Final report:
AN INVESTIGATION OF HERNIATIONS
IN GREAT LAKES ZOOPLANKTON
Report to the
MICHIGAN GREAT LAKES PROTECTION FUND
Office of the Great Lakes
Lansing, Michigan
11 June 2002
David J. Jude and Mohammed Omair, Center for Great Lakes and Aquatic Sciences,
University of Michigan, Ann Arbor, MI 48109-1090
Richard Rediske, Annis Water Resources Institute, Grand Valley State University, 740
Shoreline Drive, Muskegon, MI 49441
Bernard Naylor and Theodore Beals, Department of Pathology, University of
Michigan, Ann Arbor, MI 48109
Sonia Bellon, 784 rue de St Denis, 45560 St Denis en Val, France
TABLE OF CONTENTS
LIST OF TABLES ......................................................................................................................................... 3
LIST OF FIGURES ........................................................................................................................................ 3
ABSTRACT ................................................................................................................................................... 5
INTRODUCTION .......................................................................................................................................... 6
METHODS..................................................................................................................................................... 7
ZOOPLANKTON COLLECTION TECHNIQUES................................................................................... 7
LABORATORY COUNTING TECHNIQUES ......................................................................................... 7
BIOASSAY TECHNIQUES ...................................................................................................................... 7
Introduction ............................................................................................................................................ 7
Culture Methods for Cyclops bicuspidatus............................................................................................. 7
Exposure Media Preparation .................................................................................................................. 8
Experimental Design .............................................................................................................................. 9
RESULTS......................................................................................................................................................11
ARCHIVED SAMPLES EXAMINED FOR HERNIATIONS .................................................................11
DISTRIBUTION AND INCIDENCE .......................................................................................................13
HISTOLOGICAL FINDINGS ..................................................................................................................18
BIOASSAY TESTS ..................................................................................................................................27
Chemical Measurements........................................................................................................................27
Bioassay Test Results ............................................................................................................................27
CONCLUSIONS ...........................................................................................................................................29
LITERATURE CITED ..................................................................................................................................31
APPENDICES ...............................................................................................................................................33
Table A-1. Zooplankton food supplement preparation (blended and stored at 4 C). ................................33
Table A-2. Diatom culture solution (EPA 1993). .....................................................................................33
Table B-1. Bioassay testing schedule of events and measurements. ........................................................42
Table C-1. Chemical measurements for the bioassay test with Cyclops bicuspidatus..............................43
Table C-2. Summary of dissolved oxygen and temperature measurements in the Cyclops bicuspidatus
experiments. ..............................................................................................................................................44
2
LIST OF TABLES
Table 1. Treatments applied to Cyclops bicuspidatus in an effort to induce herniations.
Cyclops was collected from an inland pond near Muskegon, MI, 2001.
Table 2. Test conditions for conducting a 20-day bioassay test with Cyclops
bicuspidatus in order to attempt to induce herniations. Tests were run during 2001.
Table 3. Zooplankton collected from 1976, 1978-1981, and 1985 that contained one or
more herniations. Data displayed as percentage of the total number affected and split by
group, (e.g., adults (male and female) and immature individuals) based on examination of
105 samples from the following years: 1976 (n=75), 1978 (n=18), 1979 (n=1), 1980
(n=2), 1981 (n=2), and 1985 (n=7).
Table 4. Summary of the sizes of herniations measured on calanoid and cyclopoid
copepods taken from samples collected at 3 and 6 m in Lake Michigan near Muskegon,
MI during 2001. Body length was also measured along with diameter of the protrusions.
Table 5. Summary of Cyclops bicuspidatus survival and abnormality data obtained
during the 20-day bioassay tests, 2001.
Table 6. Summary of Dunnett’s Test Analysis of Cyclops bicuspidatus survival data
obtained during the 20-day bioassay tests used to attempt to induce herniations.
Tests were run during 2001. Dunnett’s critical value = 2.4800 with a 1- tailed test, α =
0.05.
LIST OF FIGURES
Figure 1. Mean yearly average percent zooplankton with herniations at 3 and 6 m based
on total counts per sample.
Figure 2. Mean yearly average percent zooplankton with parasites at 3 and 6 m based on
total counts per sample.
Figure 3. Percent zooplankton herniation incidence by depth/date.
Figure 4. Yearly mean percentage of herniations per taxa among those affected with
herniations.
Figure 5. Yearly mean percentage of “parasites” per taxa among those affected by
parasites.
Figure 6. Example of a hypothesized ellobiopsid parasite (see Bridgeman et al. 2001)
found on Eurytemora spp. collected 15 June 2001 at 3 m from eastern Lake Michigan,
3
Muskegon, MI. Note the elongated form of the “parasitic growth” emerging from a
fissure between the metasomal plates.
Figure 7. Closeup of the hypothesized ellobiopsid parasite (see Bridgeman et al. 2001)
found on Eurytemora spp. collected 15 June 2001 at 3 m from eastern Lake Michigan and
shown in Fig. 6. Note the elongated form of the “parasitic growth” emerging from a
fissure between the metasomal plates.
Figure 8. Example of the “round” form of the herniations found on Eurytemora spp.
collected 31 May 2001 at 3 m from eastern Lake Michigan. Note its appearance on the
dorsal side of the zooplankter and its emergence from between the metasomal plates.
Figure 9. Example of the “round” form of the herniations found on Epischura spp.
collected 31 May 2001 at 3 m from eastern Lake Michigan, Muskegon, MI. Note its
appearance on the lateral side of the zooplankter and its emergence from between the
metasomal plates.
Figure 10. Closeup of the “round” form of the herniations noted in Fig. 9. The herniation
was found on Epischura spp. collected 31 May 2001 at 3 m from eastern Lake Michigan,
Muskegon, MI.
Figure 11. Example of the “round” form of the herniations found on Epischura spp.
collected 31 May 2001 at 3 m from eastern Lake Michigan, Muskegon, MI. Note its
appearance on the urosome area of the zooplanker and its emergence from between the
metasomal plates.
Figure 12. Closeup of the “round” form of the herniation found on Epischura spp. (see
Fig. 11) collected 31 May 2001 at 3 m from eastern Lake Michigan, Muskegon, MI.
Note its appearance on the urosome area of the zooplanker and its emergence from
between the metasomal plates.
Figure 13. Example of the “round” form of the herniations found on Epischura spp.
collected 31 May 2001 at 3 m from eastern Lake Michigan, Muskegon, MI. Note its
emergence from between the metasomal plates and how it spread along the intersection
of the plates unlike most other lesions of this type.
4
ABSTRACT
Great Lakes zooplankton, a critical link in the food chain, have developed lesions first
referred to as exophytic tumors and later determined to be herniations, which in gross
appearance match those described in zooplankton of the Gulf of Taranto and Lago
Maggiore, Italy. We observed these herniations in samples collected as early as 1863
from Lake Superior, the 1970s, and during the late 1990s; southwestern Ohio ponds; and
from other Great Lakes. We observed two types of lesions: oval and tubular. Bridgeman
et al. (2000) suggested that lesions (long tubular ones) he found on zooplankton in a
Michigan inland lake were due to parasites (Ellobiopsidae), which are marine organisms,
postulated to have arrived here via freighters.
Incidences (percentage of the total counts of all zooplankton in a sample) of the
tubular lesions were low (ca. 0.5%), while the oval ones were more numerous and
attained highest incidences in 3 m of water (up to 5% of total zooplankton by number),
less so in 6 m, and very low in 100 m of water in eastern Lake Michigan. On average,
46% of the common zooplankton taxa were affected, including all life stages and both
sexes of copepods and rotifers. Histological analysis of the “round” lesions showed that
they are composed of apparently viable or necrotic tissue that has been extruded from the
organism through a fissure in the exoskeleton. At their base, the herniations are
continuous with viable tissue within the organism. The tumors are composed of a solid
mass of small round cells of uniform size with high nucleocytoplasmic ratios, round
nuclei, coarse chromatin and prominent nucleoli. These herniations are unique and
apparently lethal lesions (Omair et al. 2001).
