This article was downloaded by: [University of Florida] On: 17 February 2015, At: 06:01 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
A study on the lethal toxicity of Aminocarb to freshwater crayfish and its in vivo metabolism a
K.M.S. Sundaram & S.Y. Szeto
a
a
Forest Pest Management Institute,Canadian Forestry Service, Department of the Environment , Sault Ste. Marie, Ontario, P6A 5M7 Published online: 14 Nov 2008.
To cite this article: K.M.S. Sundaram & S.Y. Szeto (1979) A study on the lethal toxicity of Aminocarb to freshwater crayfish and its in vivo metabolism, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 14:6, 589-602, DOI: 10.1080/03601237909372153 To link to this article: http://dx.doi.org/10.1080/03601237909372153
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our
Downloaded by [University of Florida] at 06:02 17 February 2015
licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
J. ENVIRON. SCI. HEALTH, B14(6), 589-602 (1979)
A STUDY ON THE LETHAL TOXICI.TY OF AMINOCARB TO FRESHWATER CRAYFISH AND
Downloaded by [University of Florida] at 06:02 17 February 2015
ITS IN VIVO METABOLISM Key Words:
Aminocarb, Metabolites, Crayfish, Lethal Toxicity, Residues
K.M.S. Sundaram and S.Y. Szeto Forest Pest Management Institute Canadian Forestry Service Department of the Environment Sault Ste. Marie, Ontario, P6A 5M7
ABSTRACT Adult crayfish (Oraonetes limosus) were exposed to 0.1,
1.0, 5.0, 10.0, 25.0, 30.0, 40.0, 50.0 and 60.0 ppm of
aminocarb in water at 15°C under laboratory conditions for 144 h.
No apparent behavioral changes were observed in
crayfish exposed to 0.1, 1.0 and 5.0 ppm of aminocarb during the experiment.
Symptoms of acute toxicity were apparent at
concentrations
10 ppm, and mortality occurred at and above
25 ppm.
The L C 5 0 , 96 h, to adult crayfish was about 33 ppm. The parent compound and its metabolites, MA
(4-methylamino-m-tolyl N-methylcarbamate) and AM (4-amino-
589 Copyright © 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
590
SUNDARAM AND SZETO
m-tolyl N-methylcarbamate), were detected in crayfish 96 h after exposure to various concentrations of aminocarb.
The
primary metabolite detected was MA which accounted for 75% to 95% of the body residue.
The highest total residue
(Aminocarb + MA + AM) was 40 ppm detected in crayfish exposed
Downloaded by [University of Florida] at 06:02 17 February 2015
to 60 ppm of aminocarb for 96 h.
INTRODUCTION Aminocarb (Matacil
®
) , 4-dimethylamino-m-tolyl N-
methylcarbamate is an effective non-systemic insecticide against many lepidopterous larvae and biting insects 1 .
Its
use for spruce budwonn control in eastern Canada has increased steadily in the past few years.
In 1976, 24,000 ha in New
Brunswick and 1.5 million ha in Quebec were sprayed with aminocarb 2 .
In the following year, 120,000 ha in New Bruns-
wick and 1.4 million ha in Quebec were sprayed with the chemical 3 . Aminocarb is highly toxic to mammals and some types of fish.
The oral LD50 for rats is about 50 mg/kg, and the
LC50 (^8 h) for Atlantic salmon (Salmo solar) is 1.10 ppm 1 ' 4 . However, little is known about the toxicity of aminocarb to freshwater and marine crustaceans.
Since lobster farming is
of economic importance in the Maritime provinces and the use of aminocarb for spruce budworm control in that region is steadily increasing, information on the lethal toxicity of
LETHAL TOXICITY OF AMINOCARB
591
aminocarb to the freshwater and marine crustaceans is urgently needed. The objective of this study was to study the in vivo metabolism of aminocarb and determine its lethal toxicity to
Downloaded by [University of Florida] at 06:02 17 February 2015
crayfish, Oraonetes limosus.
I.
MATERIALS AND METHODS Crayfish Bioassay.
Adult Crayfish, Orconetes Zimosus
were trapped from the Root River at Sault Ste. Marie, Ontario and maintained in the laboratory in water collected from the same region.
The water temperature at the time of sampling
was 13°C and the subsequent bioassay was carried out in the laboratory at 15 C C.
The crayfish were fed with shredded minnow
during the experiment. Aliquots of 3,000-ml river water were fortified with amino-
carb to give the concentrations of 0.1, 1.0, 10.0, 25.0, 30.0, 40.0, 50.0 and 60.0 ppm by adding 0.003, 0.03, 0.3, 0.75, 0.9, 1.2, 1.5
and 1.8 ml of the ethanolic solution containing 100 mg aminocarb/ml. Aliquots of 1,000-ml fortified water were transferred into three polypropylene tanks (26.5 x 20.0 x 15.0 cm) and 3 to 5
crayfish were placed per tank for bioassay. was used for the control in this study.
