The bioconcentration of zinc by Gammarus pulex (L.) and the application of a kinetic model to determine bioconcentration factors

Wat. Res. Vol. 27, No. 11, pp. 1683-1688, 1993 Printed in Great Britain. All rights reserved

0043-1354/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

THE BIOCONCENTRATION OF ZINC BY G A M M A R U S PULEX (L.) A N D THE APPLICATION OF A KINETIC MODEL TO DETERMINE BIOCONCENTRATION FACTORS QIN Xu* and DAVID PASCOE• School of Pure and Applied Biology, University of Wales College of Cardiff, P.O. Box 915, Cardiff, Wales (First received December 1991; accepted in revisedform March 1993) Abstract--The bioconcentration, and subsequent elimination, of zinc by the freshwater amphipod Gammarus pulex (L.) were determined experimentally in the laboratory at five zinc exposure concentrations (0.41-2.02 mg/l). A first-order kinetic model, modified for naturally occurring trace chemicals, was then used to calculate uptake (K t ) and elimination (K2) rate constants from which the bioconcentration factors (BCF) could be predicted. Good agreement was found between the recorded and predicted BCFs and it is suggested that the model could have wider use for the study of substances naturally present at trace levels within animal tissues.

Key words--Gammarus pulex (L.), zinc, bioconcentration, toxicity, kinetic model

INTRODUCTION

model. G. pulex is a freshwater detritivorous am-

Although there have been many reports o f zinc uptake from water by freshwater fish and invertebrates, only a few of these studies provide information on bioconcentration at the steady-state and so help to explain the dynamics and fate o f zinc in

phipod occurring widely in Europe and providing an important source of food for fish. It is also a key species in a number of biotic indices for evaluating water quality and is increasingly used in laboratory and field bioassays for detecting and monitoring freshwater pollution ( M c C a h o n and Pascoe, 1988; Poulton and Pascoe, 1990). A future publication will explain the use of a

exposed organisms. The bioconcentration factor (BCF), a value that represents the concentration ratio of a chemical in an organism to that in water, at steady-state, can be determined in two ways. One method is to expose organisms for a sufficiently long period that the steady-state tissue concentration is actually observed, while the other relies on the accuracy of a kinetic model to measure uptake and elimination rate constants which can then be used to calculate the B C F assuming the chemical behaves according to the model. Since the first procedure may not only be time consuming but there are also difficulties in determining when and at what level the steady-state has been attained, the latter method appears to be more economical and accurate as it requires only a short period of exposure and relies upon actual data of both uptake and depuration to calculate the bioconcentration factor (Hamelink, 1977; Veith et al., 1979; Lohner and Collins, 1987; Walker, 1990). In this investigation the bioconcentration of zinc by Gammarus pulex (L.) from water was studied with the aid of a two-compartment (animal-water) kinetic *Present address: Department of Applied Biology and BiDtechnology, DeMonffort University, Scraptoft, Leicester LE7 9SU, England.

three-compartment model to describe the bioaccumulation (i.e. bioconcentration from water together with biomagnification via food) of zinc by G. pulex. METHODS Specimens of G. pulex collected from a tributary of the River Ely were acclimated to laboratory conditions for 1 week before the test started. The experiment was carried out using a diluter flow-through dosing system which provided 95% replacement of test solutions every 3h. Five nominal concentrations of 0.4, 0.8, 1.5, 1.8 and 2.0 mg Zn/1 and a control (dilution water) were set up, and the test solutions were run through the system for a week to establish equilibrium prior to the start of the test. Water samples were taken daily to monitor zinc concentrations and other water quality parameters (Table I). Male and non-gravid female G. pulex of body length >10mm were selected for the test and kept in plastic containers, with mesh bottoms, suspended in each exposure aquarium. 400 animals were exposed to each of the three high concentrations (mean recorded values: 1.56 + 0.05, 1.78 + 0.06, 2.02 _+0.02 mg/l), and 300 to each of the two lower concentrations (mean recorded values: 0.41 + 0.05, 0.84 + 0.06mg/1). During the experiment, G. pulex were fed horse chestnut (Aesculus hippocastanum L.) leaf discs (diameter = 8 mm, 1 leaf disc/10 animals) which were replaced daily to avoid zinc accumulation from food. At the end of the uptake test, surviving animals were rinsed with