A series of bioassays were conducted using Cyclops bicuspidatus as the test organism
to determine if the tumor-like abnormalities (e.g., herniations) previously observed in
Lake Michigan (Omair et al. 1999) could be induced in laboratory exposures. Various
exposure media were utilized including rainwater, Lake Michigan water, Grand River
water, and culture water from aquaria containing zebra mussels.
Tumor-like
abnormalities were not observed in any of the zooplankton tested with the exposure
treatments used. Data analyses using Dunnett’s Test and Tukey’s Method of Multiple
Comparisons found no difference between control and exposure groups with respect to
survival during the test. The exposures used for these experiments were designed to
screen for various causative agents. The absence of the reported abnormalities in these
experiments may indicate several possibilities:
 a labile chemical is responsible that decomposed during sample storage or
adsorbed on the filter media
 a labile organism is present that did not survive the laboratory manipulations
 a longer exposure period is required
 life cycle stages and interactions with other environmental variables may be
responsible.
 A more susceptible species may be required to show the herniation effects.
These factors could be examined by life cycle bioassays or in situ exposures. A recent
technique for in situ exposures was recently described (Pereira et al. 1999) that may be
useful for this type of investigation. Reasons for what causes these herniations remain a
mystery. Widespread induction of such lesions in zooplankton may be a worldwide
phenomenon.
5
INTRODUCTION
During examination of samples from 1995 to 1998, we found strange protruding
growths on zooplankton (Limnocalanus macrurus) collected from eastern Lake Michigan
(Omair et al. 1999). A review of the literature revealed similar “cysts” were found on
zooplankton from marine systems. One species of affected copepod in Lago Maggiore,
Italy has disappeared (Manca et al. 1996). Zooplankton play an important role in the
ecosystem as a critical intermediary in the food chain, converting algae to invertebrate
tissue. Therefore the presence of these herniations may indicate a phenomenon
portending grave consequences for the organisms themselves and those ingesting them at
higher trophic levels. In some of the Great Lakes, zooplankton populations are being
negatively affected by phosphorus declines, competition from zebra mussels, which filter
algal food resources (Bridgeman et al. 1995), and predation from the exotic zooplankters
Bythotrephes cederstroemi and Cercopagis pengoi (Charlebois et al. 2001). Zooplankton
affected by these lethal lesions will be subjected to an additional mortality vector on
populations already stressed. A noticeable decline in zooplankton has already been noted
and may be related to the yellow perch decline in Lake Michigan.
This study attempts to accomplish three major goals regarding the sudden
appearance of unusual growths or herniations in zooplankton of the Great Lakes. We
first wanted to establish whether these abnormalities had occurred in the past, so we have
attempted to examine historical samples both provided by the Smithsonian Institute and
in our laboratories from past studies in the 1970s. Second, we wanted to document the
current distribution of these herniations in samples of zooplankton in which we first
found these abnormalities, ongoing yellow perch recruitment studies in eastern Lake
Michigan. We therefore examined the samples collected from 1998 to 2001 for the
densities of all zooplankton taxa and the occurrences of herniations on these organisms.
Lastly, we have initiated a series of bioassays to attempt to induce these herniations in
cultured zooplankton by introducing a series of treatments, including rain water, zebra
mussel filtrate, and water from several different water bodies. The work plan can be
summarized as follows:
1. Document, using archived and newly collected samples, the depth distribution,
seasonal incidence, and variability among zooplankton species in the occurrence
of these tumors,
2. Perform additional histological and cytological analyses on these tumors and
lesions,
3. Conduct preliminary bioassay experiments to determine if these lesions can be
induced in the laboratory. Bioassay exposures were designed to screen for groups
of causative agents that may influence Lake Michigan zooplankton.
6
METHODS
ZOOPLANKTON COLLECTION TECHNIQUES
As part of a study of yellow perch recruitment, we collected zooplankton at a 3and a 6-m deep station south of Muskegon, MI during1998- 2001. Samples were
collected mostly during June-August, but others were collected on occasion in May and
September. Zooplankton were collected with a 0.5-m diameter, 63-μm-mesh net pulled
vertically from near bottom to the surface. A flowmeter was used to calculate volume of
water filtered and thereby densities in no./L. Samples were reduced in volume and
preserved in alcohol.
LABORATORY COUNTING TECHNIQUES
Samples of zooplankton collected in Lake Michigan were successively split until
a reasonable sub-sample of at least 100 organisms was present. These samples were
counted in the Center for Great Lakes and Aquatic Sciences Fishery Laboratory at the
University of Michigan using a circular glass counting ring, which allowed examination
and counting using a microscope. Numbers were converted to densities using flow meter
readings. Additional samples were collected alive on occasion and copepods with
herniations removed for histological analyses.
BIOASSAY TECHNIQUES
Introduction
The cyclopoid copepod, Cyclops bicuspidatus, was used as the test organism.
The bioassay testing was performed at the Annis Water Resources Institute at Grand
Valley State University. Bioassays to evaluate the incidence of tumor-like abnormalities
were conducted under laboratory conditions using a 20-day exposure. The standard
bioassay method for Ceriodaphnia spp. as described in EPA (1993) was modified for
calanoid copepods (Hook and Fisher 2001) to include the feeding of a diatom supplement
and the use of large exposure chambers. Initially, Cyclops spp., Limnocalanus spp., and
Diaptomus spp. were isolated from Lake Michigan and evaluated for their ability to grow
under laboratory cultures. Reproductive cultures for the latter two organisms could not
be maintained in the laboratory. Cyclops spp. was found to reproduce under laboratory
conditions. To eliminate the possibility of interference from herniations in the Lake
Michigan zooplankton population, Cyclops bicuspidatus was isolated from a small pond
in Allendale, MI. No incidence of tumor-like abnormalities was observed in the
population from this location.
Culture Methods for Cyclops bicuspidatus
Cyclops bicuspidatus was cultured in 8-L plastic aquaria. Culture methods were
similar to the protocol for Ceriodaphnia spp. as described in EPA (1993). The method
7
was modified for calanoid copepods (Hook and Fisher 2001) to include the feeding of a
diatom supplement. Moderately hard, well water was used for the water source. The
organisms were fed 10 mL YTC suspension and 10 mL mixed diatom culture on alternate
days. Preparation methods for both food sources are described in Appendix A. The
diatom culture was a mixed suspension of Asterionella formosa, Fragilaria crotonensis,
and Tabellaria fenestrata isolated from Muskegon Lake. The diatom culture contained
approximately 1x106 cells/mL. A temperature of 15 + 1C and a photoperiod of 16 h
light and 8 h dark were used.
Exposure Media Preparation
The following series of exposure media were prepared to screen for the causative
agent of the tumor-like abnormalities:
Table 1. Treatments applied to Cyclops bicuspidatus in an effort to induce herniations.
Cyclops was collected from an inland pond near Muskegon, MI, 2001.