Untreated water
The treated and
untreated river water were replaced every 24 h and they were aerated during the experiment.
The behavior of the crayfish
was observed four times daily and the mortality recorded at 24, 48, 72 and 96 h.
The pesticide residues in the test
animals were determined at the end of the assay.
592
SUNDARAM AND SZETO
Aliquots of the untreated and treated water were taken from each tank immediately after fortification and also prior to replacement for the determination of aminocarb concentration by GLC analysis. II.
Extraction and Clean-up.
Downloaded by [University of Florida] at 06:02 17 February 2015
1.
Water.
Aminocarb was extracted from water with
the Amberlite XAD-2 column as described by Sundaram et at.5 . The residues were dissolved in 5 ml ethyl acetate and analyzed by GLC without further clean-up. 2.
Crayfish.
Crayfish were rinsed thoroughly with
glass-distilled water to remove any aminocarb-treated water adhering to the body surface.
The animals were dried on
absorbing paper and the length and weight of each animal were determined. About 40 g (wet weight) of crayfish were homogenized three times with 150 ml of pesticide-grade ethyl acetate in a Polytron PT 10 homogenizer for 2 min; 80 g of anhydrous sodium sulfate were added in the first extraction.
The
solvent extracts were filtered through a Buchner funnel with a 3-cm layer of anhydrous sodium sulfate, and the Buchner funnel was then rinsed with 50 ml of pesticide-grade ethyl acetate.
The combined extracts were quantitatively transferred
into a graduated cylinder and made up to 500 ml with ethyl acetate. Aliquots of the crude extracts equivalent to 20 g (wet wt) of tissue were concentrated to about 2 ml in the
LETHAL TOXICITY OF AMINOCARB
593
flash evaporator at 38°C and 25 ml of aqueous phosphoric acid solution (3 ml of 85% phosphoric acid and 3 g of ammonium chloride in 1 I of aqueous solution) was added.
The
mixture was quantitatively transferred into a 250-ml separatory funnel by rinsing four times with 1 ml pesticide-
Downloaded by [University of Florida] at 06:02 17 February 2015
grade acetone and 25 ml of aqueous phosphoric acid solution each.
The aqueous solution was extracted three times with
25 ml of hexane each to remove the co-extractives and the
hexane phases were discarded.
The aqueous phase was then
neutralized with saturated sodium carbonate solution and the aminocarb and its metabolites were extracted by partitioning four times with 100, 50, 50 and 50 ml of pesticide-grade dichloromethane.
The combined extract was dried on anhydrous
sodium sulfate and evaporated just to dryness in a flash
evaporator at 38°C.
The residues were dissolved in 2 ml of
pesticide-grade ethyl acetate and analyzed by GLC for aminocarb and its metabolites. III.
GLC Analysis.
A tracor 550 gas chromatograph equipped
with a Model 702 N-P detector, and two glass columns (75 cm x 4.0 mm i.d.) were used: (1) 1.95% QFl plus 1.5% 0V17; (2) 3% carbowax 20 M TPA; all on Chromosorb W "H.P.", 80/100 mesh.
The operating parameters were: detector temperature.
240°C, injection port temperature 210°C, outlet temperature 210°C, column oven temperature 170°C, carrier gas flow (helium) 70 ml/min, hydrogen flow 2.5 ml/min, and air flow 120 ml/min.
Calibration curves were prepared daily before and
594
SUNDARAM AND SZETO
after sample detector.
analysis to confirm the stability of the
Quantification of aminocarb and its metabolites
were based on external standards. Duplicates of the untreated crayfish sample were fortified with aminocarb, AM (4-amino-m-tolyl N-methyl-
Downloaded by [University of Florida] at 06:02 17 February 2015
carbamate) at 1.0 ppm and MA (4-methylamino-m-tolyl N-methylcarbamate) at 10.0 ppm.
The recoveries were 114.9% ± 6.9% for
aminocarb, 56.8 ± 2.5% for AM and 107.5% ± 1.7% for MA. Clean extracts (1 ml = 10 g of crayfish tissue) were suitably diluted for GLC analysis if necessary.
If 8 yl of
undiluted extract gave no response, the results were reported as non-detectable (N.D.).
The detection limits were 0.1 ppm
for MA and 0.01 ppm for AM.
RESULTS AND DISCUSSION I.
Acute Toxicity and LC
Determination.
The actual con-
centrations of aminocarb in water as determined by gas chromatographic analysis are given in Table 1.
No mortality of crayfish
exposed to aminocarb at 0.1, 1.0, 5.0 and 10.0 ppm had been observed up to 144 h exposure.
However, crayfish exposed to 10.0
ppm of aminocarb showed slight changes in their behavior, e.g., loss of appetite and sluggishness in their movement after 144 h exposure.
The mortalities of crayfish 96 h after exposure to
aminocarb at various concentrations are also given in Table 1. The animals which survived after 96 h exposure to 25-50 ppm aminocarb were paralyzed.
