Propazine

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:

4 March 2010 to 28 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to internationally accepted guidelines and to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Propazine

EC Number:

205-359-9

EC Name:

Propazine

Cas Number:

139-40-2

Molecular formula:

C9H16ClN5

IUPAC Name:

6-chloro-N2,N4-bis(propan-2-yl)-1,3,5-triazine-2,4-diamine

Details on test material:

- Name of test material (as cited in study report): Propazine
- Physical state: White crystalline powder
- Analytical purity: 98.4%
- Purity test date: 23 November 2009
- Lot/batch No.: 09230119
- Expiration date of the lot/batch: 27 October 2014
- Storage condition of test material: Room temperature, in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.00358, 0.0114, 0.0366, 0.117, 0.375 mg/L
- Sampling method: 10 mL removed from medium and diluted x2 with methanol
- Reserve samples were stored in a freezer in case further analysis was required.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance was dissolved in methanol using ultrasound, then serially diluted to create solvent stock solutions at 2.34, 0.732, 0.228 and 0.0716 mg/mL. A 50 μL aliquot of the appropriate stock solution was added to 1L of dilution medium.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture and incubated for approximately three days in an orbital incubator at 21 to 25°C. Appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures which were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 1.37 x 10^6 cells/mL.

- Any deformed or abnormal cells observed: None

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.7 to 24.1 °C

pH:

7.43 to 8.88

Dissolved oxygen:

not measured

Nominal and measured concentrations:

Nominal: 0.00358, 0.0114, 0.0366, 0.117, 0.375 mg/L
Measured: 0.00351, 0.0115, 0.0355, 0.118, 0.369 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks, loosely stoppered with foam bungs
- Fill volume: 100 mL
- Aeration: Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker
- Initial cells density: 8430 cells/mL
- Control end cells density: 1272813 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 6642 to 6738 lux provided by 30 W “cool white” 1 metre fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities measured at 24 hour intervals using a haemacytometer (Improved Neubauer)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
The initial formulation trial and rangefinding test were conducted assuming an aqueous solubility of 5 mg/L and the use of solvent at a rate of 100 μL/L. Results of trials and rangefinding tests indicated that a solvent stock solution was only achievable using ultrasound treated stock solutions of methanol at a maximum concentration of 7.5 mg/mL. The solvent stock solutions were then added to OECD medium at a rate of 50 μL/L, in order to minimise bacterial growth, which was seen in the initial rangefinding tests where 100 μL/L solvent was used and had an adverse effect on algal growth. The maximum concentration then achievable using this formulation method was 0.375 mg/L.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.362 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.295-0.472 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.143 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 0.0921 - 0.241 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.118 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: biomass and growth rate

Details on results:

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

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The following values were derived from the data:

Area under the growth curve
EbC10 (72 h) : 0.0591 mg/L (95% confidence limits, 0.0156 & 0.119 mg/L)
EbC50 (72 h) : 0.143 mg/L (95% confidence limits, 0.0921 & 0.241 mg/L)
No observed effect concentration (NOEC) : 0.118 mg/L
Lowest observed effect concentration (LOEC) : 0.369 mg/L

Average specific growth rate
ErC10 (0 - 72 h) : 0.12 mg/L (95% confidence limits, 0.0504 & 0.326 mg/L)
ErC50 (0 - 72 h) : 0.362 mg/L (95% confidence limits, 0.295 & 0.472 mg/L)
No observed effect concentration (NOEC) : 0.118 mg/L
Lowest observed effect concentration (LOEC) : 0.369 mg/L

Yield
EyC10 (0 - 72 h) : 0.0479 mg/L (95% confidence limits: 0.0113 & 0.112 mg/L)
EyC50 (0 - 72 h) : 0.126 mg/L (95% confidence limits, 0.0717 & 0.216 mg/L)
No observed effect concentration (NOEC) : 0.0355 mg/L
Lowest observed effect concentration (LOEC) : 0.118 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to propazine, the EbC50, ErC50 and EyC50 respectively, were 0.143, 0.362 and 0.126 mg/L.
The “no observed effect concentration” (NOEC) for area under the growth curve and growth rate was 0.118 mg/L, and for yield was 0.0355 mg/L.

Executive summary:

In an Algal Growth Inhibition Assay, conducted in accordance with EU method C.3 (Algal Inhibition Test) and OECD Guideline 201 (Alga, Growth Inhibition Test), the unicellular green alga Pseudokirchnerella subcapitata was exposed to propazine for 72 hours.

Values for EbC50, ErC50 and EyC50 respectively, were 0.143, 0.362 and 0.126 mg/L

The “no observed effect concentration” (NOEC) for area under the growth curve and growth rate was 0.118 mg/L, and for yield was 0.0355 mg/L.