1683

1684

QZNXu and DAVmPASCOE Table 1. Mean recorded water qualities with

Changes of concentration in the animal with time are

standard errors throughout the experimental

period

Parameters

described by the differential equation:

Mean _+SE

Temperature (°C)

(2)

d C ^ / d t = Kj C w - 1(2 CA

II + 1

pH Conductive(#s/cm)

Hardness as CaCO3(mg/l)

7.70 _+0.11 284.9 + S.2 109.3 + 1.4

dechlorinated tap water transferred to control water for the elimination study, Animals were taken from zinc analysis at frequent intervals throughout the test. On each sampling occasion three replicates, each containing three animals, from each treatment were dried at 95°C for at least 48 h before being weighed and digested in 0.5 ml AristaR nitric acid in a block thermostat (95°C) overnight and then prepared for analysis, using atomic absorbance spectrophotometry, by redigesting in I0 ml of 5% AristaR nitric acid.

However, as zinc is essential for life, a basal level (Co) of 0.0933 + 0.0242 mg/g (dry weight of the animal) was detected in G. pulex by analysing samples of the control animals, and therefore, the above firstorder kinetic model was modified to provide a suitable model for the bioconcentration of a trace element naturally occurring within tissues: x~ Cw ~ C A - Co K2 d C A / d t = Kl Cw - K2(CA -- Co)

A two-compartment model (water-animal) can be used to describe the movement of a substance in and out of an animal and if the concentration of the chemical in the animal is zero (CA = 0) when

With the initial conditions of t (time) = 0, CA = Co,

Kt CA = C0 +~-~z x C w ( l - e -x2,)

(5)

for the elimination period

the test starts (t = 0), the reaction can be presented as

CA =

Co + (CA.m - -

C0) e-X2t

(6)

where

Ki

Ic2

(4)

and Cw = constant, the solutions of equation (4) are: for the uptake period

MODEL: ASSUMPTION

Cw ~

(3)

CA

(1)

K,

Ca,m= Co + ~2 X Cw(l _e-K2r )

where Cw = concentration of chemical in water (rag/l) CA = concentration of chemical in animal (mg/kg) Kt = rate constant of uptake ( h - ' ) K2 = rate constant of elimination (h-t).

2.5

=concentration at the end of uptake period T = time at the end of uptake period. As the exposure time (t), approaches infinity, from equation (4), the bioconcentration factor will be

[Zn] mg/I

2

x

x ----x

o

~

1.5

1

-~._.._m1_.m--- ~

~m---'------~

0.5 0

0

I

I

I

I

I

i

t

i

2

4

6

8 Day

10

12

14

16

2.02mg/I

I

1.78mg/I

"-~1.56mg/I

D

0.85mg/I

×

0.41mg/I

Fig. 1. Mean recorded zinc concentrations in exposure tanks throughout the experiment.

Zinc bioconccntration by Gammarus pulex

(Zn]/Dry Animal

1685

Weight (rag/g)

0.3

0.250.150.2

t

~[Zn]-O.41[mg/I

I

0.05

,

Uptake

[,

,

Elimination

,

I 0

0

I

I

I

I

I

I

I

I

I

|

I

I

I

2

4

6

8

10

12

14

16

18

20

22

24

26

Time T 95% CI

(Zn)/Dry

Animal

(days)

× Observed

Weight

• Predicted

(rag/g)

0.4

0.85 mg/I

[Zn] -

0.3 0.2

~

0.1

~

, 0

Uptake I

I

I

I

. I

,

I

Elimination I

I

I

, I

I

I

0 2 4 6 8 10 12 14 16 18 20 22 24 26 Time

T 95% cI

(days)

x Observed

Predicted

Fig. 2. Bioconcentration of zinc by G. pu/ex during and after exposure to 0.41 and 0.85 mg Zn/1--a comparison of observed with 95% CI and predicted data.

obtained from the uptake and elimination rate con- where stants: BCF = CA,~/Cw = K]/K: + Co/Cw

(7)

CA,s= concentrationof chemicalin animal body at steady-state.

