Methyl 3-oxo-2-pentylcyclopentaneacetate

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:

From 04 August 1998 to 23 October 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD Guideline 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:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-oxo-2-pentylcyclopentaneacetate

EC Number:

246-495-9

EC Name:

Methyl 3-oxo-2-pentylcyclopentaneacetate

Cas Number:

24851-98-7

Molecular formula:

C13H22O3

IUPAC Name:

methyl 3-oxo-2-pentylcyclopentaneacetate

Details on test material:

- Name of test material (as cited in study report): Methyl Dihydrojasmonate
- Substance type: pure active substance
- Analytical purity: 99.98 % (sum of 2 isomers and of a few postion isomers)
- Composition of test material, percentage of components:
- Storage condition of test material:
- Other: test material is moderately soluble (water solubility > 500 mg/l @ 20°C), has a low Vapour pressure (< 0.0001 Pa) and a logPow < 3

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not Applicable

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Not Applicable

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Correctly weighed 200 mg of the test substance and 200 mg of the carrier solvent (HCO-40) were mixed. The mixture was dissolved into and filled up to 200 ml with the test medium in order to prepare a stock solution containing the test substance at the concentration of 1000 mg/l. At the same time, another stock solution containing only the solvent (HCO-40, 1000 mg/L) was prepared in the same manner.
100 ml of fresh test medium was poured into each test flask, and from which estimated adding volume of the stock solution containing the test substance and the stock solutions containing only the solvent was removed. Then the specific volumes of stock solution containing the test substance were added into each test flask geometrically in order to prepare test solutions at the concentration of 4.00, 6.84, 11.7, 20.0, 34.2, 58.5, and 100 mg/l. At the same time, to adjust the deference of concentration of the solvent in each flask and to unify it to be the same value (100 mg/l), the specific volumes of stock solution containing only the solvent were also added into flasks. For preparing the Control, a fresh test medium was used. And, for preparing the Solvent control (concentration of the solvent: 100 mg/L), the test medium containing only the solvent was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): HCO-40
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Stock solution: 1000 mg/l, final test solutions: 100 mg/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: Selenastrum capricornutum (Pseudokirchneriella subcapitata)
- Source (laboratory, culture collection): source: ATCC; strain ATCC 22622 received on 20 June 1996 from ATCC
- Method of cultivation: In order to adapt the test algae to the test conditions, an inoculum culture in the test medium was prepared. And the inoculum culture was incubated for 5 days before starting of the test under the same conditions as the test culture.


ACCLIMATION
- Acclimation period: 5 days
- Culturing media and conditions (same as test or not): same (OECD algal medium)
- Any deformed or abnormal cells observed: none

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not Applicable

Test conditions

Hardness:

Not required in OECD 201 Guideline/Not Applicable.

Test temperature:

23 ± 2°C (the temperature within the flasks was 23.3 - 24.8 °C during the 72 hour exposure, see Table 2 in "Remarks on results incl. tables and figures" )

pH:

8.1-10.0 (See Table 3 in "Remarks on results incl. tables and figures")
Even if the pH is out of the bound of the OECD study guideline, it is not tought that it will have a negative impact on the study results.

Dissolved oxygen:

Not required in OECD 201 Guideline/Not Applicable

Salinity:

Not required in OECD 201 Guideline/Not Applicable

Nominal and measured concentrations:

Nominal concentration: 4.0, 6.84, 11.7, 20.0, 34.2, 58.5, 100 mg/l
Measured concentrations: Analytical monitoring not required according to the OECD Guideline 201 "Alga, Growth Inhibition Test" (1984). Furthermore, the test material is expected to be sufficiently stable in the test solution (half life pH 7 > 1 year and half life at pH9 > 7.8 days according to "hydrolysis in function of pH" performed on the same test material (OECD TG 111). The substance has been shown to be moderately soluble (>500 mg/l), and not volatile in the physico-chemical endpoints/studies).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (with porous silicon stoppers)
- Material, size, headspace, fill volume: 300mI glass conical flasks (with porous silicon stoppers)
- Aeration: via porous silicon stoppers, 100 rpm shaking of flasks
- Type of flow-through (e.g. peristaltic or proportional diluter): Not Applicable
- Renewal rate of test solution (frequency/flow rate): Not Applicable
- Initial cells density: 10'000 cells/ml
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes (OECD medium prepared according to OECD Guideline 201)


