Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29th November 1988 - 17th July 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The sample of cis-1,3-dichloropropene was obtained from Shell Nederland Raffinaderij B.V., Rotterdam, (batch number 1986; 88009).

Calculated mean purity of 96% m/m

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all
- Sampling method: Media samples were taken from the D. magna test system at the start (0 hour) and at the end (48 hours) of the toxicity test. No D. maina were present in the samples taken for analysis.
- Sample storage conditions before analysis: ambient temperature, approximately 20 ºC

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Quantities of a stock solution of cis-l,3-dichloropropene prepared in algal growth media were added to triplicate sets of 300 ml full volume conical flasks containing algal growth media, so that when completely full an approximately logarithmic series of nominal concentrations ranging from 2.2 - 100 mg/l was produced.

Test organisms

Test organisms (species):
Scenedesmus capricornutum
Details on test organisms:
S. capricornutum were taken from the axenic laboratory culture. The culture is derived from a strain (ATCC 22662) obtained from the American Type Culture Collection, Maryland, U.S.A.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
N/A

Test conditions

Test temperature:
25 - 28 ºC
pH:
7.3 - 7.6
Salinity:
NDA
Nominal and measured concentrations:
Nominal: 2.2, 4.6, 10, 22, 46, and 100
Mean measured: 0.82, 1.8, 3.9, 8.9, 25 and 60
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 ml connical flask with a glass stopper
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, filled to 300 ml
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): Not renewed during exposure.
- Initial cells density: 1000 cells/ml
- Control end cells density: 220,000
- No. of organisms per vessel: NDA
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: N/A

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: NDA
- Photoperiod: continuous
- Light intensity and quality: ca. 3000 lux
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter counter
- Chlorophyll measurement: NDA

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study NDA
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CL of 2.2 - 3.5
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.82 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: areas under the growth curves and average specific growth rates
Details on results:
The results of the toxicity test with Scenedesmus capricornutum are give in the attached table below. The 48 and 72 h EbC50 values calculated on the basis of the areas under the growth curves were 4.1 and 2.8 mg/l respectively. A 24 h EbC50 value could not be reliably calculated from the results of the study. 95 % fiducial limits for the 48 and 72 h EbC50 values were 2.0 - 8.3 and 2.2 - 3.5 mg/l respectively.

The ErC50 (24-48 h) and ErC50 (24-72 h) values, determined by analysis of average specific growth rates were 4.6 and 3.1 mg/l respectively.

The no observed effect concentration (NOEC) determined by analysis of both the areas under the growth curves and average specific growth rates was 0.82 mg/l

Since cis-l,3-dichloropropene was observed to be stable in a mixture of 1 + 3 v/v water + acetone for at least 10 days, it is unlikely to have decomposed to any significant extent in the aqueous test media during the tests. Losses from the test system by volatilization should have been small, since all tests were conducted in sealed test vessels with no head space. The most likely explanation for the low initial concentrations relative to the nominal values is therefore adsorption onto container surfaces during preparation and testing of media.
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
Effects were evaluated using two approaches:
(a) Comparison of areas under the growth curves.
(b) Comparison of average specific growth rates.

Calculation of EC50 values by comparison of areas under the growth curves:

Using the area under the growth curves (A), the percentage inhibition of the cell growth at each test substance concentration (IA) was calculated as the difference between the area under the control growth curve (Ac) and the area under the growth curve at each test substance concentration (At) using the formula:

IA (%) = (Ac - At / Ac) x 100

48 and 72 h IA values for each test concentration were subject to probit analysis (Finney, 1971) to determine concentrations calculated to cause a 50 % reduction in area under the growth curve (EC50 values) for 48 and 72 h exposure, together with 95 % fiducial limits.

Calculations of EC50 values by comparison of average specific growth rates:

The percentage reduction in average specific growth rate at each test substance concentration compared to the control value was plotted against the logarithm of the concentration. The EC50 values (concentrations calculated to cause a 50% reduction in growth rate) for the exposure periods over which growth rates were calculated (24-48 hours and 24-72 hours) were then determined from the plot.

To distinguish between EC50 values determined using areas under the growth curves and those determined using growth rates the symbols EbC50 and ErC50 are used respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 72 hour ErC50 to Selenastrum capricornutum of (Z)-1,3-dichloropropene was determined as 3.1 mg/L.
The no observed effect concentration (NOEC) determined by analysis of both the areas under the growth curves and average specific growth rates was 0.82 mg/l.
Executive summary:
Selenastrum capricornutum was exposed under static conditions to (Z)-1,3-dichloropropene for 72 hours at mean measured concentrations of 0.82, 1.8, 3.9, 8.9, 25 and 60 mg/L, in accordance with EU method C.3. and conducted to GLP standard.

The 72 hour ErC50 to Selenastrum capricornutum of (Z)-1,3-dichloropropene was determined as 3.1 mg/L as a mean measured concentration. The no observed effect concentration (NOEC) determined by analysis of both the areas under the growth curves and average specific growth rates was 0.82 mg/l.