Exposure to methoxychlor also increased larval mortality. This
parameter was not affected at 0.008 mg/liter; however, mortality
percentage means for larvae exposed to 0.015, 0.03, and 0.06 mg/
liter of methoxychlor were 16, 11.5, and 17, respectively. For
the seawater controls and the seawater-acetone controls, the mean
percentages for mortality were 6.0 and 3.8, respectively.

As observed in the study of Treflan, the larvae in test chambers
containing acetone completed metamorphosis more rapidly than the
larvae reared in seawater alone (Figure 6). For larvae exposed
to acetone, the number of days required for 50% of the animals to
undergo metamorphosis ranged from 11 to 17; the mean was 14 days.
The mean for seawater control larvae was 21 days.

In addition to influencing the rate of larval metamorphosis, acetone
appeared to inhibit growth. This effect also was observed in the
study of Treflan, in which acetone was used as a dispersing agent.
In the study of methoxychlor, the growth-inhibiting effect of ace-
tone was more pronounced. Metamorphosed larvae exposed to seawater
containing 50 yl/liter of acetone alone were 13% smaller than the
seawater controls. As a group, the methoxychlor-exposed larvae,
which were also exposed to 50 yl/liter of acetone, averaged 17.7%
smaller than the seawater controls, The size of the larvae did not
appear to be related to the pesticide concentration; hence, meth-
oxychlor, like Treflan, did not appear to be involved in inhibiting
the growth of the larvae in this test.

The toxicity of methoxychlor to marine bivalve molluscs has not
been studied extensively. Most of the toxicological information
on this pesticide concerns freshwater organisms, particularly fish.
Eisler and Weinstein (1967) reported that exposure of the hard-
shelled clam (M. mercenaria) to 1.1 mg/liter of methoxychlor was
not lethal, although the insecticide was highly toxic to marine
crustaceans. The 48-hour TL., estimates for the grass shrimp, sand
shrimp, and hermit crab ranged from 0,004 to 0.012 mg/liter. Ac-
cording to Butler (1963), the brown shrimp (Panaeus aztecus) is
also highly sensitive to low concentrations of methoxychlor, having
a 48-hour TLsg value of 0.006 mg/liter. Butler also reported that

exposure of C, virginica to 0.097 mg/liter for four days resulted
in a 50% growth reduction.

56


however, is 40% higher than theirs, perhaps because of the differ-
ence in method and temperature. The Oregon State University values
were obtained at 13°C; and, instead of analyzing a filtered sample
of a saturated pesticide solution prepared by mixing an excess of
the pesticide in a known volume of water, the investigators analyzed
samples of seawater that had been allowed to trickle through a bed
of pesticide-laden sand prepared by filling a column of sand with

a pesticide-acetone solution and evaporating off the acetone.

FACTORS AFFECTING THE DEVELOPMENT OF NORMAL LARVAE

Although the bay mussel embryo bioassay (Dimick and Breese, 1965)
appears to be easy to perform and should be within the capability
of many laboratories having access to a natural population of mus-
sels and seawater, problems can arise. Our major difficulty was
in defining the conditions under which acceptable percentages of
the control larvae could develop normally. Seldom are 100% of the
larvae in a sample normal; but, under the proper conditions, a
large percentage do develop normal shells. In the bay mussel em-
bryo bioassay, 85% normal larvae in a sample is considered minimum
for acceptable test results. This criterion was difficult to meet
during our early performance of the bioassay.

Measures undertaken to solve this problem culminated in several
experiments, one of which was to determine the extent to which a
delay in the time between shedding of eggs and introduction of
sperm to fertilize them affected the number of larvae developing
normal straight-hinged shells. The decision to investigate this
factor was based on our observation that different females seldom
shed their eggs simultaneously, and that there often is a consider-
able delay between the time a mussel begins to shed eggs and the
time that it has shed a sufficient number for use in the experiment.
Since several series of tests on a given pesticide usually were con-
ducted on the same day, each with eggs from a different mussel, it
was most convenient to initiate all the tests at approximately the
same time. Because of this, some egg groups were not fertilized
until several hours after they had been shed. Breese (1972) found
that a four-hour delay did not seriously affect the development of

normal larvae, but he advised limiting the delay to no more than
three hours.

22


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:

Environmental Health Effects Research
Environmental Protection Technology
Ecological Research

Environmental Monitoring

aoFwn a

Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series. This series describes research on the effects of pollution

on humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine the
fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living
organisms in the aquatic, terrestrial and atmospheric environments.

EPA REVIEW NOTICE

This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval does
not signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or
recommendation for use.


so

100 T T T T T T
— Control
22
-~- 4%
+ 88
goa e eeeccccee 176 4
8
2
2
&
3
€
a
{| sok 4
a
a
feo}
x
© aosseseeees
co} o .
=
4
F
G
Ss oe 4
0 mt !
oO 5 10 15 20 25 30 35
LENGTH OF EXPOSURE — days
FIGURE 7 EFFECT OF EXPOSURE TO 2.4-D ON PERCENTAGE OF MUSSEL POPULATION COMPLETING METAMORPHOSIS

Larvae were 29 to 30 days old when exposure was initiated. Data from the two experiments were pooled.


The study could have been aided greatly if knowledge concerning
differences in the biology of the various M. edulis populations

in the San Francisco Bay region had been available before initia-
tion of the project. This information would have been of particu-
lar value in all the phases involving use of the larvae. To assure
that a sufficient number of eggs from different females were avail
able to conduct any series of experiments requiring eggs or larvae,
several hundred mussels were usually obtained during each collection
trip. Sources close to the testing laboratory, such as Treasure
Island and St. Francis Yacht Harbor, naturally were favored. As
shown in Table 30, mussels from Treasure Island, Moss Landing, and
St. Francis Yacht Harbor did not respond well to spawn-induction
procedures, and egg viability was poor. Thirty-nine groups of
eggs, each from a different St. Francis Harbor female, were used

in the 48-hour embryo bioassays; only 7.7% of these groups contained
the minimum of 85% normal larvae in the control test. Sixty-one
egg groups from Treasure Island were tested; 11.7% produced the
acceptable number of normal larvae.

Mussels from Tomales Bay, located relatively far from the labora~
tory, proved to be much more responsive to our spawn-induction pro-
cedure, and their gametes were in better condition than those of
mussels collected at the other sites. On the average, about 35%
of the mussels subjected to spawn-induction procedures spawned.

In general, the gametes were produced in abundance. Of 42 differ-
ent egg groups used in the 48-hour embryo bioassays, about 40% pro-
duced acceptable percentages of normal larvae. Performance in the
growth and metamorphosis studies was even better.

These data illustrate the importance of knowing which available
mussel population is best suited for the contemplated study. Early
frustrations encountered during this project could have been avoided
if the Tomales Bay population had been identified as the most suit-
able for the project.

With the exception of the 48-hour embryo bioassay tests, all the
experiments performed in this investigation would have been im-
proved if continuous-flow toxicant dilution and delivery systems

as well as special larval test chambers could have been developed
and used. Although only two small adult mussels, measuring between

76


approximately the fourth day after the medium was inoculated
and continuing until about the seventh day. The culture then
was discarded, and the carboy was "sterilized" by filling it

with a 10% solution of hypochlorite, which was allowed to stand
for two to three days.

Algae used as food were separated from the culture medium in a
Sorvall continuous-flow centrifuge (Model KSB3), using an SS-34
head at 1500 rpm. The algae pellets were resuspended in uv-
treated seawater with an adjusted salinity and pH, and the cell
density was determined with a Coulter particle counter using
the procedures described by Maloney and co-workers (1962).

11


TL

Table 28.

EFFECT OF MALATHION ON MUSSEL LARVAE METAMORPHOSIS

AFTER A 40-DAY EXPOSURE INITIATED 30 DAYS AFTER FERTILIZATION OF THE EGGS
(Presence of Dissoconch Shell Used as Indication of Metamorphosis)

Pesticide concentration, mg/liter

Control 1.51 3.02 6.05 12.1
L 2 1 2 1 2 1 2 L 2
Number of larvae
Initial 100 100 100 100 100 100 100 100 100 100
Lost or crushed 36 20 28 16 18 15 10 25 14 16
Experimental 64 80 72 84 82 85 90 75 86 84
Metamorphosing,
percent 78.1%) 87.5%] 75.0%] 79.8%) 75.6%] 82.4%] 67.8%] 89.3%] 90.7%] 82.1%
Nonmetamorphosing,
percent 17.2%] 6.3%) 13.9%) 15.4%] 9.8%] 9.4%] 7.8%] 0 10) 0
Dead, percent 4.7h| 6.2%) 11.1%] 4.8%] 14.6%] 8.2%] 24.4%] 10.7%] 9.3%} 17.8%
Days to metamorphosis
of 50% of test larvae] 28 26 28 28 25 22 33 22 13 13
Shell length of juve-
nile mussels, py 456 wp 1429 uw [448 yp 1443 yp [434 yp 1439 p [417 p [413 p 4371 wp 1366 p



62

Table 2. NUMBER OF M. EDULIS LARVAE ATTACHED TO SELECTED SUBSTRATES
DURING A FIVE-DAY PERIOD

Substrate No. of No. of Mean no.
Mean no. of
Substrate surface area, tests larvae/ | of larvae 2
larvae/cm
cm per formed test attached
Silk thread 4.79 6 100 13.5 2.82
Acrylic plastic 50.24 6 100 8.2 0.16
PVC plastic 45.16 6 100 16.8 0.37
Frosted glass 51.08 6 100 10.3 0.20
Glass chamber 275.60 6 100 18.2 0.07



oot

Table 3.

Days after Fertstrzation of Eggs
(microns)

NUMBER, SEAN AND STANDARD DEVIATION OF SHELL LENGT! OP METANORPHOSED MUSSEL LARVAE EXPOSED TO METHOXYCHLOR
Exposure Initiated

Concentration ng/liter
Seawater control Solvent control 7,008 013 0,030 0.060
vay [ap iean[ sb [ofan [So [ a] itean] sD] o[ ican [Sd | w[ wean [Sp [ n[Wean [So] =] team | Sp] n[Weas[ SD | w[ Nean] SD] n [wean Sd | nw] wean [Sp] » [ivan [Sp
o fof -- 0| --- o| -- o| --- o| - o| - ° ° of -
2 fol --- of --- o| --- | a} - o| - ° ° o| -
a fol --- 0 -- o| o| --- oj - o| - 0 ° o| -
s |e] ao | is o| -- o| -- | 0} --~ of - 3} aio | 61) 2 si fiz] ser | a9] 9] ais | 55
s |s| aos | a7 aa| a] se2 | 25 | 4] 403 | 39 a3 | o| --- ]ses | aa] 1|as2 | of 2) s76 | o| al ss6 | a0] 3) s57 | a7
ro fi} saa | o a7 | 6] ass | 75 |13| aoa | 63 30 | 9| 397 | a5j1afass | 34] 1/328 | of 1} s20 | o| a| are | 62] 6} 290 | 40
az |afss2 | o 66 {17} aaa | 58 |2a| 407 | 50 a1 {1a} se9 | 28}aa] aso | 40] 7] sa | 25] of - 2] a9a | 13] 3] 366 | 70
aa fol --- 0 s1 | 6{ 42a | 90 |12] 406 | 59 so | 4} ser | a8] s] ass | a: 36] 40 | sa} o| - o| - 1}aza | o
ae falas | of} afaso | o |i2/ 399 | a2] s] ase |a21 | 6] ses | 33 sé | 9| sea | aa; 6) ass | 27| 4) soa | sa/ 7) an | 52] 8! ago | a7{ 2s] s79 | 39
is |s]4as7 j1se | 3] as9 | 86 | 9) 904 | 54 |i] as7 | sa] 9] ara | a2 sa io] se1 | 62] 10] 357 | ss jas} 04 | a5] 22] s96 | 41 Jao] saz | 34] s| 578 | 47
20 fiz} 429 | 32 aso | 22 | 4]4as | fis} asa | za] 7] 0a | 50 az | sso | 20] 2] 296 | aa |ar] saz | 47] 21] 420 | Go| 5] sm | 18] 5] sos | sa
22 fos) sea | 7a fa7| 460 | ar | 2} aor | iza} a] 495 | ss | 1/297 | of s| aca | si | a] ao | a2] of - af 403 | 0| 6] aso | ss] s|a73 | 49] o| -
2a [af ass | 0 fa2| see | oe | of --- afss0 | o | 1] aos 1|s07 } o| 3] s62 | 20} of - 3| 399 }izi| o| - o| - a] asx ] 0
26 | 9] 470 | s7 {10/550 |sa2| 1} 292 | of 1] sa | 0} 1} a2 | of 2 si | 636s | a3] 3] 377 fasz| a} ass | as| 7] soa | 52] 5| aa7 | 12] «| aos | 08
2a |9| sas | st} s| aoa | as | o| --- o} ~~ of --- o x] 320 | 32] 0| - s| 322 | sa] 6] ass | 57] 2) am | o| of -
so |a| 63a | a5 | a) ass [a6 | 0! --- of = o| --- of == of -- o| ~ 0} - o| - o| - o} >
sa fa} ass | of 1] s23 | 0 | oj --- o| --- ol --- of --- of == 0} ~ 2|273 | of o| - 3} 302 | of of -
so | 3) sas |s6 | «| 392 | 23 | 0] --- o| --- o| --- o| --- of --- o| - of = of - of - ol -
No. of
junmeta-|
juorph-
josed
larvae | 2 ° ° ° ° o ° ° ° ° ° °



no longer than 14 days. Juvenile mussels did not develop at either
concentration. Conditions in Experiment 1 (Table 6) were apparently
unsuitable for larval development, since only two of the control
larvae metamorphosed. Conditions in Experiment 2 were somewhat
better; in the control groups, a total of 159 larvae developed dis-
soconch shells. Of larvae exposed to the lowest Sevin concentration
of 0.33 mg/liter--the only pesticide-exposed group remaining--only
11 developed into juvenile mussels. Larvae in this group did not
begin to undergo metamorphosis until six days after the control
larvae had started. The juvenile larvae were also smaller in this
group than larvae in the control group.

Table 6. NUMBER, AGE, AND SIZE OF JUVENILE MUSSELS
DEVELOPING IN LARVAL CULTURES EXPOSED AT 48 HOURS
TO VARIOUS CONCENTRATIONS OF SEVIN

Experiment 1 Experiment 2
Test
concen-

. Li > No. of Length,
tration, vee Age,? ength, a Age,@ ength,
mg/liter niles days Mean | SD niles days Mean sD

0 2 37 457 | 93.0} 159 32 434 66.3
0.33 9 30 304 | 44.0 1 38 414 | 108.2
0.65 4 32 279 | 26.3 ie) -- -- --
1.30 te) -- -- [ee (0) -- -- --
2.61 le) -- -- [es (0) -- -- --

a
Number of days required by treatment group to begin metamor-
phosing.

Table 7 presents the data obtained from the second set of experi-
ments in which exposure was initiated after the larvae were 29 days
old. Although each test chamber contained an initial number of
100 swimming larvae, many were lost or crushed during renewal of
the test solutions. Because the larvae in this category were lost

36


Table 20.

PERCENTAGE OF NORMAL LARVAE DEVELOPING IN 48 HOURS

FROM MUSSEL EGGS FERTILIZED IN VARIOUS CONCENTRATIONS OF 2,4-D

Test Test number
concen-
tration, Experiment 1 Experiment 2 Experiment 3
mg/liter q) (2) (1) (2) (3) (2)
0 88.5 86.0 95.5 98.5 90.5 92.0
47 90.0 90.5 91.0 91.0 93.5 93.5
98 88.0 93.5 88.8 96.0 92.3 88.0
120 92.5 93.5 91.0 92.5 92.5 95.5
140 87.5 91.5 86.5 87.0 93.0 --
160 88.0 89.5 82.5 88.0 82.5 88.5
190 74.5 71.0 86.0 80.5 81.5 86.5
240 0 0.5 (0) ie) i) 0
48-Hour ECs,
mg/liter 210 212 213
Table 21. MEAN SHELL LENGTH OF MUSSEL LARVAE
EXPOSED TO 2,4-D FOR 10 AND 20 DAYS
Mean shell length,
Test No. of No. of
concen- larvae tests/ Day 10 Day 20
tration, | measured/ | experi-
mg/liter test ment Experi- | Experi- | Experi- | Experi-
ment 1 | ment 2 | ment 1 | ment 2
0 20 2 182 172 297 259
22.8 20 2 198 188 287 278
45.7 20 2 182 170 293 268
91.4 20 2 178 152 231 234
182.8 20 2 124 112 -- 206

61



method that a poor fit was indicated. Consequently, the method
was abandoned in favor of the one recommended by the APHA. The
estimates are based on relative percentages calculated as described
under "Material and Methods"; however, the percentages presented in
the tables are the actual percentages.

Although 20 larvae were removed every other day and measured in the
experiments on the effect of continuous exposure to the pesticides
on larval growth and metamorphosis, statistical analysis of the data
to determine the magnitude of effect was performed only on length
measurements taken on the 10th and 20th day of exposure. Appendix B
presents complete growth data. Appendix A contains the water qual-
ity data obtained during each phase of the study. Appendix C pre-
sents all data obtained in the metamorphosis experiments.

Sevin

Although five adult survival tests were performed on Sevin, only
the data from one test (Test 53, Table 3) were suitable for esti-
mating the 96-hour TL59. The pesticide concentrations in two of
the tests were not high enough to kill more than 50% of the test
animals; and in another test, 30% of the control animals died.

The 96-hour TLsq and 95% confidence limits, estimated by the method
of Litchfield and Wilcoxon (1949), were 22.7 and 15.5 to 33.4 mg/
liter, respectively.

Fewer Sevin-exposed mussles than controls attached themselves to
the test chamber. In Test 53, 70% of the mussels exposed to

11.3 mg/liter of Sevin survived; however, only 50% attached. At
the highest concentration of 30.9 mg/liter, 40% of the mussels sur-
vived, but only 10% attached. The estimated attachment ECs5q, based
on 96 hours of exposure, was 10.3 mg/liter.

Mussel embryos were considerably more sensitive than adults to
Sevin. The 48-hour ECsq ranged from 1.21 to 1.80 mg/liter for
three tests in which each concentration was tested in duplicate.
The mean was 1.5 (Table 4).

32


bo

Table 23. EFFECT OF 2,4-D ON LARVAL METAMORPHOSIS AFTER A 40-DAY EXPOSURE
INITIATED 30 DAYS AFTER FERTILIZATION OF THE EGGS
(Presence of Dissoconch Shell Used as Indication of Metamorphosis)

Test concentration, mg/liter

Control 22 44 88 176
1 2 1 2 1 2 1 2 L 2
Number of larvae
Initial 100 100 100 100 100 100 100 100 100 100
Lost or crushed 18 23 20 16 20 3 33 3 49 26
Experimental 82 77 80 84 80 97 67 120 51 74
Metamorphosing, per-
cent 79.3%] 79.2%) 71.2%] 70.3%] 77.5%] 59.8%] 73.2%] 66.7%] 80.4%] 60.8%
Nonmetamorphosing,
percent 14.6%] 13.0%] 11.3%) 21.4%] 13.7%] 22.7%] 14.9%] 24.2%] 9.8%] 20.3%
Dead, percent 6.1%] 7.8%) 17.5%] 8.3%} 8.8%] 17 5%] 11.9%} 9.1%] 9.8%] 18.9%
Days to metamorphosis of
50% of test larvae 27 28 23 25 22 27 21 24 22 31
Shell length of juvenile
mussels, 428 p, 1468 p 1446 yp 1484 p 1456 py 1463 yp 1486 yp 1463 py 1390 yy 4412 p



Table 4, NUNBER, MBAN AND STANDARD DEVIATION OF SHELL LENGTH OF METAMORPHOSED MUSSEL LARVAE EXPOSED 70 2,4-D
Exposure Initiated 30 Days after Fertilization of Eggs
(microns)

tot

Concentration, mg/liter
Seavater contro. ao To. Fema Teo
pay [w[ Wen [Sb [—n [lean [SO [-o] wean] Sb [a] wean [SO-[ a [wean] Sb | a] Wea [S| a [wean] so | awe Say ean On [ean SD
o | o}- ° 0 of == 0} --- ° o} == o} 0
2 fol- 0 ° o| --- o| - 0 o| --- 0} - °
a fol- 0 ° 0) - o|- 0 o} -- s| seo | a2 | 2 a
s |ol- 0 0 o} -~ of = o|o o| -—- o| - o
7 [ol- 0 0 o| == rj au | o 0 1}ais | o | 6] am | 66 | a] ase | 50
» |alase | o | o a}ase | o | rfasa | o | of-- 2 s6 | alas Jar | 1fsae | o | 2] aes | 0 | 5} 09 | so
un fa}oz | oo afc | sr | afer | 0 | 2/so7r jis | 0 a]sor | o | a}acs | as | afsa foo | x} 207 | o
as fof -— s}aso | 0 | 2fs2o | so | face |r | 2 ax | so | 1) 8) 0 | sfaso {so | afaos fro7 | 6} aro | a | a] as0 | so
1s | s}oo7 | a5 | 2|ase | 2 | ofaso | se | Jase | ao | afase | a2] 7} aso | or Jao faze fer | asfase | os | a] aaa | 55 | 7] 403 | 70
17 | zlase | 17 | 6490 | 86 | 6{ ssa | so | sfaae | o | sfase | 6s | 2| 206 | 25 | [scr] o | azj aaa | a7 | of - a| ace | s
as fixfaso | 7 | z]aao | sa | 7] aoe fase | o| -— r}asa] 0] aaa | o | 2]sso faso | 7] a2 | 24 | of - 1280 | 0
ax | sfazz | ee | afaoe | 6s | 10] ae | 56] 7 Jas | a0 {aafaso } or fro{ eas | 6s | a fuss} ar | 2face | a7 | 2] a25 | 9 | a] ae | so
23 | afasa Jior | s}aaz |rie | e{ ass | a6 | a8} a2 | 66 Jao] a60 | a7 | ae} aso | ao | sfaor | so | afr | ss | 6| a0 | oo | afass | 2
2s |afaor | ao | 2}ase | 25 | 5{aa7 | 2a] [aoa | 86 fax] ser | 99 | 9/496 | sx | fare] se | 2]o1 | se | jac | o | 2fase | a7
a7 | afaca | 6s {asfass | a2 | 7 aor | os | efsse | a9 | x] oaa | 0 | 2[ sae [ass | 6[ora [oa | afass | o | of - °
2a |6|aso | so | 7]/s05 | so | a} ase | o| sfasa | sz | asx | 0] sf ese jx20 | o o| -- o| - 0
a | afaso | 61 | fas | 6s | xfase | of afen | o | of-- 2} 662 fias | o o| 2 ase | io | a} ase | as
sa} s|aso | 28 | 6}aro | 75} a] s00 | o | a}ae | o | 2\eso | 0} s}asa | o 6 | sj}ace } os | 2} - s| 41 | a3
as Jalan | se | 2faa | o | o 0 1503 | 0 | o o a| aso | sa | o| - °
as |s{sss ] so | z{sue } o | 2| aor | oe | o o| = 1 o | 2}acz lex | afsas | 9 | sf sao fxea | afass | 4
No. of
phosed
Tinvae [2 10 ° 18 n 22 10 20 5 15



of 0.24 mg/liter for 96 hours killed 50% of the test population
(Table 8); however, all the mussels exposed to a measured level
of 0.1 mg/liter of Treflan survived. Test concentrations above
0.24 mg/liter or between 0.24 and 0.1 mg/liter were not evaluated.
The estimated solubility of this herbicide in seawater is 0.20 mg/
liter.

