THE EFFECT O F KRAFT PULP M I L L EFFLUENTS ON THE
FILAMENTOUS MARINE FUNGI WITH PARTICULAR REFERENCE
TO ZALERION MARITIMUM (LINDER) ANASTASIOU
L e s l i e M a r i a n churchland
B . A . , U n i v e r s i t y of B r i t i s h c o l u m b i a , 1966
A T H E S I S SUBMITTED I N PARTIAL FULFILLMENT O F THE
REQUIREMENTS FOR THE DEGREE O F
MASTER O F S C I E N C E
i n the D e p a r t m e n t
of
B i o l o g i c a l Sciences
0 L e s l i e M a r i a n C h u r c h l a n d 1 9 7 1
S i m o n Fraser u n i v e r s i t y
June, 1971
Title of Thesis: The effect of K r a f t pulp TI! 3 i F f f l u e n t s on the f llamentous marine C l l i l c ~ ~ 1 11 particular reference to zalerlon -- marl t L T ~ -- - m [ Lliider) Anastasiou
Examining Committee :
Chairman: Dr. B.P. Beirne h
Supervisor
L.J. Albri ht P
- L.D. Druehl
YW .Ffis't'i n External Examiner
Assistant Professor Simon Fraser University, British Columbia
Date Approved: fL.'*lr / 2, /97 /
The Effect of Kraft Pulp Mill Effluents on the
Filamentous Marine Fungi with Particular Reference to
.Zalerion maritimum (Linder) Anastasiou
ABSTRACT
Field studies near a Kraft pulp mill and at con-
trol stations in Howe Sound, ~ritish Columbia showed that
Kraft pulp mill effluents affected the species composition
of marine fungi. Phycomycetes were isolated as frequently
at the pulp mill as at the control stations, but Ascomy-
cetes were isolated less frequently at the pulp mill.
Certain members of the ~ungi ~mperfecti such as Mono-
dictys pelaqica (Johnson) Jones and Phialophora melinii
(~annfeldt) Conant were isolated more frequently at the
pulp mill while others such as Zalerion maritimum (Linder)
Anastasiou, were isolated less frequently.
Oceanographic measurements were carried out in an
attempt to explain the distributional effects. The pH,
salinity, and temperature values differed little when
12 m depths at the pulp mill and a control station were
compared. Dissolved oxygen at the 12 m pulp mill sta-
tion was lower than that at the control station. At a
depth of 30 cm dissolved oxygen, salinity, and pH values
iii
were both lower and more variable at the pulp mill than
at the control station. Temperature readings at a depth
of 30 cm were similar at the pulp mill and control station.
Dry weight and oxygen uptake studies showed that
Z . maritimum was tolerant to a wide range of salinity and -
pH conditions. Dry weight was higher in caustic Kraft
pulp mill effluent plus nutrients than in seawater plus
nutrients. Dry weight was lower in acidic bleach plant
effluent and alkalized bleach plant effluent plus nutri-
ents than in seawater plus nutrients.
Oxygen uptake was higher in caustic effluent plus
nutrients than in seawater plus nutrients. Oxygen uptake
was lower in acidic bleach plant effluent with and without
nutrients than in seawater with and without nutrients. Al-
kalized bleach plant effluent did not significantly affect
oxygen uptake values.
In Phialophora melinii caustic effluent and acidic
effluent without nutrients did not significantly affect
oxygen uptake. Oxygen uptake was lower in acidic bleach
plant effluent with nutrients than in seawater with nutri-
ents.
TABLE OF CONTENTS
Page
~xamining Committee Approval .................. ...................................... Abstract
Table of Contents ............................ ................................ List of Tables
List of ~igures ............................... Acknowledgements .............................. I . Introduction
A . Marine Fungal Ecology ................ B . The Pulp Process ..................... C . Pulp Mill Effluents and the ~arine
Environment .......................... I1 . Materials and Methods ....................
A . Species Composition Analysis ......... B . Oceanographic Measurements ........... C . Physiological Studies ................
................................... I11 . Results
A . Species Composition .................. B . Oceanographic ~easurements ........... C . Physiological Studies ................
IV . Discussion ............................... .................................. . V Summary
Literature Cited ..............................
iii
vii
ix
xi
1
2
5
~ppendix I ................................... curriculum Vitae .............................
Page
Table J
Table I1
Table I11
Table IV
Table V
Table VI
Table VII
Table VIII
Table IX
Table X
Table XI
Table XI1
LIST OF TABLES
Page
Species identified from panels and leaves from pulp mill and control stations
Incidence of Zalerion maritimum, Monodictys pelaqica, and Phialophora melinii on fir panels
Incidence of Zalerion maritimum, Mono- dictys pelaqica, Culcitalna achraspora and Lulworthia floridana on white pine panels
Incidence of marine Ascomycetes and ~ungi Imperfecti on hemlock, fir, and cedar pan- els submerged 6 months
Comparison of species composition of panels submerged 6 months
Incidence of marine Ascomycetes and Fungi Imperfecti on hemlock, fir and cedar panels submerged 4 months
Comparison of species composition of panels submerged 4 months
Occurrence of fungi on submerged leaves
Effect of salinity on dry weight of Zalerion maritimum grown in basal medium
Effect of pH on dry weight of Zalerion maritimum grown in seawater plus basal nutrients
Effect of Kraft pulp mill caustic efflu- ent plus basal nutrients on dry weight of Zalerion maritimum
Effect of Kraft pulp mill caustic efflu- ent (without basal nutrients) on dry weight of Zalerion maritimum
vii
Page
Table XI11
Table XIV
Effect of acidic Kraft pulp mill bleach 60 plant effluent plus basal nutrients on dry weight of Zalerion maritimum
Effect of alkalized Kraft pulp mill 6 3 bleach plant effluent plus basal nu- trients on dry weight of Zalerion mar it imum
Effect of buffered, alkalized Kraft pulp 65 mill bleach plant effluent plus basal nutrients on dry weight of Zalerion mari t imum
viii
LIST OF FIGURES
Page
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13
Location of sampling stations 13
Location of Station 1 with respect to 14 acidic and caustic sewers
Monthly salinities January, 1970- December, 1970
Monthly temperatures January, 1970- 41 December, 197 0
~onthly dissolved 0 values January, 1970- 42 December, 1970
2
Monthly pH values January, 1970-December, 43 1970
Daily salinity measurements taken at 44 Port Mellon August, 1969-August, 1970
Daily water temperature measurements 45 taken at Port Mellon August, 1969- August, 1970
The effect of salinity on oxygen uptake 49 by Zalerion maritimum without basal nu- trients
The effect of salinity on oxygen uptake 50 by Zalerion maritimum with basal nutrients
The effect of p H on oxygen uptake by 53 Zalerion maritimum in seawater without basal nutrients
The effect of p H on oxygen uptake by 54 Zalerion maritimum in seawater with basal nutrients
The effect of caustic Kraft pulp mill 55
Page
Fig. 14
Fig. 15
Fig. 16
Fig. 17
Fig. 18
Fig. 19
Fig. 20
Fig. 21
Fig. 22
effluent (without basal nutrients) on oxygen uptake in ~alerion maritimum
The effect of caustic Kraft pulp mill 59 effluent with basal nutrients on oxygen uptake in zalerion maritimum
The effect of Kraft pulp mill acidic bleach plant effluent (without basal nutrients) on oxygen uptake in Zalerion mar i timum
The effect of Kraft pulp mill acidic 62 bleach plant effluent with basal nutrients on oxygen uptake in ~alerion maritimum
The effect of Kraft pulp mill alkalized 66 bleach plant effluent (without basal nutrients) on oxygen uptake in Zalerion mar i t imum
The effect of Kraft pulp mill alkalized 67 bleach plant effluent with basal nutri- ents on oxygen uptake in Zalerion maritimum
The effect of caustic Kraft pulp mill 68 effluent (without basal nutrients) on oxygen uptake in Phialophora melinii
The effect of caustic Kraft pulp mill 69 effluent plus basal nutrients on oxygen uptake in phialophora melinii
The effect of Kraft pulp mill acidic bleach plant effluent (without basal nutrients) on oxygen uptake in PhiaL- ophora melinii
The effect of Kraft pulp mill acidic 72 bleach plant effluent plus basal nutrients on oxygen uptake in Phialophora melinii
ACKNOWLEDGEMENTS
To Dr. M. McClaren, whose continued encourage-
ment and assistance made the study possible,
to Drs. L. D. Druehl, G. H. Geen, and L. J.
Albright for their valuable suggestions and criticism,
to Mrs. B. G. Jenks and Mr. Norman Ellis for
technical assistance, and
to Dr. Ralph Patterson and Mr. Jos Macey of
canadian Forest Products for their willing supply of
effluent samples and support during the project.
I INTRODUCTION
Most of the work done on the effects of pulp mill
effluents on marine organisms has been directed towards
economically important species such as salmon (6, 39,
75-76, 89) and lobster (62, 81-82). A comprehensive
examination of the problem was carried out by the u.S.
Federal Water Pollution Control Administration in their
study of sulphite pulp mill effluents in Puget Sound.
They considered the effects of effluent on adult fish,
fish eggs, invertebrates, phytoplankton, and zooplankton
(86). The effects of Kraft pulp mill pollutants on
phytoplankton have also been studied (68); however, no
work has been done on the macroscopic algae. Moreover,
there is no information on the effects of pulp mill \
effluents on the marine decomposers, the bacteria and
fungi. Although the effects of waste products, such
as sewage, upon the distribution and biology of aquatic
fungi have been studied (17-20, 22-25, 28, 44, 79), no
work has been done on the effects of pollutants upon the
biology of marine fungi.
The aims of this study were threefold:
a) to determine the changes Kraft pulp mill effluent
caused in the distribution of the marine filamentous
fungi; b) to monitor several oceanographic parameters
to determine the effect of Kraft effluents on the physical
environment; c) to carry out physiological studies on
two species, Zalerion maritimum and Phialophora melinii,
to explain their distributional patterns.
A. parine Fungal Ecology
The pioneering study of marine mycology and marine
fungal ecology was published in 1944 by Barghoorn and
Linder (5), who described many new species of marine
fungi. The authors also determined salinity, pH, and
temperature tolerances and did a limited study of car-
bohydrate utilization for several of the more common
species.
Following this classic study, marine mycologists
concentrated on determining the geographical distributions
of marine fungi. These studies, which were mainly des-
criptive, consisted of enumeration of species from such
diverse locations as Queensland (27), Newfoundland (49),
Iceland (15), Venezuela (56), India (56), Mexico (56),
and Britain (53).
Some authors (38, 48) have attempted to correlate
distributional patterns with certain environmental factors
such as temperature and salinity. Gold (38) studied the
interaction of temperature and salinity; Hughes (48)
studied the effects of temperature, salinity, p ~ , dis-
solved oxygen, and ion ratios.
Studies on the marine fungi of the coast of British
Columbia have been carried out by Meyers and Reynolds (64),
Anastasiou and Churchland (3), Hughes (50), and Booth (8).
Meyers and Reynolds compared the fungal biota off Nanai-
mo, B.C. with fungi from other locations on the Pacific
and Atlantic coasts with respect to temperature conditions.
The other three studies (3, 50, 8) were mainly descriptive,
enumerating fungi isolated from panels (49), fungi isolated
from leaves (3), and chytrids isolated from marine muds (8).
Hughes divided his collection sites into three regions
based on salinity regimes, and noted the frequency with
which each species was isolated from each region.
Meyers' group (65-67) has investigated the role
of the fungi in degradative processes in the sea. They
have shown that certain marine fungi such as Z. maritimum
(65, 67) and ~ulworthia floridana Meyers (66) break down
cellulose when grown in seawater with added nutrients.
However, no -- in situ studies have been carried out to
determine whether the fungi, when in competition with
other marine decomposers such as the bacteria, do degrade
cellulose.
There are certain problems which the marine fungal
ecologist must consider. He must distinguish between the
"true" marine fungi (55), which grow and reproduce in the
sea, and the fungi which, although their spores occur in
the marine environment, do not complete their life cycle
in the sea. Quantitation is particularly difficult for
the marine mycologist. It is not feasible to estimate
the percentage of a substrate infected by a fungus, as
fructifications on the surface do not necessarily repre-
sent the amount of mycelium within the substrate. Data
regarding seasonal fluctuations in certain groups of
marine fungi are difficult to collect, as panels must
be submerged for several months before fruiting bodies
develop.
In summary, marine mycology is largely in the
descriptive stage, with emphasis on the enumeration of
fungi in various habitats and geographical locations.
~cological studies have been mainly autecological, re-
lating the incidence of certain species to environmental
factors such as temperature and salinity. Unexplored
areas of interest are the interaction of the marine fungi
with other marine organisms, and the role of marine fungi
in the biogeochemical cycles of the sea.
B. The Pulp Process
The pulping process removes lignin, the material
which binds together the cellulose fibres in wood, and
retains these fibres for use in pulp and paper-making.
In the Kraft, or sulphate process, wood chips are placed
in a digester to which is added "cooking liquor", con-
sisting mainly of sodium hydroxide and sodium sulphide.
