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Growth and reproduction of the earthworm Eisenia fetida exposed to sublethal concentrations of organic chemicals
Sod hl. &ochrm. Vol. 22. No. 2. pp. 175-179. 1990 Pnnted rn Great Britain. All nghts resa-wd
Copyright
0038-0717.90 53.00 + 0.00 C 1990 Rrgamon Press plc
GROWTH AND REPRODUCTION OF THE EARTHWORM ElSENZA FETlDA EXPOSED TO SUBLETHAL CONCENTRATIONS OF ORGANIC CHEMICALS EDWARD Research and Development
F. NEUHAUSER
Department. Niagara Mohawk Power Corporation. Syracuse, NY 13202. U.S.A. CLARESCE
U.S. Environmental
Protection
Agency.
300 Erie Boulevard West.
A. CALLAHAS
100 SW.
35th Street. Corvallis.
OR 97330.
U.S.A.
(Accepred 25 Augusr 1989) Summary-The determination of a short-term LC, toxicity value for a chemical is a useful indicator of the potential biological etfoct of the chemical if released into the environment. However, it is possible that adverse effects of the chemical may occur at sublethal concentrations far below LC, values. Using the earthworm Eiseniu Jvit/u (Savigny) as an indicator organism for soil ecosystems, concentrations of IO organic chemicals were evaluated for sublethal effects on earthworm growth and reproduction. Following short-term exposure to sublethal concentrations of carbaryl and dieldrin. E. @it/u was allowed to resume growth and reproduction to determine whether such ctfects were permanent. The effect on earthworm populations as a representative soil organism could be a sentinel for the effects of more destructive soil processes.
Man’s activities have rcsultcd in increasing conccn[rations of potentially-toxic materials being dclibcratcly or unintentionally distributed in soil ccosystcms. Soil can no longer bc considcrcd a waste depository without consideration of cffccts on soil ecosystems. Earthworms. one of the largest and most easily quantilicd components of soil biota, play a major role in maintaining soil fertility and structure (Edwards and Lofty, 1977). The clfcct on earthworms as a rcprcscntativc soil organism could bc a scntincl for the indication of more dcstructivc soil proccsscs. A number of laboratories have dcvclopcd earthworm toxicity tests (Davis, 1971; Stcncrsen. 1979; Lofs-Holmin, 1980, 1983; Goats and Edwards, 1982; Karnak and Hamclink, 1987). Most of thcsc tests huvc involved measuring the LC5, of various chemicals, mostly pcsticidcs. Lofs-Holmin evaluated the sublethal clTccts of pesticides on cllrthworm growth 1980) and reproduction (Lofs(Lofs-Holmin, Holmin. 191(Z). The LCs of a given chemical is often not a useful indicator of the potential ecological consequcnccs of the chcmicnl, because reproduction may be inhibited or halted at chemical concentrations far below a given LC5, (Neuhauser er al.. 1984). Other sublethal etfccts on earthworms include decreased nervous system functioning (Drcwes er al.. 1987; Callahan er cl/., 1985). To gain an insight into the sublethal effects of organic chemicals. the etTects of priority pollutants (US EPA. 1979) on earthworm growth and reproduction wcrc cxamincd. To further evaluate the persistcncc of sublethal effects of two chemicals. carbaryl and dicldrin. earthworms cxposcd to each chemical were subsequently allowed to grow and reproduce without further exposure to the chemicals. CBB
:2:-u
The test components included basic organic media (horse manure). sand. the test chemical and dcionizcd water. Glass Petri dishes. 100 by 20 mm were used as expcrimcntal contnincrs. industrial quartz sand (dominant fine sand with >50% of particles sized 0.05-0.2 mm) were used to provide drainage. Dry sand (30 g) was placed in the bottom of the dish. The sand was brought to 20 + 2% moisture by adding the proper amount of water to the dish. A mixture of the horse manure and the test substrate (20 g wet wt) was placed on top of the sand. The controls contained only horse manure, sand and deionized water. The manure contained a moisture content of 75 f 5% after the test chemical was added. If the test chemical added to the manure was not water-soluble, an organic solvent, such as acetone, was added in the same quantity to the control manure. Sequential weighings were made to insure that all of the organic solvent was evaporated before the worms were added to the manure. None of the chosen test chemicals were volatile. When the moisture content was <75% after the test chemical was added. deionized water was added to the manure to bring the moisture content up to the prescribed level. Care was exercised to insure that the test chemical was evenly distributed and mixed into the manure. Two young worms, < IO mg and < I week old, wcrc added to each of five Petri dishes for each chemical concentration tcstcd. The Petri dishes were kept at a tcmperaturc of 25 C for the duration of the test. This temperature was found to give maximum growth and reproduction for Eismia fifidu (Savigny) (Kaplan er al., 1980). Under these circumstances I75
I. The
mean
6.2’
7.1’
‘*Indicates
with
a common
significant
0.47’
induted.