OTHER TEST CONDITIONS
- Sterile test conditions: not mentionned in study report
- Adjustment of pH: none
- Photoperiod: continuous lightning
- Light intensity and quality: 4000 lux continuous lightning at the surface of the test medium in the flask
- Salinity (for marine algae): Not Applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: measured at 24h, 48h and 72h with a CDA-500 (TOA Medical Electronics)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Range finding study
- Test concentrations: 1.00, 3.16, 10.0, 31.6, 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentration below 10.0 mg/ml. Growth was observed to be reduced at 31.6 mg/l (see Table 3 in "Remarks on results incl. tables and figures"). Based on this information test concentrations of 4.00, 6.84, 11.7, 20.0, 34.2, 58.5 & 100 mg/ml were selected for the main study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 12.1-27.3 mg/l

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

49.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 41.6-58.3 mg/l

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 33.5-62.8%

Effect concentrations 5

Duration:

72

Dose descriptor:

NOEC

Effect conc.:

11.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none/not applicable

Results with reference substance (positive control):

- Results with reference substance valid? YES
- EC50: 0.363 mg/l

Reported statistics and error estimates:

Treatment of results: Growth Curves: The mean value of the cell concentration for each test substance concentration and for the controls was plotted against time to produce growth curves. Calculation of growth inhibition ratio: To determine the concentration effect relationship, both of the following approaches (“areas method” and “growth rate method”) were used:
1) Growth inhibition ratio by comparison of areas under the growth curves
2) Growth inhibition ratio by comparison of the growth rates according to the OECD 201 Guideline

No Observed Effect Concentration (NOEC) determination: After checking whether the ANOVA assumption of homogeneity of variance holds by Bartlett’s test (α=0.01), one-way layout analysis of variance (1-way ANOVA, α=0.05) and Williams’ multiple comparison test (α=0.05, both sides) were carried out. The highest test substance concentration, having no statistically significant difference in reducing effect on algal growth when compared with the control culture including the solvent, was estimated as the No Observed Effect Concentration (NOEC).
In the above mentioned static analysis, Yukms’ software Statlight #4 “The Multiple Comparisons of Groups” (Yukms Corp., Tokyo) was adopted.

Any other information on results incl. tables

Growth Curve

The average values of the cell concentration in the control culture and the solvent control culture were increased 176 times and 159 times during 72-hour cultivation respectively, and said both cultures showed normal growth under the test conditions. The growth observed for each test concentrations were as follows: 200 times in 4.00 mg/L, 184 times in 6.84 mg/L, 128 times in 11.7 mg/L, 67 times in 20.0 mg/L, 28 times in 34.2 mg/L, 4.5 times in 58.5 mg/L and 1.4 times in 100 mg/L. (See Table 1 below and see Graph Algal growth vs time in “Attached background material”).

 

Table 1 : Cell densities

Nominal Concentration (mg/l)		Cell densities (cells/ml)
		0hour	24hours	48hours	72hours
Control	Average	10000	34500	281700	1758900
	SD	0	800	6400	34600
Solvent control	Average	10000	33600	264100	1588900
	SD	0	1800	6700	111
4.00	Average	10000	31400	306400	1995600
	SD	0	6700	56300	161700
6.84	Average	10000	25500	229700	1835600
	SD	0	4100	58300	326200
11.7	Average	10000	21500	158400	1278200
	SD	0	2700	29600	273300
20.0	Average	10000	17600	96300	674200
	SD	0	1500	14100	113800
34.2	Average	10000	13600	67100	280600
	SD	0	1600	20000	36000
58.5	Average	10000	12300	28300	45000
	SD	0	200	5300	8700
100	Average	10000	13400	12800	13500
	SD	0	400	800	1200
Average : average of 3 replicates SD :standard deviation of the 3 replicates

 

EC50 and No Observed Effect Concentration (NOEC)

 

Growth inhibition concentration by comparison of the areas under the growth curves:

EbC50 (0-72): 18.2 mg/L with 95% confidence interval 12.1 - 27.3 mg/L.