The sulphur-containing chemicals, which are at a high
temperature, pressure, and pH, react with the lignin
molecule to form soluble compounds. These compounds
along with carbohydrates, resins, and minerals are
then removed by washing with freshwater.
A large quantity of the compounds used are re-
covered by concentrating and burning the residual chem-
icals (termed "black liquor") from the digestion process.
The black liquor is converted into sodium carbonate and
sodium sulphide. The sodium carbonate is then treated
with lime to form sodium hydroxide, sodium sulphide,
and calcium carbonate. Approximately 50% of the sulphur
and 90-95% of the sodium is recovered by this process (14).
The lost sulphur is replenished by the addition of sodium
s u l p h a t e , which i s reduced t o sodium su lph ide dur ing t h e
recovery process .
Af te r t h e d i g e s t i o n o r pulping p rocess , t h e c e l l u -
l o s e f i b r e s s t i l l con ta in coloured i m p u r i t i e s , which a r e
bleached wi th c h l o r i n e gas , c h l o r i n e d iox ide , o r hypo-
c h l o r i t e . These b leaching s t a g e s may be a l t e r n a t e d wi th
c a u s t i c e x t r a c t i o n s , dur ing which t h e pu lp i s washed i n a
s o l u t i o n of sodium hydroxide. The b leaching process a t
Por t Mellon ( a Kra f t pulp m i l l l oca ted i n Howe Sound, t h e
a r e a of s tudy) i s a f i v e s t a g e procedure ( 7 3 ) c o n s i s t i n g
of c h l o r i n e gas t r ea tmen t , c a u s t i c e x t r a c t i o n , c h l o r i n e
d ioxide t rea tment , c a u s t i c e x t r a c t i o n , and a f i n a l c h l o r i n e
d iox ide t r ea tmen t . The pulp i s then washed, d r i e d , and
pressed i n t o b a l e s .
The e f f l u e n t from t h e pulp-making process a t
Por t Mellon i s c a r r i e d t o t h e sea i n two p i p e s . The
c a u s t i c e f f l u e n t p ipe , exposed a t low t i d e , c a r r i e s
waste from t h e pulping and chemical recovery processes ,
and from t h e c a u s t i c e x t r a c t i o n p o r t i o n of t h e b leach
p l a n t opera t ion . The flow r a t e f o r t h i s e f f l u e n t stream
i s approximately 24 m i l l i o n g a l l o n s per 2 4 hours ( 5 9 ) .
The a c i d i c b leach p l a n t e f f l u e n t p ipe , which i s c o n s t a n t l y
submerged, d r a i n s t h e c h l o r i n a t i o n and c h l o r i n e d iox ide
t rea tment s t a g e s of t h e bleaching process . The flow r a t e
of this stream is approximately 7 million gallons per 24
hours (59).
C. Pulp Mill Effluents and the ~arine Environment -- The effluents from the Kraft pulping process may
present several hazards to organisms in the marine environ-
ment. The effluents may contain toxic compounds resulting
from the chemical additives described above. The complex
nature of lignin and other wood components has precluded a
complete analysis of the chemical composition of Kraft
pulp mill effluents. Werner (95) analyzed Kraft pulp
mill black liquor for sulphur-containing compounds, and
found evidence of sodium sulphide, sodium sulphite, sodium
thiosulphate, sodium sulphate, hydrogen sulphide, sodium
polysulphate, sulphur gas, sulphur dioxide, methyl mer-
captan, thiolignin, dimethyl disulphide, dimethylsulphide,
dimethyl sulphate, and lignin sulphonic acids. Werner
states that because black liquor is highly diluted upon
entry into the main effluent stream, only low concentra-
tions of these compounds reach the sea. Werner used
black liquor rather than effluent in order to obtain
sufficient material for analysis.
The chemicals in bleach plant wastes are more toxic
(6, 31, 47, 76) than those found in pulping and chemical
recovery wastes. None of these toxic compounds were identi-
fied until 1969, when Das et a1 (31) identified tetrachloro-
o-benzoquinone as a component of bleach plant effluent
which was toxic to salmon. These authors suggest that
the reduction of the unstable o-benzoquinones may result
in the formation of catechols, which are toxic to salmon
(Servizi et a1 (77)). In addition to the products of
lignin chlorination, bleach plant effluent also contains
traces of chlorine and chlorine dioxide (32). However,
these are very volatile, and unlikely to be traced beyond
the outfall.
The foam caused by the precipitation of lignin on con-
tact with seawater is several times more toxic than the
effluents themselves (26). These foams, if not contained,
may be transported and affect marine organisms several
miles distant from the outfall.
There are other characteristics of pulp mill efflu-
ents which may render them harmful to marine organisms.
Pulp mills are flushed out with large volumes of fresh-
water, which may create low salinity conditions in the
immediate vicinity of the effluent pipes. The addition
of freshwater may also result in the formation of an
effluent layer above the more dense seawater layer.
The pH of the effluent may be quite different from
that of seawater. The pH of the pulping process and caustic
extraction waste is high; in the case of the Port Mellon
caustic effluent, it varies from 10 to 11. The acidic
bleach plant effluent may have an extremely low pH
(varying from 2.0 to 2.5 at Port ello on). Howard and
Walden (47) suggest that abnormal pH values may be re-
sponsible for as much as 75% of the toxicity which re-
sults when fish are exposed to pulp mill wastes.
Receiving waters for pulp mill wastes may also
be depleted of dissolved oxygen by the heavy biochemical
oxygen demand of the effluent. unlike sulphite mill
effluent, which has an immediate oxygen demand for the
oxidation of reduced sulphur-containing compounds, Kraft
mill effluents require oxygen mainly for the biological
decomposition of organic material. However, there is not
necessarily a correlation between BOD and toxicity ( 4 7 ) ,
although BOD levels may be correlated with the amount of
total solids in the effluent (40).
A problem associated with pulp mill wastes is the
large quantity of particulate material which may be de-
posited on the ocean bottom near outfalls. Despite
attempts to keep fibre loss at a minimum, substantial
quantities are nonetheless carried out in the effluent
stream. The build-up of fibre on the sea floor results
in the destruction of the natural habitat for a variety
of bottom-dwelling organisms (96), and creates a suit-
able environment for the growth of aerobic and anaerobic
bacteria. These bacteria may produce such toxic gases
as methane and hydrogen sulphide (98), and deplete the
water of dissolved oxygen.
Some research has been done on the oceanographic
aspects of the dispersal of pulp mill wastes in the sea.
waldichuk (90-93) studied dispersal factors around pulp
mills and suggested 3 categories of geographical areas
where British Columbia mills were located: inlets,
partially enclosed embayments, and tide-swept channels.
The latter category, considered the most favorable for
rapid dispersal of effluents, included Port Mellon (92).
Attempts have also been made to monitor concen-
trations of Kraft pulp mill effluent after it had reached
the sea. Werner (97) developed a spectrophotometric tech-
nique based on detection of the brown coloration in lignin.
The B.C. Research Council conducted a survey for Canadian
Forest Products at Port Mellon in which the fluorescent
dye Rhodamine B was added to the caustic effluent and
traced at various depths and distances beyond the outfall
(37). However, dispersal of the bleach plant effluent
was not studied.
Materials g& Methods
A. Species Composition Analysis
Sampling stations were established in four areas
(Fig 1): Port Mellon (49'31.2'~~ 123'29.4'~~ (Station
# I)), Gambier Island (4g026.8'N, 123O26.3'~ (Station # 2)),
Keats Island (49'23.8'~~ 123'25.8'~ (Station # 3)), and
Horseshoe Bay (49•‹22.6'~, 123O16.2'~ (Station # 4)).
Fig. 2 shows the Port Mellon area in more detail, and
indicates the position of Station 1 relative to the
acidic and caustic effluent pipes.
Species composition data were acquired using a
bait sampling technique (3, 5). Baits composed of
alder leaves (during December-February arbutus leaves
were substituted) and untreated wooden panels were sub-
merged at the sampling stations. Panels were examined
immediately after collection as well as after a period
of incubation. This technique was used to distinguish
between those fungi which can develop in the sea, and
those fungi which, although their spores have been de-
posited on the panels, cannot develop until placed in
a culture chamber. The leaves were submerged to deter-
mine the occurrence of marine Phycomycetes. Two long-
term submergence studies of panels were carried out at
Fig. 1. Location of sampling stations. #1 - Port Mellon,
#2 - Gambier Island, #3 - Keats Island, #4 -
Horseshoe Bay.
Sca
Fig. 2. Location of station 1 with respect to acidic
and caustic sewers.
the pulp mill and three other sites to determine the
distribution of marine Ascomycetes.
The panels and leaves were attached to a nylon
line weighted by cement blocks which rested on the ocean
bottom. The panels were attached in groups of 10 on a
circlet of nylon line at approximately 30 cm below the
surface, and approximately 30 cm above the cement blocks.
Enough slack was left in the line so that at high tides
the cement blocks were not raised. The lines were all
attached to either buoys or to wharves which were float-
ing freely; the panels rested on the bottom except during
high tides. The lines at Port Mellon, Gambier Island,
and Horseshoe Bay were approximately 12 m in length;
the line at Keats Island was approximately 18 m in length.
Douglas fir panels, white pine panels, and leaves
were collected monthly from stations 1 and 4. The fir
panels and leaves were submerged for 1 month , whereas
the white pine panels were submerged 3 months (less in
cases where groups of panels were lost). The panels
and leaves were returned to the laboratory in poly-
ethylene bags and stored at 5 C. White pine panels were
examined within 1 to 2 days of collection. ~ i r panels
were incubated for 1 month according to techniques described
by Johnson and Sparrow (52). The leaves were sgbmerged
and incubated according to techniques developed by
Anastasiou and Churchland (3). The occurrence of fungi
on all baits were determined by the presence of repro-
ductive structures, as fungal species cannot be dis-
tinguished from one another by vegetative hyphae. The
data from the fir panels were grouped into two seasons,
a season (April-September) characterized by relatively
low salinity and high temperature, and a season (october-
March) characterized by high salinity and low temperature.
Comparisons were then made between stations for the three P
most commonly occurring species, Z. maritimum, Monodictys I
pelauica, and g. melinii. Variations in submergence time I ia ? i. of the white pine panels, and incubation time of the
leaves, precluded gr~uping and statistical analysis of
data from these baits.
For long-term submergence studies hemlock, cedar,
and fir panels were attached in groups of 10 at 30 cm
and 12 m positions at all four stations. These panels
were submerged over the periods November, 1969 to April,
1970 and May, 1970 to August, 1970. These panels were
not incubated after collection but were stored at 5 C
until examined. Sorensenls Similarity Index (801, 2C , where A + B
C = numbers of species in common, A = number of species at
Station A, B = number of.species at Station B, was used
as an index of differences in species composition between
stations.
P. melinii was frequently isolated from panels -
submerged at the pulp mill, and an attempt was made to
determine whether this fungus was present on the panels
before submergence, constituted a normal member of the
marine microbial community, or entered the sea through
the effluent pipe. To determine whether Phialophora
was present on panels before submergence, 10 fir panels
were soaked in autoclaved seawater, incubated for 1
month at 15 C,and examined for mycelium and spores.
To determine whether P. melinii could survive and grow
in the sea, the mycelium was stained with fluorescent
dye (30) and resubmerged. Two panels submerged for 1
month at the pulp mill and heavily infected with Phia-
lophora were stained with the fluorescent, vital dye
Calcofluor White ST. The panels were submerged in a
.02% solution of Calcofluor White in seawater for 8
hours, after which time the mycelium and spores were
fluorescing. The panels were then resubmerged at the 30 Cm
depth at Station 1 for one month, returned to the lab-
,oratory, and examined for new growth beyond the stained
hyphal tips. To determine whether spores of Phialophora
were ~ ~ e s e n t in the effluent, effluent samples were streaked
out on agar. Samples of each type of effluent were streaked
out on a modification of Vishniac's medium (88): glucose,
1.0 g; vitamin-free casamino acids (Difco), 1.0 g; yeast
extract (Difco), 0.2 g; liver concentrate (MC + B), 0.02
g; Bacto agar (Difco), 15 g; filtered seawater, 1000 ml.
After autoclaving, 500,000 units of penicillin G and
0.5 g of streptomycin sulphate were added to the medium.
B. oceanoqraphic Measurements
Monthly salinity, water temperature, dissolved
oxygen, and pH data were obtained from water removed at
high tide from 30 cm and 12 m depths at stations 1 and 4.
Water samples from the 12 m depths were obtained with a
2-litre capacity plastic water sampler. Salinity was
determined with a set of three specific gravity hydro-
meters and the U.S. Coast and Geodetic Survey conversion
tables (99). Water temperature measurements were taken
0 to the nearest C. For the 12 m temperature measurements,
the thermometer was immersed as quickly as possible in the
first sample drawn from the water sampler.
iss solved oxygen was measured using the Winkler
method as modified by Strickland and Parsons (83). pH
was determined upon return of the water samples to the
laboratory, a period not exceeding 4 hours. Daily measure-
ments of temperature, salinity, and pH were taken at a
depth of 30 cm, irrespective of tidal conditions, at
Station 1. Temperature and salinity were determined as
described for the monthly collections, An estimate of
daily pH values was obtained by using pH paper covering
the range 2 to 10 in units of 2.