P -z 0.05.
Ob
O.lP
P < 0.05.
4.P
0.42’
300
8.7’
0.53’
3400
1.P;
0.43’
1200
750 0.47’ 3.e
2.@
0.46
I800
7.1’
0.38’
23.600
(r
0.P
IO0
0’
7.34
0.36’
2.i
0.16’
Death
JM)
6.9
0.53’
3900
IMO
4.3’
0.43’
1000
3.7b
0.4-l’
XMiJ
,-
471:
0 3P
I50
7.ff
0 57’
8.8’
0.47’
400
69.300
hg
O.ld
I50
(mg
’)
’ 8 weeks
2.9b
0.35’
6.Y
0.54’
4900
0.3”
0.42’
IX00
Death
I500
1.2b
0.3V
2200
70.8:
0.24b
2Otl
6.3’
0.53’
80.800
8.g’
0.46’
450
Ob
9.5’
0.56’
5900
Death
zoo0
Death
24430
’ Death
94Jooo’
0. 14b
250
Lkath
7.4’
0.44’
SO0
92,300
(mg
:k14d
250
the concentration
O.?tY
200
’ for
.Y = IO for all points.
concentration 100
350 0.37’ 5.X’ 57.800 0.47’ 5.3’
Chcmrcal
worm
(.V = IO)
production
Indicated
cocoon
dificrcnt,
9.6’ 200 0.46’ 3.4b
0.48’
2900
3Ak
0.w
900
6.3’
0.51’
So-
I.‘b
o..3!P
1600
a.?
0.3Y
Il.!JcQ
3.P
0.41’
50
8.3’
0.31
5.5’
O.J?
3c0-
I.+
0.3lb
50
total
46.300
and
arc not signifiuntly
5.wb
0.5w
IO0
10.3’
‘400
4.P
0.47’
600
F value at degrees of’ freedom
m the same row
6.Y
‘Means
0.43’
Weight
0
Cocoons
Tii~~hlorophm0I
8.4’
Cocoons
2.4.6
0.w
0
Weight
Pltrfrul
7.4’
0
0.4Y
250
5.P
0.5-P
IUKI
9.3’
0.34
5900
4.6”
0.48’
25
6.4’
0-w
34,800
5.7’
O.-U
250
3.1b
0.35b
25
(g uorn-‘)
7.1’ 0 0.45’ 8.4’ 0 0.4 I’ 7.7’ 0 0.51’ 8.7’ 0 0.57’
0.51’
7.4’ 0
0.41’
0 0.47” 7.7’ 0
of E. /u/irfo
O.JY
ltttcr
weights
Weight
final
Ccxoons
4Nilrvphmol
COWMlS
Wclght
Nucwtw
Cocoons
Weight
N-,YirrosclJiphr,bmmp
Croons
Weight
Dinwrh~~lphrhal~r~
Cocoons
Weight
Didtlrin
Croons
Weight
I.2 Dirhlorc~propo~re
Ccuxms
Weight
Chlorwtw~nriclr
Cocoons
Weight
Curhur,~l
Table
500
1000
Lkath
6900
Death
50
Ckath
= l6.72**
= I .08
= 2.02 = 2.75
= 14.85.
= 2.58 = 4.43+
= 2.84
= I.41
F:, = 3.93’
F;,
b;.
fl:,=lOl
F‘L = I.01 F;,=3.13*
by* = 4.54.
c:,=2.14
F:, F;,
F;, = 7.34’
F;, = 0.49
I$
Pt. = 14.15*
I;;,
F;,
b;, = 2.45
G,
F<, = 28 09.
c,
chemicals
F v;ilw
‘) of organic
Duth
kg
Sublethal chemical effects on earthworms Table
2. The
sigmficant
IOWCSI concentration
ditTerencc
of test chemicals
in growth
or
reproduction
that
from
showed
a
the comrol
E. Jkrda Lowest (mg
concemratron kg-‘)
10 show
dltTerencc Chemical Carbaryl
control
25 25
I50
Tnchlorophcnol
for:
Reproduction
25
Dlcldnn 2.46
from
Growth
of chcmlcal slgmficant
Death
at .UXI
200
Chloroaceramide
Death
a1 loo0
NE’
Fluorenc
Death
at I500
750
J-Nltrophcnol
Death
at Zoo0
900 1400
N-Nltrorodiphcnylaminc
Death
at 2400
Phenol
Death
a~ 6900
NE
I.2 Dlchloropropane
Death
at 9:.300
NE
ar 94.400
70.800
Dimethylphthalale NE’--no
Death
ctTect observed
at the
hlghesr
non-lethal
conccntra1wn.
