,.

THIS PAPER NOT TO BE CITED WITHOUT PRIOR REFERENCE TO THE AUTHORS

International Council for the

Exploration of the Sea

Marine Environmental

Quality Committee

CM 1990/E:18

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THE TOXICITY OF DICHLORVOS TO SOME MARINE ORGANISMS

J.E. Thain, P. Matthiessen and S. Bifield.

Ministry of Agriculture, Fisheries and Food,

Directorate of Fisheries Research,

Fisheries Laporatory,

BURNHAM-ON-CROUCH, Essex CMO 8HA, England.

ABSTRACT

This paper presents information on the acute and chronic toxicity

to marine organisms of dichlorvos, an organophosphorus pesticide

used.to control lice on farmed salmon. The acute toxicity of

dichlorvos (tested as Nuvan 500 EC) to crustacean larvae Crangon

crangon and Mysidopsis bahia was 4.4 and 11.iug 1-1 respectively

(96h LC50). Molluscs were less sensitive (adult Patella vulgata

96h EC50 = 14.4 ug 1-1 , based on foot relaxation; larval

Crassostrea gigas 48h EC50 = 165 ug 1_1, based on abnormal

development). and marine algae were relatively insensitive (of

three species tested, the most sensitive was Isochrysis galbana,

whose growth over 20 days was normal at 100 ug 1-1 , but inhibited

at 1000, ug 1-1). Chronic growth tests were conducted with

juvenile bivalve molluscs, C. gigas, Venerupis decussata and

Mytilus edulis. The growth of the most sensitive species, C.

gigas, was significantly reduced over 49 days at nominal

concentrations of 33 ug'l-l.

INTRODUCTION

Dichlorvos is an organophosphorus insecticide, formulated as. .Nuvan 500 EC(Ciba-Geigy Agrochemicals). For many years it has

been widely used in agriculture, food storage and animal

husbandary as a contact and fumigant pesticide for controlling

insects. Nuvan was first used in the fish farming industry in

1976 (Rae, 1979) andan identical formulation (Aquagard) 'is now

temporarily licensed in the.U.K. as a fish medicine. Although sea

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lice occur as parasites on wild salmon they are present in

insufficient numbers to cause the salmon any ill effects.

However, on salmon farms the' fish are kept at high stocking

densities which promote intense sea lice,infestations. This can

adversely effect the health of the,salmon and may even cause, , .

death (see resume of the use of Nuvan in the salmon farming

industry by Ross and Horsman, 1988). Treatment with Aquagard

appears to be the most successful method of control currently

available for sea li6e in salmon farms. However, the'application

techniques result in large quantities of the pesticide entering

the marine environment, often in areas such as 'sea' lochs which

have a poor water exchange with the open sea. Clearly it is

essential to establish whether Aquagard adversely affects non­

target organisms. Little toxicity information exists in the

literature, particularly for species other than crustaceans."

Therefore several bioassays werecarried out to investigate acute

and chronic effects of Nuvan'on a range of marinespecies.

1. EFFECTS ON THE GROWTH OF JUVENILE BIVALVEMOLLUSCS

Three commeicially~important species of molluscs Crassostrea

gigas, Mytilus edulisand Venerupis(Tapes) decussata were each

exposed to six concentrations of dichlorvos (as Nuvan) in a flow

through dosing system for 49days.

The experimental system provided six dosing treatments at nominal

concentrations of O(control); 3.3, 10, 33~ 100 and 330 ugl-1

dichlorvos. Dichlorvos was dosed as Nuvan(containing~500g ..

dichlorvos 1-1 ), Ciba-Geigy batch no 0891CA. Stock concentrations

were prepared in distilled water and renewed daily. An upwelling

column containing the exper~mental animals was placed at the

outflow of each treatment. The water flow through each column was

maintained at a constant 200 ml min-1 and contained added algal

food at concentrations of >25 cell ul-1 of a mixture of

Tetraselmis suecica, Isochrysis galbana and Thalassiosira

pseudoriema. Throughout the experiment the water temperature was

maintained at 18.5+/- l oC.

