3'H-Cyclopropa[1,2]pregna-1,4,6-triene-3,20-dione, 1,2-dihydro-17-hydroxy-, (1.alpha.,2.alpha.)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

17-Hydroxy-1 alpha,2 alpha-methylene-4,6-pregnadiene-3,20-dione

EC Number:

606-682-1

Cas Number:

2098-65-9

Molecular formula:

C22 H28 O3

IUPAC Name:

17-Hydroxy-1 alpha,2 alpha-methylene-4,6-pregnadiene-3,20-dione

Test material form:

solid: bulk

Sampling and analysis

Analytical monitoring:

yes

Remarks:

TOC measurement

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the preparation of the test solutions a suspension with a nominal loading of 100 mg ZK 12126 per liter of demineralized water was stirred for approximately 26 hours. This suspension was filtered through a glassfibre filter. The resulting solution served as the highest concentration (saturated solution). It was further diluted 1:2, 1:4, 1:8 and 1:16 with demineralized water.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): Sammlung Algenkulturen Göttingen
- Age of inoculum (at test initiation): 3 hours

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 °C

pH:

start: 8.0 - 8.1
end: 8.7 - 9.2

Nominal and measured concentrations:

Nominal : 100 mg/L
measured: 17.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass bottles
- Control end cells density: 988000 cells/mL
- No. of organisms per vessel: 10000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell concentration of the test solutions and of the control were measured after 24, 48 and 72 hours of incubation. All measurements were made on an electronic particle counting apparatus (Coulter Counter), which was calibrated with a count of standard cell concentrations using the "Bürker'-counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 17.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 17.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no

Any other information on results incl. tables

Table 1: Inhibition of the growth rate (%) of Scenedesmus subspicatus after 72 hours exposure to 1,2 Methylen-4,6 -Dien

extrapolated concentration	Inhibition in percent
of ZK 12126 (mg/I)	Biomass (integral)	Growth rate
0,0 (control)	0	0
1,1	-19,5	0
2,2	-16,5	0,2
4,4	-58,5	-10
8,8	-86,4	-14,3
17,5	63,1	23,1

In a range-finding study 90% inhibition was observed in the saturated solution

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

1,2 Methylen-4,6-Dien had an inhibitory effect on the growth of Scenedesmus subspicatus at a concentration of 17.5 mg/L (saturated solution)
while in lower concentrations a slight increase of the algae may have occurred. It can be concluded that ZK 12126 is toxic to the alga S. subspicatus close to the solubility limit. The EbC5O (biomass) and also the ErC5O (growth rate) could
not be calculated due to the effect pattern .

Executive summary:

The purpose of this study was to determine the effects of the test compound 1,2 Methylen-4,6-Dien on the growth of the green algae Scenedesmus subspicatus. The study was conducted in agreement with the OECD guideline no. 201.

The test substance was incubated in an aqueous solution including nutrients with an algae population of Scenedesmus subspicatus for a test duration of approximately 70 hours. The nutrient solution was made up of mainly nitrate, phosphates and some trace elements. For the preparation of the test solutions a suspension with a nominal loading of 100 mg test substance per liter of demineralized water was stirred for approximately 26 hours. The resulting solution served as the highest concentration (saturated solution). It was further diluted 1 :2, 1 :4, 1:8 and 1 :16 with demineralized water. The organic carbon concentration of the test solution was analysed with a TOC analyser at the start of the incubation. The substance concentration was calculated on the basis of the molecular formula. The concentration was 17.5 mg/I. The test concentrations extrapolated are shown in the table below. The algae were exposed to each concentration in triplicate. Six vessels were prepared for the contro!. The algae were incubated in an incubator shaker under continuous light.

Control solutions were counted with an electronic particle counter ("Coulter Counter"). The increase of biomass and the growth rate were calculated on the basis of the cell counts. The calculated biomass and growth rate of each concentration were compared to those of the controls, and the inhibition was calculated.

1,2 Methylen-4,6-Dien had an inhibitory effect on the growth of Scenedesmus subspicatus at a concentration of 17.5 mg/L (saturated solution) while in lower concentrations a slight increase of the algae may have occurred. The EbC5O (biomass) and also the ErC5O (growth rate) could not be calculated due to the effect pattern .