growth and reproduction were not limited by the amount of manure (Neuhauser et al., 1980). The complete exposure was for 8 weeks although worms were also evaluated for growth and reproduction at 4 and 6 weeks. At these times, cocoons were removed and the worms were re-fed new manure. to which the appropriate concentration of test substance was added. The range of concentrations tested for each chemical included one concentration that had no sublethal etTcct, at lcast four concentrations that had a sublethal ctfcct and one concentration that had a lethal effect. Sclcction of these concentrations hclpcd to optimize the possibility of separating threshold concentrations for impairment of growth and reproduction. The final earthworm weight and mean cocoon production per worm per 8 weeks for each chemical concentration were tested for signiticant diffcrcnces using one-way analysis of variance. When a significant F value was obtained, Duncan’s new multiple range test (Steel and Torric. 1960) was used to dctcrminc ditfcrsnccs.
To evaluate the possible residual effects of persistent chemicals, the worms exposed to carbaryl and dicldrin were tested for an additional 8 weeks in manure without added chemicals. The worms were evaluated for growth and reproduction at every 2 weeks. The old manure and cocoons were removed and the worms were re-fed fresh untreated manure at each observation opportunity. The final earthworm weights and mean cocoon production per worm for weeks 8-16 were tested for significant ditferences using one-way analysis of variance When a significant F value was obtained. Duncan’s new multiple range test (Steel and Torrie, 1960) was used to dctcrmine ditfercnccs.
RFSCLTS
ASI)
DISCUSSION
The final weights and total cocoon production achieved by E. fitida cxposcd to the various concentrations of test chemicals show that only carbaryl and dicldrin caused statistically-significant reductions in both growth and cocoon production at sublethal chemical concentrations, whereas five other chemicals
177
had a significant effect on cocoon production alone (Table I). The highest sublethal concentrations of the other chemicals tested showed no significant weight reductions when compared to controls, although all tests were designed to evaluate both sublethal as well as lethal effects during the exposure period. Dimethylphthalate, N-nitrosodiphenylamine, fluorene, 4-nitrophenol and 2.4.6 trichlorophenol significantly reduced reproduction at concentrations that did not kill the worms. Chloroacetamide, 1.2 dichloropropane, and phenol caused no reduction in cocoon production or growth even at the highest concentrations tested. Carbaryl and dieldrin have been established as neurotoxins to earthworms (Drews and Vining. 1984). These two chemicals affected earthworm growth and reproduction at the lowest concentrations of the ten chemicals tested (Table 2). Venter and Reinecke (1988) demonstrated that exposing E.fitida to dieldrin concentrations of 30 mg kg-’ decreased cocoon production when compared to controls and dieldrin concentrations of 50 mg kg-’ decreased the rate of clitcllate formation.
To evaluate the possibility of rccovcry from sublethal effects after exposure to carbaryl and dieldrin, the horse-manure test-chemical substrate was replaced with horse manure only and earthworm weight and cocoon production wcrc monitored for an additional 8 weeks. Earthworm growth in the prescnce and abscncc of carbaryl (Fig. I) and dieldrin (Fig. 2) show that the worms exposed to sublethal concentrations of each chemical wcrc able to resume normal growth rates when the chemical was removed from their environment. Growth rates for both carbaryland dieldrincxposcd worms generally dccreascd with increasing concentrations of each chemical (Table 3). Upon removal of the chemical from the worms’ environment, those exposed to the highest concentrations of chemical showed the greatest recovery growth rates. At the end of the I6 weeks, the data from Figs I and 2 were analyzed and there was no significant difference in the final weight of the worms for any of the concentrations of both chemicals tcstcd, including controls. Cocoon production was severely hindered by the presence of carbaryl or dieldrin (Table 3). Removal of either chemical permitted cocoon production to begin after 8 weeks for the higher concentrations. At 25 mg carbaryl kg-’ and at 25.50 and 100 mg dieldrin kg-‘. a similar number of cocoons were produced as in the controls. It is apparent that the worms were able to start reproduction after the inhibiting chemical was removed. These results strongly suggest that even though the population density may not be immediately affected (i.e. increased mortality) exposure to sublethal concentrations results in reproductive changes that reduce the succeeding population density. Carbaryl or dieldrin at 50 mg kg-’ reduced the cocoon production by >SO% which means that the populations would be significantly reduced in 2-3 generations (Table I).
EDWMUI F. NEUHKSER and CLNIESCE A. CALLAHA.~
178
No carboryl
Carbaryl
: Too
.
tea
ab,.k.”
