293
174
165
180
22?
109
5
228
537
671
no
Plat
19
220
716
42
320
783
COV
10
586
16.
681
106
185
82
24:
43:
Date
Type
May 12
(5 Days Exposure)
Naviculoids
Large
50u
Small
50u
Licmophora sp.
Nitzschia sp.
Fragillaria sp.
May 25
(5 Days Exposure)
Naviculoids
50u
Large
23
Small
50u
29
55
Licmophora sp.
17
Nitzschia sp.
Fragillaria sp.
May 26
(6 Days Exposure)
Naviculoids
Large 50u
24
Small
50u
204
198
19
Licmophora sp.
Nitzschia sp.
Fragillaria sp.
19
May 20
(21 Days Exposure)
Naviculoids
50u
Large
Small
50u
Licmophora sp.
Nitzschia sp.
16
Fragillaria sp.
Station 5.
lost at sea" May 25.
22
TABLE III
Stations
125
49
80
892
18
50
56
745
140
29
11
15
342
37
13
256
119
12
170
966
176
28
46
321
18
17
116
31
387
85
125
20
315
54
76
650
70
21
67
6
TARLE III (Con't)
Date
Stations
TUDE
May 21
(22 Days Exposure)
Naviculoids
42
10
16
50u
Large
18
56
642
410
346
356
Small
50u
46
Licmophora sp.
64
14
Nitzschia sp.
Fragillaria sp.
May 26
(27 Days Exposure)
Naviculoids
Large 50u
74
250
29
516
Small 50u
32
148
Licmophor
sp.
Nitzschia
Sp.
14*
Fragillaria sp.
Station 5, "lost at sea" on May 25.
** Large clumps with thousands of Fragillaria sp. were seen on parts of
the plates, collected on this day.
Fig. 5
Ochromonas sp.



Raete V—V.
-st sg dee at Bee
Hamentas Harge
3. V.30-V-21
Aatos 44
Anox
CLyntomonas sp.
2

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