C. Physioloqical Studies
The culture of Z. maritimum used in physiological
studies was isolated from an alder leaf submerged 1 month
at Horseshoe Bay and collected May 29, 1969 (Collection
# H 24). The culture of 2. melinii used in physiological
studies was obtained from a fir panel submerged 1 month
at Port Mellon and collected December 18, 1969 (Collec-
tion # H 798). Stock cultures of both these species were
maintained on the medium described above, without the ad-
dition of the antibiotics. In dry weight experiments the
basal nutrients described above.were used without the
addition of agar. The components were dissolved in 1000 ml
of effluent, 1000 ml of filtered seawater, or a solution
of 500 ml effluent, 500 ml seawater. The salinity and pH
of the seawater used in physiological experiments were
28 o/oo and 8.3 respectively. The effluent-containing
media were sterilized by filtration through a .22 p
Millipore filter, and the seawater media by autoclaving
at 121 C for 15 min. It was previously established by
dry weight studies that there was no significant differ-
ence in the growth of - 2 . rnaritimum in autoclaved and
filter-sterilized media. In the experiments to deter-
mine the effect of salinity on dry weight and respiration,
the seawater was diluted with distilled water to attain
salinities of 10 o/oo and 20 o/oo. The 30 o/oo concen-
tration was attained by boiling the stock seawater solu-
tion. The nutrients added 1.9 o/oo of dissolved material
to the various concentrations of seawater. In order to
buffer the medium, 1.2 g/l of tris (2-amino-2 (hydroxy-
methyl)-1, 3 propanediol (78)) were added. In the
experiments testing the effects of pH on dry weight,
pH was adjusted with 1M NaOH or HC1 after autoclaving
or filter sterilization. In the experiment using
buffered alkalized bleach plant effluent, 1.2 g/l of
tris were added to the medium. The liquid medium used
in the dry weight studies were distributed in 50 ml por-
tions among ten 250 ml flasks for each treatment, with
the exception of the pH experiment, in which 5 flasks
were used.
The inoculum for the dry weight studies of Z.
maritimum was prepared by growing the fungus in 250 ml
flasks containing 50 ml of basal medium. The cultures
were incubated for 2 weeks at 24 C on a shaker rotating
at 60 rpm, with the exception of those used for the pH
and alkalized bleach plant effluent experiments, which
were incubated for 17 and 21 days respectively. The
mycelium from 2 flasks was washed in seawater, added
to a Sorvall "Omnimixer" containing 100 ml of autoclaved
seawater, and macerated for 20 seconds at a constant
setting. Liquid media containing a range of effluent
concentrations, pH values, or salinities were inoculated
with 1 ml of the suspension, and shaken at 60 rpm for
2 weeks at 24 C. The contents of each flask were then
filtered through a pre-dried, pre-weighed, 0.47 p
Millipore filter. The filters plus mycelium were then
dried for 1 week in a 65 2 oven, and weighed. The pH
of the filtrate was measured for each flask.
In the dry weight studies control flasks containing
seawater or seawater plus nutrients were included in each
experiment because of the difficulty when working with
th2 fungi in duplicating inoculum conditions from one
experiment to the next (16, 45). The statistical analysis
was performed on the dry weight data to determine the ex-
tent of inoculum variation within each experiment.
~espiration measurements were made with a Gilson
Model GR 20 differential respirometer according to tech-
niques described by Umbreit et a1 (85). The temperature
of the water bath was maintained at 24 C, and the vessels
shaken at 134 oscillations per minute. For measurement
of respiration with exogenous substrate, each vessel con-
tained 3 ml of medium prepared as described for the dry
weight studies. All solutions were filter sterilized.
All oxygen uptake experiments were repeated twice, once
in seawater or effluent alone, and once in once in sea-
water or effluent plus nutrients. Filtered seawater and
filtered effluent were used for those experiments in
which no other nutrients were added. Each effluent
concentration was distributed among 5 or 6 ~ilson flasks.
In the case of the salinity and pH experiments, 4 and 3
vessels respectively were used for each treatment. The
centre well of each vessel contained a small piece of
folded filter paper saturated with 0.2 ml of 20% KOH,
for c02 absorption.
The inoculum for respiration measurements was
grown as in the dry weight studies, but was lightly
macerated until the majority of the mycelial fragments
were approximately lmm or less in diameter. 1 ml ali-
quots were then transferred to 20-250 ml flasks contain-
ing 50 ml of seawater plus basal nutrients, and shaken
for 48 hours at 24 C. Mycelial fragments were next
removed with pipets and washed in two changes of filtered
seawater, sorted on filter paper (the mycelium was kept
moist with seawater), and 10 pellets of approximately
1 mm diam transferred into each Gilson flask. The
flasks were equilibrated for 15 min before oxygen uptake
measurements were begun. The experiments testing the
effects of salinity, caustic effluent, and neutralized
bleach plant effluent on oxygen uptake were duplicated.
The inoculum for respiration studies of - P. P
melinii was prepared by flooding a plate culture with
seawater and adjusting the concentration of spores to
7 10 spores/ml. One ml of this suspension was added to
each of four flasks containing 50 ml of basal nutrients
plus seawater. The flasks were incubated for 48 hours
in the manner described for - 2. maritimum. The mycelium
was then centrifuged, washed and resuspended twice in
seawater, 50% and 100% effluent solutions, and 1 ml
aliquots transferred to GilsOn vessels each containing
2 ml of liquid medium. In the experiments testing the
effects of acidic bleach plant effluent, the mycelium
had formed clumps and it was necessary to macerate the
two-day old inoculum.
The information from the fir panels and the dry
weight and respiration data were analyzed using the t-
test described by Quenoville (70). Significance at the
95% probability level was used for all comparisons be-
tween means.
Both types of effluent were collected in auto-
claved dark glass bottles and stored at 5'~. The caustic
effluent used in dry weight and respiration studies was
3 to 5 days old. The acidic bleach plant effluent was
less than 2 weeks old.
Samples were taken from both effluent pipes and
from the Port Mellon and Horseshoe Bay docks for com-
parative analysis of total sulphate, hydrogen sulphide,
sulphite, total chlorides, available chlorine, carbonate,
- 2 5 -
and bicarbonate in seawater and undilute effluent: The
analysis was carried out by a local chemical laboratory,
Coast Eldridge Professional Services, Division of War-
nock Hersey International, using the following techniques:
sulphate, gravimetric precipitation with barium chloride;
hydrogen sulphide, colourimetry based on the reaction be-
tween paraminodimethylaniline, ferric chloride, and sul-
phide ion, forming methylene blue; sulphate, reduction
of iodine and titration with thiosulfate using starch
indicator; total chloride, back titration with silver
chloride and titration of excess with sodium thiocyanate;
available chlorine, colorimetric test with orthophenotro-
lene; carbonate and bicarbonate, stoichiometric titration
with hydrochloric acid. The results of this analysis are
presented in Appendix A.
The volatile components of the effluent, C12 and
H S, might not be traced due to the period of standing 2
between time of collection and time of analysis.
Results
A. Species Composition
TableI, which lists all the species isolated
during the study , shows that certain species of Ascomy-
cetes and ~ungi Imperfecti did not-occur at the mill
and certain species of Fungi Imperfecti did not occur
at Horseshoe Bay.
Table I1 shows the incidence of those filamentous
fungi found most frequently on submerged and incubated
fir panels. Z. maritimum occurred more frequently at
both top and bottom locations at Horseshoe Bay than at
either of the Port Mellon depths. Zalericn did not occur
at the mill during the period March through June. Another
species of marine Imperfect fungus, Monodictys pelaqica,
occurred more frequently at the mill surface station
than at the other three locations. An Imperfect fungus,
Phialophora melinii -- ( 5 7 ) , was isolated only from the pulp
mill. Only infrequently were fungi isolated from the
bottom mill depth. Other species found on fir panels
but not shown in Table I1 were ~raphium sp. (Stations #1,
4 top), Lulworthia floridana (Station #4, bottom), and
Alternaria maritima (all stations).
Table I11 shows the species which occurred most
Table I Species i d e n t i f i e d from pane l s and l eaves
from pulp m i l l and c o n t r o l s t a t i o n s
Funqi imperfecti
Zalerion maritimum (Linder) Anastasiou ~onodictys pelagica (Johnson) Jones
** Phialophora melinii (Nannfeldt) Conant culcitalna achraspora Meyers and Moore
* Zalerion varia Anastasiou * Cremasteria cymatilis Meyers and Moore * ~irrenalia macrocephala (Kohlmeyer) Meyers and Moore
** Humicola alopallonella Meyers and Moore Alternaria maritima Sutherland Graphium sp.
** ~andida sp.
Ascomycetes
Lulworthia floridana Meyers * Halosphaeria circu'nvestia Kohlmeyer * Ceriosporopsis halima Linder * Nais inornata ? Kohlmeyer * Didymosomarospora euryhalina ? Johnson and Gold * Corollospora comata (Kohlmeyer) Kohlmeyer * Leptosphaeria orae-maris Linder
Phycomycetes
Nowakowskiella eleqans (Nowak.) Schroeter Phytophthora vesicula Anastasiou and Churchland Pythium sp.
Mycelia Sterilia
Papulospora halima Anastasiou
Labyrinthulae Labyrinthula sp.
NOTE: * Denotes those species isolated from control stations only
** Denotes those species isolated from pulp mill station only
Table I1 Incidence of Zalerion maritimum, Monodictys
pelaqica, and Phialophora melinii on fir
panels (submerged 1 month) at Port Mellon
and Horseshoe Bay, January-December, 1970.
Numbers indicate #/lo panels infected. 1T =
Port Mellon, top. 1B = Port Mellon, bottom.
4T = Horseshoe Bay, top. 4B = Horseshoe Bay,
bottom. Diagonal lines indicated lost panels.
Series followed by the same letter do not dif-
fer significantly from one another at the 95%
probability level.
MONTH
WA
LY
SIS
OF V
A~IANCE
Species
Station
Zalerion
1T
54
00
00
33
20
02
mar i timum
1B
23
20
00
13
00
/
4T
7
91
0 7
9
91
0 1
0 91
0 9
4B
10
41
0 7
8
81
0 /
9
6
6
/
Monodictys pelaqica
1T
05
68
90
06
03
14
1B
10
20
00
0/
10
0/
4T
~~
~~
~~
~/
~~
OO
4B
30
10
00
0/
00
0/
Phialophora melinii
1T
36
68
17
35
93
34
1B
20
00
01
3/
20
0/
4T
OO
OO
OO
.O
/O
OO
O
4B
OO
OO
OO
O/
OO
O/
commonly on submerged white pine panels, and indicates that
the frequency of Z. maritimum was higher at Horseshoe Bay - than at Port Mellon. Monodictys pelaqica was isolated
more frequently at the mill than at Horseshoe Bay, although
not with any consistency from one month to the next. p&-
citalna achraspora, another marine Imperfect, was isolated
occasionally from Horseshoe Bay but not from Port Mellon.
Lulworthia floridana, a very common marine Ascomycete in
the Howe Sound area, was not isolated from white pine
panels submerged at Port Mellon. There did not appear
to be any seasonal trends in the occurrence of the fungi
shown in Table 111. In addition to the species already
mentioned Humicola alopallonella, Graphium sp., Leptos-
phaeria orae-maris, and ~eriosporopsis halima were iso-
lated from white pine panels in Horseshoe Bay.
Tables IV and VI show the incidence of marine fungi on
cedar, hemlock, and fir panels submerged for periods of 6 and
4 months. The species on the right of the double line are
Ascomycetes; those on the left are ~ungi Imperfecti. It
appears from Tables IV and VI that cedar panels were
generally not a good substrate for marine fungi. With
the exception of ~onodictys pelaqica and ~ulcitalna
achraspora, Imperfect fungi occurred less frequently at
e I11 Incidence of Zalerion maritimum, Monodictys
pelaqica, Culcitalna achraspora and Lulworthia
floridana on white pine panels (submerged 1 -
3 months) at Port Mellon and Horseshoe Bay,
January-December, 1970. Numbers indicate
#/lo panels infected. 1T = Port Mellon,
top. 1B = Port Mellon, bottom. 4T = Horse-
shoe Bay, top. 4B = Horseshoe Bay, bottom.
Diagonal lines indicate lost panels.
Species
Station
Zalerion 1T
maritimum 1B
4T
4B
Monodictys 1T
pelaqica 1B
4T
4B
Culcitalna 1T
achraspora 1B
4T
4B
Lulworthia 1~
floridana 1B
the pulp mill than at Keats Island, Gambier Island, and
Horseshoe Bay. However, only Z. maritimum and C. achras-
pcra occurred consistently at these three stations. There
was an almost complete absence of marine Ascomycetes on
panels from either depth at the mill (Tables 111, IV, VI).
The two ascomycetous species which were isolated commonly
from control stations were ~ulworthia floridana and
Ceriosporopsis halima; the occurrence of other marine
Ascomycetes was rare.