:
l
200
m;.k;-l
l
1SOng.ke-' loo ng.lrg-’ SO mg.kg-1
:. .
l
000
, . I
100
22
i
mg.kg-’
:
control
: ;-
,o i
E t f
400
300
zoo
t
Y
100
Fig. I. The growth of E.,firicltr in the presence and ahscncc of various concentrations
The sublethal effects of pcsticidcs on earthworms can bc very disruptive of important life history cvcnts such as growth and reproduction (this rcsearch) ;md nervous system functioning (Drcwcs and Vining. 1084) which could result in ckts on important soil proccsscs. Although rccovcry of reproduction and nervous system functioning wcrc observed after dicldrin exposure (Drcwcs and Vining. IOX4). more pcrmancnt damugc hns rcsultcd from cxposurc to bcnomyl (Drcwcs et rrl.. 1987),
N = IO.
suggesting that recovery may not occur in some Glscs. Our rcscarch suggests that more consideration should bc given to evaluating sublethal cfkcts under Iicld conditions to dctcrminc ecological implications of toxic materials. 11 is obvious that LCIo cstimatcs should bc considcrcd a first approximation of toxic cffcct and that more rcfincd tests (e.g. growth and reproduction) should bc used to fully evaluate toxic substances.
No dleldrln
Dleldrin 100
of carbaryl.
280
me.
200
mg.k,,”
kg -1 i
000
100 :
z-
400
0
5 1 E
300
: I f:
200
Y 100
0
.. 2
4
4
Fig. 2. The growth of E.,/c/itkr
10
8 Wm..
12
14
14
rooke
in the presence and absence of various concentrations
of dieldrin.
N = IO
Sublethal
chemical effects on earthworms Acknowledgemenr-This
179 work
was supported
operative Agreement from the U.S. tection Agency to Cornell University.
by a CoPro-
Environmental
REFERESCES Callahan C. A.. Russell L. K. and Peterson S. A. (1985) A comparison of three earthworm bioassay procedures for the assessment of environmental samples containing hazardous wastes. Biology and Fertility of Soils 19.221-233. Davis B. N. K. (1971) Laboratory studies on the uptake of dieldrin and DDT by earthworms. Soil Biolog! & Biochemisrr? 3. ‘2 I-233. Drewes C. D. and Vinmg E. P. (198-t) In riro neurotoxic effects of dieldrin on giant fibers and escape reflex function in the earthworm Eiseniafoefidu. Pesticide Biochemisrrx and Phwolog~ 22. 93-104. Drewes C. D.. Zoran M. J. and Callahan C. A. (1987) Sublethal neurotoxic effects of the fungicide benomyl on earthwrorms (Eisenia fitida). Pesticide Science 19, 197-208. Edwards C. A. and Lofty J. R. (1977) The Biology of Eurrhworms. 2nd edn. Chapman and Hall, London. Goats G. and Edwards C. A. (1982) Testing the toxicity of industrial chemicals to earthworms. Rolhamsfed Report 1982. IOLIO5. Kaplan D. L.. Hartenstein R., Neuhauser E. F. and Malecki M. R. (1980) Physiochemical requircmcnts in the environmcnt of the earthworm Eisenia jtirida. Soil Biokogy & Biochenris/ry 12. 3J7- 352. Karnak R. E. and Hamclink J. L. (1982) A standardized method for determining the acute toxicity of chemicals to cart hworms. Ecoroxicoh~g~ und Enrironmenrul Safi/y 6, 216 -222. Lofs-flolmin A. (19X0) Measuring growth ofearthworms as a method of testing suhlcthal toxicity of pesticides. Swedish Journul of Agriculrurul Reseurch IO, 25 -33. Lofs-tlolmin A. (1982) Measuring cocoon production of the earthworm A//o/ohop/roru cuIi~gino.w (Sav.) as a method of testing sublethal toxicity of pssticidcs. Swedish JOUUICJ/04’ A,qricw/ruru/ Reseurch 12. I I7 I 19. Ncuhauser E. F.. Kaplan D. L.. Malecki M. R. and tlartcnstcin R. (19X0) Materials supporting the weight gain by the sarthworm Ei.wniu/c~ridu in waste conversion systems. A~ricdrurol U’crsrcs 2. 43 60. Neuhauser E. F.. Malecki M. R. and Loehr R. C. (1984) Growth and reproduction of the earthworm after exposure to sublethal concentrations of metals. Pedohiulogiu 27. 89 -97. Steel R. G. and Torrie J. H. (1960) Principles und Procedures of Srurisrics. McGraw-Hill. New York. Stenersen J. (1979) Action of pesticides on earthworms. Pdrt I: The toxicity of cholinestemse-inhibiting inscctitides to earthworms as evaluated by laboratory tests. Prsricide Science IO, 66-74. U.S. Envirorimental Protection Agency (1979) Waterrelated environmental hte of I29 priority pollutants. EPA -440 14-79-029. U’ushingron. D.C. Venter J. M. and Rcinccke A. J. (1988) Sublethal ccotoxicological effects of dleldrin on the earthworm Eiseniajiwfidu (Oligochaeta). In Eurrhwwms in W’usfe und Encirunmenful Alunugemen~ (C. A. Edwards and E. F. Neuhauser. Eds). pp. 337.353. SPB Academic Publishing. The Hague.