C. gigas '(0.40g), M. edulis (0.30g)and V. decussata' (l'.4g) spat

were conditioned to experimental conditions over 10 days. Ten

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animals were then placed on one of three tiers of 3.0 mm nylon

mesh in an upwelling column. At the start of the experiment the

weights of individuals were recorded. Subsequently the

measurements were recorded at weekly intervals during the 49-day

exposure period.

No mortalities occurred in any of the treatments. However, it was

observed that the clams,·V.' decussata were severely gaping at,- "

concentrations of 100 and 330 ugl-1 from week two to the end of

the experiment. Even when roughly handled the gaping persisted.

c. gigas: Figure 1a shows a growth of 550% in the control

treatment over the 49 day exposure period, typical of the growth

normally achieved in aquaculture operations. Good growth was also

shown in the 3.3 and 10 ug 1-1 treatments. At concentrations of

33, 100 and 330 ug 1-1 growth occurred but was significantly

(P<0.05 Students 'tl-test; on final ,weights)' reduced relative to

the controls. Furthermore, a dose-related reduction in growth was

observed, the 330 ugl-1 treatment showing the least weight

increase, 139% of the day 0 weight. Interestingly,' dichlorvos did

not prevent the growth of oysters at any time during the exposure

period and growth rate, although reduced in some treatments was

always linear. This suggests that the oysters were able to

eliminate and/or metabolise dichlorvossuch that during the 49

days exposure sufficient bioconcentration to cause afall-off in

tt growth-rate did not occur: Since dichlorvos acts by inhibiting

the activity of the enzyme cholinesterase in the cholinergic .

nervous system it is also possible that the reduced growth-rate

may be a direct effect on a specific physiological process ego

feeding rate.

V. decussata: A growth of between 43-58% was measured in the

controls and low level treatments over the 49 day exposure period

(Figure 1b). This was much lower than occurred·in oysters.

However,clams are much slower growing animals than oysters and in

. this study were three times larger than oysters on day O. In

treatments up to 100 ug 1-1 growth was equal to that of the

controls and at 10 and 33 ug 1-1 significantly (P<0.05) better

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than the controls. Only at 330 ug 1-1 was there a significant

(P<0.05) reduction in growthi 50% of the control value. '

M. edulis. Figure 1c shows the.growth of musseIs exposed to a

range of concentrations of dichlorvos over 42 days. A wide range

in growth (128%-256%) was measured in the controls and treatments

up to 100 ug 1-1 . At concentrations of 10 and 33 ug 1-1 there was

a marked increase in growth relative to the controls, a similar

response to that observed for clams. There is no clear

experimental cause for these observations. However, similar

effects of enhanced growth in organisms exposed to low

concentrations of chemical compounds have been described by

Stebbing (1982) and are known as hormesis. Growth inmussels was

significantly (P<0~05) reduced (approximately 50% of control

value) only in the highest concentration, 330 ug 1-1 .

At the start of this study no chemical analytical support could

be provided. Therefore, quoted concentrations are nominal and

based on the measured volume of stock solution used each day

relative tothe measured water flow. Towards the end of the

experiment two water sampIes were taken from the inflow of each

upwelling column of each treatment. Chemical analysis for

dichlorvos showed that in every treatment the actual .exposure

concentration was one-tenth the nominal concentration.