Downloaded by [University of Florida] at 06:02 17 February 2015
H O X
TABLE 1 Mortality of Crayfish (.Oraonetes limosus) 96 h after Exposure to Aminocarb.
M O M
O
Treatment (Aminocarb Cone, in ppm)
0.1 1.0 5.0 10.0
25 30 40 50 60
Concentration in water (ppm) X ± S.D •0.101 0.969 4.78 9.75 23.9 31.0 38.0 48.1 60.9
Control
0.008 0.042 0.186 0.303 2.18 2.46 2.12 3.04 0.808
Number of Crayfish Tested
12 10 9 10 10 11 13 13 13
9
Body length (m .m.) X ± S.D.
60 62
49 71 68 67 58 63 58 64
5
4 3 4 4
Observed mortality %
Corrected— mortality (%)
0 0 0 0 10
7 6 5 7
100
5
0
45.5 76.9 92.3
-
No. c" analysis = 21
—
Abbott's formula: Corrected . „ .. „ Observed mortality /£ - Control mortality z mortality % = 77^7; ^—; — 5 ~ 100 - Control mortality %
0 0 0 0 10 45.5 76.9 92.3 100 (99.2)
x 100
SUNDARAM AND SZETO
596
From the mortality data corrected for control mortality (Table 1 ) , LC
value and its 95% confidence limit were
determined according to the method described by Swaroop (Figure 1) and by computer (Hewlett Packard 9810A) probit analysis.
The LC__, 96 h, for crayfish was 32.7 ppm and its
Downloaded by [University of Florida] at 06:02 17 February 2015
upper and lower confidence limits (P = 0.05) were 40.1 ppm and 23.5 ppm respectively.
/
99-
95-
/ /A
90-
/ •2 80" m o TO-
/
1I
OL
^50-
E 3 0-
o ^ 2 01 0-
/ / / /A
14 LOG
16 PPM
1-8
2-0
FIGURE 1. REGRESSION LINE CALCULATED FROM MORTALITY OF CRAYFISH 96 H AFTER EXPOSURE TO AMINOCARB.
LETHAL TOXICITY OF AMINOCARB
597
Fenitrothion (0,0-dimethyl 0-(4-nitro-m-tolyl) phosphorothioate) has been used extensively in eastern Canada for spruce budworm control 2 ' 3 .
This organophosphorus
insecticide is extremely toxic to both marine and freshwater crustaceans.
The lethal threshold for lobster (Homarus
Downloaded by [University of Florida] at 06:02 17 February 2015
ameriaanus) is 0.015 ppb for larvae and less than 0.3 ppb for adults 7 .
The approximate L C 5 0 , 96 h are 10 ppb for small
and 30 ppb for large crayfish (Oreonetes Z-imosus)8.
In
contrast, the LC50, 96 h of aminocarb to adult crayfish determined in this study was 32,700 ppb.
By comparison, it
appeared that aminocarb is at least 1,000 times less toxic than fenitrothion to crayfish. Sundaram et al.s studied the residue concentration in waters after aerial application of aminocarb at 70 g/ha.
They
reported that 2.1 ppb and 1.9 ppb residues were detected in pond and stream water respectively, 0.6 day after application. The residue declined to trace (< 0.1 ppb) after 32 days and was no longer detected after 63 days in both pond and stream waters.
Considering the low concentration of aminocarb
residue which could be present in water following spraying and its lethal toxicity to adult crayfish, it appears that at the dose level used in spruce budworm control program, aminocarb would not pose any adverse effect on adult crayfish. II.
Metabolism of Aminocarb.
River water and crayfish
samples were analyzed prior to the experiment.
No GLC peaks
which interfere with aminocarb and its metabolites were
598
SUNDARAM AND SZETO
detected.
All the crayfish exposed to various concentrations
of aminocarb contained residues of the parent compound and its metabolites (Table 2 ) . The metabolites detected in this study were the 4-methylamino (MA) and 4-amino (AM) analogues of aminocarb.
The highest total residue (aminocarb plus the
Downloaded by [University of Florida] at 06:02 17 February 2015
4-methlyamino and the 4-amino analogues) of 40.2 ppm was detected in crayfish exposed to 60.0 ppm of aminocarb for 96 h (Table 2 ) . On the other hand, crayfish exposed to 0.1 ppm aminocarb for 144 h contained 0.007 ppm of the parent compound only and the 4-methylamino and 4-amino analogues were not detected in the sample (Table 2 ) . Considering the biological variations among samples, the total body residues appeared to reflect the concentration of aminocarb in water, i.e., it was proportional to the concentration the animals were exposed to.
However, the
concentrations
of total residue were always below the aminocarb concentration in water (Table 2) . The primary metabolic product detected in this study was MA (4-methylamino-m-tolyl N-methylcarbamate), which accounted for 75% to 94% of the total residue (Table 2 and Figure 2 ) . The non-detectability of MA in crayfish exposed to 0.1 ppm of aminocarb may have been due to the much lower detection limit required for this metabolite.
