J .A. Lindley et al. J. Exp. Mar. Biol. Ecol. 242 1999 59 –74
61
mid-estuarine species, and Acartia spp., mainly A . bifilosa which overlaps with E. affinis
but is more abundant at higher salinities e.g., Collins and Williams, 1981. These species dominated our plankton samples from the Exe and Tamar estuaries. E
. affinis carries its eggs in a sac. Diapausing eggs are shed after a few days but eggs may reach
the sediment also following predation on the egg carrying females, which are more conspicuous to predators than those without egg sacs Webb and Weaver, 1988.
Conway et al. 1994 and Flinkman et al. 1994 found that the eggs of E . affinis remain
viable after passage through fish guts. Acartia spp. spawn freely and the eggs sink.
2. Methods
2.1. Source of eggs Plankton samples were taken with a hand net of 0.5 m opening and 100 mm mesh
size. Samples were taken by streaming the net in the tidal current of the near surface waters mainly from a pontoon jetty on the Exe estuary, Devon, England. This was near
to the West Muds site described by George and Lindley 1997. Samples were taken here in April and October 1994 and May 1996. These samples were supplemented during
June and July 1996 by sampling from the shore in the Tamar estuary, again streamed in
21
the tidal current, or towed at about 1 m s for about 10 min by research vessels in
Plymouth Sound. The dates of sampling are given in Tables 1–3. Small quantities of the plankton samples were poured or transferred using a wide
mouth pipette into Petri dishes. When Acartia spp. were numerous they were concen- trated in the original sample by exploiting their positive phototaxis e.g., Stearns and
Forward, 1984. Later on the same day or on the following day adult female calanoid copepods were transferred, using Pasteur pipettes, to 0.1mm filtered sea water FSW in
Petri dishes. They were incubated in the FSW overnight |16 h. The eggs of Acartia spp. and Eurytemora affinis with egg sacs extracted were removed with pipettes. The sea
water was collected off the Eddystone Rocks 508109N, 48169W in the English Channel about 15 km from the nearest land. It has a salinity of |35‰ and the concentrations of
heavy metals and anthropogenic organic pollutants are much lower in these offshore waters than in most estuaries Marine Pollution Monitoring Management Group, 1998.
All handling of the copepods and eggs in this and subsequently described procedures was performed at room temperature approx. 208C.
Table 1 Effect of initially saturated DCB solution on hatching of Eurytemora affinis eggs; females collected 18 April
1994. FSW, filtered sea water Treatment
Eggs Hatch
Viable Control FSW throughout
132 5 .9961
.9961 DCB solution throughout
209 5 ,161
,161 FSW overnight, washed, incubated in FSW
112 3 100
100 DCB overnight, washed, incubated in FSW
239 3 36653
36653
62 J
.A. Lindley et al. J. Exp. Mar. Biol. Ecol. 242 1999 59 –74 Table 2
21
Effect of saturated PCP solution in FSW with acetone carrier and of acetone carrier 50 ml l solution in
FSW only on hatching of Acartia bifilosa eggs and viability of nauplii produced. Values in parentheses for acetone solution exclude the replicate in which eight eggs adhered. Eggs were from adult females sampled 11
October 1994. In each case except for bulk experiments there were three replicate each with about five eggs Treatment
Eggs replicates Hatch
Viable Control FSW throughout
15 3 68628
68628 PCP solution throughout
15 3
21
Acetone solution 50 ml l throughout
18 3 6065 7062
3961 4060
a
Control FSW throughout Bulk 50 2
6961 28.06,1
PCP solution throughout Bulk 20
21
Acetone 50 ml l throughout Bulk
20 90.0
65.0 FSW overnight, washed, incubated in FSW
13 3 86612
6164 PCP overnight, washed, incubated in FSW
15 3 Acetone overnight, washed, incubated in FSW
15 3 7462
6762
a
Two experiments, one with 20 eggs one with 30 eggs.
2.2. Egg incubation experiments with saturated solutions Eggs produced by the females were used in experiments carried out in Petri dishes
that were fitted with glass cover dishes but not sealed. Eggs were treated in the following
21
ways: 1 incubated in a saturated solution of DCB or PCB, the latter with 50 ml l acetone carrier solvent; 2 incubated in FSW; 3 incubated in a saturated solution of
the contaminant overnight, washed in FSW then incubated in FSW; and 4 incubated in FSW overnight, washed in FSW then incubated in FSW. When PCP solutions were used
in treatments 1 and 3, two further treatments were used to test for the effects of the acetone carrier: 5 incubated in FSW with carrier; and 6 incubated in FSW with
carrier overnight, washed in FSW then incubated in FSW. The incubations were in Petri dishes in constant light at 158C except for the initial overnight stages of treatments 3 and
4 where the samples were kept at 58C in the dark to minimize the chance of hatching during this stage. The Petri dishes were examined on the day following the start of the
incubation and then at 1–3 day intervals afterwards to find the numbers of nauplii that hatched, and whether they were still alive when examined. These data were used to
calculate percentage hatch and percentage viability, the means and standard deviations of which were calculated from arcsine-transformed data.