This suggests that the significant reduction in the growth of

oyster spat was a result of exposure to concentrations of around

3.3 ug 1-1 dichlorvos. Similarly, for musseland cla~ spat

significant reduction in growth.was the result of exposure to

dichlorvos at concentrations of around 33 ug 1-1 •

2. Effects on three marine algal species.

Three species of algae T. suecica, ~ galbana and T. pseudonema

origanally obtained from the Culture Centre of Algae and Protozoa

were initially cultured in Erdschreiber medium and artificial

media (Walne 1966). For each:trial algal cells were taken during

the exponential growth phase of these parent cultures and added

to the experimental culture flasks to give a starting

concentration of 50 cells ul-1 • The sea water used in the

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experiments was filtered to 0.2um and sterilised together with

the glassware. A stock concentration of Nuvan was prepared at

1000 ug dichlorvosl-1 in sterile sea water and subseque~tly

serially diluted in volumetric glassware to concentratins of 100,

10, 1 and 0.1 ug 1-1 • The control contained sterile seawater.

Three replicate 500ml culture flasks, were used for test

treatments. 250ml of the stock and each dilution was added to the

500ml culture flasks. No nutrients were added during the trial.

At the start of the experiment the culture flasks were placed on

a shaker under continuous fluorescent lighting. At frequent

intervals 1.0 ml sampIes were taken from each culture flask and

algal density determined using a haemocytometer.

Changes in cell'density with time in the three species are shown

in'Figures 2a-c (only data for the control and top three

concentrations are shown). Logarithmic growth was exhibited in

the control of each species tested. The growth of the flagellate

T. suecica was unaffected by 1000,ugl-1 , the highest

concentration' tested. However the flagell'ate ~ galbana showed no

growth at 1000 ug 1-1 and by day 7 all cells had died; at a

concentration of 100 ug 1-1 growth was similar to the controls.

At 1000 ug 1-1 growth of ~ pseudonema was inhibited (algistatic)

during the first 6 days of exposure but thereafter growth rate

recovered and was similar to the controls; at a concentration of

100 ug 1-1 growth was similar to the controls.

It is clear that in comparison to other phyla,algae appear to be

insensitive to dichlorvos. However,. it must be borne in mind that

even closely related species of algae can show a wide range of

response to a'chemical compound. Therefore, species otherthan

those tested here may be shown to be sensitive to lower

concentrations of dichlorvos. Furthermore, as is the standard

practice for this type of test, thetreatments were not changed

during the test and exposure concentrations were nominal.

3. ACUTE TOXICITY TO OYSTER, SHRIMP AND MYSID LARVAE

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Crangon crangon and Mysidopsis bahia: Shrimp larvae were obtained

by conditioning adults to spawn in the laboratory. Juvenile

mysids «24 hold) were obtained from a laboratory-held culture.

Tests were carried out on newly released larvae using 10 larvae

in each 50 ml test container. Test solutions were changed daily.

A stock solution of 1000 ug 1-1 was prepared daily and serially

diluted to provide a range of test concentrations. Throughout the

experiment both species were fed daily on newly hatched artemia.

Results of toxicity tests of dichlorvos on C. crangon and M.

bahia.

LC50 ug dichlorvos 1-1

1

Time hours

C. crangon

48

13.8

96

4.4

120 140

2.2

168 NOEC 96h

3.3

M. bahia 18.6 11.1 10.3 10.0 10.0

C. gigas: Pacific oyster embryos were obtained by conditioning ..

adults to spawn in the laboratory. Tests were then carried out on

these embryos using the method described by Thain and Watts

(1987) •

Embryonic development during a 48 h exposure p~riod was not

affected at and below a concentration of 50 ug 1-1 dichlorvos. At

165 ug 1-1 , the highest concentration tested, 30% of the embryos

showed abnormal development.