NOECb(0-72): 6.84 mg/L.

 

Growth inhibition concentration by comparison of the growth rates

ErC50 (24 - 48): 49.2 mg/L with 95% confidence interval 41.6 - 58.3 mg/L.

ErC50 (24 - 72): 45.9 mg/L with 95% confidence interval 33.5 - 62.8 mg/L.

NOECr(24 - 48) and NOECr (24-72): 11.7 mg/L.

 

Temperature and pH

 

Table 2: Temperature in the incubation chamber during the 72-hours exposure

Exposure period (hours)	Temperature (°C)
0 24 48 72	23.3 23.5 23.3 24.8

 

Table 3: pH values at 0- and 72-hour exposure

Nominal concentration (mg/l)	Vessel n°	pH
		0 hours	72 hour
Control Solvent Control 4.00 6.84 11.7 20.0 34.2 58.5 100	1 1 1 1 1 1 1 1 1	8.1 8.1 8.1 8.1 8.1 8.1 8.1 8.1 8.1	9.0 9.7 10.0 9.9 9.2 8.5 8.2 8.0 8.0

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The validity criteria of OECD Guideline 201 (1984) was fullfilled: Biomass increasing Factor > 16

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green alga Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata).The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga Growth Inhibition Test".

Methods. Following a preliminary range-finding test, Pseudokirchnerella subcapitata was exposed to solutions of the test material at nominal concentrations of 4.0, 6.8, 11.7, 20.0, 34.2, 58.5 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 23±2°C. The test material solutions were prepared from a stock solution of test material in HCO-40 (1000mg/l) and from a HCO-40 stock solution (1000 mg/l) to maintain final HCO-40 concentrations at 100 mg/l in all test conditions. Analytical monitoring was not performed as not required by the test guideline. Furthermore, the test material was expected to be sufficiently stable in the test conditions used according to the hydrolysis and to the physico-chemical studies performed on the same test material.

Samples of the algal populations were removed at 24, 48 and 72h and cell concentrations determined for each control, solvent control and treatment group, using a CDA-500 (TOA Medical Electronics) Particle Counter. Potassium dichromate was used as positive control.

 

Results. In terms of growth rate, exposure of Pseudokirchnerella subcapitata to the test material gave an ErC50 (24 - 72 h) value of 45.9 mg/1. The No Observed Effect Concentration based on inhibition of growth rate was 11.7 mg/l.

In terms of biomass integral (area under growth curve), exposure of Pseudokirchnerella subcapitata to the test material gave an EbC50 (0-72 h) value of 18.2 mg/1. The corresponding No Observed Effect Concentration was 6.84 mg/1.

 

Exposure of Pseudokirchnerella subcapitata to the reference material, potassium dichromate, gave an ErC50 (0 - 72 h) of 0.363 mg/l, a value in accordance with testing laboratory routine test results.

Regarding the exposure throughout the test. At pH9 the substance is known to hydrolyse rapidly. A DT50 of 7.8 days at 25°C was calculated which equates to k = 0.089 d^-1(Equation R16-10). At 25°C the substance would have lost 26.7% of the nominal concentration if the pH remained at 9 throughout the 72 hours study duration. Yet the algae study started at 8.1 and ended at 9.7 in the solvent control after 72h. In the test groups pH increased to 10 at 4 mg/L but only to between 8.0 and 8.5 for test concentrations higher than 20 mg/L where significant effects on algae growth were observed. As the substance degrades to a carboxylic acid form (see hydrolysis study) it is possible that the abiotic degradation of hedione limited the increase of pH under these test concentrations, however, only minor hydrolysis would be expected at a pH of approximately 8. Thus there is a weight of evidence to conclude that in the algae study the test substance degraded up to a maximum of 26.7%. Further it is assumed that up to 26.7% of the test concentrations of the carboxylic acid were in the test media in each test group. The degradation products are not classified for the environment according to annex I of directive 67/548/EEC. Thus it is unlikely that they contributed to the ecotoxicity observed in this study. It is concluded that it is reasonable to base the results on nominal concentrations.