Copyright
0038-0717.90 53.00 + 0.00 C 1990 Rrgamon Press plc
GROWTH AND REPRODUCTION OF THE EARTHWORM ElSENZA FETlDA EXPOSED TO SUBLETHAL CONCENTRATIONS OF ORGANIC CHEMICALS EDWARD Research and Development
F. NEUHAUSER
Department. Niagara Mohawk Power Corporation. Syracuse, NY 13202. U.S.A. CLARESCE
U.S. Environmental
Protection
Agency.
300 Erie Boulevard West.
A. CALLAHAS
100 SW.
35th Street. Corvallis.
OR 97330.
U.S.A.
(Accepred 25 Augusr 1989) Summary-The determination of a short-term LC, toxicity value for a chemical is a useful indicator of the potential biological etfoct of the chemical if released into the environment. However, it is possible that adverse effects of the chemical may occur at sublethal concentrations far below LC, values. Using the earthworm Eiseniu Jvit/u (Savigny) as an indicator organism for soil ecosystems, concentrations of IO organic chemicals were evaluated for sublethal effects on earthworm growth and reproduction. Following short-term exposure to sublethal concentrations of carbaryl and dieldrin. E. @it/u was allowed to resume growth and reproduction to determine whether such ctfects were permanent. The effect on earthworm populations as a representative soil organism could be a sentinel for the effects of more destructive soil processes.
Man’s activities have rcsultcd in increasing conccn[rations of potentially-toxic materials being dclibcratcly or unintentionally distributed in soil ccosystcms. Soil can no longer bc considcrcd a waste depository without consideration of cffccts on soil ecosystems. Earthworms. one of the largest and most easily quantilicd components of soil biota, play a major role in maintaining soil fertility and structure (Edwards and Lofty, 1977). The clfcct on earthworms as a rcprcscntativc soil organism could bc a scntincl for the indication of more dcstructivc soil proccsscs. A number of laboratories have dcvclopcd earthworm toxicity tests (Davis, 1971; Stcncrsen. 1979; Lofs-Holmin, 1980, 1983; Goats and Edwards, 1982; Karnak and Hamclink, 1987). Most of thcsc tests huvc involved measuring the LC5, of various chemicals, mostly pcsticidcs. Lofs-Holmin evaluated the sublethal clTccts of pesticides on cllrthworm growth 1980) and reproduction (Lofs(Lofs-Holmin, Holmin. 191(Z). The LCs of a given chemical is often not a useful indicator of the potential ecological consequcnccs of the chcmicnl, because reproduction may be inhibited or halted at chemical concentrations far below a given LC5, (Neuhauser er al.. 1984). Other sublethal etfccts on earthworms include decreased nervous system functioning (Drcwes er al.. 1987; Callahan er cl/., 1985). To gain an insight into the sublethal effects of organic chemicals. the etTects of priority pollutants (US EPA. 1979) on earthworm growth and reproduction wcrc cxamincd. To further evaluate the persistcncc of sublethal effects of two chemicals. carbaryl and dicldrin. earthworms cxposcd to each chemical were subsequently allowed to grow and reproduce without further exposure to the chemicals. CBB
:2:-u
The test components included basic organic media (horse manure). sand. the test chemical and dcionizcd water. Glass Petri dishes. 100 by 20 mm were used as expcrimcntal contnincrs. industrial quartz sand (dominant fine sand with >50% of particles sized 0.05-0.2 mm) were used to provide drainage. Dry sand (30 g) was placed in the bottom of the dish. The sand was brought to 20 + 2% moisture by adding the proper amount of water to the dish. A mixture of the horse manure and the test substrate (20 g wet wt) was placed on top of the sand. The controls contained only horse manure, sand and deionized water. The manure contained a moisture content of 75 f 5% after the test chemical was added. If the test chemical added to the manure was not water-soluble, an organic solvent, such as acetone, was added in the same quantity to the control manure. Sequential weighings were made to insure that all of the organic solvent was evaporated before the worms were added to the manure. None of the chosen test chemicals were volatile. When the moisture content was <75% after the test chemical was added. deionized water was added to the manure to bring the moisture content up to the prescribed level. Care was exercised to insure that the test chemical was evenly distributed and mixed into the manure. Two young worms, < IO mg and < I week old, wcrc added to each of five Petri dishes for each chemical concentration tcstcd. The Petri dishes were kept at a tcmperaturc of 25 C for the duration of the test. This temperature was found to give maximum growth and reproduction for Eismia fifidu (Savigny) (Kaplan er al., 1980). Under these circumstances I75
I. The
mean
6.2’
7.1’
‘*Indicates
with
a common
significant
0.47’
induted.
P -z 0.05.