________________________________________________________________________
________________________________________________________________________
Laboratory Exposure
Potential Causative Agent
Rain water adjusted for pH and hardness
Airborne chemical
Lake Michigan water, 0.45 µm filtered
Dissolved chemical
Lake Michigan water, 10 µm filtered
Microbiological organisms
Lake Michigan water, 60 µm filtered
Parasitic organisms
Zebra mussel culture water, 0.45 µm
Dissolved chemical excreted by zebra
filtered
mussels
Zebra mussel culture water, 60 µm filtered Organism associated with zebra mussels
Grand River Water, 60 µm filtered
Chemical or organism in a major tributary
________________________________________________________________________
In more detail, each treatment consisted of:
1. Control. Moderately hard, well water with no pretreatment.
2. Rain water. Water was collected from a rain event on 10 September 2001. The
ionic composition was adjusted using 192 mg/L NaHCO3, 120 mg/L CaSO4, 120
mg/L MgSO4, and 8 mg/L KCl. A single batch of 20 L of buffered rainwater was
prepared and stored at 40C. This exposure would screen for an airborne
contaminant.
3. Lake Michigan Water. Near-shore water from Lake Michigan near the
Muskegon Lake Channel was collected at the 20-m depth contour. The collection
depth of the water sample was 3 m. Water was filtered using a 0.45-µmmembrane filter, a 1-µm nylon mesh, and a 6-µm nylon mesh. Collections were
made on odd-numbered days and filtered for use as bioassay renewal water. The
8
filtrate from each of these aliquots would screen for the presence of dissolved
contaminants, microbiological organisms, and larger parasites.
4. Zebra Mussels.
The potential influence of zebra mussels (Dreissena
polymorpha) was examined by establishing a laboratory culture of the organism.
Approximately 100 mussels collected from Muskegon Lake were added to two
38-L aquaria. The aquaria were filled with water from Muskegon Lake and the
organisms were fed 50 mL of plankton concentrate on alternate days. The
concentrate was collected from Muskegon Lake with a 100-µm plankton net.
After 30 days, 2 L of water were removed from each aquaria and filtered using a
0.45-µm filter. A second, 2-L aliquot was removed and filtered through the 60µm-nylon mesh. Filtrates were prepared on odd-numbered days and used for
bioassay renewal water. These filtrates would screen for dissolved materials
excreted by zebra mussels and microorganisms/parasites associated with their life
cycle, respectively.
5. Grand River Water. A 2-L sample of water from the Grand River was collected
at the Eastmanville Bridge (Ottawa County, MI). The sample was filtered using a
0.4-µm filter. Filtrates were prepared on odd-numbered days and used for
bioassay renewal water. This exposure would screen for dissolved contaminants
entering Lake Michigan from a major tributary.
Experimental Design
For the bioassay testing, eight replicates per exposure media were set up for
Cyclops bicuspidatus. The experimental conditions outlined in Table 1 were used for the
bioassay evaluations.
A complete listing of the schedule of events is provided in Appendix B. On day
0, the eight replicate trials of each exposure media were prepared. Measurement of water
quality parameters was also initiated on this day. Ten first/second copepodid stage
Cyclops bicuspidatus were then added to their respective test chambers. At this time, the
organisms were fed 1.0 mL of YTC suspension and 1.0 mL of diatom culture. The glass
beakers were placed in a rack and transferred to a temperature-controlled room (15 +
1oC). The light cycle was 16 h on and 8 h off. Temperature and dissolved oxygen
measurements were taken from one randomly selected beaker for each exposure media on
even numbered days, after which the overlying water was renewed in all the beakers. The
procedure for renewal included the removal of 200 mL by aspiration using a pipette
covered with cheesecloth. Fresh exposure solution was then gently added to each beaker.
Renewal waters were collected and prepared the previous day as described above.
Feeding occurred daily. This procedure was repeated through day 20, at which point the
test was terminated. On day 1, the overlying water from the beakers was composited
from each exposure media replicate sample and 250 mL were retained for alkalinity, pH,
conductance, hardness, and ammonia analysis. On the last day, the same procedure was
performed. On day 20, the surviving test organisms were removed, counted, and
examined for abnormalities on their metasomes.
9
Table 2. Test conditions for conducting a 20-day bioassay test with Cyclops bicuspidatus
in order to attempt to induce herniations. Tests were run during 2001.
_____________________________________________________________________
1.
Test Type: ..............................Water bioassay test with renewal
2.
Temperature (C): ..................15 + 1C
3.
Light quality: ..........................Wide-spectrum fluorescent lights
4.
Illuminance: ...........................About 500 to 1000 lux
5.
Photoperiod: ...........................16 h light, 8 h darkness
6.
Test chamber size:..................300 mL high-form, lipless beaker
7.
Water volume: ........................250 mL
8.
Cover:.....................................Plastic film
9.
Renewal of overlying water: ..100-200 mL addition on even numbered days
10.
Age of test organisms: ...........First to second copepodid stage
11.
Number of organisms
per chamber:...........................10
12.
Number of replicate
chambers per treatment: .........eight
13.
Feeding:..................................YTC suspension, fed 1.0 mL daily to each test
chamber (1.0 mL contains 10.0 mg of dry solids) +
1 mL diatom suspension (1X106 cells/mL)
14.
Aeration: ................................None, unless dissolved oxygen in overlying water
drops below 40% of saturation
15.
Overlying water: ....................Well water
16.
Overlying water quality:...…. Hardness, alkalinity, conductivity, pH, and
ammonia measured at the beginning and end of a
test. Temperature and dissolved oxygen measured
daily
17.
Test duration: .........................20 days
18.
End point: ...............................Survival, with greater than 70% in the control.
________________________________________________________________________
Modified Method from EPA/600/4-90/027F (EPA 1993).
10
RESULTS
ARCHIVED SAMPLES EXAMINED FOR HERNIATIONS
Since our first observations of tumor-like abnormalities (exophytic lesions) on
Lake Michigan copepods (Omair et al. 1999, 2000), our continued attempt to trace back
such occurrences in the past revealed that such growths occurred in the year 1863 in the
Great Lakes and maybe even earlier. Nine archival Great Lakes zooplankton samples
from the Smithsonian National Museum (courtesy of Janet Reid) were examined and we
found that some calanoid copepods bore lesions similar to those we discovered in the late
1990s-2001. These samples are noted below and came from a variety of locations:
1. On 29 August 1863, samples were collected in Ontario, Canada, Lake Superior at
74 fathoms. One Epischura lacustris had a tumor-like growth on its ventral side.
2. On 29 August 1882, another calanoid zooplankter (Diaptomus spp.) had a similar
growth.
3. Epischura lacustris we examined that were collected on 20 June 1928 from Lake
Erie exhibited two tumor-like growths.
4. Another sample from the Smithsonian (no. 201045 no date) also showed
Epischura lacustris with a herniation present.
We also examined our archival zooplankton samples collected during 1973-1982 at
the D. C. Cook Nuclear Power Plant in southeastern Lake Michigan. We found two types
of anomalies on these specimens: parasitic growths (tubular or encapsulated parasites –
Bridgeman et al. 2000) and tumor-like herniations (circular) in several samples. Many
copepod taxa were affected, including various life stages: nauplii, copepodite, and adults.
All taxa in these samples were counted and identified to species and percentage of such
growths was calculated. Archived zooplankton samples from 1976, 1978-1981, and 1985
were also examined to determine (1) if herniations were present in past samples, and (2)
to determine which groups were affected and the distribution among female and male
adults and immatures.
We also collected zooplankton with herniations from a number of other areas in
our quest to determine the spatial distribution of these protrusions and perhaps assist in
determining a cause for their appearance. Sites where we collected zooplankton with
herniations during the period 2000-2002 included: Straits of Mackinaw, Lake Michigan;
a small pond near Hamilton, Ohio; the Raisin River, tributary to Lake Erie; and the St.
Clair River.
