3-methyl-6-(p-toluidino)-3H-dibenz[f,ij]isoquinoline-2,7-dione

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

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling schedule:
- Control : at 72 hours
- Test concentration/s : at 0, 24, 48 and 72 hours
- Stability of test concentration/s during exposure: Examined by chemical analysis (HPLC) at 0, 24, 48 and 72 hours

Vehicle:

no

Details on test solutions:

200.0 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. The pH was measured to be 7.8.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Strain: Strain No. 86.81 SAG
- Source: 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 (according to BRINGMANN & KÜHN (1977) was renewed once a week; cell density measurements were made using a Particle and Count Size Analyzer, Z2 (Beckman Coulter)
- 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 to make up to a final cell density of 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 mg/L CaCO3)

Test temperature:

21-24 °C

pH:

7.8-10

Dissolved oxygen:

n.a.

Salinity:

n.a.

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentration: 100 mg/L; After 72 hours the values were below or at the limit of quantification (0.0016 mg/L); Effective concentrations correspond to 0.18% of nominal values at 0 hours, ranged from 0.13 % to 0.15 % of nominal values at 24 hours, from 0.079% to 0.12 % of nominal values at 48 hours and from 0.038 % to 0.12 % of nominal values at 72 hours.

Details on test conditions:

EXPOSURE CONDITIONS:
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers, test volume 100 mL
- Culturing apparatus: Light chamber in which a temperature in the range 21 °C to 24 °C was maintained, and continuous uniform illumination was provided in the spectral range 400 to 700 nm
- Light intensity: A light intensity in the range 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lx, 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. are determined by means of a microscopic counting chamber 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 highest test concentration without algae
- Test item concentration: 100 mg/L
- Method of administration: direct weighing
- Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r], respectively, of the algal population

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/L (nominal)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/L (nominal)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/L (nominal)

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/L (nominal)

Details on results:

The results are expressed in terms of nominal concentrations and geometric mean measured values. Effective concentrations correspond to 0.18% of nominal values at 0 hours, ranged from 0.13 % to 0.15 % of nominal values at 24 hours, from 0.079% to 0.12 % of nominal values at 48 hours and from 0.038 % to 0.12 % of nominal values at 72 hours.

Reported statistics and error estimates:

After 72 h, the coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) exceeded the validity criterion of 35%.

Validity criteria fulfilled:

yes

Remarks:

Factor of the biomass parameter (120), measured in the control between 0 and 72 h, was at least 16; the mean coefficient of variation (15.3%) was not higher than 35%; the coefficient of variation of the mean specific growth rate (1.4%) did not exceed 7%

Conclusions:

No toxic effects against algae were observed at the limit of water solubility under exposure conditions. The results are expressed in terms of nominal concentrations and geometric mean measured values. Effective concentrations correspond to 0.18% of nominal values at 0 hours, ranged from 0.13 % to 0.15 % of nominal values at 24 hours, from 0.079% to 0.12 % of nominal values at 48 hours and from 0.038 % to 0.12 % of nominal values at 72 hours.

Executive summary:

The study was conducted in accordance with 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 'Alga, Growth Inhibition Test' (2006). The purpose of this method was to determine those concentrations which caused a 10 % and a 50 % adverse effect (EC 10, EC 50) or, if conducted as a limit test, to determine the adverse effects at a maximum test concentration of 100 mg/L or at the limit of water solubility. Effect data are expressed on the basis of growth rate [r] (and yield [y]). A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range finding test: 1.0, 10 and 100 mg/L (nominal). The test item was treated for 1 hour in an ultrasonic bath and stirred for 24 hours on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 -12 μm. In the main test, the algae were exposed to the test item added to dilution water at a limit concentration at the limit of water solubility under test conditions for a period of 72 hours. 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.

No toxic effect at against algae at the limit of water solubility under exposure conditions. The results are expressed in terms of nominal concentrations and geometric mean measured values. Effective concentrations correspond to 0.18% of nominal values at 0 hours, ranged from 0.13 % to 0.15 % of nominal values at 24 hours, from 0.079% to 0.12 % of nominal values at 48 hours and from 0.038 % to 0.12 % of nominal values at 72 hours. This toxicity study is classified as acceptable and satisfies the guideline requirements for the algae study.

Description of key information

No toxic effects against algae were observed at the limit of water solubility under exposure conditions. The results are expressed in terms of nominal concentrations and geometric mean measured values. Effective concentrations correspond to 0.18% of nominal values at 0 hours, ranged from 0.13 % to 0.15 % of nominal values at 24 hours, from 0.079% to 0.12 % of nominal values at 48 hours and from 0.038 % to 0.12 % of nominal values at 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.14 mg/L

EC10 or NOEC for freshwater algae:

0.14 mg/L

Additional information

"Should read: 72 h-NOEC (growth rate): ≥ 0.14 mg/L; 72 h-EC 50 (growth rate): > 0.14 mg/L"

(The results are expressed in terms of geometric mean measured values)

Solvent red 52 is highly insoluble in water with a water solubility of lower than 0.003 mg/L. For details see chapter 4.8 "Water solubility".