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:
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:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 concentrationsopen allclose all
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
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.84 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
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
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%
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.