4. ACUTE TOXICITY TO ADULT LIMPETS

Adult limpets (Patella vulgata) were collected from coastal chalk

beds near Brighton where the animals can be prised off the

substrate withoutdamage. In the laboratory, the limpets were

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,allowed to cling to Perspex plateswhich were then suspended in

. static exposure tanks. The nominal dichlorvos concentrations

tested (prepared using Nuvan 500) were 0.03, 0.1,0.3, 1.0, 3.0,

10, and 30 ugl-1 ; eac? concentration being replicated twice with

20 limpets. in each replicate. Fresh dichlorvos solutions were

made up daily, and water quality remained good throughout

·(temperature 14.7-17.7oC, pH 7.7-7.8, dissolved oxygen 7.1-9.6 mg

1-1 arid salinity >32 ppt). The·measure.of effect~as a relaxation

of the foot which prevented the animal clamping tightly to the

Perspex when genttY tapped. In severely.affected cases the foot

elongated considerably and the animal finally dropped off the

plate (these were not replaced). Although affected animals were

not dead, foot relaxation behaviour of this sort under natural

conditions would rapidly lead to death from predation and wave

action.

The 24, 48, 72 and 96h EC50 values were 26.4, 24.7, 12.8 and 14.4

ug 1-1 • The 96h value was slightly higher than the 72h figure due

to a 'small (non-significant) improvement iri the clinging

behaviour of remaining animals. The 96h no observed effect

conc~ntration(NOEC) was 3.0 ug 1-1 •

The phenomenon of limpet foot relaxation in dichlorvos solutions

was first observed by C.B •.Duggan, Fisheries Research Centre,. .

Dublin 15,Ireland, (pers. cornm. 1988) using ~ vulgata attached

.. to stones which were moved into the laboratory for testing.

However, using a similar measure of effect, Duggan found that

nominal dichlorvos concentrations (made up with Nuvan 500) aslow

as 0.1 ug 1-1 caused foot relaxation. The reason for this

disparity is not known, although the data cannot be directly

compared because Duggan did not conduct a formal EC50 test, nor

did he renew the dichlorvos solutions daily.

CONCLUSIONS

. It is clear that crustacea are more susceptible to dichlorvos

than molluscs. Acute toxicity values for the crustacea tested lay

between 2 'and .10 ug 1-1 (nominal), revealing' sensitivity similar

to that for the same species exposed to tributyl tin (TBT) , one

of the most toxic compounds known to marine organisms. However,

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molluscs appear to be much.more susceptible than previously.

thought. Published:data on molluscs (Ghetti et ale 1989, quoted

in a confidential report by Jones and Zabel, 1989) concern

Nassarius obsoletus where 96h exposure to dichlorvos inhibited

feeding at 25,000 ug 1-1 , and C. virgini~~ where the 96h

NOEC(based on normal shell growth) was 1000 ug 1-1 . It is

presumed that the tested animals were adults. Further published

d~ta on molluscs (Egidius and Moster, 1987) has shown that adult

M. edulis are also insensitive to' dichlorvos (as Nuvan), with all

animals surviving. a 24h exposure to 1000ug 1-1 • Not

unexpectedly, larval molluscsappear to be much more susceptible

than the adults.

These results show that the most sensitive organism tested was

the larval shrimp Crangon crangon (140 h'LC50 = 2.2 ug 1-1 ). Data

on other sensitive species are required before a reliable

environmental quality standard (EQS) can be determined, but the

present data suggest that the chronic EQS probably lies below 0.2

ug 1-1 . The only published field study from the vicinity of a

salmon farm (Tully·and Morrissey, 1989) found dichlorvos at 0.14

ug 1-1 in one seawater sample from BeirtreachBui Bay, Ireland,

but other samples were below the detection limit (0.02 ug 1-1 ).

Further field data will be needed before the true risks of

dichlorvos to marine life can be assessed.

REFERENCES

Egidius, E. and Moster, B1987. Effect of Neguvon and Nuvan

treatment on crabs (Cancer pagurus, ~ maenas), lobster (Homarus

gammarus) and bluemussel (~edulis). Aquaculture 60, 165-168.

Jones, A. and Zabel, T.F., 1989. Proposed provisional

environmental quality standards for dichlorvos in water. Water

Research Centre (Marlow, Bucks., SL7 2HD U.K.) Confidential

report No DOE 2249-M, 26pp + appendices.