Table 8, SURVIVAL AND ATTACHMENT DATA FOR ADULT MUSSELS
EXPOSED TO TREFLAN FOR 96 HOURS

Test Test concentration No. of | Survival, | Attached,
no. Nominal Measured | animals percent percent
15 Control -- 9 100 100
0.2 -- 6 100 33.3
21 Control -- 10 100 100
Control
(acetone) -- 10 100 89
0.2 -- 10 100 0
43 Control 0 10 100 100
Control
(acetone) 0 10 90 100
0.2 O.1 10 100 30
0.4 0.24 10 50 10

Although 0.1 mg/liter of Treflan was not lethal to the adult mus-
sels, this concentration and the higher concentration of 0.24 mg/
liter reduced the number of mussels that attached to the glass

test chamber. All mussels exposed to seawater alone or to seawater
containing 200 ul/liter of acetone attached to the chamber during
the experiment. Of the mussels exposed to 0.1 and 0.24 mg/liter
of Treflan, only 30% and 10%, respectively, attached. The 96-hour
EC59 for attachment, based on these data, is 0.35 mg/liter.

42


Spotte, S. H. Fish and Invertebrate Culture. New York, Wiley-
Interscience, 1970. 145 p.

Standard Methods for the Examination of Water and Wastewater,
13th Ed. New York, APHA, 874 p.

Stewart, N. E., R. E. Millemann, and W. P. Breese. Acute Tox-
icity of the Insecticide Sevin and Its Hydrolytic Product
l-naphthol to Some Marine Organisms. Trans Am Fisheries
Soc. 96:25-30, 1967.

Sudak, F. N., and C. L. Claff. Survival of Uca pugnax in Sand,
Water, and Vegetation Containing 2,4-dichlorophenoxyacetic
Acid, Proc Northeast Weed Control Conf. 14:508-510, 1960,

Wisely, B. Detection and Avoidance of a Cuprus Oxide Anti fouling
Paint by Bivalve and Gastropod Larvae. (Mollusca). Aust J
Marine Freshwater Res, 14:60-69, 1963.

Woelke, C. E. Development of a Receiving Water Quality Bioassay
Criterion Based on the 48-hour Pacific Oyster (Crassostrea
gigas) Embryo. State of Washington, Department of Fisheries,
Technical Report No. 9. 1972. 93 p.

Worth, H. M., and R, C, Anderson. Toxicological Studies with
Trifluralin and Metabolites. The Pharmacologist. Fail
1965. p. 150.

Zaroogian, G. E., G. Pesch, and G. Morrison, Formation of an
Artificial Seawater Media Suitable for Oyster Larvae Develop-
ment. Am Soc Zool. 9:1144, 1969.

84


by shell development in mussel embryos, byssus-thread attachment
to a substrate by adults, and adult survival.

7. A delay of more than two hours in fertilization of mussel

eggs after they have been shed can affect embryo development
markedly.

8. Use of aged natural seawater for culturing of mussel embryos
from eggs appears to be beneficial for larval development.

9, Given a choice of rigid, opaque PVC, Plexiglas, frosted
glass, or stretched silk thread for setting, a higher percentage
of the mussel larvae select PVC,

The percentage increases with
larval density.


Abstract .....

List of Figures ..

List of Tables ..

Acknowledgments ..

Instruments Used in the Study

CONTENTS

Sections

I Conclusions .

IT Recommendations

III Introduction .

IV Materials and Methods

Vv Experimental Procedures
VI Results and Discussion .
VIL References . .

VIII

Ix Appendices...

iii

Page

ii

iv

viii

13
21
81
85

86


6Ee

100

METAMORPHOSIS — cumulative percent

50 --

25

T T T T T

Control
0.36
- 0.72
+145

eeeceseee 29

! 1 1 t |

FIGURE 4

5 10 15 20 25 30 365
LENGTH OF EXPOSURE — days

EFFECT OF EXPOSURE TO SEVIN ON PERCENTAGE OF MUSSEL POPULATION COMPLETING METAMORPHOSIS

Larvae were 29 to 30 days old when exposure was initiated. Data from the two experiments were pooled.


Experiment 1, the number of juvenile mussels recovered from larval
populations exposed to 6.2 mg/liter was only 5.1% of that recovered
from the control population. In the same experiment, none of the
larvae exposed to 12.3 mg/liter survived longer than 28 days, and
during this period none of the larvae developed a dissoconch shell,

In Experiment 2, the number of control larvae metamorphosing was
low; however, the number among those exposed to 6.2 mg/liter of
malathion was even lower, amounting to about 50% of the number of
metamorphosed controls. The test chambers with 12.3 mg/liter of
malathion contained only two juvenile mussels, or 2.3% of the num-
ber found in the control chambers. Metamorphosis of the two larvae
occurred when the larvae were 22 days old. None of the other lar-
vae exposed to 12.3 mg/liter survived for longer than 28 days.

Exposure of 30-day-old larvae to malathion did not affect the num-
ber developing into juvenile mussels (Table 28). However, the
larvae exposed to the highest test malathion concentration of

12.1 mg/liter exhibited a higher rate of metamorphosis than any
other treatment group (Figure 8). On the average, about 27 days
was required for 50% of the controls to undergo metamorphosis};
however, the larvae exposed to 12.1 mg/liter required only 13 days.
In all other malathion-exposed groups, the rate of metamorphosis
did not differ significantly from that of the controls. In general,
mortality among the malathion-exposed larvae was higher than among
controls. Mortality percentages for the two tests differed con-
siderably, with some pesticide-exposed groups showing about the
same mortality as controls in one test and higher percentages in
the other test. Mortality did not appear to be related to the
malathion concentration. As a group, the malathion-exposed lar-
vae exhibited 12.6% mortality, whereas average mortality for the
controls amounted to 5.4% of the total number of experimental
animals.

EVALUATION OF THE STUDY

The bay mussel shows considerable promise as a test organism for
the laboratory evaluation of potential marine and estuarine water
pollutants. This opinion is shared by Dimick and Breese (1965).

70


It was unfortunate that the substrate preference studies were not
initiated until after completion of the definitive studies on the
effect of the pesticides on growth and metamorphosis. Information
gained from the substrate preference studies could have helped
measurably in the definitive tests. This change in the original
study schedule occurred early in August 1973 when we had to decide
whether to stay on the original schedule and, because of the un-
certainty of a supply of readily spawnable mussels during the win-
ter months, jeopardize the initiation of the growth and metamorpho-
sis studies or to conduct the growth and metamorphosis studies first,
knowing that the substrate preference studies might be abandoned.
Because the project was toxicological in nature, we decided to pro-
ceed first with growth and metamorphosis.

Information on the kinds of substrates that attract and repel larvae
that are about to undergo metamorphosis can decrease considerably
the tedium and experimental error associated with the experimental
procedures we employed during the study. Ideally, larvae that are
about to undergo metamorphosis should be presented with an attrac-
tive substrate that can be removed periodically for assessment of
the percentage of the population that have become juvenile mussels.
In addition, use of a test chamber composed of a nontoxic, repellent
material would ensure maximum use of the presented substrate.

Our substrate preference study indicated that the larvae were at-
tracted to rigid PVC plastic, particularly at larva-to-volume (ml)
ratios of 1:3 to 1:1, the highest ratio tested. Attraction to PVC
was evident in the later stages of the growth study, when larvae
left submerged in the screened PVC pipes often were difficult to
remove because of their attachment to the pipe.

Of the rigid substrates evaluated, frosted glass was the least
attractive at the higher larval densities; however, clean Plexiglas
was about as poor and probably was more suitable as a material for
test chamber construction because of its transparency. Polyethylene,
although not evaluated and generally available only in the opaque
form, may be even less attractive than Plexiglas or frosted glass.

The debris problem is one that must be considered in studies on
larval metamorphosis. The pediveliger larvae were attracted to

79


EPA-660/3-75-016
MAY 1975

Ecological Research Series



ABSTRACT

The toxicity of the insecticides Sevin, methoxychlor, and malathion
and of the herbicides Treflan and 2,4-D to the bay mussel (Mytilus
edulis) was investigated. Toxic effects were measured in terms of
survival of and byssus-thread attachment by adults, embryo shell
development, and larval growth and metamorphosis.

The results indicated that growth was the most sensitive measure
of toxicity. All the pesticides produced statistically significant
(p = 0.05) reductions in larval shell length after 10 to 20 days
of exposure. Relative to potency, methoxychlor was the most toxic,
and 2,4-D was the least toxic.

The 96-hour TL50 values for each pesticide, based on adult survival
and attachment data, were estimated, as were the 48-hour EC50
values based on data from embryo bioassays.

The effects on embryo development of delaying the time of fertil-
ization and of using seawater larval culture media of various ages
also were studied, and substrate preference by metamorphosing
larvae was investigated.

A critical evaluation of the experimental approach and procedures
is presented,

This report was submitted in fulfillment of SRI Project No. LSU-1900,
Contract No. 68-01-0190, by Stanford Research Institute under the
(partial) sponsorship of the Environmental Protection Agency. Work
was completed as of March 31, 1974.

Ms
Be


in size and shape even when turgid, and these eggs also result in
abnormal larvae.

This study was designed also to determine whether the gametes ob-
tained from mussels collected at one location were more viable than
those from mussels collected at other locations. Although the per-
centage of normal larvae developing from eggs from all four loca-
tions was poor, eggs taken from mussels collected at St. Francis
Yacht Harbor performed best. However, in subsequent toxicity tests,
the 85% figure was seldom attained when eggs from this area were
used. Eggs from Treasure Island were frequently irregular in size
and shape, and a high percentage did not develop normally. The best
results were observed with eggs from Tomales Bay.

SUBSTRATE PREFERENCE BY METAMORPHOSING LARVAE

Mussel larvae undergo morphological changes during maturation.
After fertilization, the first stage of the larvae is called a
trochophore (Field, 1922). It is free-swimming and does not have
a shell. Next is the veliger stage. Veliger larvae are shelled,
free-swimming, and posses a large, ciliated lobe or velum. As the
larva increases in size, a foot develops, and the size of the velum
diminishes. When the foot is large enough to enable the larva to
crawl as well as swim, the larva is known as a pediveliger. This
is the stage that settles or attaches to a substrate by means of
byssus threads.

Primary attachment to a solid object is made by the ciliated foot,
and "permanent" attachment occurs when the mussel secretes byssus
threads. During the early stages of attachment, the larva may
reposition itself by detaching old threads and forming new ones
after moving to a new position with its foot, or by allowing it-
self to be transported to another location by the tide. We have
on numerous occasions observed mussels break their byssus threads
and move to another site. After initial attachment, the larva
metamorphoses. Metamorphosis, the process by which the juvenile
mussel is developed, is complete when the dissoconch shell (adult
shell) appears (Bayne, 1965).

27


SECTION IX

APPENDICES

Page

Water Quality Data from Adult Survival Experiments,
48-Hour Embryo Bioassays, and Growth and Metamor-

phosis Experiments . .. Co ee 87

Mean and Standard Deviation of Shell Length of
Mussel Larvae Reared in Seawater with Various Con-
centrations of the Pesticides ........... 91

Number, Mean, and Standard Deviation of Metamor-

phosed Mussel Larvae Exposed to Various Concentra-
tions of the Pesticides ... Se ee 97

86


SECTION VII

REFERENCES

Armstrong, D. A., and R. E, Millemann. Effects of the Insecti-
cide Carbaryl on Clams and Some Other Intertidal Mud Flat
Animals, J Fisheries Res Board Can, 31:466-470, 1974.

Bayne, B. L. Growth and the Delay of Metamorphosis of the Larvae
of Mytilus edulis (L.) Ophelia. 2:1-47, 1965,

Breese, W. P. Personal communication, 1972,

Bohmont, B. L. Toxicity of Herbicides to Livestock, Fish,
Honeybees, and Wildlife. Proc Western Weed Control Conf.
21:25-27, 1967.

Butler, J. A., R. E. Millemann, And N, E. Stewart. Effects of
the Insecticide Sevin on Survival and Growth of the Cockle
Clam, Clinocardium nuttalli. J Fisheries Res Board Can. 25:
1621-1635, 1968.

Butler, P, A. Commercial Fisheries Investigations. In:
Pesticide-Wildlife Studies, Fish and Wildlife Service
Circular 167. Washington, D.C., 1963. p. 11-25.

Courtright, R. C., W. P. Breese, and H. Krueger. Formulation
of a Synthetic Seawater for Bioassays with Mytilus edulis
Embryos, Water Res. 5:877-888, 1971.

Davis, H. C., and H. Hidu. Effects of Pesticides on Embryonic
Development of Clams and Oysters and on Survival and Growth
of the Larvae. Fishery Bull. 67:393-404, 1969.

De Blok, J. W., and H.J.F.M. Geelen. The Substratum Required
for the Settling of Mussels (Mytilus edulis L.), Arch Neerl
Zool, Suppl 1. 13:446-460, 1959,

81


All pesticides, except 2,4-D, were extracted directly from the
water sample with an organic solvent. Methoxychlor and Treflan
were extracted with hexane, and Sevin and malathion were extracted
with chloroform and benzene, respectively. After the extract

was dried in the presence of nitrogen gas, the residue was dis-
solved in acetone and analyzed on the gas chromatograph. Samples
of the herbicide 2,4-D were acidified with HCl before extraction
with diethyl ether, Analysis for 2,4-D was performed after the
pesticide was esterified with diazomethane.

STABILITY AND SOLUBILITY DETERMINATIONS

To determine the behavior of the pesticides in seawater, and to
estimate the highest concentration for use in the toxicity tests,
the solubility and chemical half-life of each pesticide in sea-
water were assessed.

Solubility was determined by continuously shaking an excess
amount of pesticide in natural seawater having an adjusted salin-
ity of 25 Joo, a pH of 8 + 2°C. The filtered (Nuclepore®, 0.8 L)
samples, taken at intervals, were analyzed for the pesticide.
Concentration-time curves were constructed, and solubility was
estimated from the curves.

The stability of each pesticide was determined by shaking a
soluble amount of pesticide in seawater and analyzing samples
of the solution taken at intervals over several days. The half-
life was estimated from the resulting concentration-time curves.

PREPARATION OF TEST SOLUTIONS

All pesticide concentrations were prepared by dilution of the
highest level tested with natural seawater, A measured amount
of malathion, Sevin, and 2,4-D was added to a known volume of
seawater, sonified, and then power stirred into a larger volume
to obtain the highest concentration to be tested, Treflan and
methoxychlor first were dissolved in acetone and then were added
slowly to a constantly stirred vat of seawater. The pesticide-
acetone solution was prepared so that the saturated stock

8


Table 13.

EXPOSED TO METHOXYCHLOR FOR 96 HOURS

SURVIVAL AND ATTACHMENT DATA FOR ADULT MUSSELS

Test Test concentration No. of Survival, | Attached,
no. Nominal Measure | @nimals percent percent
14 Seawater
control -- 6 100 100
Acetone
control -- 6 100 100
0.050 -- 6 100 100
42 Seawater
control 0 10 100 90
Acetone
control 0 10 90 90
0.050 0.007 10 100 100
0.100 0.020 10 100 100
45 Seawater
control 0 10 100 100
0.050 0.055 10 100 100
0.100 0.0925 10 100 100

51



Table 2, MEAN AND STANDARD DEVIATION OF SHELL LENGTH OF MUSSEL LARVAE REARED IN SEAWATER WITH VARIOUS CONCENTRATIONS OF TREFLAN
Means Based on Measurement of 20 Larvee
(microns)

£6

Concentration, mg/liter
Seawater controls Solvent controls 0.024 7.048 0,096 0.192,
Wean [sb J wean [SD Wean | SD Mean] SD Mean] SD Wean] SD Mean [SD Mean | SD Mean | 5D Nean [SD ean SD Mean [ 5D
T
Experiment 13
105 | 2.9 | 108 | 2,9 | 105 ) 2.9 | 105 | 2. 2.9 | 105 2.9 sos | 2.9 | 105 | 2.9 | 105 2.9 | 105 | 2.9
103 | 7.0 | 107 | 7.0 | 109 | 3.7 | 106 | 6. 34 3.5 107 | 4.1 | 120 | 3.5 | 10d 10,5 | 108 | 6.5
uo | 5.3 | 108 | 7,5 | as | 8.2 | a7 | a, a7 7.6 is | 38.8 an.7 | 9 ao.s | v2 | 7a
zi }10,4 | 126 j10.7 } 133 jis.a | raz | 13. 15.7 20.7 137 | 10.0 is.o | 129 13,7 | 130 | 14.6
vai |25.5 | 141 | 12.6 | 148 {16.1 | as2 | a4. 21.9 14.6 162 | 16.2 13.9 | 155° 13.1 | 145 Jiz.s
uaz j22.s | 150 }22.2 | 152 {18.2 | 1s6 | 30.: 29.9 19. 160 | 15,7 20.4 | 166 is.s | iss S
179 177 20,8 | 179 |17.3 | 189 | 19. 31.7 29,9 178 20.4 | 176 28.1 | 179 2
186 is7 [32,8 | 177 | 25.1 | 206 | 26, 37.0 24.7 77 23.) 22.4 | 18s 0
206 200 | 37.9 | 193 | 22.8 ] 221 | 27. 20.0 21.7 194 25.1 25.5 | 177 a
230 | 35,7 | 210 | 44,0 | 230 233 | 32 29.5 38.1 187 25.8 31.8 | 177 8
27a |4o.a | 266 | 4,6 | 249 256 | 34. 40.0 29,2 188 18.3 26.4 | 183 | 98.5
260 |29.9 | 277 | 36.2 | 240 251 | 27, 23,9 44.0 230 47,7 244 | 195 | 21.5
28.9 | 268 | 43.6 | gar far. | 2a | 33, 29.6 4S 23.4
42.5 | 280 | 51,3 | 280 | 35.0 | 282 | 42. 32.2 16.2 a
271 | 56,5 | 302 | 40.6 | ass | 52. 40.3 33,5
go9 | 45.3 | 353 [43.4 | son | 42 39.7 22,0
332 | 42.2 | 345 | 30.9 | 331 | 37. 41.5
330 | 35,0 | 354 |aa.9 | su2 | 34. 43.6
340 | 30.3 | 351 |33,6 |) 362 | 28. 33.9
o ssa fan.a | --~ 334 | 42 a6 |
Expe
103 | 4.9 | 103 | 4.9 | 103} 4.9 | 103 103 uo3 | 4.9 4.9 | 103 oar)
113} 6.9 | ara] ace | tad | as | ais 113 in} $.4 74] 116 Sa
123 | 14.3 9.a | 126 | 20,5 | 122 120 13s | 3.0 10,0 | 129 7.3
141} 16.9 13.0 | 14s |is.s | 139 160 | 18.5 ao | 149 47,1
162 | 23.8 is.2 | 183 | 35.8 | 197 as7 | 22,3 19,7 | 179 20.0
193 | 19.6 En a7o | 33.8 | iss 226 | 1i.9 36.9 | 174 w.6
198 a 23.9 | 209 [25.4 | 191 219 | 32.3 28.0 | 162 214
23a | 22.5 30.8 | 202 | 338.3 | 223 243 | 32.3 24.3 | 159 29.0
222 | 16.6 49.3} gia fsa. | as 3 33.6 | 197 19.6
292 | 19.5 28. gst |2d.s | 273 a 27.8 | 216 19.3
302 | 46.8 36.9 | 305 | 35.2 | 299 5 27,8 | as 22.5
335 | 36.0 tsa | 296 [51.8 | 261 6 21.5 | 204 18.5
aca | 33.5 os.7 | 348 | 30.2 | 308 6 23,6 29 24.9
asa | 56,5 49.5 | 328 | 51.6 | 319 37.9 | 248 19.6
as6 | 93.3 go.1 | 373 | 48.2 | 278 44.0
4o.6 | 274 |4a.3 | 302 39.3
13,1 | 367 | 35.6 | 315 2d
29.0 | 353 | 39.9 | 282 31.7 -



ob

75

cumulative percent

METAMORPHOSIS,

25

FIGURE 8

T T T

Control
1.51
----- 302

eeccecces 12.1

5 10 15 20 25 30 35
LENGTH OF EXPOSURE — days

EFFECT OF EXPOSURE TO MALATHION ON PERCENTAGE OF MUSSEL POPULATION COMPLETING METAMORPHOSIS

Larvae were 29 to 30 days old when exposure was initiated. Data from the two experiments were pooled.


Table 3.

EXPOSED TO SEVIN FOR 96 HOURS

SURVIVAL AND ATTACHMENT DATA FOR ADULT MUSSELS

Test concentration

Test No. of Survival, | Attached,
no. Nominal Measured | animals percent percent
45 Control ie) 8 88 --

2.5 2.3 8 88 --

5.0 4.9 8 100 --

10.0 10.0 8 100 --

20.0 19.8 8 75 --

25 Control -- 9 100 80
Control

(solvent) -- 9 100 90

5.0 -- 9 100 80

10.0 -- 9 89 70

20.0 -- 9 78 70

40.0 -- 9 67 40

50 0 0) 10 70 70

20 9.4 10 70 60

30 13.6 10 50 40

50 34.2 10 30 20

75 25.2 10 ie) 0

53 0 0 10 100 100

20.6 11.3 10 70 50

31.7 16.0 10 70 30

48.8 20.3 10 50 20

75.0 30.9 10 40 10

33



up to 25.9% smaller than seawater control larvae and up to 15.9%
smaller than the seawater-acetone control larvae. Larvae exposed
to 0.192 mg/liter of Treflan were up to 29.6% smaller than seawater
control larvae and up to 28.1% smaller than seawater-acetone con-
trol larvae. These differences were statistically significant.

As with the data collected on metamorphosis of the larvae used in
the Sevin growth studies, the data on metamorphosis of larvae ex-
posed continuously to Treflan from the time they were 48 hours old
were inconsistent and difficult to interpret. The data from dupli-
cate experiments were pooled for analysis, but results from the two
tests were analyzed separately. These data are shown in Table 1l.
Mean age values in the table refer to the age of the larvae at the
time the first larva or group of larvae completed metamorphosis.

Table 11. NUMBER, AGE, AND SIZE OF JUVENILE MUSSELS
DEVELOPING IN LARVAL CULTURES
EXPOSED AT 48 HOURS TO VARIOUS CONCENTRATIONS OF TREFLAN

Test Experiment 1 Experiment 2
tration, No. of Age,@ Length, 2» No. of Age,4 Length, w
mg/liter juve> days Juve* days

niles Mean| SD | niles Mean] SD
0 54 24 4425 |77.0| 178 26 1456 |78.7
0 (acetone) 87 27 402 [54.3 81 26 |410 |61.0
0.024 84 28 |439 |84,0) 146 26 1444 |72.6
0.048 167 26 |379 |50.7) 150 26 |431 |80.1
0.096 1} -- Ct Rated 43 32 360 |34.4
0.192 0 -- -- |-- 0 -- -- |--

“Number of days required by treatment group to begin meta-
morphosing.

45


six freshwater arthropods and reported 48-hour TL values rang-
ing from 0.25 mg/liter for the seed shrimp (Cyprinodopsis vidua)
to 3.2 mg/liter for the grass shrimp (Paleomonetes kadiakensis)
and 50 mg/liter for the crayfish (Orconectes nais). The toxicity
of Treflan to various species of freshwater fish also has been re-
ported (Macek et al., 1969; Parka and Worth, 1965; Bohmont, 1967;
Worth and Anderson, 1965).

Although our study showed that Treflan may be lethal to adult mus-
sels exposed to 0.24 mg/liter for four days and can inhibit shell
growth in larval mussels at a concentration as low as 0.096 mg/liter
if exposure exceeds 10 days, under nonlaboratory conditions it us
unlikely that Treflan exerts serious deleterious effects on the bay
mussel or perhaps on other bivalve molluscs. Its solubility in
seawater with a salinity of 25%0, pH of 8, and temperature of

20°C is about 0.2 mg/liter. Its half-life in seawater is only

82 hours. A selective preemergence herbicide designed for use with
a variety of farm crops, particularly vegetables, Treflan is ap-
plied by mixing it into the soil. When applied at recommended lev-
els, it usually disappears in four to six months but not by leaching
(Herbicide Handbook, 1970). These characteristics suggest that,
unless Treflan is applied directly into the aquatic habitat, the
possibility of toxic levels entering the aquatic environment is low.