The other
metabolite detected was AM (4-amino-m-tolyl N-methylcarbamate (Table 2 and Figure 2 ) . The highest percentage of AM in the total residue was 1.4% detected in crayfish exposed to 30 ppm
Downloaded by [University of Florida] at 06:02 17 February 2015
TABLE 2 Residues (ppm) in Crayfish (Orconetes limosus) 96 h after exposure to aminocarb. M ' 11 w* ^\ r* + * V A r\ -r^
ireatment (Aminocarb Cone, in ppm)
0.1 1.0 5.0
Exposure (hour)
144 96 144 144 96 96 96 96 96
10.0 25.0 30.0 40.0 50.0 60.0 Control
3
Residue
^
Aminocarb % (ppm) 0.007 0.022 0.867 0.900 2.95 2.94 4.20 5.01 10.1
100 6.4 13.7 19.0 16.0 11.8 21.4 19.7 25.1
N.D.
MA 5 / (ppm) N.D.^ 0.323 5.38 3.84 15.3 21.7 15.3 20.2 30.0 N.D.
MA
=
4-methylamino-m-tolyl N-methylcarbamate
AM
=
4-amino-m-tolyl N-methylcarbamate
% 0 93.6 85.0 81.0 83.2 86.8 78.1 79.5 74.6
(ppm) N.D. N.D. 0.084 N.D. 0.189 0.329 0.058 0.166 0.130
AM^ %
0 0 1.3 0 0.8 1.4 0.5 0.8 0.3
Total-'' (ppm) 0.007 0.345 6.33 4.74 18.4 25.0 19.6 25.4 40.2
N.D.
Total =
Aminocarb + MA + AM
N.D.
Non-detectable and the detection limits were 0.1 ppm for MA and 0.01 ppm for AM.
=
o
§ n
1w5
600
SUNDARAW AND
SZETO
(1) 4-dim«thylamino-m- tolyl N* methylcarbamate (2) 4-amino-tn-tolyl N " mtthylearbamatt (3) 4 - methylamino - m - tolyl
Downloaded by [University of Florida] at 06:02 17 February 2015
N " mithylearbamat*
0
2
FIGURE EXPOSED
6
2. CHROMATOGRAM TO 3 0 - P P M
(B) UNTREATED
of aminocarb.
4
8 mln
OF (A) CRAYFISH
AMINOCARB
FOR 96 H;
CONTROL.
Although the recovery for AM was low, i . e . , 57%.
i t could only account for less than 5% of the t o t a l residue detected, even after recovery correction.
These findings
suggest that one of the metabolic routes of aminocarb in crayfish could be through the demethylation of the 4-dlmethylamino moiety, followed by deamination to form the ringhydroxylated product for conjugation.
Interestingly, MA and
LETHAL TOXICITY OF AMINOCARB
601
AM were also found in bean plants following the application of aminocarb to the leaves of the 12-day-old bean seedlings 10 . For a better understanding of the metabolism and mechanism for the elimination of aminocarb in animals, further studies
Downloaded by [University of Florida] at 06:02 17 February 2015
are required.
ACKNOWLEDGEMENT We thank Dr. J. Tibbies, Director, Sea Lamprey Control Centre, Fisheries and Environment Canada for his encouragement and Mr. Rod D. Graham for his technical assistance in this study.
REFERENCES 1.
Martin, H. and C.R. Worthing.
Pesticide Manual.
(4th ed.)
British Crop Protection Council, England (1974). 2.
Canadian Forestry Service. Control Forum 1976.
3.
Report of Annual Forest Pest
Environment Canada, Ottawa (1977).
Canadian Forestry Service.
Report of the Annual Forest
Pest Control Forum 1977.
Environment Canada, Ottawa
(1978). 4.
Nigam, P.C.
Chemical Insecticides,
Forest Insects in Canada. No. Fo 23/19/1975.
in: Aerial Control of
Ed. Prebble, M.L. Catalogue
Department of the Environment,
Ottawa, Canada (1975).
602 5.
SUNDARAM AND SZETO Sundaram, K.M.S., S. Szeto and R. Hindle.
Report FPM-X-18,
Forest Pest Management Institute, Environment Canada, Sault Ste. Marie, Ontario. 6.
Swaroop, S.
P6A 5M7.
Statistical Methods in Malaria Eradication.
WHO Monograph Series No. 51.
Downloaded by [University of Florida] at 06:02 17 February 2015
Sales Unit. 7.
(1978).
McLeese, D.W.
WHO Distribution and
Geneva, Switzerland (1966).
J. Fish. Res.
Board Can. 31: 1127-1131
(1974). 8.
McLeese, D.W.
Bull.
Environ. Contain.
Toxicol.
16(4):
411-416 (1976). 9.
Sundaram, K.M.S., Y. Volpe, G.G. Smith and J.R. Duffy. Report CC-X-116.
CCRI.
Environment Canada, Ottawa
K1A 0W3 (1976). 10.
Abdel-Wahab, A.M. and J.E. Casida. 15(2):
J. Agr. Food Chem.
479-487 (1967).