Rae, G.H., 1979. On the trail of the sea lice. Fish Farmer 2, (6),

22-25.

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Ross, A. and Horsman, P.V., 1988. The use of Nuvan 500 EC in the

salmon farming industry. Marine Conversation Soeiety, 9b,

Gloueester Road, Ross-on-Wye, Herefordshire. HR9 5BU.

Stebbing,A.R.D.,1982. Hormesis-the stimulation of growth by low

levels of inhibitors. Sei. Total Envir. 22,213-234.

Thain,J.E. and Watts,J.,1987. The use of a bioassay to measure

changes in water quality associated with a bloom of Gyrodinium

aureolum Hulbert. Rapp.P.-v. Reun. Cons. int Explor. Mer, 187­

107.

Tully,O. and Morrisey,D.,1989. Coneentrations of diehlorvos in

Beirtreaeh Bui Bay, Ireland. Mar. Poll. Bull. 20, 190-191.

Walne,P.R.,1966. Experiments in the large seale eulture of the

larve of Ostrea edulis L. Fish. Invest., Lond. Sero 2,25, 1-53.

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FIG. 1a

0/0 WEIGHT INCREASE OF C. G IGAS EXPOSEO TO OICHLORVOS

.-Control, ·D:-3.3~ .-10,0-33, .6-100, 6-330"'91"

•

7654321

04--...A~"'I""""''-'-.L.I.---,...----.........---....-----...------r---....---o

50

100

500-

550

w 35Cf)

Cl:wa:uz~

:J:

"w3:~0

TIME (weeks)

FIG. 1b

0/0 WEIGHT INCREASE OF v. DECUSSATA EXPOSED TO DICHLORVOS

• - Control, 0 - 3·3, • - 10, 0 - 33, • ·100, l::,. - 330 .,g I·

70

60

50

wCf)

<wa::(.)z 40

~

:t:C)

w3:

~ 300

20

10

o .o 1 2 3 4 5 6 7

TIME (weeks)

....---------------------------

'. ,~.

FIG. 1c

% WEIGHT INCREASE OF M· EDULI S EXPOSED TO DICHLORVOS

• - Control, 0 -3.3, • - 10, <> - 33, A. -100, 1::::. - 330 "'9 r

•

7654310;--""-""1r'---.,...---~--.....,.---....._--__r---_-

o

40

20

60

120

200

w 140Cf)

«wa:uz

TIME (wee ks)

•

FIG. 2a

EFFECT OF DICHLORVOS ON THE GROWTH OF MARINE

ALGAE

TETRASELMIS SUECICA

• - Control, • - 10, • - 100, 6. - 1000 PS '"

I1000

•• ~

• • 6.

•6. 6.

•••~.. •GIQ.

•Cf)

I-Z

6.::J0()

100...J<C)...J

<

10987654321

104----r---r-----,----r---r---,---r----.---r-----,

o

TIME (oays)

..

. \

FIG. 2b

EFFECT OF DICHLORVOS ON THE GROWTH OF MARINE

ALGAE

ISOCHRYSIS GALBANA

.-Control, +- 10, .- 100, "'_1000 P9 I·

+ ••1000 ••

•+• •+

-::..... •CIlC.

Cf) •~

z=>0 •U

100 •...I •<Cl...I

<

10987654321

104---__--.------.--....,..--"""T""--......----:ik-----.--------o

TIME (oays)

10

a•

9

•

87

•••

6

••

5432

TIME (Days)

THALASSIOSIRA PSEUDONEMA

• - Control, • - 10, • -100, .... - 1000"'9 I-

1

FIG- 2c

EFFECT OF DICHLORVOS ON THE GROWTH OF MARINE

ALGAE

10-+---.....---...------..----r----r---r-----,r---.....,..---r---,o

1000

:::l.....CIlQ.

CI) •~

z •~ •0CJ

100..J •< ,CJ..J

< •

..