I

I

95% CI

4

I

6

e

I

x Oblerved

I

n

, I

I4

0

I

2

4 95% CI

_~ i

0,2

0.3

o

i

× Observed

6 Time (days) ~

I

8

I

~

T 95% CI

4

I

~

10

i

• 2.02 mg/I

2

Predicted

[Znl

/

Uptake

I

l

,

i

x Oblerved

12

I

e

I

Predicted

io

1~

I

[ Z n ] - 1.78 mg/I

E l i m i n a t i o n ,,

Time (days)

6

I

17.n)/I~¥ Animal Weight (m0/g)

Elimination

[Znl/Dry A n i m a l WeiGht ( n a g / g )

O.li m U p t a k e

0.2

0.3

0.4

0.5

0.6

Predicted

1o

I

Elimination

Time (days)

....

[ Z n ] - 1.56 mg/i

0,4

,

Fig. 3. Bioconcentration of zinc by G. pulex during and after exposure to 1.56, 1.78 and 2.02 mgZn/l--a comparison of observed with 95% CI and predicted data.

2

Uptake

[Zn}/Dry A n i m a l WeiGht ( r a g / g )

o

0.1!

0.2

0.3

0.5

I

14

--

"~

z

0,,

Zinc bioconcentration by Gammarus pulex Table 2. Uptake (KJ and elimination (K2) rate constants (mean _+SE) and BCF for animalsexposedto eachzincconcentration(Cw) Cw(mg1-I) Kj(h-I) K2(h-I ) BCF 0.41 5.03 ± 0.8 0.01725± 0.003 519 0.85 3.38 + 0.3 0.01521± 0.001 332 1.56 3.07 _+0.3 0.01426± 0.001 275 1.78 2.54+0.3 0.00983±0.002 311 2.02

3.73 ± 0.5

0.01630 ± 0.002

275

RESULTS AND DISCUSSION

Water quality parameters remained relatively constant throughout the experimental period (Table 1) and zinc concentrations showed very little variation (Fig. 1). Due to the mortality, the bioconcentration tests lasted for 3.5 days at the highest concentration but for 15 days at the lowest concentration (Figs 2 and 3). The whole body burden of zinc of G. pulex (mg Zn per gram animal dry weight) recorded during the uptake and elimination phases of the study is shown in Figs 2 and 3 as the mean of three replicates with 95% confidence levels. A non-linear extended least squares regression programme was employed to fit the experimental data to the kinetic model described in equations (5) and (6) so that uptake and elimination rate constants, K~ and K2, could be obtained (Table 2). This allowed the prediction of whole body burdens at different times during the exposure and elimination phases (Figs 2 and 3) and the calculation of BCFs according to equation (7). Bioconcentration curves for animals from five zinc exposure tests are presented in Figs 2 and 3. Bioconcentration is clearly

1687

related to the exposure time and the zinc concerttration of the surrounding water (Fig. 4). Within 15 days exposure to zinc, at concentrations of 0.41 and 0.85 mg/l, the actual whole body burden of zinc determined experimentally in G. pulex reached a steady-state, and returned to the control level within 1 ! days after the animals were transferred to control water for elimination (Fig. 2). At concentrations of 1.56, 1.78 and 2.02 mg/I (Fig. 3), bioconcentration reached 60-75% of the steady-state level, C^.s [0.43, 0.55 and 0.45 mg/g, respectively, as predieted from equation (5)]. After 8 days of elimination, 80-85% clearance from the maximum experimentally recorded concentrations was achieved. Although many workers have used predictive models in attempts to describe the uptake and elimination kinetics of lipophilic organic toxicants by aquatic animals (Neely, 1979; Veith et al., 1979; Ktnemann and van Leeuwen, 1980; Hawker and Connel, 1986), few have employed this technique in studies of the hydrophilic heavy metals. However, van Hattum et al. (1989) used a first-order bioaccumulation model to describe the uptake of cadmium by Asellus aquaticus from food and water and reported good agreement between the predicted and measured cadmium concentrations. It was also noted that water rather than the food was the major source of cadmium. Timmermanns (1991) also reported that a first-order kinetic model gave a good description of cadmium, zinc, lead and copper uptake by fourth instar larvae of the midge Chironomus riparius (Meigen).

