Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC/MS/MS analysis

Details on sampling:

Control: at 72 hours
Test concentration: 100 mg/L (nominal) at 0 and 72 hours

Vehicle:

no

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations
- 199.8 mg of the test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer
- undissolved particles of the test item were removed by filtration using a folded filter (pore size: 7 - 12 μm) and an aseptic filter Sartobran sterile capsules (pore size: 0.45 μm + 0.2 μm)
- pH was measured to be 7.9
- to produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scene-desmus subspicatus) Strain No. 86.81 SAG
- Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium
- final cell density: about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

1.3 dH (= 22.5 CaCO3)

Test temperature:

21 - 24 °C

pH:

7.8 - 8.9

Nominal and measured concentrations:

nominal: 100 mg/L
measured: < 0.003 mg/L

Details on test conditions:

Exposure conditions
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers test volume: 100 mL
- Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily.
- Light intensity: A light intensity ranging from 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced
- Experimental design: 1 test concentration plus 1 control, 6 replicates per concentration, 6 replicates per control Initial cell density in the test cultures approximately 5000 cells per millilitre; additionally test concentration without algae
- Test item concentration: 100 mg/L (nominal)
- Method of administration: direct weighing
- Duration of exposure: 72 hours
- Criteria of effects: item-induced inhibition of yield [y] and growth rate [r] of the algal population

Calculations:
- calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10)
- for the calculations all algae counts were divided by a factor of 10000

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.003 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.003 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.003 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.003 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

nominal:
- NOEC >= 100 mg/L
- LOEC, EC10, EC50 > 100 mg/L

Validity criteria fulfilled:

yes

Remarks:

cell density (control) increased by a factor of >= 16 within 72 hours; mean coefficient of variation (CV) for section-by-section specific growth rates in control <= 35 %; CV of average specific growth rates during test period in replicate control <= 7 %

Conclusions:

No toxic effects of Macrolex Rot B against algae were observed at the limit of water solubility under exposure conditions (nominal 100 mg/L, measured < 0.003 mg/L).

Executive summary:

The toxicity of Macrolex Rot B to algae Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations was investigated in a static test according to Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006). A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test.

Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of Macrolex Rot B dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer, a folded filter and an aseptic filter. At this concentration no inhibition of algal growth rate and yield was observed at the end of the 72 hour study period. Cell densities were recorded at 24 hour intervals. All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000. During the test a temperature range of 21 - 24 °C was maintained in the test vessels. The pH was measured at the beginning of the test and after 72 hours of exposure.

The maintenance of the test item concentration was proved by analytical measurements. In order to avoid an impairment of the test system, an additional replicate was used for analysis and pH measurement at the beginning of the test. Chemical analysis and pH measurement at the end of the test were performed using a replicate of the test concentrations and the control vessels. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the limit test concentration without algae was run in parallel to the test concentration.

Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) STUDENT-t test for Homogeneous Variances. After 72 h the following values were determined [mg/L] for Growth Rate EC50 > 0.003, EC10 > 0.003, NOEC ≥ 0.003, LOEC > 0.003. The results are expressed in terms of nominal concentrations and analytical values. After 0 and 72 hours the values were below the quantification limit of the HPLC/MS/MS analysis (0.003 mg/L).

No toxic effects against algae were observed at the limit of water solubility under exposure conditions.

Description of key information

No toxic effects of Macrolex Rot B against algae were observed at the limit of water solubility under exposure conditions (nominal 100 mg/L, measured < 0.003 mg/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.003 mg/L

EC10 or NOEC for freshwater algae:

0.003 mg/L

Additional information

Should read:

EC50 > 0.003 mg/L

EC10 > 0.003 mg/L

NOEC > 0.003 mg/L