Zooplankton that we collected during the 1970s-80s from southeastern Lake
Michigan near Stevensville, MI showed that about two-thirds of the herniations observed
were found in calanoids, while the other third were found on cyclopoids (Table 3). On
calanoids, data revealed that 96.7% of the protrusions were located on the metasome
(3.3% were on the urosome). On cyclopoids, all herniations were found on the
metasome. Therefore the protrusions tended to be localized on the anterior part of
metasome for both calanoids and cyclopoids. As copepod nauplii do not have
differentiated metasomes or urosomes, we categorized the body into three areas: front,
middle, and end of the body. Our observations revealed that 80.9% of the protrusions
11
were found in the posterior end of the body, 11.1% occurred in the middle of the body,
and the rest (7.9%) were anterior.
Some individuals, including both immature and mature organisms, bore more than
one herniation. For example, 20.5% of affected adult copepods had more than one lesion,
while for immature stages, 3.2% of affected individuals had multiple lesions.
Table 3. Zooplankton collected from southeastern Lake Michigan near the D. C. Cook
Plant, 1976, 1978-1981, and 1985. Zooplankton contained one or more herniations. Data
displayed as percentage of the total number affected and split by group (calanoids,
cyclopoids), adults (male and female), and immature individuals. Results based on
examination of 105 samples from the following years: 1976 (n=75), 1978 (n=18), 1979
(n=1), 1980 (n=2), 1981 (n=2), and 1985 (n=7).
Zooplankton group
Female adults
Male adults
Immatures (C1-C5)
Cyclopoids
33.3
20
46.7
Calanoids
50
21.4
28.6
The size of the herniations on calanoids and cyclopoids collected in 2001 at 3 and
6 m showed that the diameter of the protrusions ranged from 0.06 mm to 0.13 mm (Table
4). The ratio between diameter of the protrusions and total body length revealed that
from 6.4 to 19.7% of the body length of the organisms were taken up by the tumors.
Table 4. Summary of the sizes of herniations measured on calanoid and cyclopoid
copepods taken from samples collected at 3 and 6 m in Lake Michigan near Muskegon,
MI during 2001. Body length was measured along with diameter of the protrusions.
Taxa
Cyclops spp.
Cyclops spp.
Diaptomus spp.
Diaptomus spp.
Diaptomus spp.
Diaptomus spp.
Diaptomus spp.
Diaptomus spp.
Diaptomus spp.
Diaptomus spp.
Diaptomus spp.
Diaptomus spp.
Eurytemora
spp.
Eurytemora
spp.
Eurytemora
spp.
Depth (m)
3
6
6
6
6
6
6
6
6
6
6
6
Body Length
(mm)
0.81
0.91
0.94
0.84
0.73
0.70
0.88
0.69
0.56
0.97
0.80
0.59
Diameter
of Ratio-%:Tumor
Tumor (mm)
Dia./Body Len.
0.08
9.88
0.09
9.89
0.06
6.38
0.09
10.71
0.09
12.33
0.09
12.86
0.13
14.77
0.08
11.59
0.06
11.71
0.13
13.40
0.06
7.50
0.06
10.17
6
0.47
0.06
12.77
3
0.66
0.13
19.70
6
1.00
0.13
13.00
12
DISTRIBUTION AND INCIDENCE
The lesions that we examined were found on 46% of common zooplankton taxa
and 53% of the 32 zooplankton taxa collected from the Great Lakes and contiguous
waters. Immature stages and both sexes of copepods were affected as well as two species
of rotifers. Predators had higher incidences than herbivores. Limnocalanus was the only
species affected in deep offshore waters. Herniations were usually found on the dorsal
side of the organism
Recent Lake Michigan samples collected during 1998 through 2001 as part of
yellow perch recruitment failure studies near Muskegon, MI, were also examined for
such abnormal herniations. Thirty-eight zooplankton samples were processed for the year
1998 and there were 644 copepods found with tumor-like growths. Of these 644
copepods with herniations, the calanoid group was the most adversely affected (577 had
such growths), while cyclopoids were not as severely affected (only 67 had such
growths).
In 1999, 22 samples were examined. Nauplii had the highest number of such
growths (1,256), while calanoids and cyclopoids had 250 and 102 tumor-like
abnormalities, respectively. In the year 2000, nauplii were less affected than those
examined in 1999 (658), while calanoids were leading (718) and cyclopoids had only 43
abnormalities.
In 2001, 39 samples were processed. All crustacean zooplankton were
counted and identified to species and both abnormal growths thought to be parasites and
tumor-like growths (round type) were counted for each species. There were more tumorlike growths than parasite-like growths. The latest samples we collected in September
2001 and those collected during 5 June 2002 continued to show high incidences of these
abnormalities in nearshore Lake Michigan off Muskegon, MI. The incidence of
herniations (the round forms) and the elongate forms (we will designate them parasites –
see Bridgeman et al. 2000) was documented from 1999 to 2001 at 3 and 6 m at Lake
Michigan near Muskegon, MI (Fig. 1). Herniations were found on zooplankton in almost
every sample examined (95% of the total number examined), while the occurrence of
parasites in samples was almost half that of the herniations (48%). The incidence of
herniations ranged between 0.5 and 3.5% of the total zooplankton found in a sample
(Number of samples examined as follows: 1999-22, 2000-37, 2001-33). There was an
increase over time from 1999 at both depths in the incidence of tumors found on
zooplankton; values rose from about 1% in 1999 to almost 3.5% in 2001. There did not
appear to be any differences in incidences between 3- and 6-m depth contours.
For parasites on zooplankers, their incidence on zooplankton at 3 and 6 m during
1999-2001 rose initially from values around 0.2% to around 1% during 2000, then
declined back to levels around 0.5% (Fig. 2). There was a slightly higher incidence at 6
m than 3 m.
The seasonal distribution of the mean incidence of herniations and parasites at 3
and 6 m during 2001 varied from <1% to almost 7% of the total zooplankton examined
each period (Fig. 3). There were no consistent differences in incidences between depths
of 3 and 6 m. However, there was a pattern of highest incidences in spring (May) and a
decline to the lowest levels by the end of July.
13
When the incidence of herniations (round type) was broken down by taxon among
only those found with herniations, Diaptomus spp. over the 1998 to 2001 period was
usually the most affected group, composing from 31.4 to 74.2 % of the total (Fig. 4 –
sample size was: 1998 – 38 samples, 644 herniations identified; 1999 – 22 samples, 352
herniations identified; 2000 – 37 samples, 461 herniations identified; and 2001 – 33
samples, 10,612 herniations identified). There did not appear to be much of a pattern
over the years. The next two most-affected groups included Cyclops spp. and
Eurytemora spp., which composed about 5-40% of those affected, with the exception of
Cyclops spp. in 2001, which reached an incidence rate of 65.9 %. Limnocalanus spp. and
Epischura spp. were the other two groups, which composed <10.3 % of the groups
affected.
The elongated type of herniations (parasites – see Bridgeman et al. 2000) were
found most often on Eurytemora spp. and composed from 38.5 to 57.7 % of the
incidences among groups affected (Fig. 5 – sample size was: 1999 – 22 samples, 208
protrusions identified; 2000 – 6 samples, 284 protrusions identified; 2001 – 33 samples,
913 protrusions identified). Nauplii and Diaptomus spp. were the second-most affected
groups and their incidences averaged <40% (mostly around 20%) over the years.
Cyclops spp. had low incidences (<10%). There did not appear to be any consistent
patterns over the years, except for the dominance of parasites on Eurytemora spp.
14
Avg
Percentage
per Sample
Affected
FIG. 1. Mean yearly average
percent zooplankton with
herniations at 3 and 6 m based
on total counts per sample.
4
2
0
Tumors 3m
Tumors 6m
1999 2000 2001
Avg
Percentage
Affected
FIG. 2. Mean yearly average
percent zooplankton with
parasites at 3 and 6 m based on
total counts per sample.
