2-Chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

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:

10 Mar - 27 Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Cas Number:

1211443-61-6

Molecular formula:

C14 H17 Cl N4 O

IUPAC Name:

2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for analysis were taken from all test concentrations and the control. In addition, filter containing undissolved residue was retained for analysis.
- Sampling method: Samples were taken at t=0 h and t=72 h at a volume of 2.0 mL. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/l applying a 2-day period of magnetic stirring followed by filtration through a 0.45 μm membrane filter (rc55, Whatman) to remove undissolved fraction. The obtained Water Soluble Fraction (WSF) was used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration.
- Eluate: no
- Differential loading: no
- Controls: blank control, reference control prepared with test medium
- Evidence of undissolved material: The final test solutions were all clear and colourless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. M1 was used as stock culture medium. The light intensity was 60 to 120 μE/m2/s (wavelength range of 400 to 700 nm).

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: M1 medium for culturing and M2 for test
- Any deformed or abnormal cells observed: no abnormalities observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21 - 23°C

pH:

Control: 7.9 - 8.1
Treatment: 8.0 - 7.9

Nominal and measured concentrations:

Nominal: Control, Solutions containing 1.0, 3.2, 10, 32 and 100% of the water-soluble fraction prepared at a loading rate of 100 mg/L
Measured: n.d (Control)., 1.03, 3.35, 10.1, 32.1 and 101 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Initial cells density: 1 x 10E+04 cells/mL
- Control end cells density: 238 x 10E+04 cells/mL (mean of six replicates)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Standard medium used: yes; M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: M1 medium for culturing and M2 for test
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. The temperature was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: TLD-lamps with a light intensity within the range of 91-94 µE m^-2 s^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). The spectrometer used was a Shimadzu Spectrophotometer UV-1800 including UVProbe 2.33 software.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Six replicates of exponentially growing algae were exposed to a control and a WSF prepared at a loading rate of 100 mg/L. Three replicates per concentration were exposed to solutions containing 1.0 and 10% of the WSF prepared at 100 mg/L.
- Results used to determine the conditions for the definitive study: A growth rate inhibition of 67% was observed at 100 mg/L (nominal loading rate). Based on the results of the range-finding test, the expected EC50 for growth rate inhibition was between 10 and 100 mg/L. A yield inhibition of 97% was observed at 100 mg/L (nominal loading rate). The expected EC50 for yield inhibition was between 1.0 and 10 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 17 -22 mg/L

Remarks:

analytically confirmed nominal concentration

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

6.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 5.8 - 8.1 mg/L

Remarks:

analytically confirmed nominal

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 3.2 - 5.0 mg/L

Remarks:

analytically confirmed nominal concentration

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 5.4 - 6.5 mg/L

Remarks:

analytically confirmed nominal concentration

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 3.1 - 4.1 mg/L

Remarks:

analytically confirmed nominal

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 2.3 - 3.2 mg/L

Remarks:

analytically confirmed nominal concentration

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: At the end of the final test microscopic observations were performed of the solution containing 10% of the WSF to observe for any abnormal appearance of the algae. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Concentrations: control, 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- ErC50 (72 h) = 1.3 mg/L (95% CI: 1.3 - 1.4 mg/L)

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment for growth inhibition and Williams Multiple Sequential t-test Procedure for yield inhibition, α=0.05, one-sided, smaller). Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test substance. The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Percentage inhibition of growth rate and yield (total test period) during the final test

Nominal concentration of the test substance (mg/L)	Number of replicates	Growth rate			Yield
		Mean	SD	% Inhibition	Mean	SD	% Inhibition
Control	6	1.823	0.0203	0.0	236.795	14.7325	0.0
1.0	3	1.796	0.0128	1.5*	217.888	8.3580	8.0*
3.2	3	1.773	0.0158	2.8*	203.039	9.5923	14*
10	3	1.266	0.0347	31*	43.817	4.7984	82*
32	3	0.576	0.0422	68*	4.653	0.7313	98*
100	3	0.237	0.0928	87*	1.092	0.5971	100*

* effect was statistically significant

Table 2: Concentrations of the test substance in test medium - final test

Time of sampling (h)	% of water-soluble fraction	Analysed concentration (mg/L)	Relative to initial
0	0	n.d.	-
	1.0	1.01	-
	3.2	3.30	-
	10	10.0	-
	10*	10.2
	32	31.8	-
	100	100	-
72	0	n.d.	n.a.
	1.0	1.03	102
	3.2	3.35	101
	10	10.1	101
	10*	10.5	103
	32	32.1	101
	100	101	101

* = without algae

Table 3: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	238	yes
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 must not exceed 35%	26%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.1%	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The EC50 for growth rate inhibition (72h-ERC50) was 19 mg/l with a 95% confidence interval ranging from 17 to 22 mg/l.
The EC10 for growth rate inhibition (72h-ERC10) was 4.1 mg/l with a 95% confidence interval ranging from 3.2 to 5.0 mg/l.