Ob
O.lP
P < 0.05.
4.P
0.42’
300
8.7’
0.53’
3400
1.P;
0.43’
1200
750 0.47’ 3.e
2.@
0.46
I800
7.1’
0.38’
23.600
(r
0.P
IO0
0’
7.34
0.36’
2.i
0.16’
Death
JM)
6.9
0.53’
3900
IMO
4.3’
0.43’
1000
3.7b
0.4-l’
XMiJ
,-
471:
0 3P
I50
7.ff
0 57’
8.8’
0.47’
400
69.300
hg
O.ld
I50
(mg
’)
’ 8 weeks
2.9b
0.35’
6.Y
0.54’
4900
0.3”
0.42’
IX00
Death
I500
1.2b
0.3V
2200
70.8:
0.24b
2Otl
6.3’
0.53’
80.800
8.g’
0.46’
450
Ob
9.5’
0.56’
5900
Death
zoo0
Death
24430
’ Death
94Jooo’
0. 14b
250
Lkath
7.4’
0.44’
SO0
92,300
(mg
:k14d
250
the concentration
O.?tY
200
’ for
.Y = IO for all points.
concentration 100
350 0.37’ 5.X’ 57.800 0.47’ 5.3’
Chcmrcal
worm
(.V = IO)
production
Indicated
cocoon
dificrcnt,
9.6’ 200 0.46’ 3.4b
0.48’
2900
3Ak
0.w
900
6.3’
0.51’
So-
I.‘b
o..3!P
1600
a.?
0.3Y
Il.!JcQ
3.P
0.41’
50
8.3’
0.31
5.5’
O.J?
3c0-
I.+
0.3lb
50
total
46.300
and
arc not signifiuntly
5.wb
0.5w
IO0
10.3’
‘400
4.P
0.47’
600
F value at degrees of’ freedom
m the same row
6.Y
‘Means
0.43’
Weight
0
Cocoons
Tii~~hlorophm0I
8.4’
Cocoons
2.4.6
0.w
0
Weight
Pltrfrul
7.4’
0
0.4Y
250
5.P
0.5-P
IUKI
9.3’
0.34
5900
4.6”
0.48’
25
6.4’
0-w
34,800
5.7’
O.-U
250
3.1b
0.35b
25
(g uorn-‘)
7.1’ 0 0.45’ 8.4’ 0 0.4 I’ 7.7’ 0 0.51’ 8.7’ 0 0.57’
0.51’
7.4’ 0
0.41’
0 0.47” 7.7’ 0
of E. /u/irfo
O.JY
ltttcr
weights
Weight
final
Ccxoons
4Nilrvphmol
COWMlS
Wclght
Nucwtw
Cocoons
Weight
N-,YirrosclJiphr,bmmp
Croons
Weight
Dinwrh~~lphrhal~r~
Cocoons
Weight
Didtlrin
Croons
Weight
I.2 Dirhlorc~propo~re
Ccuxms
Weight
Chlorwtw~nriclr
Cocoons
Weight
Curhur,~l
Table
500
1000
Lkath
6900
Death
50
Ckath
= l6.72**
= I .08
= 2.02 = 2.75
= 14.85.
= 2.58 = 4.43+
= 2.84
= I.41
F:, = 3.93’
F;,
b;.
fl:,=lOl
F‘L = I.01 F;,=3.13*
by* = 4.54.
c:,=2.14
F:, F;,
F;, = 7.34’
F;, = 0.49
I$
Pt. = 14.15*
I;;,
F;,
b;, = 2.45
G,
F<, = 28 09.
c,
chemicals
F v;ilw
‘) of organic
Duth
kg
Sublethal chemical effects on earthworms Table
2. The
sigmficant
IOWCSI concentration
ditTerencc
of test chemicals
in growth
or
reproduction
that
from
showed
a
the comrol
E. Jkrda Lowest (mg
concemratron kg-‘)
10 show
dltTerencc Chemical Carbaryl
control
25 25
I50
Tnchlorophcnol
for:
Reproduction
25
Dlcldnn 2.46
from
Growth
of chcmlcal slgmficant
Death
at .UXI
200
Chloroaceramide
Death
a1 loo0
NE’
Fluorenc
Death
at I500
750
J-Nltrophcnol
Death
at Zoo0
900 1400
N-Nltrorodiphcnylaminc
Death
at 2400
Phenol
Death
a~ 6900
NE
I.2 Dlchloropropane
Death
at 9:.300
NE
ar 94.400
70.800
Dimethylphthalale NE’--no
Death
ctTect observed
at the
hlghesr
non-lethal
conccntra1wn.
growth and reproduction were not limited by the amount of manure (Neuhauser et al., 1980). The complete exposure was for 8 weeks although worms were also evaluated for growth and reproduction at 4 and 6 weeks. At these times, cocoons were removed and the worms were re-fed new manure. to which the appropriate concentration of test substance was added. The range of concentrations tested for each chemical included one concentration that had no sublethal etTcct, at lcast four concentrations that had a sublethal ctfcct and one concentration that had a lethal effect. Sclcction of these concentrations hclpcd to optimize the possibility of separating threshold concentrations for impairment of growth and reproduction. The final earthworm weight and mean cocoon production per worm per 8 weeks for each chemical concentration were tested for signiticant diffcrcnces using one-way analysis of variance. When a significant F value was obtained, Duncan’s new multiple range test (Steel and Torric. 1960) was used to dctcrminc ditfcrsnccs.
