Registration Dossier

Administrative data

Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
March 1983 to May 1984.
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Data from literature where methodology and results are clearly presented, however no guideline exists for comparison. Experiment not conducted under conditions of good laboratory practice.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1989

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Laboratory reared octopuses were exposed to test solutions of the test material for 78 hours. The chromatophore expansion and locamotor response of each octopus was then assessed.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dibromoethane
EC Number:
203-444-5
EC Name:
1,2-dibromoethane
Cas Number:
106-93-4
Molecular formula:
C2H4Br2
IUPAC Name:
1,2-dibromoethane

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
not conducted.

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
other: Octopus bimaculoides, Octopus joubini & Octopus maya.

Study design

Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
LC100
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Octopus maya

Any other information on results incl. tables

Results.

In all species, the normal response to stimulation was an intense dark brown coloration (usually occurring immediately after contact) followed by escape behavior. A similar response usually involving only chromatophore expansion could often be elicited by tapping the bowl or by passing a hand above the bowl.

It was evident within one minute after the addition of the chemical that the reduction in the chromatophore expansion following MC or EDB was paralleled by a loss of locomotor response. To illustrate this association, we plotted the scores for chromatophore expansion and for the locomotor response after acute exposure to each of the chemicals tested. Figures 2 and 3 show this association in O.bimaculoides. Statistical analysis of the effects of EDB and MC on the chromatophore and locomotor ratings indicated significant change in each response. The results of Friedman nonparametric analysis of repeated measures over a 6 hour period indicated a p<0.001 for each chemical dosage administered for both measures of toxicity.

The loss of locomotor response and the reduction in chromatophore expansion intensity followed a very similar time course after MC exposure (Fig. 2). Since it was impossible to equate the scale scores for these two functions, the important comparison was the change from preexposure and its relationship to time after exposure. These two scales were sensitive to exposures as low as 0.001 mg/l of MC, with full recovery taking approximately 5 hours. At the 1.000 mg/l concentration there was 100% lethality within 3 hours.

Acute exposure to EDB produced a similar combined loss of chromatophore expansion and loss of locomotor response (Fig. 3). Detectable effects were observed within 30 minutes at a concentration of 25 rag/l, with recovery taking a minimum of 6 hours. At higher concentrations, there was a prolongation of the impaired locomotor response and associated reduction in chromatophore expansion. After MC or EDB exposure, there was a concentrationrelated reduction in both chromatophore expansion and locomotor control followed by a recovery phase. The rate of recovery after acute exposure was concentration-sensitive for both chemicals and provided further evidence of the close relationship of these two measures of motor function.

During the recovery phase, the animals often showed a mottled appearance that suggested increased control of individual chromatophore motor units. Recovery was complete within 24 hr for both locomotor and chromatophore measures.

We established the concentrations required to produce lethality in all three species (Tables 2 and 3). The lethality data for MC and EDB were similar over the size range of octopuses. For chronic EDB exposure, O. maya was the most sensitive species with respect to lethality (100% lethal at 100 mg/l within 3 hours). However, all three species demonstrated sensitivity to the lethal effects of EDB after chronic exposure at concentrations as low as 25 mg/l. The LC50 for chronic MC exposure occurred in all three species within 3 hours at 1.000 mg/l. O. bimaculoides and O. Maya were the most sensitive species to MC, with 100% lethality at 6 hours of exposure to MC at a concentration of 0.1 mg/l. However, there was evidence of lethal effects at lower concentrations with longer exposure time for O. bimaculoides and O. joubini.

Discussion.

The advantage of using octopuses for these tests is that the responses are immediate, highly visible and sensitive. Both responses-chromatophore expansion and locomotion- are controlled by the central nervous system.

The radially-arranged chromatophore muscles contract to pull out the pigment sac and thus produce color within 2/3 of a second (8). The cell bodies of the chromatophore motor units lie in the lower motor centers (chromatophore lobes) of the brain (5); thus they travel without synapse from the CNS to the skin. Cell bodies of motoneurons of the locomotor organs---arms, mantle and funnel--are found in peripheral ganglia as well as lower motor centers of the CNS (22).

However, control of whole chromatophore patterns and integrated locomotion takes place in intermediate and higher motor centers of the brain. Therefore, it is not possible for us to pinpoint the sites of action of MC and EDB. These chemicals could have a generalized toxic effect on the nervous system, and may even act solely in the periphery at the neuromuscular junction. Andrews et al.(1) demonstrated that various neurotransmitters injected into the blood of Octopus vulgaris produced a variety of chromatophore and locomotor responses; they reported that the site of action on chromatophores seemed to be in the lower motor centers, but that inking and defecation seemed to be controlled from higher motor centers. Water quality, including magnesium and calcium ion concentration, was not altered by the addition of EDB or MC. Monitoring of water quality was performed during the experiment in order to eliminate such factors from the study. Previously, anesthetic-like effects have been reported in cephalopods with agents like magnesium chloride (13) but there was no evidence from the water quality measures to suggest that this might account for the present results.

It is noteworthy that EDB is known to cause toxic effects mainly on the respiratory system, heart, liver and kidneys (17). In our experiments withOctopusits most observable effect was on the neurally controlled chromatophores and locomotion. It was clear from these studies that the sensitivity of Octopus to MC was greater than to EDB with respect to effective concentration. Similar differences in sensitivity to these two chemicals have been found in other animals. It was also observed that there were not dramatic concentration-dependent effects after acute EDB exposure. This probably reflects the narrower dosage range used for EDB compared to MC. Future experiments are planned to learn the sites of action of MC and EDB in Octopus.

Table 2. Octopus survival after chronic exposure to ethylene dibromide*.

Hours of Exposure
Species/Dosage 0 12 24 48 72
O. bimaculoides
25 6 6 6 5 0
50 6 6 4 3 0
75 6 4 3 0 0
100 6 2 0 0 0
O. joubini
25 4 4 4 4 2
50 4 4 2 0 0
75 4 4 0 0 0
100 4 2 0 0 0
O. maya
25 4 4 4 2 2
50 4 4 4 2 0
75 4 0 0 0 0
100 4 0 0 0 0

*Values represent the number of octopus alive at each time point.

Table 3. Octopus survival after chronic exposure to mercuric chloride*.

Hours of Exposure
Species/Dosage 0 2 4 6 24 48 72
O. bimaculoides
0.001 4 4 4 4 4 4 3
0.01 4 4 4 4 4 4 4
0.1 4 3 1 0 0 0 0
1 4 3 0 0 0 0 0
O. joubini
0.001 4 4 4 4 4 0 0
0.01 4 4 4 4 0 0 0
0.1 4 4 4 2 0 0 0
1 4 4 0 0 0 0 0
O. maya
0.001 4 4 4 4 4 4 4
0.01 4 4 4 4 4 4 4
0.1 4 4 2 0 0 0 0
1 4 2 0 0 0 0 0

*Values represent the number of octopus alive at each time point.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
In summary, the present findings demonstrated the utility of the octopus as a sensitive marine invertebrate for studying the effects of toxic substances. Not only is the sensitivity of the octopus similar to that of other invertebrates (9) but it is actually more sensitive than a number of vertebrate species. For example, the LC50 in the rat for EDB and MC is 117 mg/l and 37 mg/1, respectively (4,18). The generality of the effects across the three species of octopus not only yields confidence in the results, but allows substances which may have particular relevance to the natural marine environment of a particular species to be tested on any one of the species available. The use of laboratory-reared octopuses also reduces much of the inherent variability associated with using field-collected animals of unknown age and experience and often uncertain species identity.