2
1
0
Parasites 3m
Parasites 6m
1999
2000
15
2001
7/13/2001
6/29/2001
6/15/2001
6/1/2001
5/18/2001
7/27/2001
3m
6m
8.00%
6.00%
4.00%
2.00%
0.00%
5/4/2001
% affected
FIG. 3. Percent zooplankton herniation
incidence, by depth/date.
FIG. 4. Yearly mean percentage
of herniations per taxa among
those affected with herniations.
80
Cyclops
60
Diaptomus
Epischura
40
Eurytemora
20
Limnocalanus
0
1998
1999
2000
2001
16
FIG. 5. Yearly mean percentage
of parasites per taxa among
those affected by parasites.
Nauplii
Cyclops
Diaptomus
Eurytemora
100
50
0
1999
2000
2001
17
HISTOLOGICAL FINDINGS
In 1998, we presented an account of our first observation of tumors on several
species of zooplankton in the Great Lakes, which included detailed information on the
species affected and their distribution (Omair et al. 1999). Such abnormalities have also
been recorded in other species of zooplankton found in various “polluted seas” of the
world (Crisafi et al. 1977; Silina et al. 1996); and recently, calanoid copepods in Lago
Maggiore, Italy, were reported to bear exophytic lesions described as “cysts” (Manca et
al. 1996). Our recent data (Omair et al. 2000) show: 1.) herniations are the apparent
result of some type of wound or breakdown of the membrane between the metasomal
plates which burst and body tissue is forced out and then covered by a membrane, 2.)
there appear to be two types, tubular (Fig. 6, 7) and round (Fig. 8-13), 3.) the lesion tissue
originated from viable tissue(s) within the animal and was extruded through fissures in
the sutures between metasomal plates, 4) the material within the herniations contained
necrotic or apparently viable tissue, 5.) the lesions often have bacilli on their surface, but
not on the surface of the zooplankter, and 6) the lesions are thought to be lethal to the
organism.
Histological sectioning of these herniations occurred with the cooperation of the
University of Michigan medical school, Dept. of Pathology under Dr. Bernard Naylor’s
supervision. The elongated type (Fig. 6, 7) is thought to be Ellobiospis, a parasite
(Bridgeman et al. 2000), which is probably a fungus. The specimen shown in Fig. 6 and 7
is Eurytemora spp. and was caught during June 2001 in the shallow water of Lake
Michigan. The parasite is attached to the interface between the metasomal plates on the
zooplankter. The round form is the most common as noted above, and an example is
shown (Fig. 8) attached to Eurytemora spp., which was collected in May 2001. Again
this herniation is seen to be attached at the intersection of the metasomal plates and is on
the dorsal side of the zooplankter. Another example of the round form is demonstrated
for a different species, Epischura spp., which was also collected in May 2001 (Fig. 9,10).
This herniation is attached on the lateral side of the zooplankter and again at the
intersection of the metasomal plates. Epischura spp. also had one of the round types
attached to the dorsal surface of the urosome (Fig. 11, 12). We also found another subtype, which demonstrates how the herniations can spread along the interface of the plates
(Epischura spp. collected May 2001 – Fig. 13). In a few of the July 2001 samples, a
histological section of a calanoid copepod showed different kinds of cells than what had
been observed previously. These types of cells have never been seen before in our
studies, and an investigation is underway and fresh specimens are being collected to
generate a clear picture of these new cells so as to understand their nature. None of our
observations so far allow us to be certain about what type of cells these are. In all cases
for nauplii to adults, the growths mostly occur in the metasomal region of the
zooplankton carapace. The cause of these abnormalities is yet to be understood.
18
Figure 6. Example of a hypothesized ellobiopsid parasite (see Bridgeman et al. 2001)
found on Eurytemora spp. collected 15 June 2001 at 3 m from eastern Lake Michigan,
Muskegon, MI. Note the elongated form of the “parasitic growth” attached at the fissure
between the metasomal plates.
19
Figure 7. Closeup of the hypothesized ellobiopsid parasite (see Bridgeman et al. 2001)
found on Eurytemora spp. collected 15 June 2001 at 3 m from eastern Lake Michigan and
shown in Fig. 6. Note the elongated form of the “parasitic growth” attached to the fissure
between the metasomal plates.
20
Figure 8. Example of the “round” form of the herniations found on Eurytemora spp.
collected 31 May 2001 at 3 m from eastern Lake Michigan. Note its appearance on the
dorsal side of the zooplankter and its emergence from between the metasomal plates.
21
Figure 9. Example of the “round” form of the herniations found on Epischura spp.
collected 31 May 2001 at 3 m from eastern Lake Michigan, Muskegon, MI. Note its
appearance on the lateral side of the zooplankter and its emergence from between the
metasomal plates.
22
Figure 10. Closeup of the “round” form of the herniations noted in Fig. 9. The herniation
was found on Epischura spp. collected 31 May 2001 at 3 m from eastern Lake Michigan,
Muskegon, MI.
23
Figure 11. Example of the “round” form of the herniations found on Epischura spp.
collected 31 May 2001 at 3 m from eastern Lake Michigan, Muskegon, MI. Note its
appearance on the urosome area of the zooplanker and its emergence from between the
metasomal plates.
24
Figure 12. Closeup of the “round” form of the herniation found on Epischura spp. (see
Fig. 11) collected 31 May 2001 at 3 m from eastern Lake Michigan, Muskegon, MI.
Note its appearance on the urosome area of the zooplanker and its emergence from
between the metasomal plates.
25
Figure 13. Example of the “round” form of the herniations found on Epischura spp.
collected 31 May 2001 at 3 m from eastern Lake Michigan, Muskegon, MI. Note its
emergence from between the metasomal plates and how it spread along the intersection
of the plates unlike most other lesions of this type.
26
BIOASSAY TESTS
Chemical Measurements
Conductivity, hardness, alkalinity, ammonia, and pH were determined on the
culture water at the beginning and on Day 20 of each test (Appendix: Table C-1). All
water quality parameters, with the exception of ammonia, remained relatively constant (<
20% variation from start to end of test). Ammonia increased slightly ( 0.05 mg/L)
during the test due to breakdown of food and accumulation of excretory products.
Temperature and dissolved oxygen measurements were recorded on alternate days
throughout the duration of the tests (Appendix: Table C-2). Very little variation was
noted with respect to temperature.
Bioassay Test Results
Four to nine Cyclops bicuspidatus survived over the duration of all trials (Table
5). Untransformed survival data were evaluated for normality with Anderson-Darling’s
Test at  = 0.01 and the data were normally distributed. Dunnett’s Test (Table 6) showed
no statistically significant difference (α = 0.05) in mortality between the control and the
exposure media. Tukey’s Method of Multiple Comparisons was also used and no
statistical differences between control and exposure media were determined (Table 7).
No tumor-like abnormalities were observed in the control or exposure media groups at
the end of the 20-day bioassay. Since a survival rate of greater than 70% in the control
was observed, test results were considered valid.
In an effort to induce these herniations in zooplankton, Cyclops bicuspidatus was
reared in the laboratory and eight replicates were run for each treatment (control,
rainwater, zebra mussel water, Lake Michigan water, Grand River water) for 20 days.
These treatments were designed to determine if microbes, parasites, airborne or
waterborne chemicals, or some other organisms were responsible for inducing these
lesions in zooplanktons. No hernias or lesions were found in any of the control or
treatment organisms.
27
Table 5. Summary of Cyclops bicuspidatus survival and abnormality data obtained
during the 20-day bioassay tests, 2001. LKM = Lake Michigan, Zeb = Zebra mussel
treatment.