To evaluate the possible residual effects of persistent chemicals, the worms exposed to carbaryl and dicldrin were tested for an additional 8 weeks in manure without added chemicals. The worms were evaluated for growth and reproduction at every 2 weeks. The old manure and cocoons were removed and the worms were re-fed fresh untreated manure at each observation opportunity. The final earthworm weights and mean cocoon production per worm for weeks 8-16 were tested for significant ditferences using one-way analysis of variance When a significant F value was obtained. Duncan’s new multiple range test (Steel and Torrie, 1960) was used to dctcrmine ditfercnccs.
RFSCLTS
ASI)
DISCUSSION
The final weights and total cocoon production achieved by E. fitida cxposcd to the various concentrations of test chemicals show that only carbaryl and dicldrin caused statistically-significant reductions in both growth and cocoon production at sublethal chemical concentrations, whereas five other chemicals
177
had a significant effect on cocoon production alone (Table I). The highest sublethal concentrations of the other chemicals tested showed no significant weight reductions when compared to controls, although all tests were designed to evaluate both sublethal as well as lethal effects during the exposure period. Dimethylphthalate, N-nitrosodiphenylamine, fluorene, 4-nitrophenol and 2.4.6 trichlorophenol significantly reduced reproduction at concentrations that did not kill the worms. Chloroacetamide, 1.2 dichloropropane, and phenol caused no reduction in cocoon production or growth even at the highest concentrations tested. Carbaryl and dieldrin have been established as neurotoxins to earthworms (Drews and Vining. 1984). These two chemicals affected earthworm growth and reproduction at the lowest concentrations of the ten chemicals tested (Table 2). Venter and Reinecke (1988) demonstrated that exposing E.fitida to dieldrin concentrations of 30 mg kg-’ decreased cocoon production when compared to controls and dieldrin concentrations of 50 mg kg-’ decreased the rate of clitcllate formation.
To evaluate the possibility of rccovcry from sublethal effects after exposure to carbaryl and dieldrin, the horse-manure test-chemical substrate was replaced with horse manure only and earthworm weight and cocoon production wcrc monitored for an additional 8 weeks. Earthworm growth in the prescnce and abscncc of carbaryl (Fig. I) and dieldrin (Fig. 2) show that the worms exposed to sublethal concentrations of each chemical wcrc able to resume normal growth rates when the chemical was removed from their environment. Growth rates for both carbaryland dieldrincxposcd worms generally dccreascd with increasing concentrations of each chemical (Table 3). Upon removal of the chemical from the worms’ environment, those exposed to the highest concentrations of chemical showed the greatest recovery growth rates. At the end of the I6 weeks, the data from Figs I and 2 were analyzed and there was no significant difference in the final weight of the worms for any of the concentrations of both chemicals tcstcd, including controls. Cocoon production was severely hindered by the presence of carbaryl or dieldrin (Table 3). Removal of either chemical permitted cocoon production to begin after 8 weeks for the higher concentrations. At 25 mg carbaryl kg-’ and at 25.50 and 100 mg dieldrin kg-‘. a similar number of cocoons were produced as in the controls. It is apparent that the worms were able to start reproduction after the inhibiting chemical was removed. These results strongly suggest that even though the population density may not be immediately affected (i.e. increased mortality) exposure to sublethal concentrations results in reproductive changes that reduce the succeeding population density. Carbaryl or dieldrin at 50 mg kg-’ reduced the cocoon production by >SO% which means that the populations would be significantly reduced in 2-3 generations (Table I).
EDWMUI F. NEUHKSER and CLNIESCE A. CALLAHA.~
178
No carboryl
Carbaryl
: Too
.
tea
ab,.k.”