Methoxychlor

In terms of survival, methoxychlor was not toxic to adult mussels
exposed to concentrations as high as 0.092 mg/liter for 96 hours
(Table 13). This concentration is almost twice that estimated for
methoxychlor in methoxychlor-saturated, 20°C seawater. This level
of insecticide also had no effect on attachment.

In the 48-hour embryo bioassay experiments, the test solutions con-
tained measured levels of 0.011, 0.018, 0.041, and 0.075 mg/liter
of methoxychlor. Exposure to methoxychlor did not affect the de-
velopment of normal larvae significantly at any of these concentra-
tions (Table 14). The mean percentages of normal larvae found in
the seawater control and seawater-acetone control groups were 89.4
and 88.7, respectively. The mean percentage of normal larvae found

50


Larvae exposed to the highest test concentration of 0.192 mg/liter
did not undergo metamorphosis. None of the larvae exposed to this
concentration lived beyond 26 days of exposure. Nor did the lar-
vae exposed to 0.096 mg/liter of Treflan in Experiment 1 survive
longer than 26 days. Inspection of the number of larvae that did
undergo metamorphosis in Experiment 1 does not reveal any delete-
rious effects. In Experiment 1, metamorphosis appeared to have
been delayed by two to four days in groups exposed to 0.024 and
0.048 mg/liter when the age data for these groups were compared
with those for the seawater controls; however, comparison with
the age data for the seawater-acetone controls revealed no delay.

In Experiment 2, twice as many seawater control larvae developed
into juveniles than seawater-acetone control larvae; however, be-
cause this effect was not observed consistently in the two studies
(Treflan and methoxychlor) in which seawater-acetone controls were
employed, we believe the effect may be an artifact. In this ex-
periment, the number of juvenile mussels developing from larvae
exposed to 0.096 mg/liter of Treflan was only 24% of the number
of juvenile seawater control larvae and about half the number of
juvenile seawater-acetone control larvae. The Treflan-exposed
larvae also began to undergo metamorphosis six days later than
the controls and the larvae exposed to the next lower concentra-
tion, 0.048 mg/liter. In this experiment, the larvae exposed to
0.096 mg/liter were also about 21% smaller than the seawater con-
trol larvae at the time of metamorphosis.

These effects were not observed in the metamorphosis study in
which exposure to Treflan was not initiated until the larvae were
30 days old (Table 12). The concentrations tested in this study
were slightly lower than those used in the growth study, although
the same concentrations were intended. The mean and standard de-
viation of 34 determinations made on the highest test concentra-
tion, nominally set at 0.4 mg/liter, were 0.16 + 0.072 mg/liter.
The lower concentrations were estimated at 0.08, 0.04, and 0.02 mg/
liter.

Juvenile Traflan-exposed mussels were somewhat smaller than the
seawater control larvae. As a group, they were 9.6% smaller than
the seawater controls, although their size did not appear to be
related to the pesticide concentration.

46


particularly in the pediveligers and perhaps in the juveniles and
adults. According to Bayne (1965) and others (Green, 1968; De Blok
and Geelen, 1959), swimming and crawling movements of the pediveli-
ger represent efforts to locate a suitable area in which to attach
by byssus threads. Metamorphosis can be delayed by the lack of a
suitable substrate or of the proper environmental conditions (Bayne,
1965). Larvae evidently can detect subtle changes or differences
in their environment. Wisely (1963) showed that crawling mussel
larvae, Mytilus planulatus Lamarck, could detect antifouling paint
before they touched it. After detection, the mussel ceased crawl-
ing and remained closed. The shelled mussel must open its shell

to secrete byssus threads, and, in doing so, exposes itself to the
environment. Refusal to attach may indicate that the environment
is unsuitable and may reflect a defense mechanism used by the mus-
sel during periods of adversity. Reish and Ayers (1968) determined
the number of byssus threads laid down by the bay mussel under
various conditions of chlorinity and dissolved oxygen. They dis-
covered that, although the mussel can tolerate low oxygen and chlo-
rine levels, the number of byssus threads formed varies with these
two factors.

The embryo was sometimes more sensitive than, and sometimes only
as sensitive as, the adult to the pesticides. The concentration
of the insecticide Sevin that caused 50% of the embryos to develop
abnormally was 15 times less than the concentration that produced
50% mortality among the adults. However, with 2,4-D, the statis-
tics were about the same. Concentrations of methoxychlor and Tre-
flan that had no effect on adult survival also had no effect on
embryonic shell development.

Larval metamorphosis or, more specifically, the formation of the
dissoconch shell appeared to be about as sensitive an indicator of
toxic effect as shell growth; however, because of inconsistencies
in the data on metamorphosis obtained from the survivors of the
growth study, and because of the lack of information on the size
of the test population and mortality rate, the evidence is not
conclusive. Data from some of the experiments support this idea,
whereas data from others do not.

75


SECTION VIIL
INSTRUMENTS USED IN THE STUDY
1. Gas chromatograph (Microtek, Model 220) with electrolytic
conductivity detector; used in the quantitative analysis of the

pesticides.

2. Particle counter (Coulter, Model B); used in counting mussel
eggs and algal cells.

3. Refractometer (Goldberg); used in measuring salinity.

4. pH meter (Radiometer, Type PHM26).

5. Oxygen meter (Yellow Springs Instrument Co., Model 54);

used to determine dissolved oxygen levels and temperature of the
test solutions.

6. Ultraviolet liquid purifier (Ultradynamics, Model 500); used
to reduce bacterial population in natural seawater used in the
tests.

7. Sartorius analytical balance.

8. Sonifier (Branson Instruments, Inc., Model LS75).

85


The 96-hour TLs5g estimates we obtained for adult mussels are higher
than those reported for adult forms of a number of other marine in-
vertebrate organisms. The test organisms for most of the studies
on adult forms have been marine arthropods, which, perhaps because
of their close phylogenetic relationship to terrestrial insects,
show a relatively high sensitivity to Sevin. In one study using
the mollusc Clinocardium nuttalli (cockle clam), the 96-hour TL5o
was reported as 3.85 mg/liter (Butler et al., 1968). In a more
recent field study, Armstrong and Millemann (1974) observed reduc-
tions of up to 69% of controls in populations of the gaper clam
(Iresus capax) and reductions of up to 28% in populations of the
bent-nosed clam (Macoma nasuta) in field plots to which Sevin was
applied. The pesticide concentration was not measured but was
assumed to be less than 60 mg/liter.

Stewart and coworkers (1967) investigated the acute toxicity of
Sevin and its byproduct l-naphthol to a number of marine organisms.
They found that the sensitivity of some of the organisms varied
with water temperature and with the sex of the organism. The in-
vertebrate species generally were more sensitive to Sevin than to
1/naphthol. The 24-hour TLs59 for the mud shrimp (Callianassa
californiensis) was 0.13 mg/liter at 16°C, but at 20°C it was

0.04 mg/liter. Female shore crabs (Hemigrapsis oregonensis) were
more sensitive to Sevin than the male crabs: The 24-hour Tleg for
females was 0.27 mg/liter, whereas the estimate for males was

0.71 mg/liter. The sensitivity of male and female Dungeness crabs
(Cancer magister) was just the opposite of that of the shore crab.
The 24-hour TLs5q reported for male crabs was 0.60 mg/liter, whereas
the estimate for female crabs was 0.63 mg/liter. This difference
in response was not statistically significant, however. These in-
vestigators also reported an estimated 24-hour Thsg of 7.3 mg/liter
for the cockle clam. That this estimate is almost twice the one
reported by Butler and coworkers may be due to the differences in
the length of exposure.

Stewart and associates (1967) also conducted 48-hour mussel embryo
bioassays with Sevin and reported a 48-hour ECog of 2.3 mg/liter.
Several tests were performed with ECs, values ranging from 1.4 to
2.9 mg/liter. Our 48-hour EC59 estimate of 1.5 mg/liter is slightly
lower. Using the same bioassay technique, Davis and Hidu (1969)
investigated the effects of a large nuuber of pesticides to the

40


EPA-660/3-75-016
MAY 1975

TOXICITY OF SELECTED PESTICIDES TO THE

BAY MUSSEL (MYTILUS EDULIS)

By

David H. W. Liu

Jean M. Lee

Contract No. 68-01-0190
Program Element 1BA022

ROAP/TASK 16 AAR/13

Project Officer

c. S. Hegre
National Marine Water Quality Laboratory
National Environmental Research Center
South Ferry Road
Narragansett, Rhode Island 02882

NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330

For Sale by the National Technical Information Service
U.S. Department of Commerce, Springfield, VA 22151


SELECTED WATER B Rewort Noy:
RESOURCES ABSTRACTS

INPUT TRANSACTION FORM Ww

, §. Report Dace
TOXICITY OF SELECTED PESTICIDES TO THE BAY MUSSEL a

(@ytilus Edulis) 8 5 -rform: ¢ Orga, sation

Repo:t No.

Liu, David H, W.; and Lee, Jean M.

Stanford Research Institute
Menlo Park, CA 94025 68-01-0190

1 Typ “Rep. and
Perea Covered

12, Speasorin, Organ >tion Environmental Protection Agency

Environmental Protection Agency report number EPA-660/3-75-016

The toxicity of the insecticides Sevin, methoxychlor, and malathion and of the herbi-
cides Treflan and 2,4-D to the bay mussel (Mytilus edulis) was investigated. Toxic
effects were measured in terms of survival of and byssus—-thread attachment by adults,
embryo shell development, and larval growth and metamorphosis.

The results indicated that growth was the most sensitive measure of toxicity. All the
pesticides produced statistically significant (p = 0.05) reductions in larval shell
length after 10 to 20 days of exposure, Relative to potency, methoxychlor was the
most toxic, and 2,4-D was the least toxic.

The 96-hour TL59 values for each pesticide, based on adult survival and attachment data,|
were estimated, as were the 48-hour EC50 values based on data from embryo bioassays,

The effects on embryo development of delaying the time of fertilization and of using
seawater larval culture media of various ages also were studied, and substrate pref—
erence by metamorphosing larvae was investigated.

A critical evaluation of the experimental approach and procedures is presented.

Ta, Deseripcors

Pesticide Toxicity, Mussels, Mollusks, Marine Animals

IDB Tdortibeers

VC, Group 16

19. Securit Class ot oof | Send To:
Repo ages
. WATER RESOURCES SCIENTIFIC INFORMATION CENTER
Sooty B US DEPARTMENT OF THE INTERIOR
(Eve) WASHINGTON. DC 20240
David H. W. Liu I Stanford Research Institute

& US GOVERNMENT PRINTING OFFICE 1975-699-475 1&5 REGION! 10



Metamorphosis of larvae exposed from age 30 days to age 70 days
was not markedly affected by exposure to 2,4-D concentrations as
high as 176 mg/liter (Table 23). Variability in the number of
larvae that completed metamorphosis increased at concentrations
above 22 mg/liter. The mean percentages of larvae that completed
metamorphosis while exposed to 2,4-D varied from 68.6 for larvae
exposed to 44 mg/liter to 70.6 for those exposed to 22 mg/liter.
The mean for all 2,4-D-exposed groups was 70%. Of the control
larvae, 79% in both tests developed into juvenile mussels.

Generally, larvae exposed to 2,4-D exhibited higher mortality than
controls. Between-test mortality percentages varied considerably
more in the pesticide-exposed groups than in the control groups;
however, mortality did not vary with concentration. As a group,
the 2,4-D-exposed larvae had a mortality average of 12.7%, com-
pared with 7% for the controls. During the first 22 days of ex-
posure, the larvae exposed to the two higher levels of 2,4-D meta-
morposed at a faster rate than any other group (Figure 7). How-
ever, no significant differences in rate were observed by the end
of the experiments.

Few toxicity studies on the acid form of 2,4-D have been performed
on marine invertebrate organisms. Butler (1963) reported that
2.0 mg/liter of 2,4-D acid and the same concentration of the di-
ethylamine salt did not affect shell growth in adult oysters

(C. virginica); however, 3.75 mg/liter of the butoxyethanol ester
reduced shell growth by 50%, and 5 mg/liter of the 2-ethylhexyl
ester of 2,4-D reduced it by 38%. Davis and Hidu (1969) tested
two forms of 2,4-D using C. virginica and reported 48-hour ECs5o
values, based on embryo bioassays, of 8 mg/liter for the butoxy-
ethanol ester and 20.44 mg/liter for the diethylamine salt. They
also reported 14-day TL., estimates, based on larval survival, of
0.74 mg/liter for the ester and 64.29 mg/liter for the salt.
Marine crustacea appear to have much more tolerance to 2,4-D acid
than marine bivalve molluscs. The 96-hour TLsq for the fiddler
crab (Uca pugnax) is reported to be 5000 mg/liter (Sudak and Claff,
1960).

63


6b

75

METAMORPHOSIS — cumulative percent

FIGURE 5

T T T T T

—— Contro!
— — —Contro! - Acetone a=

0.02
= 0.04
+ 0.08

eeceeeeeee 0.16

1 |

5 10 15 20 25 30 35
LENGTH OF EXPOSURE — days

EFFECT OF EXPOSURE TO TREFLAN ON PERCENTAGE OF MUSSEL POPULATION COMPLETING METAMORPHOSIS
Larvae were 29 to 30 days old when exposure was initiated. Data from the two experiments were pooled.


86

‘rable 1.

Fxposuro Initiated 29 Days after Fertilization of Epes
(nicrons)

NUMBER, MEAN AND STANDARD DEVIATION OF SHELL LENGTH OF METAMORPHOSED MUSSEL LARVAR EXPOSED 70 SEVIN

Concentration, ug/liter

seawater control 3,8 am 15 z
PS TN ST TN
eo fol- a a} 0 of --- of - 0 o| -- o| - 0
2 vo]- ° of = ° 9 o| - 3 o| —- o| - a
aoe 0 o} =~ ° of o| - ° of of: °
aia o|o 0} 0 of -- rfarr foo | o a}oo | o | 2| oa | 2s | afou | o
7 Jo 0 o] ° of --- rfare fo | 1fa6s | 0 | of af aca | | 2] as8 | 27
eo fifase | of 2 nu | of -- ° ° o| - afar fs | sfaco | o | s|asr | 6 | ra) 256 | as
a fo ° s}as | o | o r{asa | o | «fase | 20 | 2faco | es] of ser | a2 | a} ser | oo | o
xa fafa fas | 2fasa | 26 | of --- ° afaso | o | afore | a | a] aca afase Jie | zfasr | 2s | 2}am | ao
rs fafaor | ar] afass | 0 | ofase Jarz | afaso | 1a | cfas | is} 2} am | s | ef acs | 20 | a] azo | 90 | afm |ss | alan | ss
xt fafvos | oa | ofai2 | ss faofsas | ox | sf sso | ao faa fars | so far} acs | so [20] asa | as | 20) ase | so |as}aao | so | 7] 220 | a
rg Jsfas | as} 2)sos fiso fo} az | co | af aos | cs | afar | se Jao} ase | ao | efas2 | so | 6) soo] so} 2] 2a | 0 | a}ass | ao
ar felsis | 26 | 6|s47 Jaor } 3] aco | a |asfaas | as | 3fe22 Jos | 1|a60 | o | 2] ass} 2 | of --~ 1} 202 | o | 2}ees } a7
aa pafaso | se fas}are | sx | fare | sx | sfaze | za] 7 [aor | or | al ass | 0 | 3) 427 faa | of --- o| - 1} 260 | 0
os }o|ase | 29 | s|ars Jasr | a2} aas faos farfsce | se | o}sas | oe | s| aa | os | 2] sia fase | 7] see | a | of - °
27 j7|sor Jas | o s|aaz | 6s | 7] soo | 27 | 7Jase | s7 | 2] 537 Joos | 2] aus | a5 | of aea | 29 | 2} aso far | a) 026 | a0
oo fafara | | sla | aa | afaso f ss | afro | o | sfas | a2 | 5] 420 | 53 | 0 a|s00 | 24 | a} as | 0 | 2[ass | 20
ar |zfaaz | 23} afacr | 8 | 6] aso | ox | 2] 558 Joa | of --~ rfae7 | o | xfse7 | of xfasr | o | of - °
aa falas: | o | {aso | 29 | of --- 2 a]sss | o | xfan | o | 1] s30 | o | sfase | o | of - 2
ae |s}aco | 2x | arf ase | so | of --- 1| s20 o| --- o| - 2/997 | 9 | af a0 | o | Jas: | 0} o
so |2}soa | s2 | afso7 | 23 | of -- 1] 507 | of 2fas2 | o |az{ a0 | ao | afas | o| 2] a7 | os | 2] - ‘ °

of

brmer ee

phosed

Thvvae [2 ° ° 6 2 a 1 2 0 0



In our investigation of this factor, we discovered that the per-
centage of normal larvae decreased markedly when fertilization was
delayed for more than two hours (Figure 1). The percentages shown
in Figure 1 are relative, calculated by dividing the percentage of
normal larvae observed in egg groups fertilized at different times
into the highest observed percentage (110 min) and multiplying by
100. There was a 45-min lapse between the time a female began to
shed eggs and the time when a sufficient number of eggs for the
experiment were produced.

The experiment was not designed for determining whether the effects
of delaying fertilization were due to the age of the eggs or to the
age of the sperm. All egg groups were fertilized by sperm from a
single male. Microscopic examination of the sperm showed that they
were active at all times.

The second factor investigated was the age of the diluent seawater.
During this study, we also evaluated Kester's artificial seawater
formulation (Kester et al., 1967) as a mussel embryo culture medium.
Personnel from the National Marine Water Quality Laboratory in West
Kingston, Rhode Island, use this formulation for culturing of ma-
rine plankton, and they suggested we use it. The formulation was
not suitable for culturing of mussel larvae, however. The mean
number of normal larvae found in 15 duplicate cultures (30 tests),
started originally from fertilized eggs taken from mussels collected
in four different geographical areas, was equivalent to 4.5% of the
larvae inspected. The range was 0.25 to 41.5%.

The unsuitability of Kester's synthetic seawater for mussel larvae
culture is probably due not to the presence of toxic components but
more likely to the absence of one or more substances essential for
larval development. Although Kester and his associates recommend
the use of reagent-grade chemicals, we substituted Leslie rock salt
for reagent-grade NaCl. Because this substitution could have in-
fluenced the results, we cannot unconditionally reject the formula-
tion. The substitution was made in an effort to include trace sub-
stances that might be beneficial to the larvae. Use of nonreagent-
grade chemicals to supply trace substances was the intent of LaRoche
and coworkers (1970) in their modification of the synthetic seawater
formulation of Zaroogian and associates (1969).

23


concentrations of each pesticide. The four levels of pesticide
were tested in duplicate, and duplicate control groups also were
used. Because acetone was required to aid dispersion of Treflan
and methoxychlor, solvent controls also were used in tests on these
pesticides.

The tests were conducted in 75 X 150 mm Pyrex crystallizing dishes.
The volume of test solution was 600 ml, and the initial number of
larvae per dish was 100. A saturated solution of each pesticide
was prepared every other day, and the desired test concentrations
were prepared by serial dilution. The test solutions in the dishes
were changed every other day.

To renew the test solution, a 3-in. length of 1/2-in. diameter PVC
pipe, with one end sealed by a piece of 53-1 Nitex screen, was
placed into the dish. The fluid in each dish was siphoned out
through the screened pipe. Fresh solution was poured into the test
dish, and the screened pipe was sprayed with a mist of seawater.

The larvae were reared in natural seawater for 29 or 30 days before
being exposed to a pesticide. The larvae population used in the
tests was composed of larvae that developed from eggs of three fe-
males collected at Tomales Bay. We selected for study only those
larval groups in which at least 85% of the larvae developed normal
shells during the first 48 hours after egg fertilization. During
the pretest rearing period, the larvae were fed M, lutheri daily at
a rate of 20,000 algal cells/ml of seawater. The seawater was
renewed every two days.

The pesticide exposure period was 40 days. During this time, all
the dishes were examined under a dissecting microscope every other
day. Larvae attached to the dish by byssus threads were removed
gently and reexamined under a compound microscope for presence of
a dissoconch shell. Those having a dissoconch shell were measured
counted, and discarded. Those without new shell growth were placed
back in the test dish. Dead larvae, as well as those that were
accidentally crushed during renewal of the test solutions, were
counted and discarded. M, lutheri was fed daily at a rate of
20,000 cells/ml of test ‘solution throughout the exposure period,

19


z01

Table 3.

Exposuro Initiated 30 Days after Fertilization of Fees
(microns)

NUMBER, MEAN AND STANDARD DEVIATION OF SHELL LENGTH OF METAMORPHOSED MUSSEL LARVAE EXPOSED TO MALATHION

Concentration, mg/liter

Scawater Control FE 3.0 “55 FER
bay [a] Wean | 9D [n [Mean] 5D [| nTwean [60 [a] wean] SO | [wean] 80 [ [wean] SD | n[wean[ 50 [ [wean] sb | a[wean [op [-n [wean | 8D
° oO oO Of --—- o O]n-- l o| - oO oO| --- oO oO
2lo ° o| --- o of --- o| - ° o| --- ° °
aio ° of --- o o| --- o| - ° o| --- ° of ---
s | o o o|--- o of --- o| - 1 o | 1 as | 0} o rfas | o
7] 0 a}as0 | o | of --- o of o| - ° 1[ a6 | o | 5 no
9 | o 2|s9 | 6 | 1fa7e | o | o |] -- 2|ai2 | sa | 2]sa0 ]2s | o| --- aa] aso | 51 | 26] 369 | 32
a} o 2} 403 frar | of 2}an | a6 | of --- tT] as | 62 | 0 3}aca [as | alan | ao} 2}an | 46
as | o asa | 76 | 2fase | aa | afar2 | a8 | 2) 350 Jai | 2] ase | o | s}art for | 10} 40s | 62 |r| ses | a7 | 1s] sao | sa
is | a] asa | aa | of --- 2feos | 2a | 6{ aes }1oo | 5| aa | 41 | af aio | vs | oe] ago fso | af a92 | .o | a2) ss7 | 54 | 1a] aco | 29
a7 | sfass Jaa] afavs | o | sear | as | s{ava | ze | sfaa7 | ao | 2] ara 190 | 6| 308 }20 | 11] 409 | 63 | 10] sas | as | 7} 30: | a8
vo | class | or] z]as2 | as | 7Jas0 | sr | 2] 500 ass | 9}ars | a9 | 10] 527 lias | 5] azo |75 | 6] 360 | 6a | a) se7 | s2 | s|a07 | o
ar | 6] ass | 29 | as}asa | oo | a]ass | oo | 3] 477 | as | a]ase | a2 | 12] 400 | a6 | 1) 409 | o | 2] 452 | a7 | s]air | es} 1] s23 | 0
2a | 5} 4a2 | on zea | 23 | 7}as2 | ar | af a36 | 62 | a|a67 | 50 | 9] ase | or | 2] asa [so | {306 | a6 | 2| asa | 28 | o
25 | afsas | o| elas | aa | s]aas | as | 6) 425 | 56 fro] asa | t2 | al asr | 28 | 5] aes Jaz | 5] 466 | 65 | 1| 34a | o °
27 | 3[4se | 60 | a0} ass [105 | a) 477 | 6a | 6{ 45a | s2 | 2] as7 | 34] 7] 401 | 09 | a{ ase Jas | 9] 413 | 50 | 1| 418 | o | 1|ssa | o
20 | z]ass | 6s | 6| 491 | x27 | 2] 498 | 7 | s]sa7 | 86 | 5] aaa }ao2 | a] az |aso | 6] 407 ]22 | 4} 261 Jase | o| - 1}440 | 0
a1 | 7] a9 | 92} s}ao7 | 4 | 6| 408 | 7a | 6) 460 | 6s | a|aa7 | or | 2] aa2 | a1 | 1] 382 | o | 7) 486 | 95 | 2| - °
a3 | 2] ass a}ass | 0 | 0] -— s| azz | ea | 1] --- o| - alan [sa | 1! 350 | 0 | of - °
as | a}aie | of] afaso | ax | a] asx | 62 | s| az | sa | of --- o| --- | 423 |22 | o| --- ol - °
as | 3}aae | 40] afase | 7s | a] aso | a6 | 3] a6 | ss | 8} axx | 22 | 2] ser a1] s76 for | of --- eo °
3a | afass | 27 | 3] 02] 17 | af asa | 30 | 2| 468 | 0 | of --- 3} a7 far | 1 o| --- of --- °

No. of

unmeta~

nor-

phosed

larvae | 12 5 19 13 8 3 2 ° ° °



30
T T T i |
—— pve
pees Glass Test Chamber
Plexiglass
25 |. ---- = Frosted Glass
Sitk Thread

3 20
5
a
«
8
fa
o 15
2
F
a
w
<
z
< 10
3

5

Oo

0 100 200 300 400 600
NUMBER OF LARVAE PER 600 mI OF SEAWATER

FIGURE 3. INFLUENCE OF LARVAL DENSITY ON LARVAL SELECTION OF SETTING
SUBSTRATE

31


GS

Table 17.