Sample
ID
Control
Rain
LKM 0.45 µm
LKM 10 µm
LKM 60 µm
Zeb 0.45 µm
Zeb 10 µm
Grand River
Abnormalities
Number of
Replicate
Survival
Present
Organisms A B C D E F G H Mean Std Dev
Initial
10 10 10 10 10 10 10 10
Final
8
Initial
10 10 10 10 10 10 10 10
Final
Initial
Final
Initial
Final
Initial
Final
Initial
Final
Initial
Final
Initial
Final
8
10
7
10
5
10
6
10
9
10
8
10
7
8
7
10
7
10
8
10
7
10
9
10
8
10
4
8
7
10
6
10
7
10
7
10
7
10
7
10
8
7
6
10
7
10
8
10
8
10
7
10
9
10
7
8
7
10
8
10
6
10
8
10
7
10
5
10
7
8
8
10
6
10
7
10
8
10
9
10
8
10
8
4
9
10
7
10
7
10
8
10
6
10
9
10
4
9
6
10
6
10
6
10
8
10
9
10
4
10
6
No
7.500
1.5119
7.250
1.0351
6.750
0.7071
6.750
1.0351
7.500
0.7559
7.875
1.2464
7.250
1.8323
6.375
1.5980
No
No
No
No
No
No
No
Table 6. Summary of Dunnett’s Test Analysis of Cyclops bicuspidatus survival data
obtained during the 20-day bioassay tests used to attempt to induce herniations. Tests
were run during 2001. Dunnett’s critical value = 2.4800 with a one-tailed test, α = 0.05.
---------------------------------------------------------------------------Dunnett's Test
Ho:Control<Treatment
---------------------------------------------------------------------------TRANSFORMED
MEAN CALCULATED IN
SIG
GROUP
IDENTIFICATION
MEAN
ORIGINAL UNITS
T STAT 0.05
----- ----------------------------------------------- ------ --1
Control
7.5000
7.5000
2
Rain
7.2500
1.0351
0.3747
3
LKM 0.45 µm
6.7500
6.7500
1.1241
4
LKM 10 µm
6.7500
6.7500
1.1241
5
LKM 60 µm
7.5000
7.5000
0.0000
6
Zeb 0.45 µm
7.8750
7.8750
-0.5621
7
Zeb 10 µm
7.2500
7.2500
0.3747
8
Grand River
6.3750
6.3750
1.6862
----------------------------------------------------------------------------
28
Table 7. Summary of Tukey’s Method of Multiple Comparisons Analysis of Cyclops
bicuspidatus survival data obtained during the 20-day bioassay tests.
___________________________________________________________
TRANSFORMED
GROUP IDENTIFICATION
MEAN
----- --------------- ----------8
Grand River
6.3750
4
LKM 10 µm
6.7500
3
LKM 0.45 µm
6.7500
7
Zeb 10 µm
7.2500
2
Rain
7.2500
5
LKM 60 µm
7.5000
1
Control
7.5000
6
Zeb 0.45 µm
7.8750
ORIGINAL
MEAN
--------6.3750
6.7500
6.7500
7.2500
7.2500
7.5000
7.5000
7.8750
0
8
\
.
.
.
.
.
.
.
GROUP
0 0 0 0 0 0 0
4 3 7 2 5 1 6
- - - - - - \
.
.
.
.
.
.
\
.
.
.
.
.
\
.
.
.
.
\
. \
. . \
. . . \
___________________________________________________________
* = significant difference (α = 0.05)
. = no significant difference
CONCLUSIONS
We found approximately half of the common zooplankton species in the Great
Lakes to have herniations; individuals of both sexes and all developmental stages were
affected. All lesions we observed (Omair et al., 1999, 2000) and those described
elsewhere (Crisafi et al., 1977; Silina et al., 1996; Manca et al., 1996) were exophytic and
occurred at various sites on the body, but usually on the dorsal surface at the intersections
of the metasomal plates. There appeared to be two types: 1.) many were large, solitary,
and smoothly round, whereas other variants were small or multiple or irregularly
contoured, and 2.) a small number were elongated and suspected by Bridgeman et al.
(2000) to be marine parasites (Ellobiopsidae). Most of our lesions were found in
preserved samples, but examination of live samples did reveal the presence of lesions on
live zooplankton, ruling out preservation techniques as a cause of the herniations, a
finding verified by Bridgeman et al. (2000).
One of the first concerns with these lesions was whether they were cancerous.
Presence of these protrusions on immature and adult zooplankton, and simultaneous
presence on many species (reminiscent of an infectious disease) are out of character for
the development of cancer. In addition, many histological cross-sectioned samples of the
herniations were observed, and all appeared to be either necrotic or viable tissue, with the
material originating from within the zooplankter. In several cases, ovarian tissue can be
clearly seen within the zooplankter (Omair et al. 2001), which was exuded into the
herniations and encapsulated with a fine membrane.
Since all but one zooplankter with lesions that we examined from unpreserved
samples were dead; whereas, those in the same sample without lesions were alive, we are
led to believe that these lesions are either lethal or lead to death by impairing movement
and feeding of affected zooplankton, thereby rendering them at a competitive
29
disadvantage and vulnerable to predation by other invertebrates and vertebrates, such as
fish.
There are several hypotheses that could be invoked to explain these herniations.
Of the two types of lesions observed, the tubular type was attributed to parasites by
Bridgeman et al. (2000), while the others remain unexplained. If in fact the initiating
agent turns out to be a parasite, it could be possible that the parasite may be responsible
for both types, being unsuccessful in attaching in some cases (round lesions), while
successful in others (elongated lesions). Epischura spp., a predatory zooplankter, has
been shown to have high incidences of lesions among species investigated in Lake
Michigan, implicating food chain bioaccumulation and the potential for ingested
anthropogenic substances adsorbed to algae and detritus to be another possible cause (see
Xuewen, et al. 1999). The recent ecosystem-wide changes in water clarity and algal
composition may have changed toxic substance accumulation pathways or allowed some
photo-induced effect on the organisms or chemicals within them. Alternatively, the
widespread occurrences of these herniations in samples collected in the 1800s as well as
in remote ponds in Ohio, suggest an airborne vector, such as a particular PCB congener
or PAH, which may act as an endocrine disrupter (Colborn et al. 1993) or in the case of
PAHs, be photo-activated in the zooplankter and cause death (Malloy et al. 1997). The
zebra mussel-induced water clarity changes would enhance this phenomenon. These
herniations may also be induced by a natural biological agent, such as a virus, parasite, or
bacterium that to date remains unidentified. In view of these findings, it is important to
investigate further the presence, composition, and cause of lesions in zooplankton in the
Great Lakes and other water bodies.
A series of bioassays were conducted using Cyclops bicuspidatus as the test organism
to determine if the tumor-like abnormalities previously observed in Lake Michigan
(Omair et al. 1999) could be induced in laboratory exposures. Various exposure media
were examined including rainwater, Lake Michigan water, Grand River water, and
culture water from aquaria containing zebra mussels. Tumor-like abnormalities were not
observed in zooplankton exposed to any of the treatments. The exposures used for these
experiments were designed to screen for various causative agents. The absence of the
reported abnormalities in these experiments may indicate several possibilities:
 the hernia-inducing agent was not present in the test media
 the hernia-inducing agent required activation by UV light
 a labile chemical is responsible that decomposed during sample storage or
adsorbed on the filter media
 a labile organism is present that did not survive the laboratory manipulations
 a longer exposure period is required
 the test organism (Cyclops bicuspidatus) was more resistant (had very low
incidence of infection in our field samples) to tumor induction than Limnocalanus
spp. or Diaptomus spp., even though it was the easiest to raise among candidate
zooplankton species
 life cycle stages and interactions with other environmental variables may be
responsible.