The data in Tables IV and VI is interpreted by
comparing similarity in species composition between sta-
tions (Table V, VII). When the pulp mill stations are
compared with the control stations, the species simil-
arity index is low, ranging from .36 to .66. When the
control stations are compared with one another, the
species similarity is high, ranging from .71 to .83.
There is also a greater similarity in species composi-
tion between depths at the control stations than there
is between depths at Port Mellon. Thus when Port Mellon,
top, is compared with Port Mellon, bottom, the species
similarity index is .57, as compared with -83, and .77
for Gambier Island, Keats Island, and Horseshoe Bay re-
Table IV Incidence of marine Ascomycetes and Fungi
~mperfecti on hemlock (H), fir (F) and
cedar (c) panels submerged 6 months. Numbers
indicate #/lo panels infected. #1 = Port Mellon,
#2 = amb bier Island, #3 = Keats Island, #4 =
Horseshoe Bay. Diagonal lines indicate lost
panels.
P
rt 0 'd
0
0
0
0
0
0
0
0
0
P 0
OD
P
P
0
I-'
0
0
0
N
tr' 0 rt rt 0 El
0
0
\
P
0
\
I-'
0
\
OD
4
\
0
4
\
0
0
\
0
I-'
0
P
IU
0
N
0
0
0
cn
N
0 0
0 0
\ I-'
P 0
P 0
\ P
0 0
0 0
\ 0
PW
UI Ul
\ N
-BZ€-
Type of Species wood
Phialophora melinii
Papulospora halima
Monodictys pelaqica
Culcitalna achraspora
Zalerion mar i t imum
Zalerion varia
Halosphaeria circumvestia
halima
Nais - inornata?
Lulworthia f loridana
fungal species isolated from Horseshoe Bay were also iso-
lated from the mill with the exception of the marine Ascomy-
cete Lulworthia floridana. Humicola alopallonella was
isolated from the pulp mill only, but would normally be
spectively (Table v). The similarity between Port Mellon
top and Port Mellon bottom is . 3 3 , compared with a value
of .83 for both depths at Gambier Island (Table VII).
Table VIII shows the occurrence of various species
of Phycomycetes, Fungi Imperfecti, and Ascomycetes on
leaves submerged at Port Mellon and Horseshoe Bay. Through-
out the study the Phycomycetes, represented in Table VIII by
Phytophthora vesicula, Nowakowskiella eleqans and Pythium
sp., were very tolerant to pulp mill effluents. However,
the latter two species were not isolated from the bottom
pulp mill station. Throughout the year of sampling, those
expected to occur on submerged leaves at Horseshoe Bay (3).
P. melinii and a species of Candida, which occurred occasion- -
ally at the pulp mill, were never isolated from Horseshoe
Bay.
A series of tests was carried out to determine the
source of the fungus - P. melinii, - frequently isolated from
Port Mellon. Of the ten fir panels which were submerged
Table V Comparison of species composition of panels sub-
merged 6 months (November, 1969 - April, 1970)
at Port Mellon, Gambier Island, Keats Island,
and Horseshoe Bay. Two stations with the identi-
cal species composition have a similarity index
of 1. Calculation of the index is described in
the Materials and Methods.
STATIONS
Port Mellon, top Gambier Island, top
Port Mellon, top Keats Island, top
Port Mellon, top Horseshoe Bay, top
Gambier Island, top Keats Island, top
Gambier Island, top Horseshoe Bay, top
Keats Island, top Horseshoe Bay, top
Port Mellon, bottom Gambier Island, bottom
Port Mellon, bottom Keats Island, bottom
Port Mellon, bottom Horseshoe Bay, bottom
Gambier Island, bottom Keats Island, bottom
Gambier Island, bottom Horseshoe Bay, bottom
Keats Island, bottom Horseshoe Bay, bottom
Port Mellon, top Port Mellon, bottom
Gambier Island, top Gambier Island, bottom
Keats Island, top Keats Island, bottom
Horseshoe Bay, top Horseshoe Bay, bottom
-34B-
SORENSEN '.S SIMILARITY INDEX -.
.55
Table VI Incidence of marine Ascomycetes and Fungi
Imperfecti on hemlock (H), fir (F), and
cedar (C) panels submerged 4 months. Numbers
indicate #/lo panels infected. #1 = Port Mellon,
#2 = Gambier Island, #4 = Horseshoe Bay. Dia-
gonal lines indicate lost panels.
* z =I (D I-' 01
I-' LC
LP
rt 0 'd
Ln
I-'
0
0
h)
0
03
03
LP
0
0
0
0
0
0
h)
03
W
h)
rt rt 0 3
0
0
0
U)
0
0
U1
03
N
Ln
I-'
0
0
0
0
h)
N
0
N
rt 0 'd
0
0
\
W
N
\
4
4
\
0
0
\
I-'
I-'
\
W
h)
\
0
LP
0
0
Ib
0
0
I-'
0
I-'
0
0
I-'
E: rt rt 0 2
0
0
0
W
W
0
0
W
0
0
0
0
0
0
0
0
0
0
Type of Species Wood
W
W
0
I-'
0
0
0
U)
0
0
0
0
0
0
0
0
0
0
Monodictys pelaqica
Culcitalna achraspora
Zalerion mari - timum
Zalerion varia
Cremasteria cymatilis
Cirrenalia macrocephala
s: Lulworthia =I f loridana
Ceriosporopsis halima
0
-?I euryhalina
1: Corollospora -?I comata 0
Table VII Coniparison of species co~?lp~sitioi? of panels
submerged 4 months (May - A u c p s t , 1970) at
Port Mellon, Gambier Island, and IIorseshoe ?
Bay. Two stations with the identical species
composition have a similarity index of 1.
Calculation of the index is described in the
Materials and Methods.
STATIONS
Port Mellon, top Gambier Island, top
Port Mellon, top Horseshoe Bay, top
Gambier Island, top Horseshoe Bay, top
Port ello on, bottom Gambier Island, bottom
Port ello on, top Port Mellon, bottom
Gambier Island, top Gambier Island, bottom
SORENSEN ' S SIMILARITY INDEX
*The station where the panels had disintegrated (#4 , bottom)
was not used in Sorensen's Similarity Index.
Table VIII Occur rence o f f u n g i on submerged l e a v e s
J u l y , 1969 - J u l y , 1970. #1 = Por t Mellon,
#4 = Horseshoe Bay.
-37B-
1 TOP 1 BOTTOM
+ -
SPECIES 4 TOP
+
4 BOTTOM
- ~owakowskiella eleqans
Phytophthora vesicula
Pythium sp.
Papulospora halima
Candida sp.
~onodictys pelaqica
~hialophora melinii
Zalerion mar i t imum
~umicola alopallonella
Graphium sp
~ulworthia floridana
Labyrinthula sp.
in sterilized seawater and incubated for 1 month, none
developed reproductive structures typical of Phialophora.
When the panels already infected with Phialophora,were
stained with fluorescent dye, resubmerged, and examined
for growth beyond the stained hyphal tips, no trace of
the fungus could be found. However, after removal from
the sea, the panels and surface detritus were still fluor-
escing. When samples of both effluent types were streaked
on antibiotic medium, no growth typical of ~hialophora
occurred. The above experiments did not determine the
source of g. melinii.
In summary, the conditions existing at Port Mellon
affected the occurrence of marine fungi in three ways.
First, certain members of the Fungi Imperfecti such as
P. melinii and Wodictys pelaqica occurred more frequently -
at the pulp mill than at control stations. Second, Fungi
~mperfecti such as - Z. maritimum and Ascomycetes such as
Lulworthia floridana occurred less frequently at Port
Mellon than at control stations. Third, the species
composition at the pulp mill was altered with the result
that there were few species in common between the pulp
mill and control stations.
-39-
B. ~ceanoqraphic Measurements
The monthly measurements of salinity, temperature,
dissolved oxygen, and pH taken at 30 cm and 12 m at sta-
tions 1 and 4 are presented in ~ i g . 3-6. There was little
difference in salinity between the 12 m depths at Port .
Mellon and Horseshoe Bay (Fig. 3). Although measurements
at 30 cm at both stations showed seasonal salinity fluc-
tuations typical of estuarine waters, the salinity at
the mill was lower than that at Horseshoe Bay (Fig. 3).
There was little difference in temperature values between
30 cm depths or 12 m depths at Port Mellon and Horseshoe
Bay (Fig. 4 ) . The dissolved oxygen values for the 30 cm
and 12 m depths at the mill were lower than those at Horse-
shoe Bay (Fig. 5). There was no indication of a serious
depletion of dissolved oxygen at the mill, as concentra-
tions rarely fell below 5 mg/l. There was little dif-
ference in pH at the 12 m depths at Port Mellon and Horse-
shoe Bay (Fig. 6). However, the pH values at the 30 cm
depth at Port Mellon were both lower and more variable
than those at Horseshoe Bay (Fig. 6).
The daily measurements of salinity and temperature
which were taken at a depth of 30 cm at Port Mellon are
presented in Fig. 7-8. Salinity (Fig. 7) followed the
Fig. 3 Monthly salinities January, 1970 - December, 1970. A---A = station 1, top. A -A = Station 1, bottom.
e-.... = Station 4, top. o . - . - . o = Station 4, bottom.
F i g . 4 Monthly temperatures January, 1970 - December 1970. A--- A = station 1, top. A -A = Station
1, bottom. e g o * * = Station 4, top. o....o= Station
4, bottom.
Fig. 5 Monthly dissolved 0 * values January, 1970 - 2
December, 1970. &---A= Station i, top,
A-A = station 1, bottom. a* - * . = sta-tion
4, top.0--..o= Station 4, bottom.
*There is a possibility that the accuracy of the Winkler
measurements taken at the pulp mill surface station were
affected by small quantities of thiosulfate or other re-
ducing agents present in the water.
Fig. 6 Monthly pH v a l u e s January, 1 9 7 0 - December, 1970.
&---A = S t a t i o n 1, t o p . A-A = S t a t i o n 1, bottom.
e.... = S t a t i o n 4 , t o p . O . . . . O = S t a t i o n 4 , bottom.
p H was determined w i t h a Corning Model 7 p H meter.
F i g . 7 D a i l y s a l i n i t y measurements t a k e n a t P o r t
Mel lon Augus t , 1969 - Augus t , 1970.
Fig. 8 ~ a i l y water temperature measurements taken at
Port Mellon August, 1969 - August, 1970.
seasonal fluctuations expected in an area influenced by
a nearby river; however, even in the winter period of
high salinity, there were values under 15 o/oo. The most
important feature to note in this graph is the large var-
iation in day to day readings. The scatter in the water
temperature graph (Fig. 8) is considerably less than that
for the salinity data (Fig. 7). Daily pH measurements
were frequently in the vicinity of pH 6 and occasionally
pH 4. ~ccasionally, presumably during the infrequent
periods when the bleach plant was shut down, readings
of pH 10 were recorded. There were no seasonal fluctua-
tions in the pH readings at the mill.
In summary, water temperature appeared not to be
affected by the effluent while salinity, dissolved oxygen,
and pH at 30 cm were lower at the mill than at Horseshoe
Bay.
C. Physioloqical Studies
Experiments were performed to test the effects of
salinity, pH, caustic effluent, and acidic effluent on
growth measured by dry weight and oxygen uptake in Z.
maritimum. Table IX shows that dry weight in distilled
water plus nutrients was significantly lower than in
water at salinities of 10, 20, or 30 o/oo plus nutrients.
Tab le I X
S a l i n i t y
ist tilled w a t e r
1 0 o/oo
20 o/oo
30 o/oo
-47-
E f f e c t o f s a l i n i t y on d r y we igh t o f Z a l e r i o n
maritimum grown i n b a s a l medium.
Dry Weiqht I n i t i a l p H (grams) ( a f t e r a u t o c l a v i n g )
F i n a l p H
- - X S X
8 .5 f.03
8 . 5 f.03
8 .5 *.03
8.4 k.03
Note: D r y w e i q h t s ~ r e c e d e d by t h e same l e t t e r do n o t
d i f f e r s i g n i f i c a n t l y a t t h e 95% p r o b a b i l i t y - -
l e v e l . x = sample mean, s x = s t a n d a r d e r r o r .
There was no significant difference in dry weight in the
three saline solutions. Fig. 9 shows the effect of sal-
inity on oxygen uptake by Z. maritimum in water without
basal nutrients. There was no significant difference
in oxygen uptake in distilled water and water at salt
concentrations of 10 o/oo, 20 o/oo, and 30 o/oo at the
end of the 2 hour period. There was also no significant
difference in oxygen uptake when nutrients were added to
distilled water and the three concentrations mentioned above
(Fig. 10).
Table X records the effects of pH on growth measured
as dry weight in z. naritimum and shows that the high buf- fering action of the seawater plus nutrient medium tended
to bring all final pH values close to 8.0. Nevertheless,
there were some significant differences in dry weights
of mycelium grown in media at different initial pH values.
The data presented in Table X indicates that the greatest
growth took place in the pH range 6 to 10. Growth was
poor from pH 2 to 3, where the pH of the media did not
rise during the course of the experiment. Growth in the
pH 4 to 5 range was good, but not as great as that in
media from pH 6 to 10.