EFFECT OF METHOXYCHLOR ON MUSSEL LARVAE METAMORPHOSIS

AFTER A 41-DAY EXPOSURE INITIATED 29 DAYS AFTER FERTILIZATION OF THE EGGS
(Presence of Dissoconch Shell Used as Indication of Metamorphosis)

Test concentration, mg/liter
Seawater Solvent 0.008 0.015 0.03 0.060
control control
1 2 1 2 L 2 1 2 1 2 1 2
Number of larvae
Initial 100 100 100 100 100 100 100 100 100 100 100 100
Lost or crushed} 21 39 7 20 18 20 0 27 20 11 1 24
Experimental 79 70 86 73 82 80 100 73 80 89 89 76
Metamorphosing,
percent 92.4%] 94.3%] 96.5%] 95.9%} 95.1%] 97.5%] 79.0%] 89.0%] 93.8%] 83.1%] 80.0%] 85.5%
Nonmetamorphos-
ing, percent 1.3%] 0 0 ie) 0 ie) 0 ie) 0 0 0 0
Dead, percent 6.3%] 5.7%) 3.5%) 4.1%) 4.9%) 2.5%] 21.0%] 11.0%] 6.2%] 16.8%) 19.1%] 14.5%
Days to meta-
morphosis of 50%
of test larvae 20 22 11 14 1 14 16 1 16 17 16 14
Shell length of
juvenile mus-
sels, 480 y [457 w [408 yp, [406 4 (386 y {411 y 1377 4376 w 4377 yw {390 y [364 yw 1400 p



We believe, however, that the full potential of the bay mussel will
not be realized until toxicological testing procedures are refined
to such a degree that most laboratories having a source of mussels
and of natural seawater can perform the studies described herein
with greater efficiency and reliability of results.

Except in the adult survival tests and the 48-hour emt yo bioassay
experiments, much of the project effort was directed toward develop-
ing methods, often resulting in considerable delays in proceeding
from one phase of the project to the next. Because of requisite
project deadlines, the procedures actually employed--particularly
in the studies on larval growth and metamorphosis--were far from
efficient.

In spite of the difficulties encountered and the limitations of the
study, the investigation revealed important information on the rela-
tive sensitivity of various stages in the life history of the bay
mussel to the pesticides evaluated. Regardless of the type of pes-
ticide, larval growth was the most sensitive indicator of toxicity--
which is readily apparent on inspection of Table 29, a summary of
the toxicity data. Table 29 presents the 96-hour Thsg and ECsg
estimates for adult survival and attachment, the 48-hour ECsg esti-
mates for embryo shell development, and the minimum effective con-
centrations that produced statistically significant reductions in
larval growth and marked reductions in the number of larvae develop-
ing into juvenile mussels. For some of the pesticides, statistically
significant reductions in larvae size did not appear until after 20
days of exposure; for others, size reductions were observable by
the tenth day. These two periods were the only ones for which ef-
fects on growth were examined in detail; it is likely that statis-
tically significant effects would have been detected earlier if all
the growth data had been evaluated. The greater sensitivity of
growth as a measure of effect is probably the outcome of longer
periods of exposure, which usually permit effects on biochemical
and physiological processes time to express themselves in grosser
terms.

The adult mussel was the least sensitive of the life history stages
investigated, and attachment by byssus-thread formation was usually
a more sensitive indicator of effect. Byssus-thread formation and
attachment to a solid substrate have a certain behavioral significance,

73


In their evaluation of various synthetic seawater formulations,
Courtright and associates (1971) found that none of the commercial
formulations tested were suitable for mussel larvae culture. They
developed the BioSea formulation, one of the essential components
of which is an L factor from Leslie coarse hide salt.

Although the percentage of normal larvae did not approach 85 in any
of the cultures used in the study of the effect of aged natural sea-
water, allowing the water to stand at room temperature for two or
more days before use as a culture medium appeared to be beneficial
(Figure 2). The percentages of normal larvae developing from eggs
taken from mussels collected in different geographical areas and
reared in seawater of a given age varied considerably; however,

the response of all larvae to seawater of different ages was about
the same, regardless of their source. On the average, only 24.7%
of the larvae reared in unaged (two-hour-old) seawater were normal.
Of those reared in water left standing for two days, 40.8% were
normal, Cultures reared in seven-day-old water contained an aver-
age of 46.8% normal larvae. It appears that allowing the water to
age for more than two days does not increase appreciably the per-
centage of normal Larvae.

These findings are contrary to those of Woelke (1972), who reported
that seawater aged for 16 and 24 hours had a statistically signifi-
cant adverse effect on development of larvae from fertilized eggs
of the Pacific oyster Crassotrea gigas. We know of no report in
which aged seawater is recommended for the culture of marine or-
ganisms. To the contrary, fresh seawater is recommended, and its
frequent renewal is emphasized (Hauenschild, 1972; Spotte, 1970).
Aging may allow heavy metals or other toxic materials to precipi-
tate or adsorb on the polyethylene container. Chemical changes in
the water were not determined.

In working with the Larvae, we observed other factors that influ-
enced the development of normal larvae. Mussels induced to spawn
may release eggs individually or in groups attached to form a clus-
ter or a string. Eggs released in attached groups produce abnormal
larvae. The size and shape of the mussel eggs are usually irregu-
lar immediately after release but become uniform after the eggs be-
come turgid; however, eggs produced by some females remain irregular

25


Appendix B

91


SECTION IIT

RECOMMENDATIONS

1. The bay mussel (Mytilus edulis L.), particularly the embryos
and larvae, should be used more frequently as a test organism
for evaluating marine and estuarine water quality.

2. A continuous-flow bioassay technique should be developed for
the bay mussel, expecially one that can be used effectively with
the larval forms.

3. Additional studies should be performed on the substrate pre-
ference of metamorphosing larvae, and the information should be
used to aid in the development of efficient, biologically mean-
ingful methods for investigating the effect of potential water
pollutants on larval metamorphosis.

4, Because of seasonal and geographical variations in natural
seawater, a standard synthetic seawater formulation suitable for
culturing of all life history stages of the bay mussel should be
adopted,


BL

Table 29.

SUMMARY OF TOXICITY DATA FROM 96-HOUR ADULT MUSSEL SURVIVAL,
48-HOUR EMBRYO SHELL DEVELOPMENT, LARVAL GROWTH, AND LARVAL METAMORPHOSIS STUDIES
(Pesticide Levels in mg/liter)

Growth Metamorphosis
96-hr 96-hr 48-br | Minimal | cont. Minimal |, ‘
Pesticide | TLs59 EC 59 Esq | effective |" "Con a8® effective |* *Centase
(adults) | (attachment)] (embryos)| concentra- ° Days concentra- of Days
. reduction . reduction
tion tion
Sevin 22.7 10.3 1.5 0.33 20.7 10 0.33 93.1 50
Treflan >0.24 0.035 >0.12 0.096 15.9 20 0.096 Death 26
Methoxychlor| >0.092 >0,092 >0.075 0.062 11.6 20 0.008 37.0 36
2,4-D (acid) }]259.0 262.0 211.7 91.4 6 10 182.8 Death 22
Malathion -- -- 13.4 6.2 0 10 6.2 94.9 32



the debris, and many were found attached by byssus threads to
fibrous substances in the debris. Many of these larvae had under-
gone metamorphosis. The source of the debris is still in question.
During the studies, all test jars were well covered with sheets of
clear polyethylene film. In view of the virtual impossibility of
avoiding formation of debris and the attractiveness of the debris
to the larvae, the usefulness of an ideal setting substrate is
somewhat decreased.

During our studies of Larval metamorphosis, we discovered that
settling--the act of attaching to a substrate by means of byssus
threads--was not a meaningful measure of the rate or extent of
metamorphosis by larval populations. It is well known and we fre-
quently observed that, in selecting a spot to settle and complete
metamorphosis, mussel larvae often secrete and break their byssus
threads several times and move to another location. Thus, the
number of larvae found attached at one time does not reflect the
number that can attach or have attached at another time.

A more accurate method for determining the number of larvae that
have undergone metamorphosis is to inspect each larva for the
presence of the dissoconch, or "adult," shell. This shell is a
lighter color than the larval shell and is readily discernible
from the latter. It appears along the entire edge of the original
shell and grows rapidly.

This evaluation of the study is included for use as a guide by

others who may be planning to perform similar studies using the
bay mussel.

80


21

22

23

24

25

26

27

28

29

30

Mean Shell Length of Mussel Larvae Exposed to
2,4-D for 10 and 20 Days . 2. 2 2s ee eee ene

Number, Age, and Size of Juvenile Mussels
Developing in Larval Cultures Exposed at 48 Hours
to Various Concentrations of 2,4-D......2+.6-.

Effect of 2,4-D on Larval Metamorphosis after a
40-Day Exposure Initiated 30 Days after Fertiliza-
tion of the Eggs . 1. 6 ee we ee we we ww ewe

Survival and Attachment Data for Adult Mussels
Exposed to Malathion for 96 Hours .....4-e.e.

Percentage of Normal Larvae Developing in 48 Hours
from Mussel Eggs Fertilized in Various Concentra-
tions of Malathion . . 1 6 ee eee eee eee

Mean Shell Length of Mussel Larvae Exposed to
Malathion for 10 and 20 Days... 2... eee eee

Number, Age, and Size of Juvenile Mussels
Developing in Larval Cultures Exposed at 48 Hours
to Various Concentrations of Malathion......

Effect of Malathion on Mussel Larvae Metamorphosis
after a ')-Day Exposure Initiated 30 Days after
Fertilization of the Eggs .. 1... see eevee

Summary of Toxicity Data From 96~Hour Adult Mussel
Survival, 48-Hour Embryo Shell Development, Larval
Growth, and Larval Metamorphosis Studies ,.....

Spawning Success and Viability of Eggs of Mussels
Collected from Various Sources .......2ee00-

vii

Page

61

62

64

67

68

69

69

71

74

77


of water remaining in the test chamber; we also found larvae caught
on the screen after it was rinsed several times. When several toxi-
cants are tested simultaneously, each concentration in duplicate,
such frequent handling of the larvae results in their accidental
loss. Even when enough manpower is available to inspect carefully
each test chamber, screen, or siphon, the task of accounting for
the 3000 larvae in each test chamber is enormous.

If further studies on growth of mussel larvae are anticipated, we
recommend that a method for applying the continuous-flow technique
be developed. In laboratories having a large supply of natural
seawater--especially those that pump seawater directly from the
ocean--continuous-flow systems should not be difficult to install.
Maintaining a sufficient food level in the larval chambers may pre~
sent a problem, although large-scale culturing of algae for constant
metering into the system is not infeasible. The major problem prob-
ably would be the design of the test chamber. Upon developing a
shell, a larva measures about 100 p, in length and somewhat less in
width. Fairly finely meshed screens are needed to retain the larvae
in the chamber in a continuous-flow system. Although screened test
chambers are used commonly in toxicity tests employing various in-
vertebrate organisms such as Daphnia magna, the use of screens with
mussel larvae could present cleaning difficulties, especially when
the larvae reach the pediveliger stage.

We found that, despite frequent renewal of the toxicant solutions,
a considerable amount of debris accumulated in the test chambers.
This debris usually was composed of a whitish, amorphous material,
lint, and other unidentifiable matter, none of which would pass
through 53-,, mesh screens. This material, especially the fibrous
type, seemed to attract the pediveligers, and this attraction made
it difficult for us to clean the screens effectively without in-
juring or killing some of the larvae. Accumulation of this debris,
in addition to feeding the larvae a continuous supply of algae,
most likely would necessitate frequent screen cleaning.

The ability of the pediveligers to attach themselves rather firmly
to the sides of the test chamber with a ciliated foot presents prob-
lems in obtaining a representative sample of the larval population
for measurements. Vigorous mixing, swirling, and agitation of the
test solution usually are not sufficient to dislodge all the Larvae.

78


Table 2, WATER QUALITY DATA FROM REPRESENTATIVE 48-HOUR EMBRYO BIOASSAY EXPERIMENTS
Means Based on 5 to 24 Determinations

68

Dissolved
Temperature, Oxygen Salinity, H
a , Pp
Pesticide °C °
mg/liter bo
Mean sD Mean sD Mean sD Mean SD
Sevin 22.0 0.55 6.6 0.22 25.9 0.20 7.80 0.10
Treflan 21.6 0.84 6.7 0.31 25.1 0.32 7.97 O.41
Methoxychlor 21.9 0.43 6.6 0.27 25.3 0.56 8.02 0.11
2,4-D 21.8 0.52 6.9 0.19 25.4 0.40 7.97 0.09
Malathion 21.6 1.09 6.7 0.31 25.7 0.36 8.02 0.08



LS

100

T T T T J

——— Control

— — — Control - Acetone

0.008
= - 0.015
Be + 0.03 7
3 seeeeeeees 0.06
&
&
3
é
3
50 4
2
@
fe)
x
=
c
°
=
<
EE
o
S25 4
.
ae
”
eo “
,
¢
eee
° pan 1 | | \
0 5 10 15 20 25 30
LENGTH OF EXPOSURE — days
FIGURE 6 EFFECT OF EXPOSURE TO METHOXYCHLOR ON PERCENTAGE OF MUSSEL POPULATION

COMPLETING METAMORPHOSIS

Larvae were 29 to 30 days old when exposure was initiated. Data from the two experiments were pooled.


Table 15. MEAN SHELL LENGTH OF MUSSEL LARVAE
EXPOSED TO METHOXYCHLOR FOR 10 AND 20 DAYS

Mean shell length, »
Test No. of | No. of
concen- larvae | tests/ Day 10 Day 20
tration, |measured/|experi-
mg/liter test ment |Experi-|Experi-|Experi-|Experi-
ment 1| ment 2 {ment 1 {ment 2
0 20 2 182 195 246 270
0 (acetone) 20 2 173 191 257 283
0.008 20 2 186 196 252 264
0.015 20 2 184 196 262 293
0.031 20 2 196 196 261 273
0.062 20 2 188 188 228 249

test indicated, however, that the seawater-acetone controls were
not significantly smaller than the seawater controls. In Experi-
ment 2, the mean sizes for all groups were similar.

After 20 days of exposure, the larvae exposed to the highest con-
centration of 0.062 mg/liter were smaller than the seawater con-
trols by about 7.5% and smaller than the seawater-acetone controls
by about 11.5%--statistically significant differences. Inhibition
of growth was not observed in any other methoxychlor-exposed group.

Marked reductions in the number of larvae that developed into juve-
nile mussels were observed when 48-hour-old larvae were exposed to
methoxychlor for 38 days (Table 16). In Experiment 1, the numbers
of metamorphosed larvae recovered from all chambers containing
methoxychlor were about the same. On the average, these chambers
contained 82.5% fewer juvenile mussels than those containing the
seawater-acetone controls. In Experiment 2, the effect appeared
to be related to the pesticide concentration. Of larvae exposed
to 0.008, 0.015, 0.031, and 0.062 mg/liter of methoxychlor, the
numbers that completed metamorphosis were 37, 50, 56, and 88% less,
respectively, than the number of seawater-acetone controls that
completed metamorphosis.

53


Beginning on day 30, the animal jars were examined daily for
attached larvae. On day 35, when the attached larvae were first
noticed, 200 swimming larvae from each of three females were
divided equally between two 75 X 150 mm Pyrex crystallizing dishes,
and one of each substrate was placed in each dish. The volume of
seawater was 600 ml. For five days, the substrates were inspected
daily for larvae attached by byssus threads. All those so attached
were counted and discarded. On the fifth day, the number of larvae
attached by byssus threads to the test container also was
determined,

The water was not renewed during the test. The larvae were fed
20,000 algal cells/ml of water every other day.

Larval Density and Larval Attachment

The four substrates used in the previously described test were
reevaluated. The procedures used in this study were the same
as those for the substrate preference study, except that the
numbers of larvae per test were 50, 100, 200, 300, and 600.
Each density was tested in duplicate. All the larvae were
hatched from eggs of one Tomales Bay female.

TOXICITY STUDIES
96-Hour Adult Survival

The survival of adult mussels exposed to various pesticide
concentrations for 96 hours was determined, and, where possible,
the 96-hour TL concentration and 95% confidence limits were
estimated by the method of Litchfield and Wilcoxon (1949).

The tests were conducted in 5-gal., wide-mouth glass jars.
Three to five pesticide concentrations plus a seawater control
were used in each test. When applicable, a second control was
used to determine the effect of the organic solvent. To mini-
mize the need to aerate the test solutions, only two mussels
were placed in each jar; however, ten mussels simultaneously
were exposed to each level of pesticide.

15


Table 10. MEAN SHELL LENGTH OF MUSSEL LARVAE
EXPOSED TO TREFLAN FOR 10 AND 20 DAYS
Mean shell length,
No. of
Test 1 No. of
concen- a tests/ Day 10 Day 10
mea-~
tration, ured/ experi
mg/liter s ment Experi- | Experi- | Experi- | Experi-
test
ment 1 | ment 2 | ment 1 | ment 2
0 20 2 146 182 270 288
0 (acetone) 20 2 154 179 252 302
0.024 20 2 178 200 222 320
0.048 20 2 176 218 255 262
0.096 20 2 168 188 200 254
0.192 20 2 160 196 190 207

exposed to all levels of Treflan was greater than that of the lar-

vae reared in both control media.
was different in the two tests.

the seawater-acetone control larvae.

The degree of growth enhancement

In Experiment 1, all the pesticide-
exposed larvae were larger than the seawater control larve (p = 0.05)
Except for those exposed to the highest Treflan concentration of
0.192 mg/liter, the pesticide-exposed larvae also were larger than

The average size of the sea-

water control larvae was less than that of the seawater-acetone
larvae, although the difference was not statistically significant.
In Experiment 2, there was no significant difference between mean
sizes of larvae exposed to 0.096 mg/liter of Treflan and of those
reared in the two control solutions; however, all other Treflan-
exposed groups were larger than the control larvae.

After 20 days of exposure, differences in response were still ap-
For example, in Experiment 2 the seawater-
acetone control larvae were larger than the seawater control larvae
(p = 0.05), but in Experiment 1 the opposite effect was observed.
The only response consistent to both tests was the reduction in mean
shell length observed in groups exposed to the two highest Treflan

parent between tests.

concentrations.

44

Larvae exposed to 0.096 mg/liter of Treflan were


An average of only 2.5% of the seawater control larvae died during
the 40-day experiment; however, an average of 18.1% died at the
lowest concentration of 0.02 mg/liter, and the same percentage of
larvae died at the highest concentration of 0.16 mg/liter. Al-
though this indicates that larval mortality is not proportional

to pesticide concentration, it also implies that mortality may not
have been caused by Treflan. Exposure to 50 l/liter of acetone
alone killed an average of 11.6% of the larvae. The high mortality
observed among the seawater-acetone controls implicates acetone;
however, mortality among the seawater-acetone controls employed in
the tests on methoxychlor was less than that of the seawater con-
trols; hence, some other factor must be involved.

The rate of metamorphosis of Treflan-exposed larvae was greater
than that of the seawater control larvae (Figure 5). The meta-
morphosis rate for Treflan-exposed larvae was not related to the
test concentration. As a group, 50% of the pesticide-exposed
larvae reached the juvenile stage in an average of 14.2 days,
whereas the average for controls was 21 days. Here acetone ap-
pears definitely to be involved. Of the seawater-acetone controls
that did undergo metamorphosis, 50% reached the juvenile stage in
in an average of 14 days. Hence, all larvae exposed to acetone
metamorphosed at a more rapid rate than those that were not ex-
posed to acetone. The same effect was observed in the experiments
on methoxychlor,.

Acetone also appeared to inhibit growth. Metamorphosed larvae that
had been exposed to 50 pl/liter of acetone alone averaged 406 y--
7.3% smaller than the controls that had been exposed to seawater
alone. Metamorphosed larvae from the methoxychlor-laden test cham-
bers averaged 401 y,, or 8.4% less than the seawater controls. These
larvae also were exposed to 50 yl/liter of acetone, which was used
as a pesticide-dispersing agent. The mean size of the larvae for
each pesticide-exposed group did not vary with pesticide concentra-
tion; hence, Treflan did not appear to be a factor in inhibiting
growth in this test.

Although the effects of exposure to Treflan have been studied in

aquatic organisms, we know of no studies using marine invertebrates.
Sanders (1970) determined the toxicity of a number of herbicides to

48


Table 30. SPAWNING SUCCESS AND VIABILITY OF EGGS
OF MUSSELS COLLECTED FROM VARIOUS SOURCES

Percentage Performance in 48-hour embryo bioassays

Mussel f£
of spawn No. of different Percentage of groups

source i ini
with minimum of

Mean | Range | e roups tested
8 88 groups Fesre 85% normal larvae

Tomales Bay {35.3 | 6-66 42 40.5
St. Francis

Yacht Harbor | 8.7 |2-15 39 7.7
Treasure

Island 18,2 | 2-39 61 11.5

Moss Landing {10.8 |1-28 Number of eggs insufficient to run
tests

3 and 4 cm, were used per 15 liters of test solution in the adult
survival tests, dissolved oxygen concentrations reached dangerously
low levels during some of the tests. Severe reductions in oxygen
levels were particularly evident whenever acetone was used as an
initial pesticide solvent. Although the situation was rectified by
aerating the test solutions, use of a continuous-flow system would
have eliminated the need for direct aeration of the test solutions,
which could have caused pesticide loss by volatilization. Use of
a continuous-flow system also would have eliminated the need to em-
ploy five 5-gal. jars per treatment group in each series of tests.

In our extended growth and metamorphosis studies, conducted for

30 to 40 days, variations in toxicant levels due to adsorption,
metabolism, volatilization, and chemical degradation were minimized
by renewing the test solutions at intervals of approximately 40 hours.
Because the larvae were extremely small, the old solution and the
larvae were siphoned frem the test chamber into a tube containing
nylon screen (53-4 mesh) to catch them. Although the test chamber
was rinsed several times with fresh seawater, and the rinse water

was poured through the screen, we know that larvae were accidentally
lost. On occasion, we discovered larvae caught in the surface film

17


solution would contain no more than 200 W1/liter of acetone.
Solutions of Treflan and methoxychlor were filtered before
use,

SOURCES OF MYTILUS EDULIS L.

Mussels used in the adult survival studies were collected from
floating docks located on Treasure Island, which lies in the
central region of San Francisco Bay. Gametes used in the study
of 48-hour embryo shell formation were obtained from mussels
collected at Treasure Island and Marconi's Cove, located along
the southeastern shore of Tomales Bay. The effects of the pesti-
cides on larval shell growth and metamorphosis, and on metamor-
phosis alone, were determined using larvae derived from mussels
collected at Marconi's Cove. Tomales Bay mussels were used in
all nontoxicological studies, except those designed to determine
the influence of artificial and aged natural seawater on larval
shell formation, For these studies, we collected mussels from
Tomales Bay, Treasure Island, and St. Francis Yacht Harbor (a
small boat-docking area along northern San Francisco).

