Iron(1+), chloro[dimethyl 9,9-dihydroxy-3-methyl-2,4-di(2-pyridinyl-kN)-7-[(2-pyridinyl-kN)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate-kN3,kN7]-, chloride

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 2004-02-09 to 2004-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The ratio between concentration is >2.2 in the second test.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,5-dimethyl 3-methyl-9-oxo-2,4-bis(pyridin-2-yl)-7-[(pyridin-2-yl)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate dichloroiron hydrate

EC Number:

610-388-9

Cas Number:

478945-46-9

Molecular formula:

C28H31N5O6FeCl2

IUPAC Name:

1,5-dimethyl 3-methyl-9-oxo-2,4-bis(pyridin-2-yl)-7-[(pyridin-2-yl)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate dichloroiron hydrate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: S2539801
- Expiration date of the lot/batch:
- Purity test date: 2004-11-12

RADIOLABELLING INFORMATION (if applicable) no

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
ambient/dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: no

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples were supplied at concentrations of 0, 10, 18, 32, 56 and 100 mg/L (test 1) and 0, 30, 10, 30, 100 and 300 mg/L (test 2) in Isomedia with and without algae
- Sampling method: duplicate aliquots of each sample were filtered through a 0.45 µm PTFE before dilution in mobile phase as follows:
0 mg/L sample were analysed after diluting 1:10 in mobile phase.
3 mg/L sample were analysed after diluting 1:10 in mobile phase.
10 mg/L sample were analysed after diluting 1:25 in mobile phase.
18 mg/L sample were analysed after diluting 1:25 in mobile phase.
30 mg/L sample were analysed after diluting 1:50 in mobile phase.
32 mg/L sample were analysed after diluting 1:50 in mobile phase.
56 mg/L sample were analysed after diluting 1:100 in mobile phase.
100 mg/L sample were analysed after diluting 1:200 in mobile phase.
300 mg/L sample were analysed after diluting 1:500 in mobile phase.
(Where mobile phase was 81% 5 mM ammonium formate pH3.0 and 19% acetonitrile)
- Sample storage conditions before analysis: not stated

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: for each defintive experiment, a nominal 1000 mg/mL stock solution was prepared by dissolving a pre-weighed aliquot of test item in purified water; 500 mg into 500 mL for the 10 to 100 mg/L concentration range, 1000 mg into 1000 mL for the 3 to 300 mg/L concentration range. Test media were prepared in volumetric flasks by adding appropriate volumes of stock, ISO concentrate and purified water to obtain the respective final nominal concentrations for each test.
- Controls: blank flasks were prepared by removing an aliquot of test media from each volumetric flask before the algae inoculum was added.
Glassware was conditioned with test compound for at least 24 hours before use in order to minimize potential losses by adsorption of compound onto the glass.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not stated.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): not stated
- Age of inoculum (at test initiation): no information, only nominal algal density 10E+4 cells/mL indicated
- Method of cultivation: not stated

ACCLIMATION
- Acclimation period: not stated
- Culturing media and conditions (same as test or not): not stated
- Any deformed or abnormal cells observed: not stated

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.9-23.2°C (10-100 mg/L)
22.9-23.0°C (3-300 mg/L)

pH:

pH at T0: 7.3 to 7.5 and at t72: from 6.9 to 8.0; variation from 0.1 to 0.5 pH units (test 1)
pH at T0: 6.8 to 7.2 and at t72: from 6.7 to 7.4; variation from 0.0 to 0.6 pH units (test 2)

Nominal and measured concentrations:

Test 1: Nominal: 0-10-18-32-56-100 mg/L. Mean measured: 0-2.72-5.56-11.14-19.68-39.43 mg/L
Test 2: Nominal: 3-10-30-100-300 mg/L. Mean measured: 0-0.86-2.77-10.65-38.65-149.56 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: not stated
- Type (delete if not applicable): open / closed not stated
- Material, size, headspace, fill volume: not stated; only indication of pre-conditionning with test compound at least 24H before use.
- Aeration: not stated
- Initial cells density: 10E+4 cells/mL
- Control end cells density: test 1: 178-fold mean increased in the cell density of control cultures over 72H; test 2: 112-fold mean increased
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes/no not stated

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium
- Culture medium different from test medium: not stated
- Intervals of water quality measurement: pH recorded at 0 and 72H

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no not stated
- Adjustment of pH: not stated
- Photoperiod: constant
- Light intensity and quality: not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] by a Z2 Coulter counter at 0, 24, 48, and 72H.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: test 1: <=2.2; test 2: approx 3
- Justification for using less concentrations than requested by guideline: not appropriate
- Range finding study: yes
Test concentrations: not reported
Results used to determine the conditions for the definitive study: yes for the determination of test concentrations of test 1; test has to be repeated as not all the required EC50 and NOEC values were obtained.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

29.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: test 2

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: test 2

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not stated
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium:not reported
Test substance analysis showed that there was a decrease in the amount of test compound available over the 72H. This loss could not be attributed to adsorption of compound onto the algal cells as the same phenomenon was observed amongst the blank test flasks (without algal cells). Hydrolysis of the principal active in test item to the associated monoester was observed in analysis by LC-MS.

Reported statistics and error estimates:

The percentage inhibitions of biomass and growth rate were calculated using the ALGAE v4.0 computer software.

Any other information on results incl. tables

Study report assessor: from the cell densities figures, it was possible to calculate the criteria as laid down in the OECD 201 guideline (2006) and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 hour tests) in the control cultures did not exceed 35%. The  coefficient  of  variation  of  average  specific  growth  rates  during  the  whole  test  period  in replicate control cultures did not exceed 7%.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

With the 3 to 300 mg/L nominal concentration range (test 2), the geometric means of the measured results were as follows: 72H-ErC50=29.40 mg/L; 72H-NOECr=2.77 mg/L:; 72H-EbC50=5.15 mg/L; 72H-NOECb=0.86 mg/L. There was 112 fold mean increase in the control cell density over 72 hours, which confirmed study validity.

Executive summary:

The purpose of the study was to determine the 72H-EC50 and NOEC values of the test item to Pseudokirchneriella subcapitata. Pseudokirchneriella subcapitata is recommended for use in the ISO freshwater algal growth inhibition test, by OECD 201 and EEC directive 92/69/EEC. Two definitive experiments were run for this study. The first experiment, using a nominal concentration based on the results of range finder tests (10 to 100 mg/L) was repeated as not all the required EC50 and NOEC values were obtained. The repeated definitive experiment, using a revised nominal concentration (3 to 300 mg/L) yielded all required values. Test solutions were prepared by diluting a concentrated stock solution with ISO media. An aliquot of Pseudokirchneriella subcapitata was added to each test solution to give a nominal algal density of 10E+4 cells/mL. Cell concentrations were determined at 0, 24, 48 and 72 hours using a Z2 Coulter counter. Test concentrations were measured by liquid chromatography/mass spectrometry. With the 3 to 300 mg/L nominal concentration range (test 2), the geometric means of the measured results were as follows: 72H-ErC50=29.40 mg/L; 72H-NOECr=2.77 mg/L:; 72H-EbC50=5.15 mg/L; 72H-NOECb=0.86 mg/L. There was 112 fold mean increase in the control cell density over 72 hours, which confirmed study validity. Test substance analysis showed that there was a decrease in the amount of test compound available over the 72H. This loss could not be attributed to adsorption of compound onto the algal cells as the same phenomenon was observed amongst the blank test flasks (without algal cells). Hydrolysis of the principal active in test item to the associated monoester was observed in analysis by LC-MS.