The effect of pH on oxygen uptake in seawater with-
~ i g . 9 The effect of salinity on oxygen uptake by
~alerion maritimum without basal nutrients.
0-0 = distilled water, 0---o = 10 o/oo,
m.. . . . = 20 0/00, IJ.....~ = 30 o/oo . Ver-
tical lines show the means f one standard
error. Means followed by the same letter
do not, at 120 minutes, differ significantly
at the 95% probability level.
~ i g . 10 The effect of salinity on oxygen uptake by
Zalerion maritimum with basal nutrients.
= distilled water, 0--- o = 10 o/oo, .. . . . = 20 o/oo, o*****o = 30 o/oo . Ver-
tical lines show the means f one standard
error. Means followed by the same letter
do not, at 120 minutes, differ significantly
at the 95% probability level.
Table X Effect of pH on dry weight of Zalerion maritimum
grown in seawater plus basal nutrients.
Initial pH (after autoclaving)
Dry Weiqht (grams - - X SX
Final pH
Note: Dry weightspreceded by the same letter do not differ
significantly at the 95% probability level. - x = sample mean, sx = standard error.
out basal nutrients is shown in ~ i g . 11. Oxygen uptake
was very low in the seawater at pH 2, higher at p~ 4
and 6, and highest at pH 8 and 10. Upon the addition
of basal medium, there was no significant difference
(P 70.05) in oxygen uptake in solutions of pH 4 through
10 (Fig. 12). Oxygen uptake at pH 2 was again very low.
It is possible that in the 2 hour time period the pH of
the medium could have been readjusted towards pH 8, as
was the case in the dry weight experiments.
Table XI shows the effect of caustic effluent plus
basal nutrients on growth in g. maritimum. Although
there was no significant difference in growth in 50%
effluent medium and seawater medium, dry weight was
greatly increased in the 100% effluent solution. After
the experiment had proceeded for one week, there was
considerably more growth in the 5@/, effluent flasks
than in the seawater flasks. However, the data in
Table XI indicates that this tendency was overcome by
the end of the two week period. I also observed that
the mycelium and spores growing in 50% and 100•‹/, effluent
solutions took an a brown hue rather than the grey or
black coloration normal for the fungus. When basal medium
was not added (Table XII), dry weight was less in lo@/,
Fig. 11 The effect of pH on oxygen uptake by Zalerion
maritimum in seawater without basal nutrients.
A---A = p H 2,.c0**~= pH 4,e-e= pH 6 , O - - - O =
pH 8, r . . . . . r = pH 10. Vertical lines show the
means + one standard error. Means followed by
the same letter do not, at 120 minutes, differ
significantly at the 95% probability level.
Fig. 12 The effect of pH on oxygen uptake by ~alerion
maritimum in seawater with basal nutrients.
A --- A = p~ 2, w * * * * * w = pH 4, @--@ = pH 6,
= pH 8, A.....A= pH 10. Vertical lines
show the means f one standard error. Means
followed by the same letter do not, at 120
minutes, differ significantly at the 95% pro-
bability level.
Table XI Effect of Kraft pulp mill caustic effluent plus
basal nutrients on dry weight of Zalerion marit-
imum .
-
Dry Weight Initial pH Final pH ( grams ) (after autoclaving or
filter sterilization)
- - % Effluent x sx
Note: Dry weights preceded by the same letter do not differ -
significantly at the 95% probability level. x = sample -
mean, sx = standard error.
-56-
effluent than in 50% effluent or seawater.
Oxygen uptake by - 2 . maritimum in caustic effluent
is summarized in Fig. 13 and 14. There was no signifi-
cant difference in oxygen uptake in seawater, 50% and
100% caustic effluent (Fig. 13). Oxygen uptake was
significantly greater in both 50% and caustic
effluent plus nutrients than in seawater plus nutrients
(~ig. 14). The studies of caustic effluent and oxygen
uptake were repeated twice, and yielded the same results.
Table XI11 shows the effect of acidic bleach plant
effluent plus basal nutrients on growth in z. maritimum. Growth in 50% and 100•‹/, solutions was very low relative
to that in seawater medium. The pH of seawater and efflu-
ent solutions dropped; the pH of both effluent concentra-
tions was < 3 at the conclusion of the experiment.
Fig. 15 and 16 show the effects of acidic bleach
plant effluent on oxygen uptake in - Z . maritimum. As
would be expected from the pH data (Fig. 11-12), oxygen
uptake in acidic bleach plant effluent with and without
nutrients was low compared to that in seawater with and
without nutrients.
Table XIV shows the.dry weight results obtained
in bleach plant effluent alkalized to pH 8 with NaOH.
Table XI1 Effect of Kraft pulp mill caustic effluent
(without basal nutrients) on dry weight of
Zalerion maritimum.
Dry Weight Initial pH Final pH (grams (after autoclaving or
filter sterilization)
% Effluent
Note: Dry weights preceded by the same letter do not differ -
significantly at the 95% probability level. x = sample -
mean, sx = standard error.
Fig. 13 The effect of caustic Kraft pulp mill effluent
(without basal nutrients) on oxygen uptake in
Zalerion maritimum. -A = seawater, e*..... = 50%
effluent, --- = 100% effluent. Vertical lines
show the means f one standard error. Means fol-
lowed by the same letter do not, at 120 minutes,
differ significantly at the 95% probability level.
Fig. 14 The effect of caustic Kraft pulp mill effluent
with basal nutrients on oxygen uptake in Zalerion
maritimum. A-A = seawater plus nutrients,
.****-@ = 5@/, effluent plus nutrients, =
100% effluent plus nutrients. Vertical lines
show the mean f one standard error. Means followed
by the same letter do not, at 120 minutes, differ
significantly at the 95% probability level.
Table X I 1 1 E f f e c t of a c i d i c K r a f t p u l p m i l l b l e a c h p l a n t
e f f l u e n t p l u s b a s a l n u t r i e n t s on d r y weight
o f Z a l e r i o n maritimum.
Dry Weight I n i t i a l pH F i n a l pH (grams) ( a f t e r a u t o c l a v i n g o r
f i l t e r s t e r i l i z a t i o n )
- - % E f f l u e n t x sx
Note: Dry weights preceded by t h e same l e t t e r do n o t
d i f f e r s i g n i f i ' c a n t l y a t t h e 95% p r o b a b i l i t y - -
l e v e l . x = sample mean, s x = s t a n d a r d e r r o r .
Fig. 15 The effect of Kraft pulp mill acidic bleach plant
effluent fwithout basal nutrients) on oxygen up-
take in Zalerion maritimum. A-A = seawater,
a..... = 50•‹/,effluent, B--- rn = lo@/, effluent . Vertical lines show the means &one standard
error. Means followed by the same letter do not,
at 120 minutes, differ significantly at the 95%
probability level.
F i g . 16 The e f f e c t o f K r a f t p u l p m i l l a c i d i c b l e a c h p l a n t
e f f l u e n t w i t h b a s a l n u t r i e n t s on oxygen up t ake i n
Z a l e r i o n maritimum. A-A = seawa te r p l u s n u t r i e n t
a..... 0 = 50% e f f l u e n t p l u s n u t r i e n t s , m--- = 10O0L
e f f l u e n t p l u s n u t r i e n t s . V e r t i c a l l i n e s show t h e
means i one s t a n d a r d e r r o r . Means fo l lowed by
t h e same le t te r do n o t , a t 120 minu tes , d i f f e r
s i g n i f i c a n t l y a t t h e 95% p r o b a b i l i t y l e v e l .
Table XIV Effect of alkalized Kraft pulp mill bleach
plant effluent plus basal nutrients on dry
weight of Zalerion maritimum.
Dry Weight Initial pH ~ i n a l p~ (grams) (after autoclaving or
filter sterilization)
- - % Effluent x sx
Note: Dry weights preceded by the same letter do not differ -
significantly at the 95% probability level. x = -
. sample mean, sx = standard error.
~uring the course of the experiment, the pH of the effluent
solutions fell, a tendency particularly apparent in the
1@3% solution, Dry weight was lower in both 50% and lo@/,
effluent medium than in seawater medium; dry weight was
considerably lower in 100% effluent than in 50% effluent.
Table XV shows the results obtained when buffer
was added to the alkalized bleach plant effluent. The
pH drop apparent in Table XIV was largely controlled.
Nonetheless, the dry weight in the 50"h and 100% effluent
plus nutrient solutions was significantly less than that
in the seawater plus nutrient solution.
~ i g . 17 and 18 show oxygen uptake in Z. maritimum
in alkalized, buffered bleach plant effluent. There was
no significant difference in oxygen uptake between sea-
water, 50"L and 100% effluent solutions with and without
the addition of nutrients. When these experiments were
repeated, 100% alkalized bleach plant effluent without
nutrients increased oxygen uptake beyond levels in sea-
water and 50% solutions without nutrients.
Fig. 19-22 show the results of oxygen uptake studies
with P. melinii. ~ i g . 19 and 20 indicate that 50% and 100%
caustic effluent with or without basal nutrients did not
significantly increase oxygen uptake levels beyond those
Table XV Effect of buffered, alkalized Kraft pulp mill
bleach plant effluent plus basal nutrients on
dry weight of Zalerion maritimum.
Dry Weight initial pH Final pH (grams) (after autoclaving or
filter sterilization)
- - % Effluent x sx
Note: Dry weights preceded by the same letter do not
differ significantly at the 95% probability level. - - x = sample mean, sx = standard error.
Fig. 17 The effect of Kraft pulp mill alkalized bleach
plant effluent (without basal nutrients) on
oxygen uptake in Zalerion maritimum. A-A =
seawater, * * * * * * * = 50% effluent, m--m= 100•‹/, ef - fluent. Vertical lines show the means 6 one
standard error. Means followed by the same letter
do not, at 120 minutes, differ significantly at the
95% probability level.
Fig. 18 The effect of Kraft pulp mill alkalized bleach
plant effluent with basal nutrients on oxygen
uptake in zalerion maritimum. A -A = seawater
plus nutrients, e0** * *e = 50% effluent plus nutri-
ents, m---rn = 100% effluent plus nutrients. Ver-
tical lines show the means f one standard error.
Means followed by the same letter do not, at 120
minutes, differ significantly at the 95% probabil-
ity level.
Fig. 19 The effect of caustic Kraft pulp mill effluent
(without basal nutrients) on oxygen uptake in
Phialophora melinii . A-A = seawater, o.*-*-o = 50%
effluent, m--- = 100% effluent. vertical lines
show the means f one standard error. Means fol-
lowed by the same letter do not, at 120 minutes,
differ significantly at the 95% probability level.
Fig. 20 The effect of caustic Kraft pulp mill effluent
plus basal nutrients on oxygen uptake in Phialo-
phora melinii. - A-A = seawater plus nutrients,
m.****e = 50% effluent plus nutrients ,w --- w= 100% effluent plus nutrients. Vertical lines show the
means f one standard error. Means followed by the
same letter do not, at 120 minutes, differ signi-
ficantly at the 95% probability level.
attained in seawater with or without basal nutrients. Oxygen
uptake values in seawater or seawater plus nutrients were
considerably higher than those recorded for - Z. maritimum.
P. melinii did not form discrete pellets, but rather formed - amorphous strands of mycelium and a large number of spores
after two days in shake culture. It is possible that larger
amounts of inoculum were deposited in each Gilson flask
using the 1 ml suspension inoculation technique. It is
also possible that - P. melinii is a fungus which grows and
metabolizes more rapidly than does Z. maritimum.
The results in Fig. 21 and 22 show the effects of
acidic bleach plant effluent on oxygen uptake in g. melinii.
~espiration rates in seawater solutions with and without
media were considerably lower than those shown in Fig. 19
and 20. Maceration of'the inoculum could have resulted in
the lower rates of oxygen consumption observed in these two
experiments. unlike - Z . maritimum, oxygen uptake values in
P. melinii were not significantly affected by 50% and 100% -
acidic bleach plant effluent without nutrients. Upon the
addition of basal nutrients, respiration rates in 100% acidic
bleach plant effluent were significantly lower than those in
seawater and 50•‹/, effluent. However, in the case of Z. mari-
Fig. 21 The effect of Kraft pulp mill acidic bleach plant
effluent (without basal nutrients) on oxygen up-
take in Phialophora melinii. A-A= seawater,
0.. . . . = 50% effluent, m--- = 100•‹/, effluent.
Vertical lines show the means f one standard
error. Means followed by the same letter do not,
at 120 minutes, differ significantly at the 95%
probability level.
Fig. 22 The effect of Kraft pulp mill acidic bleach plant
effluent plus basal nutrients on oxygen uptake in
Phialophora melinii. A-A= seawater plus nutri-
ents, e--...e = 50% effluent plus nutrients, m--- = 10O0A
effluent plus nutrients. Vertical lines show the
means 3 one standard error. Means followed by the
same letter do not, at 120 minutes, differ signi-
ficantly at the 95% probability level.
timum, both 50 and 100% acidic bleach plant effluent plus
nutrients significantly reduced respiration rates when
cbmpared with seawater plus nutrients.