The concentrations of the chemicals in the dishes were monitored by ultra-violet UV spectrophotometry at 322 nm for PCP and at 269 and 277 nm for DCB. The saturated
concentration, based on comparison with UV absorbance calibration curves prepared
21
using standard solutions of PCP, was 14 mg l . This is the same figure as quoted by
Howard 1991 for the aqueous solubility of PCP. The solubility of 1,2-dichlorobenzene
21
in pure water at 258C is 91 mg 1 Miller et al., 1984. Since the solubility of organic
chemicals in sea water is usually less than in freshwater, this calculated mass value could be an overestimate by approximately 25.
2.3. Egg incubations in low concentration solutions A series of experiments was carried out on the effects of a range of concentrations of
J .A
. Lindley
et al
. J
. Exp
. Mar
. Biol
. Ecol
. 242
1999 59
– 74
63 Table 3
Experiments with calanoid eggs in a range of initial concentrations of PCP and DCB. Dates, sampling locations with temperature, T, and salinity, S, where available species Eurytemora affinis, Acartia bifilosa or Acartia clausi , numbers of replicates and numbers of eggs
Start date: 10 5 96
Exe T 5 13.28C, 14 5 96
Exe 16 5 96
Exe
S 5 16–23‰
Species: E
. affinis PCP
E . affinis
DCB E
. affinis PCP
Replicates Total
Replicates Total
Replicates Total
eggs eggs
eggs 10
2 74
2 47
2 37
5 2
55 2
44 2
50 1
2 56
2 50
2 57
0.5 2
70 2
28 2
47 0.1
2 89
2 36
2 43
FSW 2
63 2
33 2
50 Start date:
4 6 96 Tamar
11 6 96 Tamar
11 6 96 3 7 96
Plymouth Sound T 5 13.6–14.38C,
S 5 35‰
Species: A
. bifilosa PCP
A . bifilosa
PCP A
. bifilosa DCB
A . clausi
DCB in vials Replicates
Total Replicates
Total Replicates
Total Replicates
Total eggs
eggs eggs
eggs 10
3 35
3 30
3 30
3 20
5 3
25 3
29 3
26 3
25 1
3 31
3 27
3 27
3 20
0.5 3
25 3
30 3
30 6
30 0.1
3 33
3 29
3 26
6 30
FSW 3
33 3
30 3
27 6
30
64 J
.A. Lindley et al. J. Exp. Mar. Biol. Ecol. 242 1999 59 –74
PCP and DCB and to determine whether there were interspecific variations in the effects. The saturated solutions were diluted to 10, 5, 1, 0.5 and 0.1 with FSW controls. By
expressing toxicological data for different compounds in terms of percentage saturation, comparisons between the potencies of the compounds can be made in a mechanistically
meaningful way. Since eggs cannot take up particulate material, partitioning from aqueous solution is the primary route of uptake of the toxicants. Saturated solutions thus
represent the highest level of exposure achievable. Furthermore, Ferguson 1939 proposed that narcosis the primary toxicological response caused by many organic
pollutants occurs at a constant thermodynamic activity of the chemical. Effectively, this means a constant ratio to the aqueous solubility of the chemical. In these experiments the
eggs were exposed continuously to the toxicant with no rinsing stage. In order to address the problem of the loss of DCB through evaporation, experiments were conducted in
which the eggs in their experimental medium were sealed in vials in addition to experiments in Petri dishes.
Values for EC concentrations reducing the selected response by 50 for hatching
50
and LC concentration causing 50 mortality for larval viability were calculated by
50
probit analysis. As substantially less than 100 of eggs of Acartia spp. hatched in all controls in FSW, the values for this genus were calculated from 50 of the control
values rather than 50 of the initial numbers of eggs.
2.4. Experiments with eggs in sediment samples Sediment samples for the DCB experiment were taken on 7 June 1994 at West Muds
on the Exe estuary George and Lindley, 1997 using a Perspex tube of 19 mm diameter. The experiments were started on the same day. The top 3 cm of sediment, which was not
blackened by reduction reactions, was used in the experiments described here. The sediment from each core was extruded from the tube and used in an incubation in a 100
ml beaker covered by a watch glass either under a saturated solution of the DCB or under FSW at 5–78C in the dark. At intervals of 1, 3, 6, 9, 17 and 23 days, five replicate
samples beakers containing the sediment and supernatant exposed to the toxicant and five FSW controls were removed. The sediment from each beaker was washed through a
50 mm mesh gauze for 3 min and incubated under FSW. The numbers of viable nauplii
21
hatching were determined as for the similar experiments with a solution of 4 mg l PCP
described by Lindley et al. 1998. Mortality due to the DCB was calculated by M 5 100T C where M is mortality, T the
mean numbers hatched from sediments treated with solution of the toxicant and C mean numbers hatched from FSW controls.
3. Results