Our bioassay study using rainwater, zebra mussel, Lake Michigan, and Grand River
water failed to induce herniations in Cyclops bicuspidatus. Whatever chemical,
organism, or environmental variable that is responsible for these lesions could have been
30
compromised by the selection of the test organism, the wrong season, or inadequate
environmental conditions necessary to promote them. These factors could be examined
by life cycle bioassays or in situ exposures. We recommend that future studies use in situ
bioassays (Pereira et al. 1999) or whole life cycle bioassays and consider feeding
zooplankton potentially contaminated algae from affected waters.
Lastly, we are concerned about the fate of the zooplankton populations in Lake
Michigan. Our past and present zooplankton data show that the densities of zooplankton
have declined dramatically from mean July density of 50,000-60,000/m3 in the 1970s and
1980s to 13,000-14,000/m3 during early 2000s (Jude, unpublished data). Along with this
decline in densities is also an alarming trend of increases in the incidences of the
abnormal growths on copepods (still ongoing in June 2002), which are key food items for
larval fish and which are probably limiting the survival of several pelagic fish species in
Lake Michigan (e.g., yellow perch). The potential loss of additional zooplankters to
death or predation due to the additional mortality source of these lesions may result in
substantial harm to organisms higher in the trophic web, such as larval fish, and has the
potential to disrupt ecosystem function. Our challenge now is to identify etiological
aspects and the pathogenesis of these lesions to help design strategies to combat or
obviate any adverse effects associated with their presence.
LITERATURE CITED
Bridgeman, T., Fahnenstiel, G. L., Lang, G. A., and Nalepa, T. F. 1995. Zooplankton
grazing during the zebra mussel (Dreissena polymorpha) colonization of Saginaw
Bay, Lake Huron. J. Great Lakes Res. 21: 567-573.
Bridgeman, T., G. Messick, and H. Vanderploeg. 2000. Sudden appearance of cysts and
ellobiopsid parasites on zooplankton in a Michigan lake: a potential explanation of
tumor-like anomalies. Can. J. Fish. Aquat. Sci. 57:1539-1544.
Charlebois, P., M. Raffenberg, and J. Dettmers. 2001. First occurrence of Cercopagis
pengoi in Lake Michigan J. Great Lakes Res. 27:258-261.
Colborn, T., vom Saal, F.S., and Soto, A.M. 1993. Developmental effects of endocrinedisrupting chemicals in wildlife and humans. Environ. Health Perspectives 101: 378384.
Crisafi, P., and Crescenti, M. 1977. Confirmation of a certain correlation between
polluted areas and tumorlike conditions as well as tumor growths in pelagic copepods
provening from numerous seas of the world. Rapp. Comm. Int. Mer. Médit. 24:155.
EPA. 1993. Methods for measuring the acute toxicity of effluents and receiving waters to
freshwater and marine organisms. 4th Ed. U. S. Environmental Protection Agency,
Cincinnati, Ohio. EPA/600/4-90/027F.
Great Lakes Water Quality Board. 1991.
Cleaning up our Great Lakes, A report on
toxic substances in the Great Lakes basin ecosystem. Report on Great Lakes water
quality to the International Joint Commission. Windsor, Ontario, Canada, August
1991, 47 pp. xx22
Hook, S.E. and N.S. Fisher 2001: Sublethal effects of silver in zooplankton: importance
of exposure pathways and implications for toxicity testing. Environ. Toxicol. Chem.
20(3):568–574.
31
Malloy, K.D., Holman M.A., Mitchell D., and Detrich, H.W. 1997. Solar UVB-induced
DNA damage and photoenzymatic DNA repair in Antarctic zooplankton. Proc. Nat.
Acad. Sci. USA 94: 1258-1263.
Manca, M., Beltrami, M., and Sonvico, D. 1996. On the appearance of epibionts on the
crustacean zooplankton of a large subalpine lake undergoing oligotrophication (L.
Maggiore, Italy). Mem. 1st. Ital. Idrobiol. 54: 161-171.
Manno, J., Myers, S. and Riedel, D. 1995. Proceedings of the State of the Lakes
Ecosystem Conference (SOLEC), Ottawa, Ontario, Canada, 1995, U.S.
Environmental Protection Agency, Chicago, IL.
Omair, M., B. Naylor, D. Jude, T. Beals, and H. Vanderploeg. 2001. Histology of
herniations through the body wall and cuticle of zooplankton from the Laurentian
Great Lakes. J. Invert. Pathology 77:108-113.
Omair, M., Vanderploeg, H.A., Jude, D.J., and Fahnenstiel, G. 1999. First observations
of tumor-like abnormalities (exophytic lesions) on Lake Michigan zooplankton. Can.
J. Fish. Aquat. Sci. 56:1711-1715.
Pereira, A.M.M, A.M. Velho da Maia Soares, F. Gonçalves, and R. Ribeiro, 1999. Test
chambers and test procedures for in situ toxicity testing with zooplankton. Environ.
Toxicol. Chem. 18(9):1956–1964.
Silina, N.I. and Khudolei, V.V. 1994. Tumor-like anomalies in planktonic copepods.
Hydrobiological J. 30:52-55.
Vanderploeg, H. A. 1999. Tumors in Zooplankton of Lake Michigan, in Random
Samples, ed. Constance Holden. Science 284:1613.
Xuewen, M., Bruner, K.A., Fisher, S.W. and Landrum, P.F. 1999. Absorption of
hydrophobic contaminants from ingested Chlamydomonas rheinhardtii and Chlorella
vulgaris by zebra mussels, Dreissena polymorpha. J. Great Lakes Res. 25:305-317.
ACKNOWLEDGEMENTS
The Michigan Great Lakes Protection Fund, Office of the Great Lakes, provided
funding for this study for which we are thankful. Zooplankton samples, from which
we removed affected individuals, were collected as part of a Great Lakes Fishery
Trust (a grant to John Dettmers, Ill. Nat. Hist. Survey, John Janssen, Univ. Wisc.Milwaukee, David Jude, Center for Great Lakes and Aquatic Sciences, U. MI, and
Scott McNaught, Central MI Univ.) study on yellow perch recruitment. We
gratefully acknowledge Stephen Hensler for assistance in collection of the
zooplankton samples, procurement of the Smithsonian zooplankton samples, removal
of some of the affected zooplankton, advice on the writing of the report, and editing
duties. We thank N. Andresen, R. Bixby, M. Edlund, and M. Julius for help with the
specimens and photography. D. L. Banka assisted with typing, while M. Deming, B.
Smola, L. M. St. Dennis, and S. Stamper provided histological and cytological
support. Rebecca Hayes provided assistance with the graphs, data processing, and
final report preparation.
32
APPENDICES
Table A-1. Zooplankton food supplement preparation (blended and stored at 4 c).
Constituent
Tetrafin® Fish Food
CEROPHYLL ®
Yeast
YTC Preparation**
Concentration
4.0 g/L
3.0 g/l
3.0 g/l
Table A-2. Diatom culture solution (EPA 1993).
Macronutrient
NaNO3
MgCl2.6H2O
CaCl2.2H2O
MgSO4.7H2O
K2HPO4
NaHCO3
Na2 SiO3.9H20
Micronutrient
H3BO3
MnCl2.4H2O
CoCl2.6H2O
CuCl2.2H2O
Na2MoO4.2H2O
FeCl3.6H2O
Na2EDTA.2H2O
Na2SeO4
Concentration
(mg/L)
25.5
12.2
4.41
14.7
1.04
15.0
20
Element
B
Mn
Co
Cu
Mo
Fe
Concentration
(mg/L)
4.20
2.90
1.20
1.91
0.186
11.0
2
0.469
2.14
Concentration
(ug/L)
32.5
115
0.354
0.004
2.88
33.1
Se
1.00
N
Mg
Ca
S
P
Na
Si
K
C
Element
Concentration
(ug/L)
185
416
1.43
0.012
7.26
160
300
2.39
* EPA. 1993. Methods for measuring the acute toxicity of effluents and receiving
waters to freshwater and marine organisms. 4th Ed. U. S. Environmental Protection Agency, Cincinnati,
OH. EPA/600/4-90/027F.