To summarize, - 2. maritimum was tolerant to a wide
range of salinity and pH conditions, as shown in studies
of oxygen uptake and growth measured as dry weight. Caustic
effluent increased oxygen uptake and dry weight relative to
those observed in seawater, and acidic bleach plant effluent
decreased dry weight and oxygen uptake relative to seawater.
When the bleach plant effluent was alkalized, a toxic effect
was demonstrated in dry weight studies but not in oxygen up-
take experiments. In g. melinii, neither caustic effluent
with or without nutrients nor acidic bleach plant effluent
without nutrients significantly affected oxygen uptake.
Oxygen uptake in 100% acidic bleach plant effluent with
added nutrients was approximately half that in seawater
and 50% solutions.
cent to a pulp mill and in "control" areas to determine the
effects, if any, of Kraft pulp mill effluents on the species
I composition of filamentous marine fungi. The principal "con-
I
troll8 area chosen, Horseshoe Bay, was a site distant from both
Howe Sound pulp mills. Panels were frequently lost from other
areas which were tested as control sites. The Keats Island and
Gambier Island stations were both considerably closer to the
pulp mill. However, oceanographic and species composition data
from these sites and Horseshoe Bay were very similar.
The field survey indicated some differences in species
composition between the pulp mill and control stations. There
were a small number of species in common at the pulp mill sta-
tion and control stations, a fact supported by Sorensents Sim-
ilarity Index. Comparison of the species composition at the
three control stations indicated a high degree of similarity.
However, the small number of isolations of certain species
limits the statistical validity of Sorensents Similarity Index.
All groups of fungi. were poorly represented on the
bottom panels at Port Mellon, This is difficult to explain
because the pH, salinity, and temperature at this depth
-75-
differed little with that at the same depth at Horseshoe
Bay. There was some variation in dissolved oxygen levels
at the Port Mellon bottom station; however, marine fungi
are tolerant of low oxygen pressures (52). Moreover, the
concentration of effluent reaching this depth is low (37).
The best explanation for the paucity of marine fungi at this
depth is that the panels which rest on the bottom are buried
in the fibre bed and fungal spores are physically prevented
from landing and germinating on the wood substrate. When
the fibres themselves were examined, the only fungal struc-
tures observed were biflagellate zoospores.
Phycomycetes, Ascomycetes, and ~ungi Imperfecti were
all isolated from the pulp mill surface station and control
areas during the course of the study. The Phycomycetes,
which were isolated from leaves only, occurred at the pulp
mill as frequently as at Horseshoe Bay. This may reflect
the fact that the marine Phycomycetes can usually grow
under low salinity conditions (2, 52). The data would also
suggest that these fungi may tolerate high concentrations of
acidic and caustic effluent as well as low pH conditions.
Labyrinthula - was found on leaves from both levels
at Port Mellon and Horseshoe Bay. This is not unexpected,
as Labyrinthula has been shown to be tolerant to a wide range
of salinity, temperature, and pH conditions (52).
The results from the species composition data indi-
cate an almost complete absence of marine Ascomycetes at the
pulp mill station. Other authors (5, 38, 48) have studied
the two Ascomycetes most commonly isolated from the control
stations, Ceriosporopsis halima and ~ulworthia floridana,
and related their growth and distribution to certain en-
vironmental parameters. Although Barghoorn and Linder (5)
found that Ceriosporopsis halima grew more readily on fresh-
water agar than on seawater agar, Hughes (48) isolated this
species from medium and high salinity areas only. Labora-
tory studies by Barghoorn and Linder (5) showed that Cer-
iosporopsis halima grew best over a pH range of 5.2 - 9.2
(the highest pH tested) and a temperature range of 15 - 25'~.
Lulworthia floridana, the only representative of
the marine Ascomycetes isolated from Port Mellon, was iso-
lated from the bottom sampling depth where conditions of
temperature, salinity, and pH were closest to those at the
control stations. The temperature and salinity tolerances
of Lulworthia floridana were studied by Gold (38) who
found that this fungus did not occur in water of salinities
under 2 0 o/oo . The absence of Ascomycetes at the pulp mill surface
station in the summer months may be explained by low salinity
values. This explanation would not apply from October through
March as during this time salinity generally ranged from 15 -
30 o/oo. Nor can the lack of Ascomycetes be explained in
terms of competition from other species, as there was no
heavy fungal growth on panels removed from the mill.
A point to note in regard to Ascomycetes and other
marine fungi relates to marine borers such as Teredo and
~imnoria. ~arine borer cavities were conspicuously absent
on panels submerged at t k pulp mill. Although panels sub-
merged 6 months at Horseshoe Bay or other control stations
were thoroughly penetrated and close to disintegration
from borer attack, panels at Port ello on were still intact
after a submergence period of 1 year. There is some evi-
dence that attack by Limnoria may be facilitated by the -
presence of marine fungi, upon which they may feed (52).
The corollory, that fungi infect cavities formed by marine
borers, is also true although the cavities are not a nec-
essary prerequisite to fungal attack. Perhaps the inabil-
ity of borers, bacteria, and other marine organisms to de-
compose the panels at the pulp mill could make infection
and breakdown of wood materials more difficult for the marine
fungi .
The Fungi Imperfecti isolated were a heterogeneous
I group and showed different degrees of tolerance to the con-
ditions existing at the pulp mill surface station. Mono-
dictys pelaqica, a very common marine Imperfect species,
appeared to grow more readily at Port Mellon than at Horse-
shoe Bay. This phenomenon could have resulted from an abil-
ity to utilize some component of Kraft mill effluent. On the
other hand, Monodictys pelaqica could have become established
as a result of the limited competition from other marine
fungi such as Z. maritimum, a pioneer species which heavily
infects submerged panels.
Z. maritimum, perhaps the most common wood-inhabit- -
ing species in the Pacific Northwest (49, 6 3 ) , was isolated
less frequently from Port Mellon than from Horseshoe Bay.
Although physiological studies by Barghoorn and Linder ( S ) ,
Gustafsson and Fries (41) and the present study show that
Zalerion is capable of growing in media made with distilled
water, H6hnk (46) found Z. maritimum to be absent in waters
below 7 o/oo in salinity. Hughes (50) found that - Z. mari- -
timum occurred as frequently in areas of low salinity as ---
high salinity. Ritchie (71) showed that the higher the
temperature, the higher the salinity optimum for this fungus.
The results from the salinity measurements in the
field suggest that in the months May through September low
salinity conditions could have prevented the occurrence of
this species at the pulp mill station. However, salinity
conditions did not explain the lower frequency of occur-
rence of this fungus in the winter months. Temperature
did not appear to be a contributing factor, as temperature
conditions at the mill were not seriously affected by the
effluent. Moreover, Barghoorn and Linder (5) demonstrated
that - Z . maritimum could grow over a wide range of temperature
levels. They also investigated pH tolerances, and found
that - Z. maritimum could grow over the whole range tested,
4.4 - 9.2. It would appear that none of the oceanographic
parameters monitored could explain the decreased frequency
of occurrence of - Z. maritimum at the pulp mill station. For
this reason the physiological experiments, which will be dis-
cussed in a later section, were carried out.
The Imperfect fungus which occurred exclusively at
the pulp mill was Phialophora melinii, a species not comrnon-
ly isolated from marine habitats. studies have been carried
out on fungi which may occur in slime accumulations within
pulp mills (9-13, 34-36, 69, 72, 94), and several members
of the genus ~hialophora, - P. fastiqiata (Lagerb. & Melin)
Conant, g. richardsiae (Nannf.) Conant, - P. lignicola (Nannf.)
Goidanich, and P. alba van Beyma, have been isolated from
this source. Phialophora spp. have also been recorded as
occurring on stored wood, wood pulp, and wood chips (4, 57,
63, 72). The species isolated from Port Mellon was very
closely related to g. fastiqiata, differing only in the
size of the conidia (87).
My attempts to determine the source of Phialophora
melinii at the pulp mill station were inconclusive. The
results suggest that the spDres of this fungus were deposit-
ed on the panels in the field, but did not develop in the
sea. ~xperiments indicated that the source of the spores
was not the panels themselves, or either of the two effluents,
although it is possible that the spores were present in the
effluent in such low concentrations that they were not found
in the samples tested. The temperature of the effluent ranged
from 5 to 10 C in the winter and 20 to 25 in the summer (60),
neither low nor high enough to kill the spores of this fungus.
Aerially born spores are another p~ssible means of infection.
Some species of ~hialophora grow in wood chip piles ( 5 8 ) ,
and spores from such a source could have been carried by
wind and deposited in the sea. Whatever the source of
inoculum, the spores of this fungus were abundant in waters
in the vicinity of the pulp mill.
The oceanographic data was collected in an attempt
to explain the species composition differences. The measure-
ments in this survey were taken in the zone of highest ef-
fluent concentration, at a point approximately 100 yards
from each effluent pipe. Farther from the effluent pipes
salinity, temperature, and dissolved oxygen quickly approach-
ed values common in local coastal waters (37).
The B.C. Research Council (37) surveyed at different
depths as well as distances from the mill and found that the
effluent had little effect on salinity, temperature, dis-
solved oxygen, and pH at a depth of 30 feet.
The measurement of effluent concentrations under-
taken by the B.C. Research Council (37) indicated that
dependent upon tides, currents, and wind conditions .05%
to 100% effluent (only caustic effluent was monitored in
this study) was found at the closest station to the point
where the panels were submerged. Only low concentrations,
.5% or less, could be traced at a distance of 1 mile from
t h e e f f l u e n t p ipe . The e f f l u e n t tended t o move i n a north-
e r l y o r sou the r ly d i r e c t i o n depending on t i d a l cond i t ions ;
s i t e s t e s t e d very c l o s e l y t o t h e m i l l could show no t r a c e
of e f f l u e n t . The a r e a of 5 t o 100% e f f l u e n t concent ra t ion
extended l e s s than 2000 f e e t from t h e o u t f a l l , whereupon
t h e e f f l u e n t was r a p i d l y d i l u t e d a s t h e d i s t a n c e beyond
t h e m i l l i nc reased . Ef f luen t concent ra t ion decreased rap-
i d l y wi th inc reas ing depth , p a r t i c u l a r l y beyond t h e immediate
a r e a around t h e o u t f a l l s . Concentrat ions measured a t t h e
30 f o o t depth a t t h e s t a t i o n n e a r e s t where t h e panels were
loca ted were 6%, 5.4% and 0.2%.
The v a r i a b i l i t y which can be seen i n e f f l u e n t con-
c e n t r a t i o n s was a l s o c h a r a c t e r i s t i c of t h e oceanographic
parameters which were measured a t t h e s u r f a c e pulp m i l l
s t a t i o n . S a l i n i t y , d i s so lved oxygen and pH were not only
v a r i a b l e from day t o day, but a l s o could change cons iderably
over a 24 hour pe r iod . Hardon (43) conducted a survey a t a
s i t e very c l o s e t o where t h e panels were submerged and found
t h a t temperature, s a l i n i t y , d isso lved oxygen, and p H va r i ed
from 9 t o 1 2 C , 15 t o 2 1 o/oo, 3 mg/l t o 7 rng/l, and 6.0 t o
8.5 r e s p e c t i v e l y i n 24 hours . Low readings u s u a l l y co r res -
ponded t o low t i d e l e v e l s , when e f f l u e n t d i l u t i o n c l o s e t o
-83-
the outfalls was at a minimum.
When the results from the oceanographic measurements
at Port Mellon are interpreted, it should be kept in mind
that certain parameters such as salinity were considerably
affected by the nearby Rainy River. Thus, the low salinity
values in the summer months should be to a large degree
attributed to runoff conditions. The low temperature of
the freshwater may also have compensated for the higher temp-
eratures of the effluent stream. However, pH and dissolved
oxygen effects were mainly due to the presence of the efflu-
ents. Perhaps the most acute effect of the pulp mill efflu-
ent was to consistently lower pH values.
It was difficult to correlate the species composition
data with salinity, pH, dissclved oxygen, temperature, or
effluent conditions as these factors varied daily at the
mill. In an effort to understand the distribution of Z.
maritimum and g. melinii, physiological experiments on growth
and oxygen uptake were carried out.
Although there are some unique problems associated
with manometric techniques as applied to the filamentous
fungi (1, 16), I felt that the results from such studies
would be useful when considered in conjunction with the re-
sults from dry weight experiments. One problem with res-
piration studies in the fungi has been the high rates of
endogenous respiration, caused by the accumulation of re-
serve materials from the overly rich media usually employed
in studies of fungal physiology (1, 16). The media used in
this study therefore employed the relatively low concentra-
tion of lg/l glucose. Some factors which should be con-
sidered in respect to inoculation techniques are that mac-
eration may partially destroy the cells (16, 29), that the
dry weight of mycelium may change during the course of the
experiment (29), and that oxygen uptake should not be
correlated to dry weight unless the ratio of respiring to
non-respiring protoplasm is constant (29).