PRETEST TREATMENT OF THE MUSSELS AND SPAWNING TECHNIQUE

Adult mussels used in 96-hour survival experiments were trans-
ported from the collection site moist in plastic bags. Upon
arrival at the laboratory, the shells were scraped of extraneous
material and then transferred to 45-gal. aquaria containing re-
circulating seawater maintained at 20°C. The mussels were used
within two days after collection. Mussels collected as a source
of gametes were stored moist at about 53°C after being scraped
free of debris. They were induced to spawn within 24 to 48
hours after collection.

Several methods of inducing spawn were tried, However, the only
consistent method involved placing the chilled mussels in individ:
ual glass bowls containing enough 20°C natural seawater to cover
them. The mussels usually began to produce gametes within an
hour.


Table 14, PERCENTAGE OF NORMAL LARVAE
DEVELOPING IN 48 HOURS FROM MUSSEL EGGS
FERTILIZED IN VARIOUS CONCENTRATIONS OF METHOXYCHLOR

Test Test number

concen~ Experi- Experi- Experi- Experi-

tration, ment_1 ment 2 ment 3 ment_4

mg/liter qd) } (2) | @M 7] 2) |] @M | @ | M 7] (@
0 88.5 | 84.5 | 91.5 | 91.0 | 89.3 | 86.0 | 94.5 | 89.5
0 (acetone) 86.0] 89.5 | 89.0] 90.0] 89.0 | 89.5 } 90.5 | 85.8
0.011 89.5 | 88.0 | 90.5 | 87.0} 86.0 | 86.5 | 93.4 | 93.0
0.018 87.5 | 90.0] 89.0] 88.5 | 91.5 | 86.0 | 94.0 | 93.5
0,041 80.5 | 83.0 | 90.5 | 90.0 | 89.3 | 95.3 | 84.0 | --
0.075 94.0 | 98.5 | 84.0 | 90.0 | 91.3 | 75.1 | 88.0 | 91.3
48-Hour ECsq,
mg/liter >0.075 >0.075 >0.075 >0.075

in the containers with the highest methoxychlor concentration of
0.075 mg/liter was 89.0,

The mean and standard deviation for the highest test concentration
used in the growth study was 0.062 + 0.0078 mg/liter, based on

43 analyses. Estimates of the lower test concentrations are 0.031,
0.015, and 0.008 mg/liter. Seawater control and seawater-acetone
control groups were employed in each series of tests. The acetone
concentration in all test chambers except the seawater control cham-
bers was 50 yl/liter.

The results obtained from the two experiments were slightly differ-
ent (Table 15). In Experiment 1, the mean size of the larvae ex-
posed to all levels of methoxychlor for 10 days was similar to that
of the seawater control larvae, except for those reared in 0.032 mg/
liter of methoxychlor. These larvae were 7.7% larger than the
seawater controls, The difference was statistically significant

(p = 0.05). All larvae exposed to methoxychlor were larger than
the seawater-acetone controls. Analysis of the data by applying
the t-test showed that this difference was significant. The same

52


FIGURES

Page
Effect of Time of Fertilization on Number of Normal
Larvae Developing from Eggs of the Bay Mussel... 24
Effect of Age of Natural Seawater on the Number of
Normal Larvae Developing from Eggs of the
Bay Mussel . 6 6 ee ee tt ee ee we ee ee 26
Influence of Larval Density on Larval Selection
of Setting Substrate . 1. 6 ee ee we ee eee 31
Effect of Exposure to Sevin on Percentage of
Mussel Population Completing Metamorphosis .... 39
Effect of Exposure to Treflan on Percentage of
Mussel Population Completing Metamorphosis .... 49
Effect of Exposure to Methoxychlor on Percentage
of Mussel Population Completing Metamorphosis ... 57
Effect of Exposure to 2,4-D on Percentage of
Mussel Population Completing Metamorphosis .... 65
Effect of Exposure to Malathion on Percentage of
Mussel Population Completing Metamorphosis .... 72

iv


Table 26.

MEAN SHELL LENGTH OF MUSSEL LARVAE
EXPOSED TO MALATHION FOR 10 AND 20 DAYS

Mean shell length, yw
Test No. of No. of
concen- larvae tests/ Day 10 Day 20
tration, | measured/| exper-
mg/liter test iment | Experi- | Experi- | Experi- | Experi-
ment 1 | ment 2 | ment 1 | ment 2
0 20 2 156 159 214 262
1.5 20 2 155 162 2i1 248
3.1 20 2 164 154 220 264
6.2 20 2 148 151 174 198
12.3 20 2 126 130 160 158
Table 27. NUMBER, AGE AND SIZE OF JUVENILE MUSSELS

DEVELOPING IN LARVAL CULTURES
EXPOSED AT 48 HOURS TO VARIOUS CONCENTRATIONS OF MALATHION

Experiment 1 Experiment 2
Test
concen-

L h
tration, | Total | Age,# Length, w Total | Age,4 ength, pb
mg/liter | number | days Mean | sp number | days Mean sp

0 276 20 393 | 76.1 87 20 415 72.0
1.5 168 20 396 71.6 140 22 451 71.2
3.1 166 20 397 | 82.5] 228 20 461 98.4
6.2 14 22 369 | 87.9 43 24 392 | 100.0
12.3 0 -- -- | -- 2 22 410 --

“Number of
phosing.

days required by treatment group to begin metamor-

69



100

relative percent

50

40

NORMAL LARVAE

30

20

° | | | | |

0 50 100 150 200 250 300
TIME OF FERTILIZATION AFTER SHEDDING OF EGGS — minutes

FIGURE 1 EFFECT OF TIME OF FERTILIZATION ON NUMBER OF NORMAL
LARVAE DEVELOPING FROM EGGS OF THE BAY MUSSEL

24


Table 24. SURVIVAL AND ATTACHMENT DATA
FOR ADULT MUSSELS EXPOSED TO MALATHION FOR 96 HU. RS

Test | Test concentration | No, of | Survival, | Attached,
no. | Nominal | Measurea | animals percent percent
49 0 i) 10 90 80

5 9.0 10 20 20

10 17.0 10 0 0

20 23.5 10 0 0

40 48.0 10 0 i}

80 69.0 10 0 0

52 0 0 10 100 100
0.75 0.74 10 50 50

1.25 1.4 10 60 50

2.50 2.8 10 80 80

5.0 6.9 10 100 90

10.0 13.0 10 80 80

56 0 i) 10 100 100
1.25 1.2 10 100 90

2.5 2.5 10 100 90

5.0 5.0 10 100 100

10.0 8.6 10 100 90

20.0 18.5 10 100 30

59 ie} 10) 10 100 90
6.25 0.34 10 90 90

12.5 6.3 10 100 100

25.0 12.2 10 90 30

50.0 19.4 10 70 0

100.0 39.4 10 100 0

67



Table 19. WATER QUALITY DATA FOR 96-HOUR ADULT MUSSEL
SURVIVAL TEST NO. 12 ON 2,4-D

Test Temper- Dissolved Salinity,
concen- ature, oxygen, bo pH
tration, °C mg/liter
mg/liter | Mean sD Mean sD Mean sD Mean sD
0 18.1 0.72 6.4 0.74 23.3 0.74 7.8 0.11
-- 18.0 | 0.67 7.6 1.54 | 23.4 | 0.25 7.9 0.11
142 18.0 | 0.66 7.8 1.55 22.9 0.75 7.8 0.11
-- 17.9 0.68 | 8.0 1.54 | 23.5 0.41 7.7 0.01
274 18.0 0.56 8.2 0.91 24.0 0.00 7.4 0.54
-- 17.2 0.00 9.1 0.08 24.5 0.00 7.6 0.01
331 18.4 | 0.35 | 8.7 0.43 { 24.5 0.00 | 6.5 0.22
334 18.6 | 0.35 8.7 0.14 24.5 0.00 | 6.4 0.07

to the test chamber was less than that of the controls at concen-
trations above 142 mg/liter. The 96-hour EC5g for attachment,
based on data from Test 12, was 262 mg/liter.

The trochophore larvae were slightly more sensitive to 2,4-D than
the adults. The 48-hour ECsg estimates ranged from 210 to 213 mg/
liter for three embryo bioassay experiments in which each treatment
group was tested in duplicate (Table 20). The mean was 211.7 mg/
liter.

The 2,4-D concentrations evaluated in the growth study were 182.8,
91.4, 45.7, and 22.8 mg/liter. The standard deviation of the high-
est test concentration--the only test level monitored throughout
the experiment--was 10.2 mg/liter. Only seawater controls were
employed. Table 21 presents data from the study.

After 10 days of exposure, a statistically significant reduction

in growth was observed in larvae exposed to a concentration of

91.4 mg/liter of 2,4-D (Experiment 2). These larvae were 11.6%
smaller than the controls. Reduction in growth was not observed

at this concentration in Experiment 1, At 182.8 mg/liter, inhibition

60


Larval Growth and Metamorphosis

Effects of the pesticides on larval growth and metamorphosis were
investigated by exposing the larvae continuously to various consen-
trations of each pesticide for periods of up to 40 days. During
the exposure period, the shell length of the larvae was measured
every other day, and the number of larvae undergoing metamorphosis
was recorded.

The gametes used in the tests were obtained from mussels collected
at Tomales Bay during August and September 1973. Before exposure
to the pesticides, eggs were placed in 5-gal. glass jars containing
15 liters of natural seawater and fertilized. Eggs were kept in
separate groups according to female at a concentration of 30,000
per liter.

Forty-eight hours after fertilization, the contents of each jar
were mixed thoroughly by a stream of air. During mixing, a 20-ml
aliquot was removed from each jar, and the number of normal-shelled
larvae was determined from a group of 200. The contents of all
jars containing less than 85% normal-shelled larvae were discarded
Larvae from three females then were pooled. All larvae in a l-ml
sample were counted so equal numbers from the three females could
be combined. After thoroughly mixing the larvae, we determined the
number again.

Exposure to the pesticides was initiated within approximately 52
hours after fertilization of the eggs. The experiments were con-
ducted in 1.25-gal. Pyrex animal jars, each containing 3 liters of
test solution and approximately 3000 normal-shelled larvae. Four
levels of each pesticide were used, Control groups were reared in
natural seawater.

In tests on Treflan and methoxychlor, seawater and solvent groups
were included. The solvent control groups were reared in natural
seawater containing 50 }1/liter of acetone--the same level present
in all chambers containing pesticide. Each pesticide was tested
twice, and each treatment level was run in duplicate.

17


Table 4. PERCENTAGE OF NORMAL LARVAE DEVELOPING
IN 48 HOURS FROM MUSSEL EGGS FERTILIZED
IN VARIOUS CONCENTRATIONS OF SEVIN

Test number
Test
concentration, Experiment 1 Experiment 2 Experiment 3
mg/liter dd) (2) qd) (2) () (2)
0 93.5% | 90.0% | 90.0% | 96.0% | 88.0% | 83.5%
0.19 87.5 82.5 81.7 84.5 68.5 84.0
0.26 89.7 88.5 -- 76.3 -- 79.5
0.65 72.1 71.0 72.5 74.3 67.0 78.3
1.3 65.5 35.5 “- 62.0 36.5 40.5
3.3 6.5 2.0 0 2.5 0 2.0
48-hour ECsq,
mg/liter 1.48 1.80 1.21

Although the effect of the pesticides on larval growth was studied
by measuring 20 larvae from each treatment group every other day
for a minimum of 30 days (Appendix B), we elected to analyze sta-
tistically the data collected on the 10th and 20th days. This
period covers growth of the veliger and pediveliger larvae. During
the study, larval measurements were discontinued in several test
jars because of insufficient numbers of survivors. Since only a
few jars were terminated during this period, size comparisons were
possible among nearly all the treatment levels. The difference be-
tween the means of the control larvae and the pesticide-exposed
larvae were tested for significance by subjecting the data to a
Student's t-test, using a 95% level of significance. The concen-
trations of Sevin used were 0, 0.33, 0.65, 1.30, and 2.61 mg/liter.
Only the highest concentration was measured; the lower concentra-
tions were estimated using the mean of the highest concentration
as a starting point. The mean of 2.61 mg/liter for the highest
concentration was calculated using values from 45 determinations.
The standard deviation was 0.39 mg/liter.

Table 5 presents the mean shell lengths of larvae exposed to Sevin
for 10 and 20 days. Growth was a more sensitive measure of toxic

34


Table 16. NUMBER, AGE, AND SIZE OF JUVENILE MUSSELS
DEVELOPING IN LARVAL CULTURES EXPOSED AT 48 HOURS
TO VARIOUS CONCENTRATIONS OF METHOXYCHLOR

Experiment 1 Experiment 2
Test
concen-

No. of Length No. of Length,
tration, juve. Age, Bins juve- Age, Bb
mg/liter niles days Mean | SD niles days Mean | SD

0 24 26 395 | 64.9] 126 28 448 | 74.3
0 (acetone) 99 26 427 | 71.4 68 26 419 | 68.4
0.008 14 26 401 | 71.4 43 26 373 | 55.0
0.015 27 26 412 61.9 34 26 389 62.6
0.031 12 26 345 | 48.0 30 26 395 | 50.0
0.062 15 26 385 59.5 8 28 358 27.2

Exposure to methoxychlor also appeared to inhibit growth at all
pesticide levels. The degree of inhibition did not appear to be
related to the pesticide concentration. In both experiments, the
pesticide-exposed larvae were about 9.5% smaller than the seawater-
acetone controls.

In the metamorphosis study in which exposure to methoxychlor was
delayed until the larvae were 29 days old (Table 17), the concen-
trations tested were insignificantly different from those used in
the growth and metamorphosis experiments. The mean and standard
deviation of the highest test concentration were 0.0595 + 0.0081 mg/
liter based on 31 analyses. The larvae were exposed to the pesti-
cide for 41 days.

A slight decrease in the number of larvae that completed metamor-
phosis was observed at concentrations of 0.015 mg/liter or higher.
For larvae exposed to 0.015, 0.03, and 0.06 mg/liter, the percen-
tages of metamorphosed larvae, averaged for the two tests, were
84, 88.4, and 82.8, respectively. For the seawater controls and
the seawater-acetone controls, the percentages were 93.4 and 96.2,
respectively,

54


Water temperature, pH, salinity, and dissolved oxygen content

were measured daily. Two samples of the highest test level were
analyzed for pesticide every other day. The samples were
collected immediately after the solution was prepared.

20


Appendix A

87


American oyster (Crassostrea virginica) and the hard-shell clam
(Mercenaria mercenaria). Their estimates of the 48-hour EC59 for
oyster and clam larvae were 3.0 and 3.82 mg/liter, respectively.

We were unable to locate any published reports on the effects of
Sevin on mussel larvae growth or metamorphosis. Other bivalve
molluscs have been studied, however. Butler and coworkers (1968)
reported that three-day exposure to 0.8 mg/liter of Sevin is lethal
to the larvae of the cockle clam. At concentrations as low as

0.1 mg/liter, Sevin inhibited the growth of clam larvae. In

C. virginica, Davis and Hidu (1969) found that exposure to 2.0 mg/
liter of Sevin for 12 days produced a marked reduction in larval
growth.

We encountered many difficulties in our efforts to determine the
effects of the five pesticides on metamorphosis, using survivors
of the growth study. Thus, the data obtained are questionable.
The study in which initiation of exposure was delayed until the
larvae were 29 days old was better controlled and produced more
meaningful information.

Our study indicates that adult forms of M. edulis may be more re-
sistant to Sevin than other species of bivalve molluscs and are
decidedly more resistant to the insecticide than marine anthropods.
The early trochophore larvae of the mussel are as sensitive to
Sevin as the larvae of several other molluscan bivalves; however,
they are about 15 times less tolerant of the pesticide than the
adult mussel.

Inhibition of shell growth was observed at a concentration 45%
less than that which caused 50% of the larvae used in the 48-hour
embryo bioassay experiments to develop abnormally. This concentra-
tion (0.65 mg/liter) also reduced the number of larvae that de-
veloped dissoconch shells and inhibited their growth rate.

Treflan

Treflan, also known as trifluralin, is a selective preemergence
herbicide. Exposure of adult mussels to a measured concentration

41


Dimick, R. E., and W. P, Breese. Bay Mussel Embryo Bioassay.
Proc. 12th Pacific Northwest Industrial Waste Conf, College
of Engineering, University of Washington, 1965. p. 165-175.

Droop, M. R. On the Ecology of Flagellates from Some Brackish
and Freshwater Rock-Pools of Finland. Acta Botan Finnica.
51:1-52, 1953.

Eisler, R., and M. P. Weinstein. Changes in Metal Composition
of the Quahaug Clam, Mercenaria mercena » after Exposure
to Insecticides. Chesapeake Sci, 8:253-258, 1967.

Field, I. A. Biology and Economic Value of the Sea Mussel
Mytilus edulis, Bull U.S. Bureau Fisheries. 38:127-259,
1922.

Green J. The Biology of Estuarine Animals, Seattle, University
of Washington Press, 1968. 401 p.

Herbicide Handbook of the Weed Society of America. WSSA Mono-
graph 3, 2nd Ed. 368 p.

Hauenschild, C. Animals: Invertebrates. In: Research Methods
in Marine Biology, Schlieper, C. (ed.), Seattle, University
of Washington Press, 1972. p. 216-247.

Kester, D. R., I. W. Duedall, D. N. Connors, and R. M. Pytkowicz.
Preparation of Artificial Seawater. Limmol and Oceanog.
12:176-179, 1967.

LaRoche, G., R. Eisler, and C. M. Tarzwell. Bioassay Procedures
for Oil and Oil Dispersant Toxicity Evaluation. J Water
Pollut Control Fed, 42:1982-1989, 1970.

Lichtenstein, E. P., and K. R. Schultz. Effect of Moisture and
Microorganisms on the Persistence and Metabolism of Some
Organophosphorus Insecticides in Soils, with Special Em-
phasis on Parathion, J Econ Entomol, 57:618-127, 1964.

82


Table 18.

SURVIVAL AND ATTACHMENT DATA

FOR ADULT MUSSELS EXPOSED TO 2,4-D (ACID) FOR 96 HOURS

Test | Test concentration | No. of | Survival, | Attached,
no. Nominal Measured animals percent percent
7 0 -- 6 100 --

50 -- 6 83 --
100 -- 6 100 --
200 -- 6 83 --
400 -- 6 33 --
600 -- 6 0 --
12 0 (0) 12 100 92
100 -- 6 100 100
200 142 6 100 100
300 -- 12 92 83
400 274 6 50 33
450 -- 12 42 25
500 331 6 0 0
600 334 12 0 0
41 0 0 10 100 80
250 240 10 100 50
300 275 10 100 80
350 340 10 30 10
400 395 10 0 0
450 445 10 0 0
40 0-450 -- 10 All animals died

within 24 hours.

59



Litchfield, J, T., Jr., and F. Wilcoxon. A Simplified Method
of Evaluating Dose-Effect Experiments. J Pharmacol Exp
Therap. 96:99-113, 1949,

Macek, K. J., C. Hutchinson, and 0. B, Cope. Effects of Tempera-
ture on the Susceptibility of Bluegills and Rainbow Trout
to Selected Pesticides, Bull Environ Contam Toxicol.
4:174-183, 1969.

Maloney, T. E., E. J. Donovan, Jr., and E. L. Robinson. Deter-
mination of Numbers and Sizes of Algal Cells with an Elec-
tronic Particle Counter. Phycologia. 2:1-8, 1962.

Matthiessen, G. C., and R. C. Toner. Possible Methods of Im-
proving the Shellfish Industry of Martha's Vineyard, Duke's
County, Massachusetts, Marine Research Foundation, Inc.,
Edgartown.

Millemann, R. E., and R. S. Caldwell. Biological Effects of
Pesticides on the Dungeness Crab. Oregon State University,
Corvallis. Progress Report. EPA Contract 68-01-0188.

May 1973. 63 p.

Parka, S. J., and H. M. Worth. The Effects of Trifluralin to
Fish. Presented at the Southern Weed Conf., Dallas, Texas.
January 1965.

Reish, D, J., and J. L. Ayers, Jr. Studies on the Mytilus edulis
Community in Alamitos Bay, California. III. The Effects
of Reduced Dissolved Oxygen and Chlorinity Concentrations
on Survival and Byssus Thread Formation. The Veliger.
10:384-388, 1968.

Rumker, R. V., R. M. Matter, D, P, Clement, and F. K. Erickson,
The Use of Pesticides in Suburban Homes and Gardens and
Their Impact on the Aquatic Environment. EPA-OWP, Pesticide
Study Series No. 2. May 1972.

Sanders, H. 0. Toxicities of Some Herbicides to Six Species of
Freshwater Crustaceans. J Water Pollut Control Fed.
42 1544-1550, 1970.

83


In this project, substrate preference experiments were conducted
to provide information that would aid in the design of test pro-
cedures for determining the effects of the five pesticides on lar-
val growth and settling. The substrates evaluated were polyvinyl
chloride (PVC) sheet, frosted glass, acrylic plastic sheet (Plexi-
glas®), and silk embroidery thread, Table 2 presents the mean
number of larvae attached to the various substrates in six tests.
The differences between the means were analyzed statistically by
analysis of variance followed by the Duncan multiple-range test.

Substrate preference in order of rank was:

PVC is greater than frosted glass and Plexiglas
Silk is greater than Plexiglas

PVC is equal to silk

Silk is equal to frosted glass

Frosted glass is equal to Plexiglas.

The number of larvae settling on the glass test chamber was sig-
nificantly greater than that settling on any of the test substrates.
Bayne (1965) found that although a few larvae attach to smooth
glass when this is the only substrate provided, most do not and
eventually die.

In our experiments, all four substrates and the glass chamber were
simultaneously available to each group of 100 larvae. The average
number of larvae settling on all available surfaces was 67 per 100
larvae. Of those that settled, about 27% settled on the smooth
glass chamber, 25% settled on PVC, 22% settled on silk thread,

15% settled on frosted glass, and about 12% settled on acrylic
plastic. Larval density was greatest on silk thread, followed in
order by PVC, frosted glass, acrylic plastic, and the glass test
chamber.

De Blok and Geelen (1959) evaluated 13 different substrates rela-
tive to selection by settling M. edulis larvae. They found that
the larvae preferred filamentous substrates to solid substrates,
silk embroidery thread being the most preferred Bayne (1965)
also found silk embroidery thread to be superior to all the sub-
strates he tested except for natural algae fibers.

28


SECTION I

CONCLUSIONS

1. The insecticide Sevin is toxic to the bay mussel at concen-
trations lower than its estimated solubility in seawater. Toxic
effects were observed in adults as well as in larvae.

2, Although adult mussels can tolerate exposure to a saturated
solution of Treflan, they appear to be able to detect extremely
low levels of this herbicide. Embryo shell development is not
affected by Treflan at a concentration half that of its estimated
solubility in seawater; however, larval growth and metamorphosis
are reduced.

3. Adult mussels can tolerate and do not appear to detect meth-
oxychlor concentrations approaching twice the estimated solubility
of this insecticide in seawater. Mussel eggs incubated in a
saturated solution of methoxychlor develop normally; however,
larval growth is depressed, and metamorphosis is inhibited.

4. All the life history stages of the bay mussel can be affected
adversely by exposure to 2,4-D at levels lower than 25% of its
estimated solubility in seawater.

5. Malathion is toxic to the bay mussel at all life history
stages, producing abnormal shell development in embryos, depres-
sion of growth, and inhibition of metamorphosis at concentrations
lower than 20% of its estimated solubility in seawater, The
response of adult mussels to this insecticide was inconsistent,
the same concentration being lethal at times and nonlethal at
others.