Received:
March 1, 1979.
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
A study on the lethal toxicity of Aminocarb to freshwater crayfish and its in vivo metabolism a
K.M.S. Sundaram & S.Y. Szeto
a
a
Forest Pest Management Institute,Canadian Forestry Service, Department of the Environment , Sault Ste. Marie, Ontario, P6A 5M7 Published online: 14 Nov 2008.
To cite this article: K.M.S. Sundaram & S.Y. Szeto (1979) A study on the lethal toxicity of Aminocarb to freshwater crayfish and its in vivo metabolism, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 14:6, 589-602, DOI: 10.1080/03601237909372153 To link to this article: http://dx.doi.org/10.1080/03601237909372153
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our
Downloaded by [University of Florida] at 06:02 17 February 2015
licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
J. ENVIRON. SCI. HEALTH, B14(6), 589-602 (1979)
A STUDY ON THE LETHAL TOXICI.TY OF AMINOCARB TO FRESHWATER CRAYFISH AND
Downloaded by [University of Florida] at 06:02 17 February 2015
ITS IN VIVO METABOLISM Key Words:
Aminocarb, Metabolites, Crayfish, Lethal Toxicity, Residues
K.M.S. Sundaram and S.Y. Szeto Forest Pest Management Institute Canadian Forestry Service Department of the Environment Sault Ste. Marie, Ontario, P6A 5M7
ABSTRACT Adult crayfish (Oraonetes limosus) were exposed to 0.1,
1.0, 5.0, 10.0, 25.0, 30.0, 40.0, 50.0 and 60.0 ppm of
aminocarb in water at 15°C under laboratory conditions for 144 h.
No apparent behavioral changes were observed in
crayfish exposed to 0.1, 1.0 and 5.0 ppm of aminocarb during the experiment.
Symptoms of acute toxicity were apparent at
concentrations
10 ppm, and mortality occurred at and above
25 ppm.
The L C 5 0 , 96 h, to adult crayfish was about 33 ppm. The parent compound and its metabolites, MA
(4-methylamino-m-tolyl N-methylcarbamate) and AM (4-amino-
589 Copyright © 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
590
SUNDARAM AND SZETO
m-tolyl N-methylcarbamate), were detected in crayfish 96 h after exposure to various concentrations of aminocarb.
The
primary metabolite detected was MA which accounted for 75% to 95% of the body residue.
The highest total residue
(Aminocarb + MA + AM) was 40 ppm detected in crayfish exposed
Downloaded by [University of Florida] at 06:02 17 February 2015
to 60 ppm of aminocarb for 96 h.
INTRODUCTION Aminocarb (Matacil
®
) , 4-dimethylamino-m-tolyl N-
methylcarbamate is an effective non-systemic insecticide against many lepidopterous larvae and biting insects 1 .
Its
use for spruce budwonn control in eastern Canada has increased steadily in the past few years.
In 1976, 24,000 ha in New
Brunswick and 1.5 million ha in Quebec were sprayed with aminocarb 2 .
In the following year, 120,000 ha in New Bruns-
wick and 1.4 million ha in Quebec were sprayed with the chemical 3 . Aminocarb is highly toxic to mammals and some types of fish.
The oral LD50 for rats is about 50 mg/kg, and the
LC50 (^8 h) for Atlantic salmon (Salmo solar) is 1.10 ppm 1 ' 4 . However, little is known about the toxicity of aminocarb to freshwater and marine crustaceans.
Since lobster farming is
of economic importance in the Maritime provinces and the use of aminocarb for spruce budworm control in that region is steadily increasing, information on the lethal toxicity of
LETHAL TOXICITY OF AMINOCARB
591
aminocarb to the freshwater and marine crustaceans is urgently needed. The objective of this study was to study the in vivo metabolism of aminocarb and determine its lethal toxicity to
Downloaded by [University of Florida] at 06:02 17 February 2015
crayfish, Oraonetes limosus.
I.
MATERIALS AND METHODS Crayfish Bioassay.
Adult Crayfish, Orconetes Zimosus
were trapped from the Root River at Sault Ste. Marie, Ontario and maintained in the laboratory in water collected from the same region.
The water temperature at the time of sampling
was 13°C and the subsequent bioassay was carried out in the laboratory at 15 C C.
The crayfish were fed with shredded minnow
during the experiment. Aliquots of 3,000-ml river water were fortified with amino-
carb to give the concentrations of 0.1, 1.0, 10.0, 25.0, 30.0, 40.0, 50.0 and 60.0 ppm by adding 0.003, 0.03, 0.3, 0.75, 0.9, 1.2, 1.5
and 1.8 ml of the ethanolic solution containing 100 mg aminocarb/ml. Aliquots of 1,000-ml fortified water were transferred into three polypropylene tanks (26.5 x 20.0 x 15.0 cm) and 3 to 5
crayfish were placed per tank for bioassay. was used for the control in this study.
Untreated water
The treated and
untreated river water were replaced every 24 h and they were aerated during the experiment.