Preliminary studies were carried out with Z. mari-
timum to determine what inoculation technique would yield
the smallest variation in oxygen uptake values between
replicate flasks. These experiments showed that oxygen
uptake rates were most uniform when pellets or mycelial
fragments of the same diameter were used. Dry weight of
the inoculum among replicate flasks was also fairly uniform,
averaging 2.1 k 0.3 mg. However, those flasks with the
highest oxygen uptake values did not necessarily correspond
to those flasks with the highest dry weights at the con-
clusion of the experiment. Therefore, the direct oxygen
uptake method in which total oxygen uptake is plotted
against time but not related to dry weight (85) was used
to present the respiration data.
All dry weight and respiration studies on Zalerion
maritimum were carried out at 2 4 ' ~ , found by Barghoorn and
Linder (5) and G.C. Hughes (51) to be the optimal temperature
for growth. Although salinity and pH optima (5) have been
derived for this fungus by measuring radial growth on agar,
I felt that these experiments should be repeated using
aerated liquid media. The dry weight and respiration ex-
periments show that Z. maritimum has a broad tolerance to
varying salinity conditions; however, this principle does
not necessarily apply under field conditions, where the
concentration of essential nutrients may be low. The results
from the salinity experiments agree with those of Barghoorn
and Linder, who found that ~elicoma maritimum could grow
on freshwater medium, although not as well as on seawater
medium. The dry weight and respiration studies indicate
that Z. maritimum is tolerant to a wide range of pH levels
under laboratory conditions, as was also shown in the work
of Barghoorn and Linder (5). unlike many fungi, the optimum
pH for growth of Z. maritimum is in the alkaline range,
suggesting an adaption to growth in the marine environment.
However, when supplied with basal nutrients, this fungus
can grow to a limited extent at pH 3. The pH and salinity
studies in the laboratory do not explain the limited occur-
rence of Z. maritimum in the pulp mill area.
When growth and oxygen uptake of Z. maritimum were
measured in caustic effluent, caustic effluent with added
basal nutrients stimulated rather than inhibited fungal
metabolism. Perhaps growth and oxygen uptake increased
owing to utilization by the fungus of the sugars and/or
lignin content of the effluent. Caustic effluent alone
did not stimulate dry weight or oxygen uptake. This con-
firms the findings of ~nkvist (33) and others (61, 84)
who found that certain basal nutrients were required for
growth of fungi in pulp mill effluents. Perhaps one of
the most interesting aspects of the present study was the
discovery that one of the organisms natural to the marine
environment could utilize some component in caustic pulp
mill effluent. Suggested areas of future research are:
to determine what nutrients should be added to the effluent
for optimal growth; to determine whether lignin and lig-
nosulphonic acids are utilized; and to determine
whether this organism could be used in a microbial waste
treatment process.
other authors have investigated the growth of fungi
in pulp mill effluents. Some fungal species may grow in
sulphite and sulphate liquors if certain nutrients are
supplied (33, 61, 84). The fungi may use the sugars in
the effluent, reducing the biochemical oxygen demand (33)
and in some cases producing as end products economically
useful compounds such as fumaric acid (74). The use of
pulp mill wastes as a source of nutrition for commercial
yeast has also been investigated (74). Certain fungi
have the ability to degrade lignin (21, 42) and it has
been shown that some species may break down the lignin in
pulp mill liquors (7, 33, 54, 61, 84).
A series of experiments was carried out to deter-
mine whether the acidic bleach plant effluent could cause
the lower frequency of occurrence of 3. maritimum at Port
Mellon. The low pH (2.0 - 2.4) of the bleach plant efflu-
ent was probably responsible for the toxic effect of un-
alkalized media. However, bleach plant effluent adjusted
to the pH of seawater still retained some toxicity for the
fungus. In the unbuffered medium, the drop in pH which
-88-
occurred as growth proceeded could have been partially respon-
sible for the toxic effects. Perhaps the toxic component
had a more pronounced effect on the fungus under conditions
of low pH. When tris buffer was added to the alkalized bleach
plant effluent, the toxic effect shown in the unbuffered
medium was partially, but not completely overcome. The
toxic component of the alkalized bleach plant effluent did
not affect normal oxygen uptake by the fungus. When the
experiment testing the effects of alkalized bleach plant
effluent plus nutrients on oxygen uptake was repeated, dif-
ferent results were obtained. Two different samples of ef-
fluent do not necessarily have the same chemical composition,
and perhaps the effluent used in the second experiment con-
tained a higher percentage of carbohydrate and lignin, and
a lower percentage of the toxic component.
The results from the physiological studies suggest
that the combination of high concentrations of bleach plant
effluent and low pH conditions may be at least partially
responsible for the lower frequency of occurrence of 2.
maritimum at the pulp mill site during the winter months.
Oxygen uptake studies were carried out with - P.
melinii to determine whether this fungus showed a greater
tolerance to pulp mill effluents than did Z. maritimum.
Unlike Z. maritimum, oxygen uptake in g. melinii was not
stimulated by some component of the caustic effluent. The
results from the acidic bleach plant effluent studies indi-
cated that high concentrations of unneutralized bleach plant
effluent had no significant effect on oxygen uptake when
compared to seawater. With the addition of basal nutrients,
oxygen uptake was decreased only in lo@/, unneutralized bleach
plant effluent. The frequent isolation of - P. melinii from
Port ello on may be due to the tolerance of this fungus to
low pH conditions and to high concentrations of bleach
plant effluent.
V SUMMARY
I effluents cause certain changes in the species composition
I of marine fungi in an area very close to a pulp mill efflu-
ent discharge. Certain fungi such as Zalerion maritimum - were isolated less frequently at the pulp mill station than
at other stations. However, certain fungi such as Phialo-
phora melinii appeared to develop more readily on panels
submerged at the pulp mill station.
Physiological studies with g. maritimum indicated
that this fungus utilized some component or components of
the caustic effluent, resulting in greater growth and oxygen
uptake than found in control flasks. Although other fungi !
have been demonstrated to have this ability, this is the
first report of a marine fungus able to utilize pulp mill
I effluent . other work done on Z. maritimum indicated that the
salinity values prevailing at the pulp mill might inhibit
the growth of this fungus during the summer months. However,
it was difficult to correlate the absence of Zalerion during
the winter months with any of the measured physical parameters.
~ h y s i o l o g i c a l experiments ind ica ted t h a t t h e b leach p l a n t
e f f l u e n t might con ta in a substance t o x i c t o ~ a l e r i o n , par-
t i c u l a r l y a t low pH va lues . I t i s t h e r e f o r e suggested t h a t
t h e b leach p l a n t e f f l u e n t combined wi th low pH values may
be re spons ib le f o r t h e lower frequency of t h i s fungus a t t h e
pulp m i l l .
S tud ies wi th Phialophora - d i d not i n d i c a t e t h e source
of t h e spores of t h i s fungus. ~ h y s i o l o g i c a l experiments
showed t h a t c a u s t i c e f f l u e n t d i d no t a f f e c t oxygen uptake,
bu t t h a t t h i s s p e c i e s was p a r t i c u l a r l y t o l e r a n t t o high
concent ra t ions of a c i d i c bleach p l a n t e f f l u e n t . I t i s
suggested t h a t t h i s spec ies developed we l l on pane l s sub-
merged a t 30 cm a t t h e m i l l because of i t s t o l e r a n c e t o low
pH l e v e l s and high concen t ra t ions of b leach p l a n t e f f l u e n t .
I ~ LITERATURE CITED
Ainsworth, G.C. and A.S. Sussman. 1965. - The Funqi: An - Advanced Treatise. Volume T_. The Funqal Cell. Academic Press, New York - and London.
Ainsworth, G.C. and A.S. Sussman. 1968. The Funqi: An Advanced - Treatise. Volume z. The Fungal Population Ecoloqy. Academic - Press, New York and London.
Anastasiou, C.J. and L.M. churchland. 1969. Fungi on decaying leaves in marine habitats. Can. J. Bot. 47: 251-257.
Atwell, E .A. 1931. Occurrence of Cadophora fastisiata in Canada. Phytopathology 21: 761.
Barghoorn, E.S. and D.H. Linder. 1944. Marine fungi: their taxonomy and biology. ~arlowia 1: 395-467. -
Betts, J.L. and G.G. Wilson. 1966. New methods for reducing the toxicity of Kraft mill bleachery wastes to young salmon. Jour. ~ish. Res. Bd. Can. 23: 813-824.
~ilai, V.F., 0 .M. Andropova and R.V. Voitsokhavskyi. 1970. The use of lignin by microfungi (English summary) . Akad. Nauk. USSR Kiev 32: 557-560.
Booth, T. 1969. ~arine fungi from British columbia: monocentric chytrids and chytridiaceous species from Coastal and ~nterior halomorphic soils. Syesis 2: 141-161.
Brewer, D. 1958. Studies on slime accumulations in pulp and paper mills. I. Some fungi iso- lated from mills in New Brunswick and Newfound- land. Can. J. ~ o t . 36: 941-946.
Brewer, D. 1959. Studies on slime accumulations in pulp and paper mills. 11. Physiological studies of Phialophora fastiqiata and - P. richardsiae. Can. J. Bot. - 37: 339-343.
Brewer, D. 1959. Studies on slime accumulations in pulp and paper mills. 111. The stimulation of growth by "white watert'. Can. J. Bot. - 37: 517-521.
Brewer, D. 1960. Studies on slime accumulations in pulp and paper mills. IV. Fungal floras of slime accumulations. Tappi 43: 609-611.
Brewer, D. 1961. Studies on slime accumulations in pulp and paper mills. V. preliminary ob- servations on the effect of extracts of spruce and fir on the growth of Phialophora fastiqiata. Can. J. Bot. - 39: 1579-1583.
Casey, J.P. 1960. Pulp and Paper Chemistry - and Chemical Technoloqy. Interscience Publishers, London.
Cavaliere, A.R. 1968. Marine fungi of Iceland: a preliminary account of Ascomycetes. Mycologia 60: 475-479. -
Cochrane, V .W. 1958. Physioloqy of Funqi . John Wiley & Sons, New York, London, Sydney.
Cooke, W.B. 1954. Fungi in polluted water and sewage. I. Literature review. Sewage and Industrial Wastes ~ngineering 26: 539-549.
Cooke, W.B. 1954. Fungi in polluted water and sewage. 111. ~ungi in a small polluted stream. Sewage and Industrial Wastes Engin- eering 26: 790-794.
Cooke, W.B. 1957. Nutritional requirements of nine common sewage fungi. Sewage and Industrial Wastes 29: 1243-1251.
Cooke, W.B. and K.A. ~usch. 1957. Activity of cellulose-decomposing fungi isolated from
sewage-polluted water. Sewage and Industrial Wastes - 29: 210-217.
21. Cooke, W.B. 1957. Natural and induced fungal degradation of lignin. Tappi 40: 301-306.
22. Cooke, W.B. and A.F. Bartsch. 1959. Aquatic fungi in water with high waste loads. Sewage and Industrial Wastes 31: 1316-1322.
23. Cooke, W.B. and A.F. Bartsch. 1960. Aquatic fungi in some Ohio streams. Ohio Jour. Sci. 60: 144-148. -
24. Cooke, W.B. 1967. Fungal populations in relation to pollution of the Bear River, Idaho-Utah. Utah Acad. Proc. - 44: 298-315.
25. Cooke, W.B. and G.S. Matsuura. 1969. Distribu- tion of fungi in a waste stabilization pond system. Ecology 50: 689-694.
26 ." Courtright, R.C. and C.E. Bond. 1969. Potential toxicity of Kraft mill effluent after oceanic discharge. The progressive Fish Culturist - 31: 207-212.
27. Cribb, A.B. and J.W. Cribb. 1960. Marine fungi from Queensland-IV. Pap. Univ. Queensland Dep. Bot. - 4: 39-44.
28. curtis, E.J.C. 1969. Sewage Fungus: its nature and effects. Water Research 3: 289-311.
29. Darby, R.T. and D.R. Goddard. 1950. Studies of the respiration of the mycelium of the fungus Myrothecium trerrucaria. Arner. Jour. Bot. - 37: 379-387.
30. Darken, M.A. 1962. Absorption and transport of fluorescent brighteners by microorganisms. Appl. Microbiol. 10: 387-393.
31. Das, B .S., S .G. Reid, J.L. Betts, and K. Patrick. 1969. Tetrachloro-o-benzoquinone as a component
in bleached Kraft chlorination effluent toxic to young salmon. Jour. ~ish. Res. Bd. Canada 26: 3055-3067. -
Earwaker, Jack, Technical Control Department, Canadian Forest Products, Port Mellon, B.C. 1969 (October). Personal communication.
33. " Enkvist, T. and Backlund, C. 1955. Some bio- chemical experiments with spent liquors (English summary). Paper and Timber, Finland 37: 53-60. -
Eveleigh, D.E. and D. Brewer. 1963. Studies on slime accumulations in pulp and paper mills. VI. Isolation of thermophilic and thermotol- erant fungi from paper mills. Can. J. Bot. 41: 1377-1382. -
~veleigh, D.E. and D. Brewer. 1964. Ecological observations on the fungi and bacteria in slime accumulations in a paper mill. Can. J. Bot. - 42: 35-43.