6. Larval growth is probably the most consistently sensitive
indicator of toxicity, followed in order of decreasing sensitivity


In the 48-hour embryo bioassay experiments, Treflan had no effect
on shell development in the trochophore larvae at the highest mea-
sured test concentration of 0.12 mg/liter (Table 9). The mean of
the percentages of normal control larvae for three duplicated ex-
periments (six tests) was 88.6%. For larvae exposed to 0.12 mg/
liter of Treflan, the mean was about 82%. The difference between
the means was not statistically significant (p = 0.05).

Table 9, PERCENTAGE OF NORMAL LARVAE
DEVELOPING IN 48 HOURS FROM MUSSEL EGGS
FERTILIZED IN VARIOUS CONCENTRATIONS OF TREFLAN

Test Test number

concentration, Experiment 1 Experiment 2 Experiment 3

mg/liter OTe 7a 7 ea oye
0 82.0 75.0 91.2 94.5 95.5 93.5
0 (acetone) 84.5 85.0 93.3 91.5 -- 93.5
0.018 85.5 86.5 -- 77.8 90.0 93.8
0.036 75.5 88.5 79.2 90.5 90.5 90.0
0.070 88.0 83.5 83.5 90.5 89.0 91.5
0.120 81.5 79.5 87.5 75.3 83.5 84.5
48-Hour EC. 45
mg/liter 0.12 0.12 0.12

In the larval growth study, the concentrations tested were 0.024,
0.048, 0.096, and 0.192 mg/liter. Seawater control and seawater-
acetone control groups also were included. The acetone concentra-
tion was 50 ul/liter in all test chambers except those containing
the seawater control groups. Thirty-five determinations were per-
formed on the highest test concentration. The mean and standard

deviation were 0.192 + 0.037 mg/liter.

Reduced shell length was not observed in any of the Treflan-
exposed treatment groups during the first 10 days of exposure

(Table 10). To the contrary, the average shell length of larvae

43


ACKNOWLEDGMENTS

Stanford Research Institute gratefully acknowledges for their par-
ticipation in this project Dr. Gordon W. Newell, Director, Tox-
icology Department, and Project Manager; and Dr. Dale M. Coulson,
Manager, Inorganic Analytical Physical Chemistry. Ms. Karen Martin,
Invertebrate Biologist, and Mr. Howard Bailey, Fisheries Biologist,
contributed considerable advice and assistance during the project.

The Institute also thanks Mr. Wilbur J. Breese, Oregon State Uni-

versity, who, as a consultant, provided expert advice and guidance
throughout the project.

viii


Le

Table 7. EFFECT OF SEVIN ON MUSSEL LARVAE METAMORPHOSIS AFTER A 40-DAY EXPOSURE
INITIATED 29 DAYS AFTER FERTILIZATION OF THE EGGS
(Presence of Dissoconch Shell Used as Indication of Metamorphosis)

Test concentration, mg/liter

Control 0.36 0.72 1.45 2.9
1 2 1 2 1 2 1 2 1 2
Number of larvae
Initial 100 100 100 100 100 100 100 100 100 100
Lost or crushed 16 19 22 35 27 24 35 16 40 31
Experimental 84 81 78 65 73 76 65 84 60 69
Metamorphosing,
percent 94.0% | 92.6%] 91.0%] 84.6%] 82.2%] 88.2%] 81.6%] 83.3%] 80.0%] 75.4%
Nonmetamorphosing,
percent 1.2%] 0 6.4%| 9.2%) 2.7%) 3.9%] 1.5%] 2.4%] 0 0
Dead, percent 4.8%] 7.4%] 2.6%] 6.2%] 15.1%) 7.9%] 16.9%] 14.3%] 20.0%] 24.6%
Days to metamorpho-
sis of 50% of test
larvae 23 24 21 24 19 23 17 24 16 17
Shell length of
juvenile mussels, y, [455.4 y!458.6 1499.2 483.5 11J450.8 1406.5 }391.6 1393.3 1350.2 1339.3



The mussels were inspected daily, and the number dead or attached
to the jars was recorded. Mussels were considered dead if, upon
being prodded with a glass rod, those with gaping valves showed no
valvular or body movement.

Larval Shell Development

The procedures described by Dimick and Breese (1965) were used to
determine the effect of the pesticides on larval shell development.
The mussels from which the gametes were obtained were collected
from Treasure Island and Tomales Bay. Treasure Island mussels were
used in tests on Sevin, In tests on Treflan, methoxychlor, 2,4-D,
and malathion, the mussels were collected from Tomales Bay,

The tests were conducted in 250-ml Pyrex beakers containing about
6000 eggs in a volume of 200 ml. The larvae were cultured for 48
hours after fertilization of the eggs and then killed. The number
of normal larvae (straight-hinged shells) in a group of 200 consec-
utive larvae was determined and expressed in relative percentage
units, which were calculated by dividing the percentage of normal
larvae in a pesticide-exposed group by the percentage of normal
larvae in the control group and multiplying by 100. Only data ob-
tained from tests in which 85% of the control larvae were normal
were considered acceptable.

Four to seven different pesticide levels were tested. In tests on
methoxychlor and Treflan, two control groups were used. One group
was exposed to seawater alone, and the other was exposed to sea-
water containing 50 W1/liter of acetone. All treatment levels,
including the controls, were tested in duplicate.

Upon initiation of a series of tests on a given pesticide, a
sufficient volume of each test level of the pesticide was prepared
to fill four extra beakers. These beakers did not contain larvae.
The contents of two beakers were analyzed for pesticide at the
beginning of the test, and the contents of the other two were
analyzed at the end of the test. Water temperature, pH, dissolved
oxygen, and salinity also were measured in samples taken from the
same four beakers initially and after 48 hours.

16


DILUENT WATER

The natural seawater used in all pesticide toxicity studies was
obtained from the Steinhart Aquarium in San Francisco, California,
The aquarium facility draws the water from a point approximately
one-half mile offshore in the Pacific Ocean through a Raney
collecting system. The intake is buried by about 20 ft of sand.
The suitability of this water for maintaining marine life has
been proven by Steinhart personnel's consistent success in rear-
ing a myriad of marine organisms.

The water was transported weekly from the aquarium to SRI's test-
ing laboratory in 400-gal., black polyethylene containers. Before
use, the water was passed through an ultraviolet irradiator (Ultra-
dynamics Model 500). When necessary, the salinity was reduced to
25°/o0 by adding deionized tapwater (1 megohm), and the pH was
adjusted to 8 + 0,2 by the addition of HCl or NaOH.

ALGAE CULTURE

Cultures of Monochrysis lutheri Droop (1953) were maintained to
feed the larvae used in the growth and metamorphosis study.
Starting cultures were obtained from the Department of Botany,
Indiana University, Bloomington, Indiana, The culture medium
of Matthiessen and Toner (1966) was used.

Sterile cultures were maintained in 250- and 2000-ml Erlenmeyer
flasks set on a rotary shaker and were illuminated with Sylvania
Gro-lux fluorescent lamps. The temperature of the culture room
was maintained at 18 + 1°C, Algae in the 250-ml flasks were
used to inoculate the 2000-ml flasks, which in turn were used
to inoculate the 5-gal. carboys from which we took the cells
used to feed the mussel larvae.

Cultures maintained in the 5-gal. carboys were not kept sterile.
Agitation was accomplished by vigorous aeration. Population
growth was monitored daily in cultures set up early in the proj-
ect until sufficient data were accumulated to serve as a guide
for predicting population growth rate. Algae used to feed the
larvae were removed from the carboy cultures starting at

10


TABLES

Solubility and Half-Life of Five Pesticides in
Natural Seawater at 20°C . 1 ee ee ee eee eee

Number of M, edulis Larvae Attached to Selected
Substrates During a Five-Day Period ........

Survival and Attachment Data for Adult Mussels
Exposed to Sevin for 96 Hours . 4. 2.4 es ee eee

Percentage of Normal Larvae Developing in 48 Hours
from Mussel Eggs Fertilized in Various Concentra-
tions of Sevin . 6 1 1 ww ee eee we we ewe we

Mean Shell Length of Mussel Larvae Exposed to
Sevin for 10 and 20 Days... ee ee ee ee ene

Number, Age, and Size of Juvenile Mussels Developing
in Larval Cultures Exposed at 48 Hours to Various
Concentrations of Sevin .. 1. ee ee eee eee

Effect of Sevin on Mussel Larvae Metamorphosis after
a 40-Day Exposure Initiated 29 Days after Fertiliza-
tion of the Eggs . 6 6 ee ee ee ee ee ee ee

Survival and Attachment Data for Adult Mussels
Exposed to Treflan for 96 Hours ......2..ees

Percentage of Normal Larvae Developing in 48 Hours
from Mussel Eggs Fertilized in Various Concentra-
tions of Treflan. . 16. ee ee ee ee ene

Page

21

29

33

34

35

36

37

42

43


Malathion

Malathion is an organophospate insecticide that degrades relatively
rapidly upon application to soil or water. In a silt-loam soil,
3.2 ppm persisted for eight days, with about 0.1 ppm remaining
(Lichtenstein and Schultz, 1964). In water, 10% of 10 ug/liter
remained after 14 days (Rumker et al., 1972).

The insecticide appears to be much more toxic to aquatic crustacea
than to aquatic molluscs. The 24-hour TLs5q for the grass shrimp,
sand shrimp, and hermit crab ranges from 0.118 to 0.246 mg/liter
(Eisler, 1969), whereas the clam, M. mercenaria, tolerated four
days of exposure to a concentration of 37 mg/liter (Eisler and
Weinstein, 1967). Using shell growth as a measure of effect,
Butler (1963) found that exposure of C. virginica to 0.097 mg/liter
of malathion reduced growth by 50%. The number of normal larvae
developing from eggs of the same species of oyster, fertilized in
water containing 9.07 mg/liter of malathion, also was reduced by
50% in 48 hours (Davis and Hidu, 1969). The same investigators
reported a 14-day They of 2.66 mg/liter for oyster larvae survival.

Although several tests were performed, we were unable to determine
the Tso for adult mussels using survival or attachment as the
measured parameter. Response was inconsistent within and between
tests (Table 24). For example, in one test only 20% of the mussels
survived at a measured concentration of 9.0 mg/liter, and 100%
mortality was observed at 17 mg/liter. In another test, all the
mussels survived at a measured concentration of 39.4 mg/liter.

The response pattern for attachment was also inconsistent.

The response of the embryos to malathion was much more consistent
than that of the adults. The 48-hour ECsg values, estimated from
the data from four duplicated embryo bioassays, were 13.5, 17.2,
12.5, and 10.5 mg/liter. The mean was 13.4 mg/liter. Table 25
presents data from these tests.

In the growth tests, the highest test concentration for malathion
averaged 12.3 4 2.3 mg/liter. The mean and standard deviation were
calculated from 35 determinations. Estimates of the lower test
concentrations are 6.2, 3.1, 1.5, and 0 mg/liter.

66


SECTION VI

RESULTS AND DISCUSSION

PESTICIDE SOLUBILITY AND STABILITY IN SEAWATER

Table 1 presents the solubility and half-life of each pesticide
used in this study. The salinity, pH, and temperature of the
natural seawater were 25 Y,,, 8 + 0.2, and 20 + 2°C, respectively.
The half-life of malathion was not determined.

Table 1. SOLUBILITY AND HALF-LIFE OF FIVE PESTICIDES
IN NATURAL SEAWATER AT 20°C

los Solubility, Half-life,
Pesticide .

mg/liter hours
Methoxychlor 0.05 Stable
Treflan 0.20 82
Sevin 60 82
Malathion 110 --
2,4-D (acid) 1100 Stable

The true equilibrium solubility of most "insoluble" pesticides in
water is essentially unknown. Although the values in Table 1 are
approximate, they provide an estimate for maximum test concentra-
tions that should be employed in toxicological experiments in which
the presence of undissolved pesticide is undesirable.

Our estimate for the solubility of methoxychlor in seawater is the
same as that reported by Millemann and Caldwell (1973) of Oregon
State University. Our estimate of the solubility of Treflan,

21


SECTION IIT

INTRODUCTION

Pesticides are chemical compounds formulated specifically to
kill or control undesirable life forms, be they protists, plants,
or animals, Although the chemical industry has been endeavoring
to develop selective pesticides, most currently in use are broad-
spectrum formulations that may adversely affect nontarget life
forms. In addition, the methods for applying pesticides, par-
ticularly those used in agriculture, provide considerable oppor-
tunity for pesticides to enter nontarget areas and thus become

a hazard to many organisms.

This report describes the results of an investigation of the
effects of five commonly used pesticides on Mytilus edulis, an
estuarine mussel. This mussel inhabits many bays and estuaries
throughout the world, and in some regions is esteemed as a food
for human consumption. The pesticides selected for study were
malathion, methoxychlor, and Sevin, which are broad-spectrum
insecticides; Treflan, a selective preemergence weed killer; and
2,4-D, a selective herbicide used particularly in the control
of broad-leafed weeds. These pesticides were studied with respect
to their effects on the survival of mature mussels, on shell
development in 48-hour-old larvae, and on shell growth and
metamorphosis in developing larvae.


8 T T T
St. Francis Yacht Harbor

+ Coyote Point

eS Treasure Island

Tomales Bay

60

percent
a g
3 3

NORMAL LARVAE
®
3

2 4

10 f= +

0 \ L i

oO 50 100 160 200
AGE OF NATURAL SEAWATER — hours

FIGURE 2. EFFECT OF AGE OF NATURAL SEAWATER ON THE NUMBER
OF NORMAL LARVAE DEVELOPING FROM EGGS OF THE
BAY MUSSEL

26


$6

Table 4

HEAN AND STANDARD DEVIATION OF SHELL LENGTH OF MUSSEL LARVAE REARED IN SEAWATER WITH VARTOUS CONCENTRATIONS OF 2,4—D

Means Based on Measurement of 20 Larvae
(microns)

Concentration, mg/liter

Seawater control 28 aa 182.8
bay |Wean | sp Mean | 60 Mean [SD | Wean | 5p ican |_SD Mean] 80] wean | SD ean | 80 ean [80
o --- — _ —
2 faa | va | aaa | so | at | as | ase | 5.5 | as | 5.7 | a2 | sa | 109 | 4.6 | 206 5.2 | 113 7
a | aes | 76 | ar [soa | 223 | arr | aza | 7.2 | a2e |are | amr | 79 ] as | 209 | a4 aos | s.r | soa | oe
6 | 130 | 8.5 | ass | 7a | x96 | ar.7 | 439 J as.e | iso fas.a | a2 | 10.9 | a0 | 17.9 | 327 us | so | us | 9.9
s | ass | 22.8 | isi | 20.5 | x52 | 23.8 | 151 | 26.2 | 150 | 22.3 | 150 | 23.1 | as2 | a8 | 139 ne |uro | no a
so | ste Jars | res | s2.2 | 198 | sta | rez | 25.3 | aor jars | 1a | ars | ato | aas | ave aa | sgn
a2 | 207 | 27.8 | ais [ass | 2i7 | 24.6 | 22a | 25.6 | 205 |] a5.8 | 205 | 23.6 | im | 25.9 | ass -
1s} aa | 26.6 | 227 | 30.2 | 257 | as.s | 223 | 29.4 | 225 | 22,7 | 255 | 30.3 | 201 | 42.7 | aaa -
1s | 255 | 33.3 | 283 | 36.9 | 288 | 30.9 | 26a | 26.9 | 265 | ise | 2m | 22.5 | 203 | 24.6 | 216 -
is | 273 | ai.o | 287 | s4.5 | 205 | 29.4 | 250 } 5.5 | 237 | 58.2 | 261 | ave | 242 | ao.2 | 262 -
20 | 280 | 59.2 | sia | 32.6 | 30a | 28.2 | 270 | 36.1 | 293 | 4.8 31.9 | 240 | ss.6 | 223
22 - -- - - —
2s | saa [agar | s20 | ara | 373 | s2.5 | 282 | saa ais | 292 | 28.9 s | a8.2 | 2a9 .| 35,7
26 | a29 | as.s | s40 | 95.9 | 363 | a6.2 | sii | sa.0 | 294 | 39.4 | 286 | 98.7 66.5
2s | za | sas | si7 | 1.0 | a7a | 48.6 | 204 | 47.0 | 307 | 60.4 | 309 | a7.8 avs | 224 | 58.2
ao | asa fata | asa |as.7 | aga | 24.0 | asa | 25.9 | a6 | w2.6 | 206 | sao | 201 | 16% | 2 37.0
Ey sav fas.o | as | sas | 28 | 23.4 | soe | 27.9 oma jane | ar | as.

Experiment 2
o faa | oa | ae Joa | ase fae fata [oa ]oasa [oa | ase | we fo asa | oar fara | az fase | az | aaa | ar
2 |i | 69 | 12s | 5.7 | aa | a9 | aes aa | 5.3. | uo | ss | 9 | ws | aa | 6.9 | ate | 65
a | asr | 7.9 | 227 | 9.0 | 25 | a9 | 129 aa | aa | ass wo | 6s | ae | 7s] a6 | so] ur | 39
6 | a | oa | as7 | 9.6 f aaa ae | 139 asr [16.5 | 151 v7 | iso | ast faze | a6 | ss | ie | sa
s | aay fags | asa favs | asa | 23.7 | tas ass | is.6 | 160 ais) asa | age fasa | aa7 | 6.9 | ata | oa
w | aes faz.o | 77 |as.e | 195 | 20.9 | asz as | 22.6 | 166 ro | 15.6 | 15a | 9.7 | ase fae | a | 9.0
12 | 199 | 26.8 | 193 | 20.9 | 206 | 22.9 | ara asa | 20.8 | s07 are | 19.4 | 200 | 28.6 | 163 iss | ie | 9.3
ra | a7 | 2t.7 | 20a f 26.9 | 233 | 2s.a | an 26.8 | 238 ass} 16.9 | isa [7.2 | 194 | to. | 12s | 14.9
re | 225 f2s.a | 221 fas. | 257 | ao.7 | 242 33.9 | 239 | 24.0 | 210 | 2a.7 | 2x6 | 22.0 | 205
ws | cas Jos. | 252 | s4.8 ) 272 | sea | zao a3.a | 250 | 39,4} 245 | 30.5 | 258 | 28,6 | 2az
20 | 255 | arc | 263 | as.8 | 280 | sas | 277 2ei_} 29.8 | 274 | aso | 232 | 26.9 | 235 | a2.2 | 206
oa | soo | 28.5 | 305 | 35.8 | aie | st | sor | 35.1 | a | seo | 298 | ao.5 | ear | a2a | 25s | 39.5 | 162 | 16.2
os | sas | 20.6 | 323 | a6,0 | sas | 37.6 | sav | a4.s | 282 | as.7 | ato | 50.1 ] 227 | sea | 265 | asa
2s | sas | 90.8 | 206 | as.8 | saz | 33.8 } 328 | 50.7 s72 | as fava | 256 | a | 269 | 38,9
ao | aca | 67.6 | 298 | 50.7 | 350 | 72.2 | 29 | sas | sao | s1.6 | sus | 42.7 | 275 | 63.0 | 270 | aaa
on aaa | as.o | 36a | a2a | sss | s9.9 | ass [27.9 | sa | ao.0 | 272 | as | 276 | 38.2



Table 1. WATER QUALITY DATA FROM REPRESENTATIVE 96-HOUR ADULT SURVIVAL EXPERIMENTS

88

Dissolved oxygen,
T t °C Salinity, % HH
Pesticide* emperarare , mg/liter alinity, Yoo P
Mean SD | Analyses | Mean sD Analyses | Mean sD Analyses | Mean SD | Analyses
Sevin 19.5 | 1.62 125 7.2 0.45 125 25.4] 0,24 20 7.95] 0.14 20
(53)
Treflan 20.2 | 0.30 100 4,9 2.11 100 26.0] 0.10 24 7.83 | 0.10 36
(43)
Methoxychlor | 19.1 | 0,60 75 7.1 0.50 75 26.1 | 0.49 6 8.09 | 0.10 6
(45)
2,4-D 18.0 | 0.63 42 8.0 1.30 42 23.6 | 0.67 25 7,50 | 0.52 26
(12)
Malathion 19.5] 1.18 150 6.7 0.38 150 24.2) 0.36 24 7,87 | 0.09 12
(56)

*
Experiment numbers in parentheses,



SECTION IV

MATERIALS AND METHODS

PESTICIDES

The pesticide formulations evaluated, their source, and their
purity as specified by the manufacturer were the following:

© 2,4-D, 2,4-dichlorophenoxyacetic acid; The Dow
Chemical Company; production lot No. 092440;
purity, 94.8%.

®
* Sevin , l-naphthyl N-methylcarbamate; Union Carbide
Corporation; production lot No. 72055; purity, 99.7%.

e Malathion, 0,0-dimethyl phosphorodithioate of diethyl
mercaptosuccinate; American Cyanamid Co.; produc-
tion lot No. IV 90715; purity, 95%.

®

¢ Treflan , %,@,a-trifluoro-2,6-dinitro-N,N-dipropyl-
p-toluidine; Eli Lilly and Co.; production lot
No. X-14788; purity, 99%.

* Methoxychlor, 2,2-Bis(p-methoxyphenyl)-1,1,1-
trichloroethane; E. I. du Pont de Nemours & Co.,
production lot number not indicated; purity,
minimum of 88% p,p' isomer.

ANALYTICAL METHODS

Test concentrations of the pesticides were monitored routinely
throughout each test. All analyses were performed on a Microtek
Model 220 gas chromatograph equipped with an electrolytic con-
ductivity detector in the oxidative mode. The colum, 2 mm

ID by 120 cm, was packed with 6% SE 30 on 100/200-mesh Gas Chrom Q.