The behavior of the crayfish
was observed four times daily and the mortality recorded at 24, 48, 72 and 96 h.
The pesticide residues in the test
animals were determined at the end of the assay.
592
SUNDARAM AND SZETO
Aliquots of the untreated and treated water were taken from each tank immediately after fortification and also prior to replacement for the determination of aminocarb concentration by GLC analysis. II.
Extraction and Clean-up.
Downloaded by [University of Florida] at 06:02 17 February 2015
1.
Water.
Aminocarb was extracted from water with
the Amberlite XAD-2 column as described by Sundaram et at.5 . The residues were dissolved in 5 ml ethyl acetate and analyzed by GLC without further clean-up. 2.
Crayfish.
Crayfish were rinsed thoroughly with
glass-distilled water to remove any aminocarb-treated water adhering to the body surface.
The animals were dried on
absorbing paper and the length and weight of each animal were determined. About 40 g (wet weight) of crayfish were homogenized three times with 150 ml of pesticide-grade ethyl acetate in a Polytron PT 10 homogenizer for 2 min; 80 g of anhydrous sodium sulfate were added in the first extraction.
The
solvent extracts were filtered through a Buchner funnel with a 3-cm layer of anhydrous sodium sulfate, and the Buchner funnel was then rinsed with 50 ml of pesticide-grade ethyl acetate.
The combined extracts were quantitatively transferred
into a graduated cylinder and made up to 500 ml with ethyl acetate. Aliquots of the crude extracts equivalent to 20 g (wet wt) of tissue were concentrated to about 2 ml in the
LETHAL TOXICITY OF AMINOCARB
593
flash evaporator at 38°C and 25 ml of aqueous phosphoric acid solution (3 ml of 85% phosphoric acid and 3 g of ammonium chloride in 1 I of aqueous solution) was added.
The
mixture was quantitatively transferred into a 250-ml separatory funnel by rinsing four times with 1 ml pesticide-
Downloaded by [University of Florida] at 06:02 17 February 2015
grade acetone and 25 ml of aqueous phosphoric acid solution each.
The aqueous solution was extracted three times with
25 ml of hexane each to remove the co-extractives and the
hexane phases were discarded.
The aqueous phase was then
neutralized with saturated sodium carbonate solution and the aminocarb and its metabolites were extracted by partitioning four times with 100, 50, 50 and 50 ml of pesticide-grade dichloromethane.
The combined extract was dried on anhydrous
sodium sulfate and evaporated just to dryness in a flash
evaporator at 38°C.
The residues were dissolved in 2 ml of
pesticide-grade ethyl acetate and analyzed by GLC for aminocarb and its metabolites. III.
GLC Analysis.
A tracor 550 gas chromatograph equipped
with a Model 702 N-P detector, and two glass columns (75 cm x 4.0 mm i.d.) were used: (1) 1.95% QFl plus 1.5% 0V17; (2) 3% carbowax 20 M TPA; all on Chromosorb W "H.P.", 80/100 mesh.
The operating parameters were: detector temperature.
240°C, injection port temperature 210°C, outlet temperature 210°C, column oven temperature 170°C, carrier gas flow (helium) 70 ml/min, hydrogen flow 2.5 ml/min, and air flow 120 ml/min.
Calibration curves were prepared daily before and
594
SUNDARAM AND SZETO
after sample detector.
analysis to confirm the stability of the
Quantification of aminocarb and its metabolites
were based on external standards. Duplicates of the untreated crayfish sample were fortified with aminocarb, AM (4-amino-m-tolyl N-methyl-
Downloaded by [University of Florida] at 06:02 17 February 2015
carbamate) at 1.0 ppm and MA (4-methylamino-m-tolyl N-methylcarbamate) at 10.0 ppm.
The recoveries were 114.9% ± 6.9% for
aminocarb, 56.8 ± 2.5% for AM and 107.5% ± 1.7% for MA. Clean extracts (1 ml = 10 g of crayfish tissue) were suitably diluted for GLC analysis if necessary.
If 8 yl of
undiluted extract gave no response, the results were reported as non-detectable (N.D.).
The detection limits were 0.1 ppm
for MA and 0.01 ppm for AM.
RESULTS AND DISCUSSION I.
Acute Toxicity and LC
Determination.
The actual con-
centrations of aminocarb in water as determined by gas chromatographic analysis are given in Table 1.
No mortality of crayfish
exposed to aminocarb at 0.1, 1.0, 5.0 and 10.0 ppm had been observed up to 144 h exposure.
However, crayfish exposed to 10.0
ppm of aminocarb showed slight changes in their behavior, e.g., loss of appetite and sluggishness in their movement after 144 h exposure.
The mortalities of crayfish 96 h after exposure to
aminocarb at various concentrations are also given in Table 1. The animals which survived after 96 h exposure to 25-50 ppm aminocarb were paralyzed.
Downloaded by [University of Florida] at 06:02 17 February 2015
H O X
TABLE 1 Mortality of Crayfish (.Oraonetes limosus) 96 h after Exposure to Aminocarb.