33
Table B-1. Bioassay testing schedule of events and measurements.
Test Schedule Cyclops bicuspidatus
Day
Temp & DO
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
X
X
Renewal
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Feeding
Water quality
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Table C-1. Chemical measurements for the bioassay test with Cyclops
bicuspidatus.
Summary of Initial and Final Water Quality Parameters For
Cyclops bicuspidatus
Sample
Control
Rain
Parameter
(%)
pH
8.09
7.96
2
Conductivity (umhos/cm)
757
751
1
Alkalinity (mg/l CaCO3)
192
203
6
Hardness (mg/l CaCO3)
Ammonia (mg/l NH3)
150
150
0
0.01
0.04
300
pH
8.33
8.00
4
Conductivity (umhos/cm)
540
530
2
Alkalinity (mg/l CaCO3)
156
193
24
Hardness (mg/l CaCO3)
Ammonia (mg/l NH3)
142
157
10
0.01
0.03
200
pH
8.16
8.24
1
Conductivity (umhos/cm)
540
560
4
182
191
5
Hardness (mg/l CaCO3)
Ammonia (mg/l NH3)
155
150
3
0.01
0.04
300
pH
8.35
7.97
5
Conductivity (umhos/cm)
665
729
10
161
190
18
LKM 10 u m Alkalinity (mg/l CaCO3)
Hardness (mg/l CaCO3)
Ammonia (mg/l NH3)
156
159
2
0.01
0.03
200
pH
8.18
8.32
2
Conductivity (umhos/cm)
540
520
4
188
202
7
LKM 60 u m Alkalinity (mg/l CaCO3)
Hardness (mg/l CaCO3)
Ammonia (mg/l NH3)
154
152
1
0.01
0.02
100
pH
8.22
8.27
1
Conductivity (umhos/cm)
689
705
2
183
190
4
Zeb 0.45 u m Alkalinity (mg/l CaCO3)
Grand River
Difference
10
LKM 0.45 u m Alkalinity (mg/l CaCO3)
Zeb 10 u m
Day
0
Hardness (mg/l CaCO3)
Ammonia (mg/l NH3)
147
156
6
0.12
0.25
108
pH
8.05
8.34
4
Conductivity (umhos/cm)
726
678
7
Alkalinity (mg/l CaCO3)
190
191
1
Hardness (mg/l CaCO3)
Ammonia (mg/l NH3)
155
154
1
0.13
0.28
115
pH
8.05
8.34
4
Conductivity (umhos/cm)
726
678
7
Alkalinity (mg/l CaCO3)
190
191
1
Hardness (mg/l CaCO3)
Ammonia (mg/l NH3)
155
154
1
0.08
0.13
63
Table C-2. Summary of dissolved oxygen and temperature measurements in the Cyclops
bicuspidatus experiments.
Test No:
Toxicant:
Organism:
Cyclops bicuspidatus
Table C-2. Summary of Daily Temperature and Dissolved Oxygen Measurements For Cyclops bicuspidatus In The B
DO
%
71.50
Day
10
DO
Temp
o
%
C
75.60
15.4
12
DO
Temp
o
%
C
71.30
15.1
DO
%
68.60
Day
10
DO
Temp
o
%
C
75.90
15.2
12
DO
Temp
o
%
C
81.00
15.1
DO
%
74.00
Day
10
DO
Temp
o
%
C
94.40
15.7
12
DO
Temp
o
%
C
83.40
14.3
DO
%
72.50
Day
10
DO
Temp
o
%
C
74.70
15.8
12
DO
Temp
o
%
C
77.60
15.7
DO
%
70.00
Day
10
DO
Temp
o
%
C
81.30
14.8
12
DO
Temp
o
%
C
76.30
15.4
Sample:
Control
DO
%
93.70
Temp
o
C
15.3
DO
%
80.40
Temp
o
C
15.1
DO
%
61.20
Temp
o
C
15.3
8
6
4
2
0
DO
%
71.30
Temp
o
C
15.2
Temp
o
C
15.0
Sample:
Rain
DO
%
98.20
Temp
o
C
15.0
DO
%
86.50
Temp
o
C
15.0
DO
%
63.10
Temp
o
C
15.9
8
6
4
2
0
DO
%
76.60
Temp
o
C
15.9
Temp
o
C
15.9
Sample:
LKM 0.45 u m
DO
%
78.50
Temp
o
C
15.0
DO
%
76.50
Temp
o
C
15.7
DO
%
67.20
Temp
o
C
15.0
8
6
4
2
0
DO
%
73.70
Temp
o
C
14.0
Temp
o
C
15.7
Sample:
LKM 10 u m
DO
%
92.30
Temp
o
C
15.6
DO
%
91.70
Temp
o
C
15.9
DO
%
81.40
Temp
o
C
15.3
8
6
4
2
0
DO
%
80.10
Temp
o
C
15.6
Temp
o
C
15.2
Sample:
LKM 60 u m
Temp
o
C
15.8
DO
%
96.60
Temp
o
C
15.4
DO
%
86.80
Temp
o
C
15.5
8
6
4
2
0
DO
%
66.00
Temp
o
C
14.0
DO
%
73.50
Temp
o
C
14.2
14
Temp
o
C
15.7
DO
%
72.70
Tem
DO
%
78.70
Tem
DO
%
79.50
Tem
DO
%
75.10
Tem
DO
%
75.60
Tem
o
C
15
14
Temp
o
C
15.8
o
C
15
14
Temp
o
C
14.2
o
C
15
14
Temp
o
C
15.1
o
C
15
14
Temp
o
C
15.7
o
C
15
Test No:
Toxicant:
Organism:
Ana
Te
Cyclops bicuspidatus
Te
Table C-2 (Cont). Summary of Daily Temperature and Dissolved Oxygen Measurements for Cyclops bicuspidatus in th
Day
10
Sample:
Zeb 0.45 um
0
Temp
2
DO
o
%
C
15.9
97.20
Temp
4
DO
o
%
C
15.8
85.20
Temp
6
DO
o
%
C
15.0
68.00
Temp
8
DO
o
%
C
15.3
73.40
Temp
DO
o
%
C
15.7
74.10
Temp
o
C
15.6
0
Temp
2
DO
o
%
C
15.0
97.70
Temp
4
DO
o
%
C
14.9
81.90
Temp
6
DO
o
%
C
15.1
62.40
Temp
8
DO
o
%
C
15.1
73.70
Temp
DO
o
%
C
15.0
0
Temp
o
C
14.8
2
DO
Temp
%
C
14.8
85.00
o
4
DO
Temp
%
C
15.8
73.40
o
6
DO
Temp
%
C
15.9
63.50
o
Temp
%
C
15.9
71.10
o
o
72.20
C
15.2
Temp
%
C
15.3
65.60
o
%
C
15.0
%
78.90
Temp
o
Temp
o
83.90
DO
Temp
Temp
%
C
15.7
76.30
o
Temp
o
78.90
C
15.6
DO
Temp
14
o
1
%
84.80
C
15.2
DO
Temp
%
C
15.1
12
DO
1
DO
%
C
15.7
%
C
15.9
Day
10
DO
14
DO
12
DO
o
8
DO
79.70
Temp
Sample:
Grand River
%
Temp
Day
10
Sample:
Zeb 10 um
12
DO
o
80.10
C
15.4
DO
Temp
%
C
15.5
14
82.30
o
1
72.30
o