16

Teble 3, MEAN

AND STANDARD DEVIATION OF SHELL LENGTH OF MUSSEL LARVAE REARED IN SEAWATER WITH VARIOUS CONCENTRATIONS OF METHOXYCHLOR

Means Based on Measurement of 20 Larvae

(microns)
Concentratzon, mg/liter
Scawater control Solvent control 0,008 0.019 0.05 0.082
days [Mean [ SD | Moan] Sb [wean] 5b [ Wean] SO | wean] 8D] wean] SD | Wean[ Sp | Mean] SD | Wean] Sb | Nean[ So | Mean] SO | Nean[ 9D
T
i T
Experanent 1
o us fora |oas ]oza fas | va | ais fora | aaa faa] aaa fo za fuss J 7a] ass f 7a | ass faa | aaa | 7a | sas | 7a
2 a16 [33/0 } 122 | 9/0 } tas } 912 | a9 | eis | 122 | acs | ai7 fuz.9 | ata Jao.a | ai | 9.7 | 1x7 | 7.0 | a9 | aa | ize | 9.6
4 135 [1010 | 120 | 9.2 | 135 j 10.9 | 192 | 91a | 13a Jaze | 141 fisce | 136 | 9.7] 146 [x07 | 139 |ar.6 | 242 [15.5 | rar | 9.5
6 aaa fig:3 | 147 [17.2 | 1az | 2010 | raz | rao | 157 [18:0 | 146 | 19.8 | 150 } 16.5 | 160 |ra.a | is3 | 20.2 | 150 | 19.4 | 197 | 15.6
8 ass | 2111 | 153 | 20.1 | 171 | 22.3 | 167 | 15.3 | 164 | 17.4 | 178 | 22-6 | 168 | 22.9 | 199 | 24.5 | 176 | 26.5 | 169 |18.4 | 173 | 15.5
10 ass | 2419 | 159 | 27-8 | 187 | 12:8 | 188 | 20.8 | 186 | 26.5 | 186 | 23.7 | 181 | 19.8 | 194 | 22.4 | 197 |as.6 | 190 12.5 | 187 | 15.0
12 190 | 23:0 | iso | 31.9 | 191 | 28.9 | 175 | 31.5 | 181 | 28.6 | 201 | 20.1 | 193 | 24.6 | 183 | 28.7 | aaj 27.4 | a72 | 17.9 | 167 | 19.2
4 225 | 22.4 | 217 | 25.6 | 232 | 36.7 | 215 | 30.7 | 189 | 34.0 | 21: | 29.5 | 204 | 20.6 | 224 | 38.7 | aia [24.7 | isa | 22.9 | 197 | si.9
16 207 | 34.6 25.1 | 230 | 342 | 211 | 33.3 | 205 | 25.7 | 219 | 39.1 | 227 | 31.6 | 204 | 44.6 | zis | 40.5 | 189 | 29.0 | 210 | 26.6
38 226 | 26.3 30.2 | 2a3 |a7.7 | 242 | 26.1 | 216 | 29.3 | 258 | aso | 240 | 1.9 | 228 | 33.9 | 224 |aa.a | 199 | as.s | 225 | 41.6
20 26a | 32.2 26.5 | 266 | 27.7 | 261 | 50.4 | 244 | 45.9 | 267 | 90.8 | 298 | 36.0 | 281 | 52.8 | 241 | 38.0 | 239 |ai.e | 221 | 42.2
22 291 | 39.7 agla | 230 | 30:3 | 233 | 35.0 } 269 | 41.8 | 289 | 28.9 | 290 ) 49.7 | a7 |4o.a | 239 | 33.7 | 226 | 33.0
2a 306 | 23.9 55.0 | 256 | 38.5 | 262 | 58.3 | 260 | 54.9 | 268 | 40.1 | 283 | 45.9 | 268 | 50.1 | 242 | 39.7 | 299 | 50.6
26 323 | 34.7 41.2 | 283 | 38.1 | 223 | 44.7 | 323 | 24.6 | 327 | 28.2 | 276 | 48.5 | 9a | as.0 | 280 | 31.4 | 299 | 45.9
28 340 | 34.6 ss.7 | 288 | 45.4 | 260 {57.1 | 322 | 38.2 | aio | 60.8 | 273 | 50.2 | 294 | 54.0 | 265 | 53.7 | 262 | soa
30 a7 | 25.1 so. | 281 | 48.5 | 277 | 46.6 | 347 | 30.4 | 334 | 43.5 | 207 | 55.0 | 331 | 50.6 | 287 | 38.8 | 281 | 38,6
32 gar | 28.6 eas | 334 | 26.3 | 247 | 60.4} 319 | 42.2 | saa | 47.5 | 317 | 43.7 | 328 | 36.2 | 259 | 37.6 | 25s | 35.2
34 33 | 40.1 si.7 | 321 | 42.9 | 258 | 59.7 | 307 | 5a.3 | 321 | 68.1 | 323 | 49.1 | 328 | 56.4 | 271 | 42.2 | 27a | a7.2
ne | 373 383 | 63.3 50.7 | 316 | 50.5 | 269 | 39.9 | 349 | 47.3 | 356 | 58.7 | 317 | 92.8 | 348 | 40.4 | 281 | 33.6 | 260 | 40.1
' Experiment 2

o [120 | 7.9 | 10 7.9 | 110 | 7.9 | 10 | 7.9 | a0 | 7.9 | aro | 7.9 | at0 | 7.9 | a20 | 7.9] a20 | 7.9] ato | 7.9 | a10 | 7.0 | a10 | 7.9
2 | 109 [10.0 | 113 f2z.3 | 110 Ja3.s | 113 j12.8 | 120 Jae.1 | 120 f1s.o | 120 Jao.4 | 126 }14.6 | 127 Joa | ais [az.9 | 127 |a2.2 | ais fasia
a [ars fuze | 116 |ae.s / 115 | 10.7 | 116 | 18.8 | 106 | 9.5 | 215 |a7.0 | 130 J 16.7 | 127 Jara | 135 [21.3 | 134 fi6.3 | 11 |20.8 | 137 Jie.
6 | 173 |26.2 | 161 | 3813 | 136 | 22.0 | 141 | 19.4 | 153 Jag.7 | 136 Jor.7 | 1as | 28.4 | 162 |a3:7 | 153 |a70 | isa }a7.1 | aas jain | 153 |is.6
3 | 246 | 31.2 | 152 | 2sis | 149 | 23.3 | 16a | 19.3 | 171 ]28.3 | 165 | 19.4 | 175 | is.0 | 172 | 20:3 | 16a |21i9 | 181 | 19.8 | 167 |22-0 | 187 |as.6
10 | 197 |4al6 | 193 | 26:6 | 105 | 21.6 | 187 }1s:1 | 207 |20.1 | 185 |25:6 | 190 | 24:5 | 201 | 28:6 | 198 |i9.9 | iss |23:7 | 17s |23.3 | 202 Joa.
12 | 189 |33.1 | 174 | 30.9 | 192 | 26.6 | ise | 33.9 | 189 | 39.9 | 212 |26.0 | 20a | 29.0 | 213 |a1.1 | 196 | 19.2 | 192 22.6 | 178 |20.4 | 178 |is.a
a4} 197 [30.9 | 191 | 32.6 | 189 | 49.3 | 208 | 41.0 | 203 | 99.6 | 21a |as.3 | 213 | 37.7 | are |2a7 | 228 | 221 | 215 |28-9 | 196 |26.4 | 202 Jain
as | 228 J17.0 | 225 |29.8 | 231 | 36.2 | 28 219 |ar.2 | 240 | 30.5 | 235 | 24.4 | 254 |2a0 | 230 | 2515 | 292 |aa.a | 205 J25.9 | 220 | 26.7
is | 224 |28.6 | 244 | 26.7 | 256 | 30.2 | 267 } 37.3 | 235 |3a.o | 238 |ao.a | 271 | 28.7 | 265 |ar.s | 262 | 2013 | 252 | aro | 220 Jaro | 218 | 35.0
20 | 259 | 25.0 | 280 | 17.6 | 264 | 50.6 | 303 | 39.7 | 267 | 39.3 | 262 Jar.6 | 292 | 35.4 | 294 |a7.8 | 265 | 30.4 | 281 | 24.7 | 236 |as.7 | 262 | 19.9,
22 | 282 |27.9 | 280 |47.6 | 257 | 58.0 | 209 | 53.6 | 276 | 45.8 | 252 | 59.0 | 293 | 43.4 | 302 |30.7 | 277 | 4114 | 279 | 37-5 | 275 |20.0 | 28a | ai.9
24 | 275 | 31.5 | 295 |se.s | 324 | aes } 317 | 36.8 | 280 |47.7 | 283 | 42.6 | 311 | 39.5 | 252 | 39.6 | 277 | a1.7 | 293 | 40.4 | 275 | 46.7 | 261 | 34.5
26 | 306 | 35.0 | 209 | a9.9 | si9 | 38.0 | 347 |44a.a | 303 | 38.1 | 306 | 42.9 | 325 | 33.0 | 328 | 32.8 | 302 | 35.6 | 301 |a2.2 | 273 |39.2 | 201 | a1.9
zs | sia [49.8 | 303 |sa.2 | 316 | 44.6 | 336 | 42.5 | 33a |38.3 | 329 |a5.a | 346 | 34.5 | 348 [28.9 | s19 | 33.6 | 310 | 37.1 | 2a | 32.6 | 208 | 38.1
30 | 301 | 46:5 | 330 |4o.8 | 338 | 33.4 | 326 | 56.2 | 313 | 54.2 | 322 | 50.4 | 323 |s2.2 | 349 | 40.7 | 302 |aa.9 | 299 | 43.0 | 298 | 41.0 | 298 | 49.1
32 | as: |as.7 | 338 [63.7 | 330 | 61.6 | 381 | 30.5 | 369 | 38.5 | 329 | 53.9 | 354 | 37.8 | 359 47.9 | 310 | 56's | 338 | 24.0 | ai2 |sa.a | 206 | 42.8
sa_| 358 | 37.9 339. | 48.6 | == 399 | 49.8 303 | 56.5 | 361 fos.2 | 306 |as.6 | 325 | 44.0 | 278 |at.o | 280 | 35.4



66

Table 2, NUNBER, MEAN AND STANDARD DEVIATION OF SHELL LENGTH OF METAMORPHOSED MUSSEL LARVAE EXPOSED TO TREFLAN
Exposure Initiated 20 Days after Fertilization of Eges
(microns)
Concentration, mg/liter
Seavater control Solvent control 0.08 0,040, 2.080 PRT
pay [a fMean] SD] nf wean [S| n [Wear] sb [ n[ Mean [90 [o[Wean [S| n[ wean] S| n[uemn[ Sb] n Neon [SO] a] ean [Sb] n[ wean] Sp [a] wean | SO] a] Neon] So
o | of- 0} - o| --- of = o| --- ° ° ° ° °
2 | ol o| - o| -- o| --- of ° ° ° ° °
a | of--- o| - 0} -- o| --- o} --- ° ° ° ° o
5 | o|--- | - o| --- o| --- o| --- ° ° ° ° °
7 | ol- of - 2} 330 | 7 | o| --- 3} aa7 | 92 27 {a} a7a | ss as | 5 ae} 4}a00 | as] 2{ are | so] a}az5 | 23
o | 1}4ss | of 1] 302 | 0 | a] 4oa } 20 \14] 999 | se fas} aso | aa 2s | 6] a67 | 44 ae| 6] ca | 36 [1s] sez | 28 | a] ars | as] 4} oa | aa
n ]afsse | of a} a60 | a2 | of -—- 1s} axa | 94 |15| aoa | 68 a7 {1s} 400 | 60 so} s{a70 | 93}16| 406 | a5] 3] 377 | 72} 7) a80 | ot
13 | {aa | aa] a] a07 | 12] 2] aae | 7a] 6] as7 | ss frsfses | ax se | 3fasa | ua] o nifan | 49] af 425 | 24] 5] aoa | a9] | ae | os
1s | 6422 {aos farfaro | 05] s| 397 | a7| 7] 403 | 2 | 7] ase | 20 so! 2]s02 | 68) 2 sea | 23] sf az | 72] 0 2] sea | a1| a| 70 | 6s
ar | {as | a8] 2] 469 } ae [ss] axz | a2} 6) 959 | a8} 7] as8 | 5a a0 |oa]a77 | as {er sis | eo Joa] a77 | 55 {os as }a2] sz | as | 29] 360 | 33
ao | 7)aso | as] af ave | a3 {21 | a02 | 79] 1} 960 | o| alass | 2s a|6faa | se] 1 492 | o| of 5 az |is] 10 | 60] 9] 400 | aa
ai |aifar7 | os aa] aia | 62 | fase | o| 2) aaa ifaw | o o| if | of o --~ 2|ae | s]o a|ase | us] 8] sa7 | 08
2a | alaso | a8] 5] 40a | 72 | 2] 579 [299] 1] ase | o | o| --- of = 2 se: | of zara | of o a}ser | o| 3| ae7 [asa
za | alaao | s6{ s| 4s: | a9 | 6] 08 Jar2 | a] a2 | 60 | 1| 480 | 0 12a) 2}ae6 | e@| 2 azz | 78] o| ~~ ° °
26 | 2}sos | es} s| aaa | a3] efor Ja20| af an fas} 2] asa | o alas | 96] 2 a60 | 0} a] ass |era] o rfaaa | of 3| 993 | 32
za | afan | o| s}ase | a7] 1] aos | o| 1}s60 | o | o| — a}a6a | a0] 0 --- of --- 6| 400 | 90 °
30 | 2{4e0 | ae |1o| 422 | so | of --- of = ° of --- o ° o| --- °
3 ato | 70] 4{ 399 | as | of az | ofo afass | of o =~ ° ° °
39 |1afasa }ro2} 2| 20 | 0 | of --- o| --- of o} --- — ° ° °
No. of
onmeta=
prosed
Torwae | 8 3 ° 1 ° ° 1 ° 1 2 ° 1



of growth was observed in both tests. In Experiments 1 and 2,
respectively, larvae were 31.9 and 34.9% smaller than the controls.
Continued exposure of the larvae for 20 days resulted in statisti-
cally significant decreases in larval size at 91.4 and 182.8 mg/
liter. In Experiments 1 and 2, the larvae exposed to 91.4 mg/

liter of 2,4-D were smaller than the controls by 22.5 and 9.6%,
respectively. All the Larvae exposed to 182.8 mg/liter of 2,4-D

in Experiment 1 died within 12 days of exposure. Larvae exposed

to this concentration in Experiment 2 were 20.5% smaller than the
controls. The differences were statistically significant (p = 0.05).

The growth study was extended to 32 days to determine the effect of
2,4-D on larval metamorphosis, The data, presented in Table 22,
varied considerably between the two tests and did not follow any
readily identifiable trend. Fewer controls than pesticide-exposed
larvae developed into juvenile mussels. The only observation con-
sistent to both experiments was that the larvae exposed to 182.8 mg/
liter failed to undergo metamorphosis. At this concentration, the
four larval groups tested did not survive longer than 22 days.

Table 22, NUMBER, AGE, AND SIZE OF JUVENILE MUSSELS
DEVELOPING IN LARVAL CULTURES EXPOSED AT 48 HOURS
TO VARIOUS CONCENTRATIONS OF 2,4-D

Test Experiment 1 Experiment 2

concen- L h

tration, | Total | Age,# ength, Total | Age,# Lemgth,
/lit bi d

mg/liter | number | days |4/.. | gy | number | days Mean | sp
0 14 26 372 | 57.6 25 22 386 | 56.7
22.8 99 22 453 | 81.7 72 22 444 | 77.6
45.7 124 24 361 | 52.3 67 24 356 | 48.1
91.4 3 24 339 | 81.3 43 26 343 | 41.6

182.8 0 -- -- |-- 0 -- -- | --

a
Number of days required by treatment group to begin meta-
morphosing.

62


Table 25. PERCENTAGE OF NORMAL LARVAE DEVELOPING IN 48 HOURS
FROM MUSSEL EGGS
FERTILIZED IN VARIOUS CONCENTRATIONS OF MALATHION

Test number
Test 7
concentration, Experi~ Experi 5 aan meron
. ment ment men men
meter Fo Toal@a!latw)a) a]
0 95.5] 98.5] 88.0] 91.0] 85.5] 87.0] 85.0] 86.5
1.95 96.5] 93.0) -- |-- |-- |-- |-- | --
2.75 -- -- 80.0] 83.5] 73.5] 73.0] 82.0] 78.8
3.95 93.5] 96.0; -- |-- |-- |-- |-- | --
5.50 -- -- 85.8 | 87.3] 70.3] 70.8] 64.0] 58.5
8.15 82.5) 94.0|-- |-- |J-- |-- |-- |--
12.0 -- -- 77.5) 79.5 | 42.3 | 48.0] 37.0] 37.5
15.5 36.0] 32.5|-- |-- |-- |-- J] -- | --
24.0 --_|--
48-Hour EC5q,
mg/liter 13.5 17.2 12.5 10.5

Larvae exposed to concentrations of up to 3.1 mg/liter for 10 and
20 days exhibited a growth rate similar to that of the controls
(Table 26). At the higher concentrations, the larvae exposed to
6.2 mg/liter for 10 days were about 5% smaller than the controls

in both experiments; at 12.3 mg/liter, the larvae were 18 to 19%
smaller than controls. After 20 days of exposure, the differences
in size increased. The greatest difference was observed in Experi-
ment 2, in which the larvae were 24.4% smaller than the controls
at 6.2 mg/liter and 39.7% smaller at 12.3 mg/liter. The percentage
difference noted for these two concentrations at 10 and 20 days was
statistically significant (p = 0.05).

Continued exposure for a total of 30 days after shell development
of the larvae used in the growth study resulted in a marked decrease
in the number of larvae developing into juvenile mussels. This ef-
fect on metamorphosis was particularly evident in larval populations
exposed to 6.2 and 12.3 mg/liter of malathion (Table 27). In

68


SECTION V

EXPERIMENTAL PROCEDURES

EXPLORATORY STUDIES

Time of Fertilization and Larval Shell Development

Approximately 6000 eggs, obtained from a single Tomales Bay
female, were placed in each of six 250-ml beakers containing
200 ml of natural seawater. Sperm from a single Tomales Bay
male was introduced into two beakers at a time 45, 110, 130,
165, and 260 min after the first eggs were shed. About 47 hours
after the sperm were introduced, a few drops of neutral red
stain--enough to impart a light orange color to the water--were
added to each beaker, One hour later, the contents of each
beaker were transferred to 250-ml plastic Falcon culture flasks
containing 50 ml of 10% buffered formalin. Two-hundred consec-
utive larvae in each flask were examined through a dissecting
microscope, and the number with straight-hinged shells was
recorded,

Seawater Type and Larval Shell Development

The effect of artificial seawater (Kester et al., 1967) and of
2-, 48-, and 168-hour-old natural seawater on shell formation of
larvae was investigated using the gametes of mussels collected
in July 1973 from two areas in San Francisco Bay (St. Francis
Yacht Harbor and Treasure Island) and from Marconi's Cove in
Tomales Bay.

The artificial seawater was prepared with reagent-grade chemi-
cals and deionized water (1 megohm) and stored overnight. The
natural seawater, obtained from the Steinhart Aquarium, was
allowed to age in polyethylene containers at room temperature,
The salinity and pH were adjusted, and the adjusted water was
exposed to ultraviolet light before use,

13


96

Table 5.

NEAN AND STANDARD DEVIATION OF SHELL LENGTH OF MUSSEL LARVAE REARED IN SEAWATER WITH VARIOUS CONCENTRATIONS OF MALATHLON

Means Based on Measurement of 20 Larvae

(microns)
[ Concentration, ng/liter
Seawater control Ts Ent ce i.
vay [Wear] sp [7 wean [Sp [wean [sp [wean [0 | Nean | SD | wean [SD | Wean [Sp [wean [8 | wean | 8 | wean | SD
Experiment 1

o | wen - _

2 | 12 | 6.9 | x20 | 7.3 | ue | 7.2 | a7 | 75 | aso | os | ue | co | us | sc | us | 92 | 6 | 69 6.9

4 | 13a | 7.6 | 398 | az.3 | ise | 6.9 | 128 | 10.7 | 133 | 10.2 | 135 | 8.6 | 132 | 8.0 } ass | 9.0 | aa | 9.5 | a7 | 25

6 | a2 [ase | xa [aria | sao 0.5 | aso | 9.0 | aaa | ars | asa | as.2 | 196 Juss | ass fus.a | ar | 9.2 | aaa | a2

a | ast | 12.0 | 146 | as.9 | 150 j 14.9 | 146 | 1s.8 | a66 | as.7 | ver ) aaa | rt fase | 247 | ate | ae7 | asa | a0 | 12.9
ro | 150 | a7.8 | 152 | 16.4 | 154 ]as.e | 185 | 15.9 | 189 | 14.9 | 168 | 1.0 | 14a | ae.o | x47 | aso | r2e fas.2 | ae | aaa
wz | az | ars | 176 | 20.2 | 1 | 16.1 | 163 | 18.2 | 168 | a7.s | 70 | 21.9 | re | 268 | 152 | 13.2 | 166 j x7 ) 141 | 19.5
1a | 19s | t9.6 | 172 | 29a | 167 | 18.7 | 168 | 28.6 | 17s | 21.8 | x72 [asa | sao | 22a | ra | o7.2 | ase | a2.0 | 192 | 13.0
re | ize | 27.7 | 180 | 23.8 | 17s | 26.8 | 177 | 27.0 | ave | 27.7 | 1e7 [azo | 167 | 26.4 | as: | 1s.6 | 142 | sar | 190 | 1.2
rs | 210 | 21.6 | 188 | 27.9 | 186 | 92.6 | a1 | 28a | 195 ] as. | 208 jas.1 | ree | 20.1 | 16s | 19,9 | a7 | 235 | 194 | 12.2
20 | 225 | 31.6 | 204 | 32.3 | 210 | 40.1 | 212 | 26,6 | 225 | s4.8 | 216 | 22.2 | 190 | 20.6 | 157 | 121 | 105 | 46.7 | ass | 15.6
22 | 242 | ara | 227 | 35.7 | 223 | s1.a | 230 | ass | 2 | 23.8 | 242 | 39.9 | ara | 25.7 | 207 | 20.7 | ar | ava | 4s | 74
2a | 263 | 30.3 | 226 | 20.6 | 234 | 25.8 | 230 | 49.1 | 243 | aaa | 25a | 32.9 | 194 | 27.2 nie | 16s | rae | aso | aa
26 | 297 | 30.7 | 204 | 33.9 | asa | 41.6 | 250 | 42.7 | 26a | 39.6 | 268 | 20.4 | 296 | 35.9 163 | 37.3
2s | 206 | 37.5 | 201 | 32.2 | 262 | 95.1 | 246 | aso | 265 | 36.2 | 27 | 36,0 | 230 | 25.4 — zz | 39.9
ao | azo | 52.1 | 326 | 33.4 | 30s | 4s.e | 286 | 4e.1 | 279 | 33.9 } 323 | 30.3 | 262 | 35.0 -- -

se | see | 66.9 | saa | so.6 | si1 | 55.3 | 301 | 32.3 | 279 | 33,0 | se2 | a8.a | 201 | 25.2 _ -

Experiment 2

o | - — - _ - — -

2 | 16 | 60 | 16 | so | 14 | 5.9 | 109 | 5.6 | 113 | 6a | 107 | 8.3 | as | 7.6 | 13 | so | a2 | 5.9 | 107 | 5.9

a | aa | 95 | ase | 9.4 | 136 | 7.8 | 138 Jao | 224 | a2 | tas | 7.4 | azs | 7.3 | a7 | as | ts | oe | uz | a7

6 | 46 fase | ss: | ras | 1sa fiz.e | 160 fas.s | ra | are | 24s ]rs.a | aaa | asa | 140 | as.2 | ax | 7.7 | a9 | a0.9

s | ser [26s | 147 | 28.6 | x42 } 22.6 | 157 |aae | rao | 1.0 | 198 | 29.4 | ass | rs. | sr | 9.3 | 1s2 | 15.2 | 132 | 18.7
ro | aes | a7.e | 152 ]as.0 | a60 ]as.o | 165 | 20.8 | 1a5 | rea | x62 | 2x4 | 247 | 2s.9 | ass | asa | iss Jase | as | se
12 | 196 | 43.0 | 179 | 37.2 | 186 | 25.2 | 145 | s41 | 158 | 26.6 | ass | 25,6 | 186 | 26.1 | aa6 | 37.9 | 14s | 23.4 | 134 | a7.9
ua | 2x6 | 50.9 | ss | 28.2 | 206 ] 29.6 | x81 | 22.0 | 157 | 10.7 | 174 | 29.2 | 160 Jars | 15a | 21.6 | 152 J as.s | 146 | 148
re | 216 [40.9 | 179 | a1. | 200 | 33,3 | 218 | as.6 | 17: | 23.8 | 229 | aaa | 16s |is.e | x66 | 16.5 | 149 | 23.5 | 157 | 24.7
is | 237 | 46.2 | 24a | 41.0 | 226 | 30.2 | 223 | 28.8 | 199 | 30.8 | 215 | aa.0 | 178 | 23.8 | 180 | 18.0 | 153 | 24.4 | 155 | 27.8
20 | 273 | aaa | 252 | ara | 220 | 92.9 | 266 | s1.a | 267 | si.9 | 261 | 26.3 | aro | s7.0 | 1e5 | 12.2 | 159 | as | iss | a9.9
22 | 27s | aa.6 | 201 | 24.9 fave | ar.9 | 273 | 36.6 | 2390 | 54.0 | 246 | 60.9 | 224 | 22.8 | 217 | s1.9 | a6 | 24.5 | 163 | 22.2
24 | 309 | 97.7 | 290 | 2a.a | soo | a1.5 | sie | 2s.a | 269 | 47.7 | 207 | 21.0 | 255 | 26.2 | 223 | 24,7 | 170 | 36.4 | 208 | az.7
26 | su fas.s | si9 | 33.6 | s06 | 43.8 | 30 | a1.6 | 227 | os.s | sis | 38.0 | 24 | o7.3 | 238 | ata | --- 200 | 54.8
2s | 289 | 59.9 | 333 | 36.1 | 297 | sc.6 | 333 | se.s | sos | 56.0 | sur | 42.2 | 253 | ar.9 | 259 | 38,0 | --- 268 | 74.9
30 | 330 | 66.0 | a35 | 52.2 | 362 | as.6 | 365 | 3.0 | sos | 40.4 | 294 | 50.2 | 905 | 42.2 | 268 | 36,9 | --- --

sz | sss |e. | ase | eas | a7s | a7.a | ase | aaa | 299 | 40.0 | sa2 | 35.2 | 298 | 36.4 | 264 | aac | --- -



The test solutions were renewed every other day, and the pesticide
solutions were prepared from freshly mixed stock. The larvae were

separated from the test solutions by collecting them on 53-l) Nitex’

screens.