M O M
O
Treatment (Aminocarb Cone, in ppm)
0.1 1.0 5.0 10.0
25 30 40 50 60
Concentration in water (ppm) X ± S.D •0.101 0.969 4.78 9.75 23.9 31.0 38.0 48.1 60.9
Control
0.008 0.042 0.186 0.303 2.18 2.46 2.12 3.04 0.808
Number of Crayfish Tested
12 10 9 10 10 11 13 13 13
9
Body length (m .m.) X ± S.D.
60 62
49 71 68 67 58 63 58 64
5
4 3 4 4
Observed mortality %
Corrected— mortality (%)
0 0 0 0 10
7 6 5 7
100
5
0
45.5 76.9 92.3
-
No. c" analysis = 21
—
Abbott's formula: Corrected . „ .. „ Observed mortality /£ - Control mortality z mortality % = 77^7; ^—; — 5 ~ 100 - Control mortality %
0 0 0 0 10 45.5 76.9 92.3 100 (99.2)
x 100
SUNDARAM AND SZETO
596
From the mortality data corrected for control mortality (Table 1 ) , LC
value and its 95% confidence limit were
determined according to the method described by Swaroop (Figure 1) and by computer (Hewlett Packard 9810A) probit analysis.
The LC__, 96 h, for crayfish was 32.7 ppm and its
Downloaded by [University of Florida] at 06:02 17 February 2015
upper and lower confidence limits (P = 0.05) were 40.1 ppm and 23.5 ppm respectively.
/
99-
95-
/ /A
90-
/ •2 80" m o TO-
/
1I
OL
^50-
E 3 0-
o ^ 2 01 0-
/ / / /A
14 LOG
16 PPM
1-8
2-0
FIGURE 1. REGRESSION LINE CALCULATED FROM MORTALITY OF CRAYFISH 96 H AFTER EXPOSURE TO AMINOCARB.
LETHAL TOXICITY OF AMINOCARB
597
Fenitrothion (0,0-dimethyl 0-(4-nitro-m-tolyl) phosphorothioate) has been used extensively in eastern Canada for spruce budworm control 2 ' 3 .
This organophosphorus
insecticide is extremely toxic to both marine and freshwater crustaceans.
The lethal threshold for lobster (Homarus
Downloaded by [University of Florida] at 06:02 17 February 2015
ameriaanus) is 0.015 ppb for larvae and less than 0.3 ppb for adults 7 .
The approximate L C 5 0 , 96 h are 10 ppb for small
and 30 ppb for large crayfish (Oreonetes Z-imosus)8.
In
contrast, the LC50, 96 h of aminocarb to adult crayfish determined in this study was 32,700 ppb.
By comparison, it
appeared that aminocarb is at least 1,000 times less toxic than fenitrothion to crayfish. Sundaram et al.s studied the residue concentration in waters after aerial application of aminocarb at 70 g/ha.
They
reported that 2.1 ppb and 1.9 ppb residues were detected in pond and stream water respectively, 0.6 day after application. The residue declined to trace (< 0.1 ppb) after 32 days and was no longer detected after 63 days in both pond and stream waters.
Considering the low concentration of aminocarb
residue which could be present in water following spraying and its lethal toxicity to adult crayfish, it appears that at the dose level used in spruce budworm control program, aminocarb would not pose any adverse effect on adult crayfish. II.
Metabolism of Aminocarb.
River water and crayfish
samples were analyzed prior to the experiment.
No GLC peaks
which interfere with aminocarb and its metabolites were
598
SUNDARAM AND SZETO
detected.
All the crayfish exposed to various concentrations
of aminocarb contained residues of the parent compound and its metabolites (Table 2 ) . The metabolites detected in this study were the 4-methylamino (MA) and 4-amino (AM) analogues of aminocarb.
The highest total residue (aminocarb plus the
Downloaded by [University of Florida] at 06:02 17 February 2015
4-methlyamino and the 4-amino analogues) of 40.2 ppm was detected in crayfish exposed to 60.0 ppm of aminocarb for 96 h (Table 2 ) . On the other hand, crayfish exposed to 0.1 ppm aminocarb for 144 h contained 0.007 ppm of the parent compound only and the 4-methylamino and 4-amino analogues were not detected in the sample (Table 2 ) . Considering the biological variations among samples, the total body residues appeared to reflect the concentration of aminocarb in water, i.e., it was proportional to the concentration the animals were exposed to.
However, the
concentrations
of total residue were always below the aminocarb concentration in water (Table 2) . The primary metabolic product detected in this study was MA (4-methylamino-m-tolyl N-methylcarbamate), which accounted for 75% to 94% of the total residue (Table 2 and Figure 2 ) . The non-detectability of MA in crayfish exposed to 0.1 ppm of aminocarb may have been due to the much lower detection limit required for this metabolite.