:
l
200
m;.k;-l
l
1SOng.ke-' loo ng.lrg-’ SO mg.kg-1
:. .
l
000
, . I
100
22
i
mg.kg-’
:
control
: ;-
,o i
E t f
400
300
zoo
t
Y
100
Fig. I. The growth of E.,firicltr in the presence and ahscncc of various concentrations
The sublethal effects of pcsticidcs on earthworms can bc very disruptive of important life history cvcnts such as growth and reproduction (this rcsearch) ;md nervous system functioning (Drcwcs and Vining. 1084) which could result in ckts on important soil proccsscs. Although rccovcry of reproduction and nervous system functioning wcrc observed after dicldrin exposure (Drcwcs and Vining. IOX4). more pcrmancnt damugc hns rcsultcd from cxposurc to bcnomyl (Drcwcs et rrl.. 1987),
N = IO.
suggesting that recovery may not occur in some Glscs. Our rcscarch suggests that more consideration should bc given to evaluating sublethal cfkcts under Iicld conditions to dctcrminc ecological implications of toxic materials. 11 is obvious that LCIo cstimatcs should bc considcrcd a first approximation of toxic cffcct and that more rcfincd tests (e.g. growth and reproduction) should bc used to fully evaluate toxic substances.
No dleldrln
Dleldrin 100
of carbaryl.
280
me.
200
mg.k,,”
kg -1 i
000
100 :
z-
400
0
5 1 E
300
: I f:
200
Y 100
0
.. 2
4
4
Fig. 2. The growth of E.,/c/itkr
10
8 Wm..
12
14
14
rooke
in the presence and absence of various concentrations
of dieldrin.
N = IO
Sublethal
chemical effects on earthworms Acknowledgemenr-This
179 work
was supported
operative Agreement from the U.S. tection Agency to Cornell University.
by a CoPro-
Environmental
REFERESCES Callahan C. A.. Russell L. K. and Peterson S. A. (1985) A comparison of three earthworm bioassay procedures for the assessment of environmental samples containing hazardous wastes. Biology and Fertility of Soils 19.221-233. Davis B. N. K. (1971) Laboratory studies on the uptake of dieldrin and DDT by earthworms. Soil Biolog! & Biochemisrr? 3. ‘2 I-233. Drewes C. D. and Vinmg E. P. (198-t) In riro neurotoxic effects of dieldrin on giant fibers and escape reflex function in the earthworm Eiseniafoefidu. Pesticide Biochemisrrx and Phwolog~ 22. 93-104. Drewes C. D.. Zoran M. J. and Callahan C. A. (1987) Sublethal neurotoxic effects of the fungicide benomyl on earthwrorms (Eisenia fitida). Pesticide Science 19, 197-208. Edwards C. A. and Lofty J. R. (1977) The Biology of Eurrhworms. 2nd edn. Chapman and Hall, London. Goats G. and Edwards C. A. (1982) Testing the toxicity of industrial chemicals to earthworms. Rolhamsfed Report 1982. IOLIO5. Kaplan D. L.. Hartenstein R., Neuhauser E. F. and Malecki M. R. (1980) Physiochemical requircmcnts in the environmcnt of the earthworm Eisenia jtirida. Soil Biokogy & Biochenris/ry 12. 3J7- 352. Karnak R. E. and Hamclink J. L. (1982) A standardized method for determining the acute toxicity of chemicals to cart hworms. Ecoroxicoh~g~ und Enrironmenrul Safi/y 6, 216 -222. Lofs-flolmin A. (19X0) Measuring growth ofearthworms as a method of testing suhlcthal toxicity of pesticides. Swedish Journul of Agriculrurul Reseurch IO, 25 -33. Lofs-tlolmin A. (1982) Measuring cocoon production of the earthworm A//o/ohop/roru cuIi~gino.w (Sav.) as a method of testing sublethal toxicity of pssticidcs. Swedish JOUUICJ/04’ A,qricw/ruru/ Reseurch 12. I I7 I 19. Ncuhauser E. F.. Kaplan D. L.. Malecki M. R. and tlartcnstcin R. (19X0) Materials supporting the weight gain by the sarthworm Ei.wniu/c~ridu in waste conversion systems. A~ricdrurol U’crsrcs 2. 43 60. Neuhauser E. F.. Malecki M. R. and Loehr R. C. (1984) Growth and reproduction of the earthworm after exposure to sublethal concentrations of metals. Pedohiulogiu 27. 89 -97. Steel R. G. and Torrie J. H. (1960) Principles und Procedures of Srurisrics. McGraw-Hill. New York. Stenersen J. (1979) Action of pesticides on earthworms. Pdrt I: The toxicity of cholinestemse-inhibiting inscctitides to earthworms as evaluated by laboratory tests. Prsricide Science IO, 66-74. U.S. Envirorimental Protection Agency (1979) Waterrelated environmental hte of I29 priority pollutants. EPA -440 14-79-029. U’ushingron. D.C. Venter J. M. and Rcinccke A. J. (1988) Sublethal ccotoxicological effects of dleldrin on the earthworm Eiseniajiwfidu (Oligochaeta). In Eurrhwwms in W’usfe und Encirunmenful Alunugemen~ (C. A. Edwards and E. F. Neuhauser. Eds). pp. 337.353. SPB Academic Publishing. The Hague.