Body Burden of Zinc (rag/g) 0.35

0.3 ,

0.25

,-,iIiiiil. .................. ,_ii .iiii i;ii; ... I •

0.15 0.1 0.05 0

I 0

0.5

T

95% CI

I 1 Concentration []

12 hours

J

I

1.5 of Zinc in Water (rng/I) '~-48

hours

--~-72

2

hours

Fig. 4. The relationship between zinc uptake (with 95% CI) and exposure time, for G. pulex exposed to five zinc concentrations.

2.5

1688

QIr~ Xu and DAVIDP~coE Table 3. Z2 goodness-of-fit test for the adequateness of the kinetic model Cw(mg I-~) d.f. Z2 X02~s 0.41 13 0.026 < 3.565 0.85 14 0.023 < 4.075 1.56 11 0.041 < 2.063 1.78 9 0.049 < 1.735 2.02 9 0.090 < 1.735

In the present investigation with G. pulex the tissue concentrations o f zinc predicted from the model and those recorded in the experiment were not significantly different (Table 3) and it can be concluded that the first-order kinetic model employed in the study gives a good indication of zinc bioconcentration by G. pulex. It is likely that the model will also be applicable to bioconcentration studies with other chemicals which occur normally at trace levels in animal tissues.

Acknowledgements--We are grateful to Dr Zhi Zhang and Dr Glyn Taylor for advice on the use of kinetic models. This work was supported by the British Council and the Education Ministry of the People's Republic of China. REFERENCES Hamelink J. L. (1977) Current bioconcentration test methods and theory. In Aquatic Toxicology and Hazard Assessment STP 634 (Edited by Mayer F. L. and Hamelink J. L). ASTM, Philadelphia, Pa.

van Hattum B., De Voogt P., van den Bosch L., van Straalen N. M. and Joose E. N. G. (1989) Bioaccumulation of cadmium by the freshwater isopod Asellus aquaticus (L.) from aqueous and dietary sources. Envir. Pollut. 62, 129-151. Hawker D. W. and Connel D. W. (1986) Bioconcentration of lipophilic compounds by some aquatic organisms. Ecotoxic. Envir. Saf I1, 184-197. K6nemann H and Van Leeuwen K. (1980) Toxicokineties in fish: accumulation and elimination of six chlorobenzenes by guppies. Chemosphere 9, 3-19. Lohner T. W. and Collins W. J. (1987) Determination of uptake rate constants for six organochlorines in midge larvae. Envir. Toxicol. Chem. 6, 137-146. McCahon C. P. and Pascoe D (1988) Use of Gammarus pulex (L.) in safety evaluation tests: culture and selection of a sensitive life stage. Ecotoxic. Envir. Saf 15, 245-252. Neely W. B. (1979) Estimating rate constants for the uptake and clearance of chemicals by fish. Envir. Sci. Technol. 13, 1506-1510. Poulton M. J. and Pascoe D (1990) Disruption of precopula in Gammarus pulex (L)--development of a behavioural bioassay for evaluating pollutant and parasite induced stress. Chemosphere 20, 403-415. Timmermans K. R. (1991) Cadmium, zinc, lead and copper in larvae of Chironomus riparius (Meigen) (Diptera: Chironomidae): uptake and effects. In Trace Metal Ecotoxicokinetics of Chironomids, Chap. 4. Unpublished Ph.D. thesis, University of Amsterdam. Veith G. D., de Foe D. and Bergstedt B. V. (1979) Measuring and estimating the bioconcentration factor of chemicals in fish. J. Fish. Res. Bd Can. 36, 1040-1048. Walker C. H. (1990) Kinetic models to predict bioaccumulation of pollutants. Funct. Ecol. 4, 295-301.