The other
metabolite detected was AM (4-amino-m-tolyl N-methylcarbamate (Table 2 and Figure 2 ) . The highest percentage of AM in the total residue was 1.4% detected in crayfish exposed to 30 ppm
Downloaded by [University of Florida] at 06:02 17 February 2015
TABLE 2 Residues (ppm) in Crayfish (Orconetes limosus) 96 h after exposure to aminocarb. M ' 11 w* ^\ r* + * V A r\ -r^
ireatment (Aminocarb Cone, in ppm)
0.1 1.0 5.0
Exposure (hour)
144 96 144 144 96 96 96 96 96
10.0 25.0 30.0 40.0 50.0 60.0 Control
3
Residue
^
Aminocarb % (ppm) 0.007 0.022 0.867 0.900 2.95 2.94 4.20 5.01 10.1
100 6.4 13.7 19.0 16.0 11.8 21.4 19.7 25.1
N.D.
MA 5 / (ppm) N.D.^ 0.323 5.38 3.84 15.3 21.7 15.3 20.2 30.0 N.D.
MA
=
4-methylamino-m-tolyl N-methylcarbamate
AM
=
4-amino-m-tolyl N-methylcarbamate
% 0 93.6 85.0 81.0 83.2 86.8 78.1 79.5 74.6
(ppm) N.D. N.D. 0.084 N.D. 0.189 0.329 0.058 0.166 0.130
AM^ %
0 0 1.3 0 0.8 1.4 0.5 0.8 0.3
Total-'' (ppm) 0.007 0.345 6.33 4.74 18.4 25.0 19.6 25.4 40.2
N.D.
Total =
Aminocarb + MA + AM
N.D.
Non-detectable and the detection limits were 0.1 ppm for MA and 0.01 ppm for AM.
=
o
§ n
1w5
600
SUNDARAW AND
SZETO
(1) 4-dim«thylamino-m- tolyl N* methylcarbamate (2) 4-amino-tn-tolyl N " mtthylearbamatt (3) 4 - methylamino - m - tolyl
Downloaded by [University of Florida] at 06:02 17 February 2015
N " mithylearbamat*
0
2
FIGURE EXPOSED
6
2. CHROMATOGRAM TO 3 0 - P P M
(B) UNTREATED
of aminocarb.
4
8 mln
OF (A) CRAYFISH
AMINOCARB
FOR 96 H;
CONTROL.
Although the recovery for AM was low, i . e . , 57%.
i t could only account for less than 5% of the t o t a l residue detected, even after recovery correction.
These findings
suggest that one of the metabolic routes of aminocarb in crayfish could be through the demethylation of the 4-dlmethylamino moiety, followed by deamination to form the ringhydroxylated product for conjugation.
Interestingly, MA and
LETHAL TOXICITY OF AMINOCARB
601
AM were also found in bean plants following the application of aminocarb to the leaves of the 12-day-old bean seedlings 10 . For a better understanding of the metabolism and mechanism for the elimination of aminocarb in animals, further studies
Downloaded by [University of Florida] at 06:02 17 February 2015
are required.
ACKNOWLEDGEMENT We thank Dr. J. Tibbies, Director, Sea Lamprey Control Centre, Fisheries and Environment Canada for his encouragement and Mr. Rod D. Graham for his technical assistance in this study.
REFERENCES 1.
Martin, H. and C.R. Worthing.
Pesticide Manual.
(4th ed.)
British Crop Protection Council, England (1974). 2.
Canadian Forestry Service. Control Forum 1976.
3.
Report of Annual Forest Pest
Environment Canada, Ottawa (1977).
Canadian Forestry Service.
Report of the Annual Forest
Pest Control Forum 1977.
Environment Canada, Ottawa
(1978). 4.
Nigam, P.C.
Chemical Insecticides,
Forest Insects in Canada. No. Fo 23/19/1975.
in: Aerial Control of
Ed. Prebble, M.L. Catalogue
Department of the Environment,
Ottawa, Canada (1975).
602 5.
SUNDARAM AND SZETO Sundaram, K.M.S., S. Szeto and R. Hindle.
Report FPM-X-18,
Forest Pest Management Institute, Environment Canada, Sault Ste. Marie, Ontario. 6.
Swaroop, S.
P6A 5M7.
Statistical Methods in Malaria Eradication.
WHO Monograph Series No. 51.
Downloaded by [University of Florida] at 06:02 17 February 2015
Sales Unit. 7.
(1978).
McLeese, D.W.
WHO Distribution and
Geneva, Switzerland (1966).
J. Fish. Res.
Board Can. 31: 1127-1131
(1974). 8.
McLeese, D.W.
Bull.
Environ. Contain.
Toxicol.
16(4):
411-416 (1976). 9.
Sundaram, K.M.S., Y. Volpe, G.G. Smith and J.R. Duffy. Report CC-X-116.
CCRI.
Environment Canada, Ottawa
K1A 0W3 (1976). 10.
Abdel-Wahab, A.M. and J.E. Casida. 15(2):
J. Agr. Food Chem.
479-487 (1967).
Received:
March 1, 1979.
