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
3.1. Egg incubation experiments with saturated solutions Eurytemora affinis dominated a plankton sample taken on 18 April 1994 temperature
7.58C, salinity 8–20‰ and two egg sacs, either detached or attached to a dead female,
J .A. Lindley et al. J. Exp. Mar. Biol. Ecol. 242 1999 59 –74
65
were used in each replicate experiment with DCB and FSW controls. Five replicates were used for each of the treatments 1 and 2. Three replicates were used for each of
treatments 3 and 4. The results are given in Table 1. Only one egg failed to hatch in the FSW controls and all the nauplii were alive when examined, whereas three hatched in
the DCB solution, none of which survived. Temporary immersion in DCB reduced the percentage hatch but all the nauplii that hatched were alive. The percentage hatch varied
between the three replicates for the temporary immersion, the results were 0 of 37 eggs, 12.5 of 40 eggs and 100 of 50 eggs. Typically, 90 of the DCB was lost
through evaporation from solution in the Petri dishes within the first 24 h, and none was detectable after 48 h. All hatching took place within 4 days of the start of incubation.
Adult female Acartia bifilosa were extracted from a plankton sample taken on 11
October 1994 temperature 13.28C, salinity 26–29‰ in which that species was dominant. The eggs that these produced overnight i.e. in approximately 16 h were used
in three replicates of each of the treatments described above but with PCP with acetone carrier as the toxicant. Three replicates of each of two additional treatments were carried
21
out, continuous incubation in 50 ml l acetone solution in FSW and initial incubation in
acetone solution followed by transfer to FSW after |24 h. In each replicate five eggs were used except for one acetone replicate where eight eggs adhered to each other and
could not be separated without damage. Remaining eggs were used in bulk experiments with 20 or 30 eggs per Petri dish. The results are listed in Table 2 with the values for the
acetone replicates calculated for including and excluding that in which the eggs were clumped. Both continued immersion and overnight immersion in initially saturated PCP
solution completely suppressed hatching. PCP concentrations fell to approximately 80
21
of the saturated concentration, approximately 11 mg l , within the first 24 h of an
experiment. The acetone carrier had no consistent effect. All hatching took place within 4 days of the start of incubation. A single Eurytemora affinis egg sac containing seven
21
eggs was incubated in 50 ml l acetone solution. All the eggs hatched and the nauplii
were viable. 3.2. Egg incubations in low concentration solutions
The experiments on a range of concentrations of toxicants and to determine interspecific variability were carried out on eggs from females collected between May
and July 1996. The species, numbers of replicate experiments and the numbers of eggs are listed in Table 3 and the percentages of eggs which hatched and the percentages
which yielded viable alive when first examined nauplii are illustrated in Figs. 1 and 2.
Eurytemora females were extracted from samples taken from the Exe in May
temperature 11–138C, salinity 12–25‰. No Eurytemora affinis eggs hatched success- fully at a dilution of 10 of a saturated solution of DCB and only three hatched of a
total of 111 eggs in PCP. Overall, 40 hatched in 5 PCP and 42 in 5 DCB. Solutions of 0.5 PCP or less and 1 DCB or less had little effect on hatching. The
calculated EC
values for hatching and LC values for viability are summarised in
50 50
21 21
Table 4. The EC values were equivalent to 291 mg l
or 1.1 mmol l PCP and 3.5
50 21
21
mg l or 22 mmol l
DCB. Viable nauplii were found only in solutions of 1 or less with no significant differences between FSW and the 0.1, 0.5 and 1 solutions. The
66 J
.A. Lindley et al. J. Exp. Mar. Biol. Ecol. 242 1999 59 –74
Fig. 1. Percentage hatch and percentage viable nauplii from eggs of Eurytemora affinis and Acartia bifilosa in a range of dilutions of initially saturated solutions of PCP.
21 21
calculated LC were equivalent to a maximum of 96 mg l
or 0.37 mmol l PCP and
50 21
21
a maximum of 15.9 mg l or 74.1 mmol l
DCB initial concentration. The hatching success of A
. bifilosa did not differ significantly between 10 of a saturated solution or lower concentrations of both toxicants and filtered sea water but all dilutions of PCP
reduced viability in comparison with filtered sea water and none survived in the 10
21 21
solution. These data gave a calculated LC equivalent to 980 mg l
or 3.7 mmol l .
50
The effects on viability were not significant at 1 and below. At DCB concentrations of 10 dilution viability was greatly reduced in experiments in Petri dishes and no Acartia
clausi nauplii were viable in experiments in vials where loss of DCB through evaporation was eliminated. Because of the rapid loss of DCB from dishes, reliable
quantitative data could only be obtained from experiments carried out in vials but in
J .A. Lindley et al. J. Exp. Mar. Biol. Ecol. 242 1999 59 –74
67
Fig. 2. Percentage hatch and percentage viable nauplii from eggs of Eurytemora affinis and Acartia bifilosa in Petri dishes and Acartia clausi in sealed vials in a range of dilutions of initially saturated solutions of DCB.
68 J
.A. Lindley et al. J. Exp. Mar. Biol. Ecol. 242 1999 59 –74 Table 4
Eurytemora affinis, Acartia bifilosa and A . clausi. Calculated EC
and LC levels initial concentrations for
50 50
PCP and DCB solutions expressed as saturation. Nt, no downward trend in the range 0–10 Species
Compound Vessel
EC LC
50 50
Eurytemora affinis PCP
Petri dishes 2.1
0.7 DCB
Petri dishes 3.2
1.7 Acartia bifilosa
PCP Petri dishes
nt 0.8
a
DCB Petri dishes
nt 14.2
A . clausi
DCB Vials
nt 2.0
a
Extrapolated.
these experiments the viability of control nauplii of A . clausi was less than 50. The
21 21
LC was equivalent to a maximum concentration of 2.1 mg l
or 16.6 mmol l .
50
Some of the dead nauplii which hatched from eggs in the higher concentrations of PCP and DCB were malformed as Tester and Costlow 1981 found with nauplii
hatched from eggs from Acartia tonsa that had been exposed to Diflubenzon DFB, marketed as Dimlin. No deformed nauplii were alive when examined.
3.3. Experiments with eggs in sediment samples The results of the incubations following treatments with DCB and the respective
controls are shown in Fig. 3. DCB was rapidly lost, presumably mainly due to evaporation, and was not detectable in the samples after 1 day. No hatched nauplii were
recovered from samples which had been incubated in the DCB solution at low temperature and in darkness for 1 day. Some nauplii were found in samples that had
been incubated in initially saturated DCB solution for longer periods. This created the apparent anomaly of estimated mortality declining from 100 to lower levels at later
Fig. 3. a Means numbers and their standard deviations of nauplii hatched during sediment incubations following exposure to a saturated solution of DCB in sea water for 1–23 days with FSW. d With light
line5control FSW, m with heavy line5DCB solution. b Estimated percentage mortality due to treatment DCB for each time interval is shown to the right.
J .A. Lindley et al. J. Exp. Mar. Biol. Ecol. 242 1999 59 –74
69
dates, but it should be noted that the value for each treatment duration interval was calculated from a separate set of treated and control samples. The differences between
the treated and control samples in numbers of nauplii recovered were significant t . 6.00, P , 0.001 except after 1 day, t 5 4.51, P , 0.01 and after 17 days t 5 3.13,
P , 0.05 on each occasion. The value of t for the results for samples treated for 6 days, when the number of nauplii recovered from the DCB-treated samples was greatest, was
7.75, the second highest value. All hatching occurred within 14 days of the start of incubation at 158C and within 28 days from collection of the samples.
4. Discussion
