1H,5H-Pyrazolo(1,2-a)pyrazol-1-one, 2,3-diamino-6,7-dihydro-, dimethanesulfonate

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 December 2006 to 28 June 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): R0054002C, synonym IMEXINE OBH
- Physical state: beige to yellow powder
- Purity: 99.7%
- Impurities (identity and concentrations): not indicated
- Purity test date: 20 July 2006
- Lot/batch No.: R0054002C 013 L 001, synonym 0604070001
- Expiration date of the lot/batch: 01 August 2007
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature and under nitrogen gas

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At T0 hour, samples were taken from each test solution before addition of algae, except for the control.
At T24, T48 and T72 hours, samples were taken from all test replicates at each concentration (except for the control), pooled by concentration. Before analysis, these samples containing algae were centrifuged and the supernatant was analyzed separately to verify the test item concentration in solution throughout the test.

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

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

Details on test organisms:

Strain No.: CCAP 278/4.
Species commonly used in Europe for aquatic toxicity testing and recommended in OECD and EEC guidelines.
Origin: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN, Argyll PA37 1QA, Scotland.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

35.7 mg/L as CaCO3

Test temperature:

between: 21.8°C and 22.2°C

pH:

pH: 5.87 to 8.37

Dissolved oxygen:

Not measured

Salinity:

Not measured

Nominal and measured concentrations:

2.47, 7.41, 22.2, 66.7, 200 and 600 mg/L nominal corresponding respectively to 0.730, 0.910, 3.40, 5.00, 108 and 315 mg/L espressed as geometric means of measured concentrations.

Details on test conditions:

Test water: reconstituted water (OECD medium). Four primary stock solutions were made up every month and sterilized by autoclaving or filtration. The final culture medium, prepared from these four stock solutions, has a stock life of one week (at +4°C away from light). Before use, its pH was verified at 8.1 ± 0.1 or adjusted until this pH was obtained. The hardness of this medium is approximately 34 ± 17 mg/L as CaCO3.

Temperature: controlled daily in a flask containing test water but no algae, run alongside the test. Between 21°C and 24°C and did not vary by more than ± 2°C during the test.

Illumination: continuous. The light intensity was approximately 7000 lux. The number of lux received by each culture flask was specified in the raw data.

Duration of test: 72 hours.

Transfer: the number of cells in the algal pre-culture was counted and the quantity of pre-culture to be added to the test solutions was calculated to give a concentration of 1 x 104 cells/mL. The algal pre-culture was then added where appropriate to the 100 mL test solutions and all the 250 mL conical flasks stoppered with sterile cotton wool wrapped with lint and transferred to the agitator.

Culture homogeneity: the solutions were agitated throughout the test.

pH: between 6± 0.2 and 9± 0.2 in all test item solutions after pH adjustement when relevant ( i.e. < 5.8 or > 9.2) .

Hardness: measured once in OECD reconstituted water before the start of the test.

Forced aeration: not used during the test but the cultures were constantly agitated.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

84.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 72 h: 66.8 - 106 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL 72 h: 17.2 - 27.2 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

other: growth rate and yield

Details on results:

According to the results in the test solutions, it was demonstrated that the test item induced an important inhibition algal growth from 22.2 mg/L nominal. This inhibition was due for one part to the reduction of the light intensity by the test item. This impact of light absorption by the test item became statistically significant at the highest concentration (i.e. 600 mg/L nominal).
After 72 hours, inhibition of growth rate in the test solution at 600 mg/L nominal minus inhibition of growth rate in the control solution with shading by this test solution was > 10%. The same effect was observed after 72 hours for inhibition of yield.

As inhibitions of growth rate and yield observed were therefore due to a combined impact of shading effect and inherent toxic effect, toxicity endpoints were based on inhibition of algal growth in test solutions.

Results with reference substance (positive control):

EC50 72 h (potassium dichromate) = 1.18 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a 72-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the EC50 (calculated from geometric means of measured concentrations) of IMEXINE OBH, was 84.6 mg/L based on average specific growth rate (ErC50) and 21.5 mg/L based on yield (EyC50).
The No Observed Effect Concentration (NOEC) was 3.40 mg/L (geometric mean of measured concentrations).