Eveleigh, D.E. and D. Brewer. 1964. Nutritional requirements of the microflora of a slime accu- mulation in a paper mill. Can. J. Bot. 42: 341-350.
Fraud, G.C., Walden, C.C. and T.E. Howard. 1970. Effluent study-Port Mellon. B.C.R.C. Project No. 1316. (unpublished).
38. Gold, H.S. 1959. ~istribution of some lignicolous Ascomycetes and Fungi ~mperfecti in an estuary. Jour. Elisha Mitchell Sci. Soc. - 75: 25-28.
39. ' Grande, M. 1964. Water pollution studies in the river Otra, Norway. Effects of pulp and paper mill wastes on fish. Int. J. Air. Wat. Poll. 8: 77-88. -
Greer, B.A., R.E. ~illespie and P.C. Trussel. 1956. ~iochemical oxygen demand of total effluent from
full-bleach Kraft mill. Tappi 39: 599-603.
Gustafsson, U. and N'. Fries. 1956. Nutritional requirements of some marine fungi. Physiologia Plantarum - 9: 462-465.
42. ' Haider, K. and K.H. Domsch. 1969. Decomposition and transformation of lignified plant material by microscopic soil fungi (English summary). Arch. Mikrobiol. Berlin - 64: 338-348.
43. Hardon, M.S. 1970. Response of the intertidal crab Hemiqrapsus oreqonensis to Kraft pulp mill effluent in the coastal marine environment. B. Sc. Thesis, University of British Columbia.
Harvey, J.V. 1952. elations ship of aquatic fungi to water pollution. Sewage and Industrial Wastes Engineering - 24: 1159-1164.
Hawker, L.E. 1950. Physioloqy of Funqi. Cramer, New York .
Hchnk, W. 1956. Studien zur brack-und seewasser- mykologie VI. tjber die pilzliche Besiedlung verschieden salziger submerser standorte. Ver6ff. Inst. Meeresforsch. Bremerhaven - 4: 195-213.
47 .Y' Howard, T.E. and C.C. walden. 1965. Pollution and toxicity characteristics of Kraft pulp mill effluents. Tappi 48: 136-141.
48. Hughes, G.C. 1960. Ecoloqical aspects -- of some liqnicolous funqi in estuarine waters. Doctoral Dissertation: Florida State University, Tallahassee, Florida. cited in: Johnson, T.W. and F.K. Sparrow. Funqi in Oceans and Estuaries. - Weinheim, J: Cramer, 1961.
Hughes, G.C. 1968. ~ntertidal lignicolous fungi from Newfoundland. Can. J. Bot. 46: 1409-1417.
Hughes, G.C. Marine fungi from British Columbia: occurrence and distribution of lignicolous
species. Syesis 2: 121-140. - Hughes, G.C. 1969 (May). Personal communication.
Johnson, T.W. and F.K. Sparrow. 1961. ~ungi in Oceans and Estuaries. J. Cramer, Weinheim.
Jones, E.B.G. 1963. Marine fungi. 11. Ascomy- cetes and Deuteromycetes from submerged wood and drift Spartina. Trans. Brit. Mycol. Soc. 46: 135-144. -
d 54. Kleinert, T.N. and C.S. Joyce. 1955. Lignosul-
phonic acids as mold nutrients. Pulp and Paper Magazine of Canada 56: 206. -
55. Kohlmeyer, J. and E. Kohlmeyer. 1964. Synoptic Plates of Hiqher Marine ~unqi. J. Cramer, - Weinheim.
Kohlmeyer, J. 1968. Marine fungi from the tropics. Mycologia 60: 252-270. -
Lagerberg, T., G. Lundberg, and E. eli in. 1927. ~iological and practical researches into blueing in pine and spruce. Svenska skogsvforen. tidssk. 25: 56-739.
58. Lindgren, R.M. and W.E. Eslyn. 1961. ~iological deterioration of pulpwood and pulp chips during storage. Tappi 44: 419-429.
59. Macey, J. ~ e a d , ~echnical Control Department, Cana- dian Forest Products, Port Mellon, B.C. 1969 (~ay). Personal communication.
60. Macey, J. Head, Technical Control Department, Cana- dian Forest Products, Port Mellon, B.C. 1971 (~ebruary). Personal communication.
61. Marton, J., A.M. Stern and T. Marton. 1969. De- colorization of Kraft black liquor with Poly- porus versicolor, a white rot fungus. Tappi 52: 1975-1981. -
McLeese, D.W. 1970. ~ e h a v i o u r o f l o b s t e r s exposed t o bleached K r a f t m i l l e f f l u e n t . J o u r . F i s h . Res. Bd. Canada - 27: 731-736.
Melin, E. and J . A . Nannfeldt . 1934. Researches i n t o t h e b l u e i n g o f ground wood-pulp. Svenska Skogs. T ids sk r . - 32: 397-616.
Meyers, S.P. and E.S. Reynolds. 1960. Occurrence o f l i g n i c o l o u s f u n g i i n n o r t h e r n A t l a n t i c and P a c i f i c marine l o c a l i t i e s . Can. J. Bot. - 38: 217-226,
65. J Meyers, S.P. , P r i n d l e , B. and E.S. Reynolds. 1960. C e l l u l o l y t i c a c t i v i t y of marine f u n g i . Degrada- t i o n o f l i g n o - c e l l u l o s e m a t e r i a l . Tappi 43: 534-538.
Meyers, S.P. and E. S c o t t . 1968. C e l l u l o s e de- g r a d a t i o n by Lulworthia f l o r i d a n a and o t h e r l i g n i c o l o u s marine f u n g i . Marine B i o l . 2: 41-46.
Moore, R.T. and S.P. Meyers. 1962. Thalass iomycetes 111. The genus Za le r ion . Can. J. ~ i c r o b i o l . - 8 : 407-416.
68. o k a i c h i , T. and Y. ~ k i t a k a . 1969. The e f f e c t s of s u l f i d e p u l p was t e s on t h e growth o f marine phytop lankton . B u l l . Plankton Soc. Japan 16: 126-132.
P rende rgas t , A.G. 1948. The s l i m e f l o r a o f B r i t i s h paper and board m i l l s . The Paper-maker and ~ r i t i s h Paper Trade J o u r n a l 116: TS 21-26.
70. ~ u e n o v i l l e , M.H. 1950. ~ n t r o d u c t o r y s t a t i s t i c s . But te rwor thyspr inger , London.
71. ~ i t c h i e , D . 1957. S a l i n i t y optima f o r marine f u n g i a f f e c t e d by tempera ture . Amer . Jour . Bot . 44: 870-874.
72. Robak, H. 1932. ~nvestigations regarding fungi on Norwegian ground wood pulp and fungal infection of wood pulp mills. Nytt. Mag. Naturv. 2: 185-330.
73. Roberts, D. 1965. Expansion at Port Mellon. Pulp and Paper Magazine of Canada 66: 77-86.
74. Romano, A.H. 1958. ~umaric acid fermentation of spent sulphite liquor. Tappi - 42: 687-689.
75. Schaumberg, F.D., T.E. Howard, and C.C. Walden. 1967. A method to evaluate the effects of water pollutants on fish respiration. Water Research - 1: 731-737.
76. Servizi, J.A., E.T. Stone and R.W. Gordon. 1966. Toxicity and treatment of Kraft pulp bleach plant waste. International Pacific Salmon Fisheries Commission Progress Report #3.
77. Servizi, J.A., R.W. Gordon and D.W. Martens. 1968. Toxicity of two chlorinated catechols, possible components of Kraft pulp mill bleach waste. In- ternational Pacific Salmon Fisheries Commission Progress Report #l7.
78. Sguros, P.L. and J. Sirnms. 1963. Role of marine fungi in the biochemistry of the oceans. 11. Effect of glucose, inorganic nitrogen, and TRIS (hydroxymethyl) aminomethane on growth and pH changes in synthetic media. ~ycologia 55: 728- 741.
79. Sladeckova, A. 1963. Aquatic Deuteromycetes as indicators of starch campaign pollution. Int. Revue ges. Hydrobiol. 48: 35-42.
80. ~drensen, T. i948. A method of establishing groups of equal amplitude in plant sociology based on similarity of species content. Det. ~ongelige danske vidensk. selsk. Biol. skc. 9 . V . Nr. 4 34 p.
Sprague, J.B. and D.W. McLeese. 1968. Toxicity of Kraft pulp mill effluent for larval and adult lobsters, and juvenile salmon. Water Research 2: 753-760.
Sprague, J.B. and D.W. McLeese. 1968. Different toxic mechanisms in Kraft pulp mill effluent for two aquatic animals. Water Research - 2: 761-765.
Strickland, J.D.H. and Parsons, T.R. 1968. & Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada Bulletin #l67, Ottawa.
Tono, T., Y. Tani and K. Ono. 1968. Microbial treatment of agricultural and industrial wastes. I. Adsorption of lignins and clarification of lignin-containing liquor by moulds. J. Ferment. Tech. 5: 569-570.
Umbreit, W.W., R.H. Burris and J.F. Stauffer. 1964. Manometric --- Techniques. Burgess, Minneapolis.
U.S. Department of the Interior Federal Water Pol- lution Control Administration. 1967. Pollution- a1 Effects of Pulp and Paper Mill Wastes in - - -- Puqet Sound. Washington State Pollution Control Commission, olympia, Washington.
Van Beyma thoe Kingma, F.H. 1943. Beschreibung der im Centraalbureau voor schimmelcultures vorhand- enen arten der gattungen Phialophos Thaxter und Marqarinomyces Laxa nebst schks.sel zu ihrk best- imrnung. Antonie van Leeuwenhoek Jour. Microbial. and Serology,g: 51-76.
vishniac, H.S. -1956. On the Ecology of the Lower Marine Fungi. 0 1 . Bull : 410-414.
Walden, C.C. and T.E. Howard. 1968. A cooperative research program on Kraft pulp mill effluent quality. Pulp and Paper Magazine of Canada 69: 3-7.
90. Waldichuk, M. 1956. Pulp mill pollution in Alberni Harbour, British columbia. Sewage and Industrial Wastes 28: 199-205.
91. Waldichuk, M. 1958. Some Oceanographic characteris- tics of a polluted inlet in British Columbia. Jour. Mar. Res. 17: 536-551.
92. Waldichuk, M. 1962. Review of water pollution research in British Columbia. Presented at the annual meeting of the ~isheries Research Board of Canada, Jan 4-6, 1962.
93. Waldichuk, M. 1968. Waste disposal in relation to the physical environment-oceanographic aspects. Syesis 2: 4-27.
94. Wang, C.J.K. 1961. preliminary report on the fungous flora of pulp and paper in New York. Tappi 44: 785-788.
95. 1 Werner, A.E. 1963. Sulphur compounds in Kraft pulp mill effluents. Canadian Pulp and Paper Industry 16: 35-43. -
96. J Werner, A.E. 1965. Suspended solids from mill efflu- ents. Canadian Pulp and Paper Industry - 66: 109- 114.
97. Werner, A .E. and w .F. Hyslop. 1967. Distributions of Kraft mill effluent in a British Columbia harbour. Jour. Fish. Res. Bd. Canada 24: 2137- 2153.
98. Werner, A.E. 1963. Gases from sediments in polluted coastal waters. Pulp and Paper Magazine of Canada 69: 61-70. -
99. zerbe, W.B. and C.B. Taylor. 1953. Coast and Geo- detic Survey. U.S. Dept. Commerce, Special Publication #298.
APPENDIX I
Table A-1 . Analysis of effluent and water samples for
total sulphate, hydrogen sulphide, sulphite,
total chlorides, available chlorine, car-
bonate and bicarbonate. Sample No. 1 = Port
Mellon near outfall, sample No. 2 = Horseshoe
Bay, sample No. 3 = acid effluent, sample
No. 4 = caustic effluent.* - indicates less
than 1.0 ppm.
Sample 1 Sample 2 Sample 2 Sample 4
Total sulphate 910 ppm 1,383 ppm 88 PPm 72 PPm
Hydrogen sulphide
Sulphite - - - 5.5 ppm
Total chlorides 5,600 ppm 9,275 ppm 1,050 ppm - ~vailable chlorine - - - - Carbonate
Bicarbonate
* Analysis carried out by Coast Eldridge Professional
Services Division, Warnock Hersey International.
CURRICULUM VITAE
Name :
Place and Year of Birth:
ducat ion :
Experience:
Awards :
Leslie Marian Churchland
Vancouver, British Columbia, 1945
University of British Columbia, B.A., 1966.
Simon Fraser University, ~ i o - logical Sciences, Graduate Studies, 1969-1971.
Research Assistant, Department of Botany, University of Brit- ish Columbia, 1963-1968.
Teaching Assistant, Simon Fraser University, 1969-1970.
B.C. Government Scholarship, 1962-1966.
National Research Council Scholarship, 1969-1971.
publications:
Anastasiou, C.J. and L.M. churchland. 1968. An Olpidiopsis parasitic on a marine fungus. Syesis 1: 81-85.
Anastasiou, C.J. and L.M. Churchland. 1969. ~ungi on decaying leaves in marine habitats. Can. J. Bat. 47: 251-257.