Before the larvae were returned to them, and before they were

refilled, the jars were scrubbed and rinsed with deionized fresh
water; no detergent was used. During renewal of the test solu-
tions, 20 to 40 larvae were removed from the submerged screens,
fixed in neutral buffered formalin (10%), and measured under a
compound microscope equipped with a calibrated ocular micrometer.
As soon as the larvae approached 250 in size, the jars were in-
spected for metamorphosed larvae on the days of solution renewal,
All larvae with a dissoconch shell were counted, measured, and
discarded. Metamorphosed larvae were processed in addition to
those removed for growth measurements. Each test was terminated
when none of the jars contained enough larvae to proceed further.

During the tests, the larvae were fed M, lutheri daily at a rate of
20,000 cells/mi of test solution per day. Algal cell counts were
made on aliquots removed from each jar on the days between solution
renewal, and algal cells were added to maintain the nominal number.
However, if less than 20% of the cells had been consumed, additional
rations were not provided. On the days of solution renewal, the
full nominal number of algal cells was provided to each test jar.

Each level of pesticide was prepared by serial dilution. The
highest nominal test concentration was equal to, or slightly higher
than, the EC determined in the 48-hour embryo shell formation
tests; if the EC could not be determined, the nominal concentra-~
tion was equivalent to the maximum solubility of the pesticide in
the diluent seawater. The highest test concentration was deter-
mined analytically each time the test solutions were renewed.
Salinity, pH, temperature, and dissolved oxygen were measured in
each jar immediately after and just before solution renewal,

Metamorphosis

The effect of exposure to the five pesticides on metamorphosis was
investigated by exposing 29- to 30-day-old larvae to four different

18


Lv

Table 12. EFFECT OF TREFLAN ON MUSSEL LARVAE METAMORPHOSIS AFTER A 40-DAY EXPOSURE
INITIATED 30 DAYS AFTER FERTILIZATION OF THE EGGS
(Presence of Dissoconch Shell Used as Indication of Metamorphosis)

Test concentration, mg/liter
Seawater Solvent 0.02 0.040 0.080 0.160
control control
1 2 1 2 1 2 1 2 1 2 1 2
Number of Larvae
Initial 100 100 100 100 100 100 100 100 100 100 100 100
Lost or crushed} 24 16 22 31 27 29 6 16 23 12 20 2
Experimental 76 84 78 69 73 71 94 84 77 88 80 98
Metamorphosing,
percent 89.5%] 92.8%] 89.7%] 87.0%] 84.9%] 78.9%] 85.1%] 86.9%] 79.2%] 81.8%] 87.5%] 75.5%
Nonmet amorphos-
ing, percent 7.9%) 4.8%] 0 0 0 1) 1.1%] 0 1.3%] 2.3%] 0 1.0%
Dead, percent 2.6%) 2.4%] 10.3%] 13.0%} 15.1%] 21.1%] 13.8%] 13.1%] 19.5%] 15.9%] 12.5%] 23.5%
Days to metamor-
phosis of 50%
test larvae yaa 21 16 12 12 4 12 11 16 16 16 17
Shell length of
juvenile mus-
sels, 449 pp 1427 p 1424 py 1387 yp [383 yp }403 py 1412 p [424 wp 1397 uw 1383 p [402 uy [406 p



6

STANDARD DEVIATION OF SHELL LENGTH OF MUSSEL LARVAE REARED IN SEAWATER WITH VARIOUS CONCENTRATIONS OF SEVIN

Table 1, MRAN AND
Means Based on Measurement of 20 Larvae
(m.erons)
Concentration, mg/liter
Seawater control 0.35 0.85 v5 2.6
vay [eam] so Mean [SD Wean [3 Wean [50 Hea ]_Sd_ | Mean [5D Mean [85 ean [8D ean [8D ean [50
1.6 | oa | 10,6 | roa | r0.6 | 104 | 10.6 | 104 | 10.6 | 104 | 10.2 | s04 | 10.6 | 104 | 10.6 | 10a | 20.6 | 104 } 10.6
zo.1 | 192 | 22.5 97 | 16.9 a9 j 15.0 | 207 | 22.4 | 437 | as.e | 113 | 11.2 | 107 | 13.2 | 105 | 13.8 | 103 | 14.0
sax [| isa [15.7 | a25 | a7.9 | aaa | 222 | ate | ree | te | 23:0 | 100 | 15.8 | 109 | 181 | 105 | a5.5 ER
as.4 | 141 | 28.7 is.3 | 139 | 30.5 | 127 | 24.7 | 113 | 29.8 | 16 | 19.5 | asa | 22.4 | 109 | 19.4 23.6
a7.9 | 159 | 51.9 26.9 as.8 | 147 | 22.8 | 129 | 28:3 | 13: | 16.4 | 116 | 20.8 | x20 | 17.0 144
zo.s | 162 | 67-4 4.6 sis | are | ao1 | 141 | 35:2 | 135 | 29.6 | 109 | 22-4 | iz | ats 21.6
50.8 | 233 | 43.5 46.6 30.0 | as7 | 33.0 | 177 | 17.0 27.2 | 127 | 19.5 17.5 20.0
78.6 | 174 | 68.4 49.6 as.2 | isa | 37.8 | 180 | 37.5 21.0 22.9
19.7 | 272 | 30.4 34.9 33.5 | 216 | 26.3 | 197 | 23.5 314 23.4
56.3 | 300 | 65.0 34.4 26.2 | 189 | 3a.a | ass | 33.9 aia 16.2
32.7 | 322 | 26.9 an 30.9 | 181 | 2510 | 227 | 19.6 23.0 in
a2io | 342 | 2214 82.3 as.2 | 196 | 16.0 | 207 | 25:8 47.7 19.6
si.3 | 251 | 59.7 26.4 27.7 | 200 | 20.2 | 227 | ga.2 19.0
e1.0 | 318 | 45.7 B74 24.0 | 179 | 29.4 | 219 | ats 8a
g6.4 | 373 | 45.8 66.0 30.6 | 228 | 8.3 | 250 | 26.1 28.4
0.2 | 386 | 21.8 22.7 azis | 2a. | 2012 | 258 | 31.5 34.3
307 | as.7 | 420 | aaa | 235 | 23.6 2s.1 | 267 | 24.4 | 277 | 33.2
393 | 30.8 | 425 | 33.0 | s17 | 89.3 is.4 | 213 | galas | 208 | ila
372 | 74.2 | 431 | 35.7 | 263 | 36.7 ais | 286 | sa2 | 317 | 22.6
Experiment 2
o 9s | 8.0 ss | 8.0 os | 8.0 ss | 8.0 ss | 8.0 os | 8.0 8.0 a | 8.0 8.0 8.0
2 jare Jasco | us |aea | ue [aaa |oam Jars | a7 Jasia | a7 | 8.7 37.3 99 | 20.0 out 13.5
a | aes [ase | 109 | 19.2 | 113 |asi2 | ase | 94 | a9 | ize | a6 | 14.8 11.3 so | 15.1 12.7 13.3
6 | 123 |ss.. | avs |2ae | ais | 22.0 | 4 |asc7 | aa (aria | tae} 22.0 13.2 | a7 | asia 10.5 14.7
a | 160 | 22-0 | 140 | 17.6 | 115 | 20.0 | 107 | 21:9 | 107 | 1:9 | aa2 | izis 15.1 99 fara 10.0 1s
zo | 173 | 30.0 | 179 | 25.2 | 132 | 26s | 143 }ae.3 | a32 | 219 | 120 | 18.2 12,3 | 107 | 13.0 16.4 8.7
12 | 206 |s5a.6 | 176 | 51.3 | 162 j 25.1 | 1s3 | 33.8 | i42 | 35.1 | 143 | 31.9 19.2 | 10 | 7.2 13.7
aa f ize favs | 187 | 49.3 | ies | 46.1 | 171 | aais | isi | 383 | 181 | 2314 20.1 | 123 | 22.9
ie | 21s |ss.2 | 213 | 46.5 | 1s: | 38.2 | 16s |2s.7 | 169 | 38.2 | 160 | 20.0 za. | 122 | 15,7 22.4
is | 21 |4s.2 | 3182 | 42.0 | 1s2 | 34.2 | 195 | 26.4 | 160 | 25.2 | 170 | 25.3 15.3 | 127 | 10.6 a1.
zo | 26a |sa.2 | 225 | 36.4 | a79 | 35.4 | 208 | ss.a | 175 | 18.0 | 159 | 28.0 13.7 | 139 | 16.4
22 | 297 | 33.0 | 273 | ars | 207 | 38.1 | 219 | 36.5 | 195 | 38.0 | 173° | 21.2
za | 301 | 33.4 | 272 | ar.2 | 223 | 27.5 | 22a | 0.2 | ai7 | ai.7 | 201 | 26.5 2er | 26.6
26 | sto 33.5 | 263 | 33.8 | 179 | a9.2 | 179 | 33.3 | 160 | 42.5 | 162 | ar8 229 | 33.5
2s | 348 | 20.3 | s09 | 39.3 | 219 | aio | 256 | 37.1 | 246 | 39.5 | 179 | 2615 2e7 | 4a.
30 | 347 |3s.9 | a15 | 46.3 | 235 | 35.3 | 224 | 54.9 | 232 | 60.0 | 212 | aoe aur | 37.3
sa | = == o--
34 | 387 {33.8 | sa9 | 49.9 | 201 | 62.8 | 256 | 57.8 | 273 | a2.s 64.5 269 | 46.0
36 | 370 |2aa | 367 | 30.7 | 233 | 2a.a | 273 | 40.4 | 225 | 24a 43.7 280 | 61.8
as | 423 | 41.9 | 377 | 36.9 | 308 | 59.0 | 306 | 39.5 | 324 | 28.7 B14
40 | aoa | 40.2 | 361 | 5s.2 | 299 | 80.9 | 299 | 64.9 45.5
a2 | 43s |36.1 | 367 | 52.0 | 315 |as.s | 334 | 38.6 46.6
“4 --- —-
as | 9585 |28.9 | 399 | 37.5 | 407 | a1.2 42.5,
as | a59 | 56.0 | 401 | 29.4 | 368 | 32.3
so | 350 |s3.2 | 388 |sa.s | as | 4s



The number of larvae in the test chambers had a considerable effect
on substrate selection by the larvae. As the number of larvae per
600 ml of seawater was increased above 100, the number of larvae
settling on PVC also increased, while the number selecting the
other test substrates as well as the glass chamber decreased (Fig-
ure 3). At the highest larval density tested (600 per chamber) ,
50% of the larvae that settled were attached to the PVC substrate.

Although we cannot explain why preference for PVC increased with
larval density, the negative phototaxic response of pediveliger
larvae (Bayne, 1965) may explain their preference for PVC. The
PVC substrate was the only one that did not transmit light. The
experiments were conducted under well-illuminated conditions, and
it is possible that the dark color of the PVC substrate as well as
its opaqueness attracted the larvae.

TOXICITY STUDIES
General Considerations

In the following sections, the data obtained on each pesticide are
reported and discussed separately. Except where specifically noted,
all pesticide concentrations are measured concentrations, and the
TLs5q and EC59 values have been estimated on the basis of the mea-
sured levels. In the study of pesticide effects on growth and
metamorphosis and on metamorphosis alone, only the highest test
concentration was monitored throughout the exposure period. The
lower concentrations were estimated on the basis of a 0.5 dilution
factor. Actual measurements of the lower concentrations were per-
formed once or twice during the test and were found to be very simi-
lar to the estimated values.

The 48-hour ECs5q values estimated from the data obtained in the em-
bryo bioassay experiments were estimated by the graphical method
described in Standard Methods (1973). Although the line of best
fit, drawn visually through the data points obtained in some of
the tests, touched virtually all the points, the large number of
larvae counted for each test concentration increased the power of
Chi2 sufficiently during application of the Litchfield-Wilcoxon

30


Each seawater type was tested using eggs from four females from
each collection site. The eggs from different females were
tested separately and in duplicate, The number of normal-
shelled larvae was determined after 48 hours, as described in
the section on time of fertilization and larval shell
development,

Substrate Preference

The following substrates were evaluated: frosted glass; smooth
rigid, opaque polyvinyl chloride (PVC) sheet; smooth, clear
acrylic plastic sheet; and white silk thread, Each substrate
was constructed in the shape of an "L,”
imately the same dimensions. The horizontal and vertical
sections of the rigid substrates were attached to each other
with Dow-Corning silicone adhesive. Excess adhesive was
removed from the joints, The silk thread was wound vertically
over a frame made of a 1/8-inch-diameter glass rod. Each frame
contained the same number of strands. The approximate areas
exposed to the larvae were 4.6, 3.9, 4.5, and 4.8 cm", respec-
tively, for the acrylic sheet, PVC sheet, frosted glass plate,
and silk thread substrates. The surface area of the silk
thread was obtained by measuring the diameter of a length of
wetted thread at ten different points and averaging the
diameters. Fine filaments extending from the main body of the
thread were not included,

each leg having approx-

Mussels used in the study were obtained from Tomales Bay in
September 1973. The eggs from several females were incubated
in separate groups according to female, and two samples from
each group were examined after 48 hours. All groups in which
less than 85% of the larvae in both samples had straight-hinged
shells were discarded. The remaining groups were reared for at
least 30 more days in 1,25-gal. Pyrex” animal jars, each con-
taining 3 liters of natural seawater and 3000 larvae. During
this period, the water was renewed every other day. M. lutheri
was provided daily throughout the experiment at a rate of
20,000 cells/ml. Daily algae cell counts indicated that a
higher feeding rate was not necessary

14


Our study indicates that the bay mussel has considerable tolerance
to methoxychlor. Survival and attachment by byssus threads in
small adult mussels (3 to 4 cm) were not affected by exposure for
96 hours to methoxychlor concentrations as high as 0.092 mg/liter,
which is almost twice the estimated solubility of the pesticide

in 20°C seawater. Nor did the pesticide have an adverse effect

on the percentage of normal larvae developing from eggs fertilized
in water containing as much as 0.075 mg/liter. Exposure of 48-hour-
old larvae to concentrations as high as 0.062 mg/liter for 10 days
did not reduce growth. Only after relatively prolonged exposure

to methoxychlor were adverse effects observed. Exposure to 0.062 mg/
liter of the pesticide reduced growth by 11.6% after 20 days. The
same concentration also reduced by 88% the number of larvae devel-
oping into juvenile mussels during exposure for 38 days.

The chemical stability of methoxychlor and, thus, its relatively
high environmental persistence suggest that, in spite of its rel-
atively low acute toxicity to the bay mussel, the pesticide cannot
be considered nonhazardous to the mussel or other bivalve molluscs.

2,4-D

The acid form of 2,4-D was used in this study. Several other forms
are commercially available, including the amine salt, the butoxy-
ethanol ester, and the acetamide salt. The solubility of the acid
form in distilled water is 600 to 700 mg/liter (Herbicide Handbook,
1970). Our estimate of solubility in 20°C seawater was about

1100 mg/liter.

Exposure of small adult mussels (3 to 4 em) to measured concentra-
tions of 331 and 334 mg/liter of 2,4-D for 96 hours resulted in
100% mortality (Table 18). Mortality may have resulted from low
pH, which averaged 6.5 at 331 mg/liter and 6.4 at 334 mg/liter
(Table 19), The 96-hour TLsg> estimated from data obtained from
Test 12, was 259 mg/liter, with 95% confidence limits of 232 to
289 mg/liter. Pesticide determinations were not performed on the
test solutions used in Test 7, The 96-hour TL,,, based on the
nominal concentrations, was 290 mg/liter. [The 95% confidence
limits could not be calculated using the method of Litchfield and
Wilcoxon (1949). ] The number of mussels that attached themselves

58


10

1

12

13

14

15

16

17

18

19

20

Mean Shell Length of Mussel Larvae Exposed
to Treflan for 10 and 20 Days .. +4 + ee ee eee

Number, Age, and Size of Juvenile Mussels
Developing in Larval Cultures Exposed at 48 Hours
to Various Concentrations of Treflan . 1... + 6 ©

Effect of Treflan on Mussel Larvae Metamorphosis
after a 40-Day Exposure Initiated 30 Days after
Fertilization of the Eggs . 2. 6 +e ee ee eevee

Survival and Attachment Data for Adult Mussels
Exposed to Methoxychlor for 96 Hours . « «6 «es «

Percentage of Normal Larvae Developing in 48 Hours
from Mussel Eggs Fertilized in Various Concentra-
tions of Methoxychlor . «2. eee ee eee eevee

Mean Shell Length of Mussel Larvae Exposed to
Methoxychlor for 10 and 20 Days . 1... eee eee
Number, Age and Size of Juvenile Mussels Developing
in Larval Cultures Exposed at 48 Hours to Various
Concentrations of Methoxychlor ......e+.eeee
Effect of Methoxychlor on Mussel Larvae Metamorphosis
after a 41-Day Exposure Initiated 29 Days after
Fertilization of the Eggs ... 2... 2.2 ee eee
Survival and Attachment Data for Adult Mussels
Exposed to 2,4-D (Acid) for 96 Hours... .....4.
Water Quality Data for 96-Hour Adult Mussel

Survival Test No. 12 on 2,4-D ...... 2. ee .
Percentage of Normal Larvae Developing in 48 Hours

from Mussel Eggs Fertilized in Various Concentra-
tions of 2,4-D......

vi

Page

44

45

47

51

52

53

54

55

59

60

61


06

Table 3,

WATER QUALITY DATA FROM GROWTH

AND METAMORPHOSIS EXPERIMENTS

Dissolved Oxygen

Tv t °c Salinity, °% HH
Pesticide emperature y mg/liter alinity, "Yoo P
Mean sD Analyses | Mean sD Analyses | Mean sD Analyses | Mean sD Analyses
Sevin 20.8] 1.09 774 6,80 | 0.41 533 Not recorded 8.04] 0.11 502
Treflan 19.0 | 0.81 797 6.57 | 0.80 599 25.4 | 0.87 239 8.03 | 0.08 569
Methoxychlor | 19.8 | 1.04 873 6.30 | 0.98 645 25.3 | 1.00 321 7.98) 0.97 644
2,4-D 20.4] 0.93 670 6.87 | 0.40 445 Not recorded 7.89 | 0.29 419
Malathion 20.0] 0.99 627 6.80 | 0.13 484 25.3] 1.11 132 8,02] 0.10 477
Table 4. WATER QUALITY DATA FROM METAMORPHOSIS EXPERIMENTS
Dissolved Oxygen
Tv. t °c Salinity, % HH
Pesticide emperature, mg/liter alinity, Yo, P
Mean sD Analyses | Mean sD Analyses } Mean sD Analyses | Mean sD Analyses
Sevin 19.7] 1.76 260 7,00 | 0.51 180 25.3 | 0.34 175 8.04] 0.12 175
Treflan 19.6] 1.29 282 6.40 | 0.91 195 25.5 | 0.59 180 8.01] 0.11 189
Methoxychlor | 19.6] 1.12 240 6.60 | 0,94 181 25.2 10.33 181 8.00] 0.12 181
2,4-D 19.6] 1.29 240 6.70 | 0.55 160 25.4] 0.50 153 8.06] 0.15 160
Malathion 19.7] 1.27 240 6.70 | 0.47 160 25.4] 0.51 153 8.06] 0.10 155



Table 5. MEAN SHELL LENGTH OF MUSSEL LARVAE
EXPOSED TO SEVIN FOR 10 AND 20 DAYS

Mean shell length, pu

Test No. of No. of
concen- larvae . Day 10 Day 20

. tests/
tration, |measured/ . tle ile ile ile 7
mg/liter test experiment| Experi xperi-|Experi-|Experi-

ment 1] ment 2| ment 1| ment 2

0 20 2 170 174 309 244
0.33 20 2 162 138 218 194
0.65 20 2 128 126 204 165
1.30 20 2 122 109 164 130
2.61 20 2 124 99 -- --

effect than embryo shell development. After 10 days of exposure,
0.33 mg/liter of Sevin reduced larval size by 20.7% in one of the
tests (Experiment 2). In the same experiment, a 29.5% reduction
in growth was observed at the same concentration after 20 days of
exposure. The lowest concentration that inhibited growth in both
experiments was 0.65 mg/liter. As much as a 27.5% reduction in
size was observed after 10 days of exposure, and as much as a
32.6% reduction in size was observed after 20 days of exposure.

At the highest concentration of 2.61 mg/liter, the duplicate jars
used in both tests did not contain enough larvae to warrant their
continued use after 14 days. By the tenth day, the larvae exposed
to this concentration of Sevin were about 46% smaller than controls.

The effect of Sevin on metamorphosis to the juvenile stage was
studied in two ways. First, the effect of continuous exposure to
Sevin from the 48-hour stage to larval metamorphosis was investi-
gated by continuing the growth experiments beyond 30 days. Second,
the effects were studied by delaying exposure until the larvae were
29 days old.

In the first set of experiments, larvae exposed to a measured Sevin

concentration of 1.3 mg/liter did not survive longer than 39 days.
Larvae exposed to the highest test level of 2.61 mg/liter survived

35


Appendix C

97


through procedural error, they were not included in the analysis
of the data. Percentages of metamorphosing, nonmetamorphosing,
and dead larvae presented in Table 7 are based on the number of
experimental larvae, not on the initial number of larvae. Only
the highest concentration was monitored throughout the test. The
mean and standard deviation of the highest test concentration,
based on 31 determinations, were 2.9 + 0.76 mg/liter.

Sevin-exposed larvae as a group did not undergo metamorphosis as
extensively as the controls. An average of 93.3% of the control
larvae developed into juveniles, whereas the average for all groups
exposed to Sevin was 83.3%. The percentages of metamorphosing lar-
vae decreased with increasing levels of Sevin. The mean percentages
of metamorphosed larvae found in the test chambers containing 0.36,
0.72, 1.45, and 2.9 mg/liter of Sevin were 93.3, 87.8, 85.2, 82.4,
and 77.7, respectively.

Mortality also increased with increasing levels of Sevin. An aver-
age of 6.1% of the controls died during the course of the 40-day
exposure period. A smaller percentage died in the chambers with
0.36 mg/liter of Sevin; however, at greater concentrations, a
higher percentage of the larvae died. Mortality among larvae ex-
posed to the highest test concentration of 2.9 mg/liter amounted
to an average of 22.3% of the experimental population.

In general, the pesticide-exposed larvae metamorphosed at a faster
rate than the control larvae. This enhanced rate was particularly
evident in larvae exposed to 2.9 mg/liter of Sevin (Figure 4). Of
those that metamorphosed, 50% had metamorphosed at this concentra-
tion in about 16.5 days, whereas the controls required an average
of 23.5 days.

The average size of the larvae at metamorphosis also was affected
by exposure to Sevin. The mean size of the control larvae was
457 wu. Larvae exposed to 0.36 mg/liter of Sevin were about 10 uy
larger; however, at concentrations above 0.36 mg/liter, the larvae
decreased progressively in size as the pesticide concentration in-
creased. Larvae exposed to 0.72, 1.45, and 2.9 mg/liter were 6.2,
14.1, and 24.6% smaller than controls.

38